labeled cytotoxic t-cells: Topics by Science.gov

Sample records for labeled cytotoxic t-cells

HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

PubMed

Lewis, D E; Yang, L; Luo, W; Wang, X; Rodgers, J R

1999-06-18

To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.
Cytotoxic Mechanisms Employed by Mouse T Cells to Destroy Pancreatic β-Cells

PubMed Central

Varanasi, Vineeth; Avanesyan, Lia; Schumann, Desiree M.; Chervonsky, Alexander V.

2012-01-01

Several cytotoxic mechanisms have been attributed to T cells participating in β-cell death in type 1 diabetes. However, sensitivity of β-cells to these mechanisms in vitro and in vivo is likely to be different. Moreover, CD4+ and CD8+ T cells may use distinct mechanisms to cause β-cell demise that possibly involve activation of third-party cytotoxic cells. We used the transfer of genetically modified diabetogenic T cells into normal, mutant, and bone marrow chimeric recipients to test the contribution of major cytotoxic mechanisms in β-cell death. We found that 1) the killing of β-cells by CD4+ T cells required activation of the recipient’s own cytotoxic cells via tumor necrosis factor-α (TNF-α); 2) CD8+ T-cell cytotoxic mechanisms destroying β-cells were limited to perforin and Fas ligand, as double knockouts of these molecules abrogated the ability of T cells to cause diabetes; and 3) individual CD8+ T-cell clones chose their cytotoxic weaponry by a yet unknown mechanism and destroyed their targets via either Fas-independent or Fas-dependent (∼40% of clones) pathways. Fas-dependent destruction was assisted by TNF-α. PMID:22773667
An early history of T cell-mediated cytotoxicity.

PubMed

Golstein, Pierre; Griffiths, Gillian M

2018-04-16

After 60 years of intense fundamental research into T cell-mediated cytotoxicity, we have gained a detailed knowledge of the cells involved, specific recognition mechanisms and post-recognition perforin-granzyme-based and FAS-based molecular mechanisms. What could not be anticipated at the outset was how discovery of the mechanisms regulating the activation and function of cytotoxic T cells would lead to new developments in cancer immunotherapy. Given the profound recent interest in therapeutic manipulation of cytotoxic T cell responses, it is an opportune time to look back on the early history of the field. This Timeline describes how the early findings occurred and eventually led to current therapeutic applications.
Characterization of CD4+ T cell-mediated cytotoxicity in patients with multiple myeloma.

PubMed

Zhang, Xiaole; Gao, Lei; Meng, Kai; Han, Chunting; Li, Qiang; Feng, Zhenjun; Chen, Lei

2018-05-01

Multiple myeloma (MM) is an incurable cancer characterized by the development of malignant plasma cells. The CD8 T cell-mediated cytotoxicity is considered a major player in antitumor immunity, but in MM patients, the CD8 T cells displayed senescence markers and were functionally impaired. To investigate whether cytotoxic CD4 T cells could act as a treatment alternative in MM, we examined the frequency and function of naturally occurring cytotoxic CD4 T cells in MM patients. The cytotoxic CD4 T cells were identified as granzyme-A, granzyme B-, and perforin-expressing CD4 T cells, and their frequencies were significantly upregulated in MM patients when compared with healthy controls. The frequencies of cytotoxic CD4 T cells in MM patients were not associated with the frequencies of cytotoxic CD8 T cells, but were negatively associated with disease severity. Interestingly, the expression levels of inhibitory molecules, including PD-1 and CTLA-4, were significantly lower in cytotoxic CD4 T cells than in cytotoxic CD8 T cells. When co-incubated with autologous CD38 + CD138 + plasma cells, CD4 T cells were capable of eliminating plasma cells with varying degrees of efficacy. In MM patients, the frequency of circulating plasma cells was negatively correlated with the frequency of cytotoxic CD4 T cells. Therefore, CD4 T cell-mediated cytotoxicity existed naturally in MM patients and could potentially act as an option in antitumor therapies. Copyright © 2018 Elsevier Inc. All rights reserved.
HBV-specific CD4+ cytotoxic T cells in hepatocellular carcinoma are less cytolytic toward tumor cells and suppress CD8+ T cell-mediated antitumor immunity.

PubMed

Meng, Fanzhi; Zhen, Shoumei; Song, Bin

2017-08-01

In East Asia and sub-Saharan Africa, chronic infection is the main cause of the development of hepatocellular carcinoma, an aggressive cancer with low survival rate. Cytotoxic T cell-based immunotherapy is a promising treatment strategy. Here, we investigated the possibility of using HBV-specific CD4 + cytotoxic T cells to eliminate tumor cells. The naturally occurring HBV-specific cytotoxic CD4 + and CD8 + T cells were identified by HBV peptide pool stimulation. We found that in HBV-induced hepatocellular carcinoma patients, the HBV-specific cytotoxic CD4 + T cells and cytotoxic CD8 + T cells were present at similar numbers. But compared to the CD8 + cytotoxic T cells, the CD4 + cytotoxic T cells secreted less cytolytic factors granzyme A (GzmA) and granzyme B (GzmB), and were less effective at eliminating tumor cells. In addition, despite being able to secrete cytolytic factors, CD4 + T cells suppressed the cytotoxicity mediated by CD8 + T cells, even when CD4 + CD25 + regulator T cells were absent. Interestingly, we found that interleukin 10 (IL-10)-secreting Tr1 cells were enriched in the cytotoxic CD4 + T cells. Neutralization of IL-10 abrogated the suppression of CD8 + T cells by CD4 + CD25 - T cells. Neither the frequency nor the absolute number of HBV-specific CD4 + cytotoxic T cells were correlated with the clinical outcome of advanced stage hepatocellular carcinoma patients. Together, this study demonstrated that in HBV-related hepatocellular carcinoma, CD4 + T cell-mediated cytotoxicity was present naturally in the host and had the potential to exert antitumor immunity, but its capacity was limited and was associated with immunoregulatory properties. © 2017 APMIS. Published by John Wiley & Sons Ltd.
Role of T-bet, the master regulator of Th1 cells, in the cytotoxicity of murine CD4+ T cells.

PubMed

Eshima, Koji; Misawa, Kana; Ohashi, Chihiro; Iwabuchi, Kazuya

2018-05-01

Although CD4 + T cells are generally regarded as helper T cells, some activated CD4 + T cells have cytotoxic properties. Given that CD4 + cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4 + T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4 + T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4 + T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4 + T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4 + T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas - target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells. © 2018 The Societies and John Wiley & Sons Australia, Ltd.
Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

PubMed

Akhmetzyanova, Ilseyar; Zelinskyy, Gennadiy; Schimmer, Simone; Brandau, Sven; Altenhoff, Petra; Sparwasser, Tim; Dittmer, Ulf

2013-02-01

The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.
Peptide-Induced Antiviral Protection by Cytotoxic T Cells

NASA Astrophysics Data System (ADS)

Schulz, Manfred; Zinkernagel, Rolf M.; Hengartner, Hans

1991-02-01

A specific antiviral cytotoxic immune response in vivo could be induced by the subcutaneous injection of the T-cell epitope of the lymphocytic choriomeningitis virus (LCMV) nucleoprotein as an unmodified free synthetic peptide (Arg-Pro-Gln-Ala-Ser-Gly-Val-Tyr-Met-Gly-Asn-Leu-Thr-Ala-Gln) emulsified in incomplete Freund's adjuvant. This immunization rendered mice into a LCMV-specific protective state as shown by the inhibition of LCMV replication in spleens of such mice. The protection level of these mice correlated with the ability to respond to the peptide challenge by CD8^+ virus-specific cytotoxic T cells. This is a direct demonstration that peptide vaccines can be antivirally protective in vivo, thus encouraging further search for appropriate mixtures of stable peptides that may be used as T-cell vaccines.
Mechanisms of Dihydroartemisinin and Dihydroartemisinin/Holotransferrin Cytotoxicity in T-Cell Lymphoma Cells

PubMed Central

Zhao, Xindong; Zhao, Chunting; Zhao, Hongguo; Huo, Lanfen

2015-01-01

The validated therapeutic effects of dihydroartemisinin (DHA) in solid tumors have encouraged us to explore its potential in treating T-cell lymphoma. We found that Jurkat cells (a T-cell lyphoma cell line) were sensitive to DHA treatment with a IC50 of dihydroartemisinin. The cytotoxic effect of DHA in Jurkat cells showed a dose- and time- dependent manner. Interestingly, the cytotoxic effect of DHA was further enhanced by holotransferrin (HTF) due to the high expression of transferrin receptors in T-cell lymphoma. Mechanistically, DHA significantly increased the production of intracellular reactive oxygen species, which led to cell cycle arrest and apoptosis. The DHA treatment also inhibited the expression of protumorgenic factors including VEGF and telomerase catalytic subunit. Our results have proved the therapeutic effect of DHA in T-cell lymphoma. Especially in combination with HTF, DHA may provide a novel efficient approach in combating the deadly disease. PMID:26502166
IFNγ enhances cytotoxic efficiency of the cytotoxic T lymphocytes against human glioma cells.

PubMed

Shao, Shengwen; Risch, Eric; Burner, Danielle; Lu, Lingeng; Minev, Boris; Ma, Wenxue

2017-06-01

Cytotoxic T lymphocytes (CTLs) are a key player in cancer immunotherapies, and MHC class I molecules on the cell surface are crucial for cellular recognition. However, the aberrant expression of MHC class I molecules is frequently found in various malignancies. IFNγ has dual functions in cancer progression, and its effect on tumor immunity is controversial. To investigate whether IFNγ can enhance cytotoxic efficiency of the tumor antigen-specific CTLs, we generated the CTLs using modified human dendritic cells as antigen presenting cells, then studied the activities of CTLs on human leukocyte antigen (HLA)-A2 positive glioma cells treated with, or without IFNγ. The results from both ELISpot and cytotoxicity assays demonstrated that the CTLs recognized and eliminated the HLA-A2 positive glioma cells treated with IFNγ more effectively when compared to the glioma cells deprived of IFNγ treatment. In addition, in vitro experiments showed that the levels of MHC class I molecules were upregulated in all of the HLA-A2 positive glioma cells. Using the publicly accessed TCGA data of low-grade glioma, we found significantly positive associations between IFNγ and both MHC class I molecules and CD8 + T cell activation score (p<0.0001). Furthermore, we found a significantly reduced risk of death in the glioma patients with high T cell activation score in comparison to those with low score (p=0.022). These findings suggest that a clinical application of IFNγ treatment may have potential benefits. Copyright © 2017. Published by Elsevier B.V.
The mechanism of T-cell mediated cytotoxicity. VI. T-cell projections and their role in target cell killing.

PubMed Central

Sanderson, C J; Glauert, A M

1979-01-01

Electron micrographs of material fixed during the first 10 min of a T-cell cytotoxic system showed T-cell projections and T-cell burrowing into target cells. These observations were made possible by using a system with a very high rate of killing. The projections vary in shape and size, and can push deeply into the target cell, distorting organelles in their path, including the nucleus. The projections contain fine fibrillar material, to the exclusion of organelles. They push the target cell membrane in front of them to form pockets approximating to the shape of the projection. Areas of close contact occur between the projections and the target cell membrane, particularly at the leading edges. The likelihood that these projections develop as a result of contact with specific antigen, and are involved in the cytotoxic mechanism is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 PMID:311336
CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma.

PubMed

Zhou, Yong; Zha, Jie; Lin, Zhijuan; Fang, Zhihong; Zeng, Hanyan; Zhao, Jintao; Luo, Yiming; Li, Zhifeng; Xu, Bing

2018-01-15

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4 + T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19 + cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19 + cells than patients who did not show recurrence. Examining cytotoxic CD4 + T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4 + T cells. Also, frequency of cytotoxic CD4 + T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4 + T cells against autologous CD19 + cells was investigated. We found that the cytotoxic potential of CD4 + T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19 + cells presented a significant reduction after longer incubation with cytotoxic CD4 + T cells, suggesting that cytotoxic CD4 + T cells preferentially eliminated MHC II-expressing CD19 + cells. Blocking MHC II on CD19 + cells significantly reduced the cytolytic capacity of CD4 + T cells. Despite these discoveries, the frequency of cytotoxic CD4 + T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4 + T cells presented an MHC II-dependent cytotoxic potential against autologous CD19 + cells and could potentially represent a future treatment option for DLBCL. Copyright © 2017 Elsevier Inc. All rights reserved.
Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators.

PubMed

Sørensen, Rikke Baek; Hadrup, Sine Reker; Svane, Inge Marie; Hjortsø, Mads Christian; Thor Straten, Per; Andersen, Mads Hald

2011-02-17

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in normal and pathologic settings. Here, we describe that spontaneous cytotoxic T-cell reactivity against IDO exists not only in patients with cancer but also in healthy persons. We show that the presence of such IDO-specific CD8(+) T cells boosted T-cell immunity against viral or tumor-associated antigens by eliminating IDO(+) suppressive cells. This had profound effects on the balance between interleukin-17 (IL-17)-producing CD4(+) T cells and regulatory T cells. Furthermore, this caused an increase in the production of the proinflammatory cytokines IL-6 and tumor necrosis factor-α while decreasing the IL-10 production. Finally, the addition of IDO-inducing agents (ie, the TLR9 ligand cytosine-phosphate-guanosine, soluble cytotoxic T lymphocyte-associated antigen 4, or interferon γ) induced IDO-specific T cells among peripheral blood mononuclear cells from patients with cancer as well as healthy donors. In the clinical setting, IDO may serve as an important and widely applicable target for immunotherapeutic strategies in which IDO plays a significant regulatory role. We describe for the first time effector T cells with a general regulatory function that may play a vital role for the mounting or maintaining of an effective adaptive immune response. We suggest terming such effector T cells "supporter T cells."
Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy.

PubMed

Yoshikawa, Toshiaki; Takahara, Masashi; Tomiyama, Mai; Nieda, Mie; Maekawa, Ryuji; Nakatsura, Tetsuya

2014-11-01

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3‑specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.
Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

NASA Astrophysics Data System (ADS)

Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

1994-06-01

IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.
Modulation of CD4(+) T cell-dependent specific cytotoxic CD8(+) T cells differentiation and proliferation by the timing of increase in the pathogen load.

PubMed

Tzelepis, Fanny; Persechini, Pedro M; Rodrigues, Mauricio M

2007-04-25

Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.
Epidermal Cadm1 expression promotes autoimmune alopecia via enhanced T cell adhesion and cytotoxicity.

PubMed

Giangreco, Adam; Hoste, Esther; Takai, Yoshimi; Rosewell, Ian; Watt, Fiona M

2012-02-01

Autoimmune alopecia is characterized by an extensive epidermal T cell infiltrate that mediates hair follicle destruction. We have investigated the role of cell adhesion molecule 1 (Cadm1; Necl2) in this disease. Cadm1 is expressed by epidermal cells and mediates heterotypic adhesion to lymphocytes expressing class 1-restricted T cell-associated molecule (CRTAM). Using a murine autoimmune alopecia model, we observed an increase in early-activated cytotoxic (CD8-restricted, CRTAM-expressing) T cells, which preferentially associated with hair follicle keratinocytes expressing Cadm1. Coculture with Cadm1-transduced MHC-matched APCs stimulated alopecic lymph node cells to release IL-2 and IFN-γ. Overexpression of Cadm1 in cultured human keratinocytes did not promote cytokine secretion, but led to increased adhesion of alopecic cytotoxic T cells and enhanced T cell cytotoxicity in an MHC-independent manner. Epidermal overexpression of Cadm1 in transgenic mice led to increased autoimmune alopecia susceptibility relative to nontransgenic littermate controls. Our findings reveal that Cadm1 expression in the hair follicle plays a role in autoimmune alopecia.
Interleukin-17A Promotes CD8+ T Cell Cytotoxicity To Facilitate West Nile Virus Clearance.

PubMed

Acharya, Dhiraj; Wang, Penghua; Paul, Amber M; Dai, Jianfeng; Gate, David; Lowery, Jordan E; Stokic, Dobrivoje S; Leis, A Arturo; Flavell, Richard A; Town, Terrence; Fikrig, Erol; Bai, Fengwei

2017-01-01

CD8 + T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic-mediator gene expression and promoting CD8 + T cell cytotoxicity against WNV infection in mice. We find that IL-17A-deficient (Il17a -/- ) mice are more susceptible to WNV infection and develop a higher viral burden than wild-type (WT) mice. Interestingly, the CD8 + T cells isolated from Il17a -/- mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 postinfection, significantly reduces the viral burden and increases survival, suggesting a therapeutic potential for IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8 + T cell cytotoxicity, which may have broad implications in other microbial infections and cancers. Interleukin-17A (IL-17A) and CD8 + T cells regulate diverse immune functions in microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while CD8 + T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8 + T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8 + T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces the viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8 + T cell axis may also have broad implications for immunity to other microbial infections and cancers, where CD8 + T cell
Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

PubMed

Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.
Effect of irradiation on human T-cell proliferation: low dose irradiation stimulates mitogen-induced proliferation and function of the suppressor/cytotoxic T-cell subset.

PubMed

Gualde, N; Goodwin, J S

1984-04-01

Unfractionated human T cells exposed to 10-50 rad of X irradiation incorporated less [3H]thymidine than nonirradiated T cells when subsequently cultured with PHA or Con A. The cytotoxic/suppressor T-cell subset, isolated as either OKT8(+) or OKT4(-) cells, demonstrated significantly enhanced [3H]thymidine incorporation in PHA- or Con A-stimulated cultures after exposure to 10-50 rad, compared to unirradiated cells, while the proliferation of the OKT4(+) helper/inducer subset was inhibited by low dose irradiation. It has been previously reported that approximately 30% of the cytotoxic/suppressor subset also stains with OKM1. When the cytotoxic/suppressor subset was further subdivided into OKT4(-), OKM1(+), and OKT4(-), OKM1(-) cells, proliferation of the OKT4(-), OKM1(+) population was inhibited by exposure to 25 rad while proliferation of the OKT4(-), OKM1(-) population was stimulated. The increase in proliferation of the cytotoxic/suppressor T-cell subset after low dose irradiation is paralleled by an increase in suppressor activity of these cells. T cells exposed to 25 rad and then cultured with Con A for 48 hr caused greater inhibition of IgG production when added to fresh autologous lymphocytes stimulated by pokeweed mitogen than did unirradiated cells. Thus, low dose irradiation enhances both the proliferation and function of the human suppressor T-cell subset.

Fighting Viral Infections and Virus-Driven Tumors with Cytotoxic CD4+ T Cells

PubMed Central

Muraro, Elena; Merlo, Anna; Martorelli, Debora; Cangemi, Michela; Dalla Santa, Silvia; Dolcetti, Riccardo; Rosato, Antonio

2017-01-01

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors. PMID:28289418
CRTAM determines the CD4+ cytotoxic T lymphocyte lineage

PubMed Central

Takeuchi, Arata; Badr, Mohamed El Sherif Gadelhaq; Miyauchi, Kosuke; Ishihara, Chitose; Onishi, Reiko; Guo, Zijin; Sasaki, Yoshiteru; Ike, Hiroshi; Takumi, Akiko; Tsuji, Noriko M.; Murakami, Yoshinori; Katakai, Tomoya; Kubo, Masato

2016-01-01

Naive T cells differentiate into various effector T cells, including CD4+ helper T cell subsets and CD8+ cytotoxic T cells (CTL). Although cytotoxic CD4+ T cells (CD4+CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4+ T cells that express class I–restricted T cell–associated molecule (CRTAM) upon activation possesses the characteristics of both CD4+ and CD8+ T cells. CRTAM+ CD4+ T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM+ T cells are the precursor of CD4+CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4+CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM+ T cells traffic to mucosal tissues and inflammatory sites and developed into CD4+CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4+CTL through the induction of Eomes and CTL-related gene. PMID:26694968
Effect of irradiation on human T-cell proliferation: low dose irradiation stimulates mitogen-induced proliferation and function of the suppressor/cytotoxic T-cell subset

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gualde, N.; Goodwin, J.S.

1984-04-01

Unfractionated human T cells exposed to 10-50 rad of X irradiation incorporated less (/sup 3/H)thymidine than nonirradiated T cells when subsequently cultured with PHA or Con A. The cytotoxic/suppressor T-cell subset, isolated as either OKT8(+) or OKT4(-) cells, demonstrated significantly enhanced (/sup 3/H)thymidine incorporation in PHA- or Con A-stimulated cultures after exposure to 10-50 rad, compared to unirradiated cells, while the proliferation of the OKT4(+) helper/inducer subset was inhibited by low dose irradiation. It has been previously reported that approximately 30% of the cytotoxic/suppressor subset also stains with OKM1. When the cytotoxic/suppressor subset was further subdivided into OKT4(-), OKM1(+), andmore » OKT4(-), OKM1(-) cells, proliferation of the OKT4(-), OKM1(+) population was inhibited by exposure to 25 rad while proliferation of the OKT4(-), OKM1(-) population was stimulated. The increase in proliferation of the cytotoxic/suppressor T-cell subset after low dose irradiation is paralleled by an increase in suppressor activity of these cells. T cells exposed to 25 rad and then cultured with Con A for 48 hr caused greater inhibition of IgG production when added to fresh autologous lymphocytes stimulated by pokeweed mitogen than did unirradiated cells. Thus, low dose irradiation enhances both the proliferation and function of the human suppressor T-cell subset.« less
Cytotoxic T cells use mechanical force to potentiate target cell killing

PubMed Central

Basu, Roshni; Whitlock, Benjamin M.; Husson, Julien; Le Floc’h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C.; Huse, Morgan

2016-01-01

SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. PMID:26924577
Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

PubMed

Basu, Roshni; Whitlock, Benjamin M; Husson, Julien; Le Floc'h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C; Huse, Morgan

2016-03-24

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. Copyright © 2016 Elsevier Inc. All rights reserved.
In vitro cytotoxicity of CD8+ T cells in multi-drug-resistant tuberculosis. A preliminary report.

PubMed

Sada-Ovalle, Isabel; Torre-Bouscoulet, Luis; Valdez-Vázquez, Rafael; Lascurain, Ricardo

2009-05-01

Specific CD8+ T-cell cytotoxicity has been recognized as being involved in the elimination of drug-susceptible tuberculosis (DS-TB). Given that there is currently no information on the cytotoxic effector functions of CD8+ T cells in multi-drug-resistant tuberculosis (MDR-TB), our objective was to analyse the cytotoxic activity, both basal and stimulated, of CD8+ T cells from MDR-TB patients and compare it with that of DS-TB patients, as well as purified protein derivative (PPD)+ and PPD- subjects. Cytotoxic activity of CD8+ T cells from MDR-TB patients, DS-TB patients, PPD+ and PPD- subjects was measured by a colorimetric assay, using H37Rv culture filtrate protein as the antigenic stimulus. Twenty-eight subjects were studied (7 MDR-TB patients, 7 DS-TB patients, 7 PPD+ subjects and 7 PPD- subjects). In the presence of the antigenic stimulus, the cytotoxic activity of CD8+ T cells from MDR-TB patients (% lysis) increased from 6.7% to 59.6% (P < 0.001). In DS-TB patients lysis increased from 3.2% to 22.5% (P < 0.001), whereas in PPD+ subjects it increased from 2.7% to 12.0% (P < 0.001) and in PPD- subjects from 1.3% to 3.2% (P < 0.001). Basal cytotoxic activity was significantly higher for MDR-TB patients than PPD+ and PPD- subjects (P = 0.003), but not compared with that for DS-TB patients (P = 0.05). Stimulated cytotoxic activity was highest for MDR-TB patients. CD8+ T cells from MDR-TB patients showed an exaggerated cytotoxic activity after antigenic stimulation. Further studies are required to elucidate the role of this response in the immunopathogenesis of MDR-TB.
T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

PubMed

Chapuis, Aude G; Roberts, Ilana M; Thompson, John A; Margolin, Kim A; Bhatia, Shailender; Lee, Sylvia M; Sloan, Heather L; Lai, Ivy P; Farrar, Erik A; Wagener, Felecia; Shibuya, Kendall C; Cao, Jianhong; Wolchok, Jedd D; Greenberg, Philip D; Yee, Cassian

2016-11-01

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8 + T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses
Hepatocellular apoptosis associated with cytotoxic T/natural killer-cell infiltration in chronic active EBV infection.

PubMed

Nomura, Yuko; Kimura, Hiroshi; Karube, Kennosuke; Yoshida, Shiro; Sugita, Yasuo; Niino, Daisuke; Shimizu, Kei; Kimura, Yoshizo; Aoki, Ryosuke; Kiyasu, Junichi; Takeuchi, Masanori; Hashikawa, Keiko; Hirose, Shinichi; Ohshima, Koichi

2009-07-01

The aim of the present study was to identify the mechanism of hepatocellular apoptosis induced by EBV-infected cytotoxic T/natural killer (NK) cells in chronic active EBV infection (CAEBV). Eight patients with CAEBV were studied, and infected T-cell expansion and NK-cell expansion were detected in four patients each. Biopsy or necropsy was performed on lymph node, liver, or spleen, and each specimen was subjected to immunohistochemical double staining of CD3 plus caspase-3 with the addition of cytotoxic markers of T-cell restricted intracellular antigen-1 (TIA-1), perforin, and granzyme B, as well as EBV in situ hybridization (EBV-ISH). In the liver, some of the infiltrating CD3-positive lymphocytes stained positively for EBV-ISH and cytotoxic markers. Double staining of CD3 plus caspase-3 indicated caspase-3 positive hepatocytes with apoptotic features, accompanied by extensive infiltration of CD3-positive cells, which were directly attached to the apoptotic caspase-3 positive hepatocytes. In contrast, far fewer cells stained positive for caspase-3 in lymph node and spleen than in liver. The present findings suggest that in patients with CAEBV, cytotoxic T/NK cells may directly induce hepatocytes to undergo apoptosis more frequently than they do cells in other organs of the reticulo-endothelial system.
T-Cell Therapy Using Interleukin-21–Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression

PubMed Central

Chapuis, Aude G.; Roberts, Ilana M.; Thompson, John A.; Margolin, Kim A.; Bhatia, Shailender; Lee, Sylvia M.; Sloan, Heather L.; Lai, Ivy P.; Farrar, Erik A.; Wagener, Felecia; Shibuya, Kendall C.; Cao, Jianhong; Wolchok, Jedd D.; Greenberg, Philip D.

2016-01-01

Purpose Peripheral blood–derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti–CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses
Mitomycin C-treated or irradiated concanavalin A-activated T cells augment the activation of cytotoxic T cells in vivo.

PubMed

Moyers, C; Pottmeyer-Gerber, C; Gerber, M; Buszello, H; Dröge, W

1984-10-01

The activation of cytotoxic T lymphocytes (CTL) in vivo after immunization of normal or cyclophosphamide-treated mice with allogeneic cells was strongly augmented by the administration of mitomycin C-treated or irradiated concanavalin A-activated spleen cells (Con A-spl). This effect of the Con A-spl was abrogated by treatment with Anti-Thy 1 antibody plus complement, and was therefore presumably mediated by activated "helper" T cells. (The term "helper" cell is only operationally defined in this context and indicates that the augmenting irradiation resistant T cells are obviously not CTL precursor cells). These observations indicated (i) that even the cytotoxic response against allogeneic stimulator cells suffers in vivo from insufficient "helper" T cell activity, and (ii) that the injection of Con A-spl may serve as a simple procedure to apply this "helper" activity in vivo. This procedure was at least as effective as the repeated injection of interleukin 2 (IL-2)-containing cell supernatants with up to four 30-unit doses of IL-2 per mouse. IL-2-containing cell supernatants were found to mediate similar effects only if injected into the footpads but not intravenously. This was in line with the reported observation that IL-2 has an extremely short half-life in vivo. The injection of Con A-spl was also found to augment the proliferative response in the regional lymph nodes.
Characterization, in vitro cytotoxicity assessment, and in vivo visualization of multimodal, RITC-labeled, silica-coated magnetic nanoparticles for labeling human cord blood-derived mesenchymal stem cells.

PubMed

Park, Ki-Soo; Tae, Jinsung; Choi, Bongkum; Kim, Young-Seok; Moon, Cheol; Kim, Sa-Hyun; Lee, Han-Sin; Kim, Jinhyun; Kim, Junsung; Park, Jaeberm; Lee, Jung-Hee; Lee, Jong Eun; Joh, Jae-Won; Kim, Sungjoo

2010-04-01

Live imaging is a powerful technique that can be used to characterize the fate and location of stem cells in animal models. Here we investigated the characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, MNPs@SiO2(RITC). We also conducted various in vivo-uptake tests with nanoparticle-labeled human MSCs. MNPs@SiO2(RITC) showed photostability against ultraviolet light exposure and were nontoxic to human MSCs, based on the MTT, apoptosis, and cell cycle arrest assays. In addition, MNPs@SiO2(RITC) did not affect the surface phenotype or morphology of human MSCs. We also demonstrated that MNPs@SiO2(RITC) have stable retention properties in MSCs in vitro. Furthermore, using optical and magnetic resonance imaging, we successfully detected a visible signal from labeled human MSCs that were transplanted into NOD.CB17-Prkdc(SCID) (NOD-SCID) mice. These results demonstrate that MNPs@SiO2(RITC) are biocompatible and useful tools for human MSC labeling and bioimaging. The characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, RITC were investigated in this study. RITC showed photostability against ultraviolet light exposure and was nontoxic to human MSCs. Using both optical and magnetic resonance imaging, successful detection of signal from labeled human MSCs transplanted into mice is demonstrated. Copyright 2010 Elsevier Inc. All rights reserved.
Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

PubMed Central

Knies, Diana; Medenhoff, Sergej; Wabnitz, Guido; Luckner-Minden, Claudia; Feldmeyer, Nadja; Voss, Ralf-Holger; Kropf, Pascale; Müller, Ingrid; Conradi, Roland; Samstag, Yvonne; Theobald, Matthias; Ho, Anthony D.; Goldschmidt, Hartmut; Hundemer, Michael

2013-01-01

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency. PMID:23717444
Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells

PubMed Central

Backer, Ronald; Schwandt, Timo; Greuter, Mascha; Oosting, Marije; Jüngerkes, Frank; Tüting, Thomas; Boon, Louis; O’Toole, Tom; Kraal, Georg; Limmer, Andreas; den Haan, Joke M. M.

2009-01-01

The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for cross-presentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8+ DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8+ DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8+ T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity. PMID:20018690
Cytotoxic activity of ethanolic extract of the marine sponge Aaptos suberitoides against T47D cell

NASA Astrophysics Data System (ADS)

Nurhayati, Awik Puji Dyah; Prastiwi, Rarastoeti; Sukardiman, Wahyuningsih, Tri

2018-04-01

Aaptos suberitoides marine sponge produce many kinds of secondary metabolites. The purpose of this study were to examine the cytotoxic, proliferation inhibition and apoptosis induction of marine sponge A.suberitoides. The sponge was extracted with 96 % ethanol. Ethanol extract cytotoxicity assay were performed with MTT method (Microculture Tetrazolium) against to cell line of T47D. The proliferation inhibition were done by doubling time. The apoptosis induction by observing the treated cell morphology after staining with acrydine orange. The results show that cytotoxic activity of the ethanol extract was 153.109 µg/mL, inhibits cell proliferation cell lines of T47D at 24 hours of incubation and apoptosis induction.
Primary cutaneous CD8+ cytotoxic T-cell lymphoma involving the epidermis and subcutis in a young child.

PubMed

Wang, Lei; Gao, Tianwen; Wang, Gang

2015-04-01

CD8+ cytotoxic T-cell lymphoma involving the skin represents a heterogeneous group of diseases that include subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma, and 'type D' lymphomatoid papulosis. In this report, we describe a case of CD8+ cytotoxic T-cell lymphoma involving both the epidermis and subcutis. The patient was a 6-year-old girl who presented with a 3-year history of multiple plaques on her trunk and legs. The lesions had relapsed twice but responded well to prednisone. Histopathologic examination showed the proliferation of atypical lymphocytes in the epidermis, dermis and subcutaneous tissue. On immunohistochemical analysis, the atypical lymphocytes were positive for βF1, CD3, CD8, perforin, granzyme B and TIA-1, but negative for T-cell receptor (TCR) γ, CD4, CD30 and CD56. It was difficult to classify this tumor in terms of the known types of cutaneous lymphoma, and this case should be differentiated with subcutaneous panniculitis-like T-cell lymphoma and primary cutaneous aggressive epidermotropic CD8+ T-cell lymphoma. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

DOE PAGES

Chen, Yen-Wei; Drury, Jeanie L.; Moussi, Joelle; ...

2016-11-30

Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue ® assay was employed tomore » assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes.« less
Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

PubMed Central

Drury, Jeanie L.; Moussi, Joelle; Taylor-Pashow, Kathryn M. L.

2016-01-01

Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes. PMID:28044136
Downmodulation of the Inflammatory Response to Bacterial Infection by γδ T Cells Cytotoxic for Activated Macrophages

PubMed Central

Egan, Paul J.; Carding, Simon R.

2000-01-01

Although γδ T cells are involved in the regulation of inflammation after infection, their precise function is not known. Intraperitoneal infection of T cell receptor (TCR)-δ−/− mice with the intracellular bacterium Listeria monocytogenes resulted in the development of necrotic foci in the livers. In contrast, the peritoneal cavities of infected TCR-δ−/− mice contained an accumulation of low density activated macrophages and a reduced percentage of macrophages undergoing apoptosis. γδ T cell hybridomas derived from mice infected with Listeria were preferentially stimulated by low density macrophages from peritoneal exudates of infected mice. Furthermore, primary splenic γδ T cells isolated from Listeria-infected mice were cytotoxic for low density macrophages in vitro, and cytotoxicity was inhibited in the presence of antibodies to the γδ TCR. These results demonstrate a novel interaction between γδ T cells and activated macrophages in which γδ T cells are stimulated by terminally differentiated macrophages to acquire cytotoxic activity and which, in turn, induce macrophage cell death. This interaction suggests that γδ T cells regulate the inflammatory response to infection with intracellular pathogens by eliminating activated macrophages at the termination of the response. PMID:10859339
Interleukin 2 and interleukin 10 function synergistically to promote CD8+ T cell cytotoxicity, which is suppressed by regulatory T cells in breast cancer.

PubMed

Li, Xiaogang; Lu, Ping; Li, Bo; Zhang, Wanfu; Yang, Rong; Chu, Yan; Luo, Kaiyuan

2017-06-01

The precise role of interleukin (IL)-10 in breast cancer is not clear. Previous studies suggested a tumor-promoting role of IL-10 in breast cancer, whereas recent discoveries that IL-10 activated and expanded tumor-resident CD8 + T cells challenged the traditional view. Here, we investigated the role of IL-10 in HLA-A2-positive breast cancer patients with Grade III, Stage IIA or IIB in-situ and invasive ductal carcinoma, and compared it with that of IL-2, the canonical CD8 + T cell growth factor. We first observed that breast cancer patients presented higher serum levels of IL-2 and IL-10 than healthy controls. Upon prolonged TCR stimulation, peripheral blood CD8 + T cells from breast cancer patients tended to undergo apoptosis, which could be prevented by the addition of IL-2 and/or IL-10. The cytotoxicity of TCR-activated CD8 + T cells was also enhanced by exogenous IL-2 and/or IL-10. Interestingly, IL-2 and IL-10 demonstrated synergistic effects, since the enhancement in CD8 + T cell function when both cytokines were added was greater than the sum of the improvements mediated by each individual cytokine. IL-10 by itself could not promote the proliferation of CD8 + T cells but could significantly enhance IL-2-mediated promotion of CD8 + T cell proliferation. In addition, the cytotoxicity of tumor-infiltrating CD8 + T cells in breast tumor was elevated when both IL-2 and IL-10 were present but not when either one was absent. This synergistic effect was stopped by CD4 + CD25 + regulatory T cells (Treg), which depleted IL-2 in a cell number-dependent manner. Together, these results demonstrated that IL-2 and IL-10 could work synergistically to improve the survival, proliferation, and cytotoxicity of activated CD8 + T cells, an effect suppressible by CD4 + CD25 + Treg cells. Copyright © 2017 Elsevier Ltd. All rights reserved.
The cytotoxic effect of Elephantopus scaber Linn extract against breast cancer (T47D) cells

NASA Astrophysics Data System (ADS)

Sulistyani, N.; Nurkhasanah

2017-11-01

Breast cancer is one of the main cause of death. Elephantopus scaber Linn (ES) which has been used as a traditional medicine contains an antitumor compounds. This study aimed to explore the active fraction from ethanolic extract of ES as anticancer and to determine its inhibition effect on the cell proliferation cycle of breast cancer (T47D) cells. The ES leaf was macerated with ethanol and then evaporated to get the concentrated extract. The extract was fractionated using petroleum ether, chloroform, and methanol respectively. The cytotoxic activity of each fraction was carried out with MTT method, and the inhibition of cell cycle test were observed by flowcytometry method. The result showed that ES and the fractions have cytotoxic activity against T47D cell lines with IC50 values of extract, petroleum ether, chloroform, and methanol fractions were 58.36±2.38, 132.17±9.69, 7.08±2.11, and 572.89±69.23 µg/mL. The inhibition effect of ethanol extract on the lifecycle of cells was occured in sub G1 phase. There was no prolonging of G1, S, G2/M and polyploidy phase of T47D cell lines. The chloroform fraction of ES is the most cytotoxic fraction against T47D cells without prolonging the cell lifecycle.

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

PubMed

Iwanowicz, Luke R; Densmore, Christine L; Ottinger, Christopher A

2004-02-01

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (Pcytotoxic cell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.
Vav1-phospholipase C-γ1 (Vav1-PLC-γ1) pathway initiated by T cell antigen receptor (TCRγδ) activation is required to overcome inhibition by ubiquitin ligase Cbl-b during γδT cell cytotoxicity.

PubMed

Yin, Shanshan; Zhang, Jianmin; Mao, Yujia; Hu, Yu; Cui, Lianxian; Kang, Ning; He, Wei

2013-09-13

T cell antigen receptor γδ (TCRγδ) and natural killer group 2, member D (NKG2D) are two crucial receptors for γδT cell cytotoxicity. Compelling evidences suggest that γδT cell cytotoxicity is TCRγδ-dependent and can be co-stimulated by NKG2D. However, the molecular mechanism of underlying TCRγδ-dependent activation of γδT cells remains unclear. In this study we demonstrated that TCRγδ but not NKG2D engagement induced lytic granule polarization and promoted γδT cell cytotoxicity. TCRγδ activation alone was sufficient to trigger Vav1-dependent phospholipase C-γ1 signaling, resulting in lytic granule polarization and effective killing, whereas NKG2D engagement alone failed to trigger cytotoxicity-related signaling to overcome the inhibitory effect of Cbl-b; therefore, NKG2D engagement alone could not induce effective killing. However, NKG2D ligation augmented the activation of γδT cell cytotoxicity through the Vav1-phospholipase C-γ1 pathway. Vav1 overexpression or Cbl-b knockdown not only enhanced TCRγδ activation-initiated killing but also enabled NKG2D activation alone to induce γδT cell cytotoxicity. Taken together, our results suggest that the activation of γδT cell cytotoxicity requires a strong activation signal to overcome the inhibitory effect of Cbl-b. Our finding provides new insights into the molecular mechanisms underlying the initiation of γδT cell cytotoxicity and likely implications for optimizing γδT cell-based cancer immunotherapy.
Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells.

PubMed

Lee, Pei-Ling; Chen, Bo-Chia; Gollavelli, Ganesh; Shen, Sin-Yu; Yin, Yu-Sheng; Lei, Shiu-Ling; Jhang, Cian-Ling; Lee, Woan-Ruoh; Ling, Yong-Chien

2014-07-30

Zinc oxide nanoparticles (ZnO NPs) exhibit novel physiochemical properties and have found increasing use in sunscreen products and cosmetics. The potential toxicity is of increasing concern due to their close association with human skin. A time-of-flight secondary ion mass spectrometry (TOF-SIMS) and confocal laser scanning microscopy (CLSM) imaging method was developed and validated for rapid and sensitive cytotoxicity study of ZnO NPs using human skin equivalent HaCaT cells as a model system. Assorted material, chemical, and toxicological analysis methods were used to confirm their shape, size, crystalline structure, and aggregation properties as well as dissolution behavior and effect on HaCaT cell viability in the presence of various concentrations of ZnO NPs in aqueous media. Comparative and correlative analyses of aforementioned results with TOF-SIMS and CLSM imaging results exhibit reasonable and acceptable outcome. A marked drop in survival rate was observed with 50μg/ml ZnO NPs. The CLSM images reveal the absorption and localization of ZnO NPs in cytoplasm and nuclei. The TOF-SIMS images demonstrate elevated levels of intracellular ZnO concentration and associated Zn concentration-dependent (40)Ca/(39)K ratio, presumably caused by the dissolution behavior of ZnO NPs. Additional validation by using stable isotope-labeled (68)ZnO NPs as tracers under the same experimental conditions yields similar cytotoxicity effect. The imaging results demonstrate spatially-resolved cytotoxicity relationship between intracellular ZnO NPs, (40)Ca/(39)K ratio, phosphocholine fragments, and glutathione fragments. The trend of change in TOF-SIMS spectra and images of ZnO NPs treated HaCaT cells demonstrate the possible mode of actions by ZnO NP involves cell membrane disruption, cytotoxic response, and ROS mediated apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.
Cytotoxicity of arctigenin and matairesinol against the T-cell lymphoma cell line CCRF-CEM.

PubMed

Su, Shan; Cheng, Xinlai; Wink, Michael

2015-09-01

Arctigenin and matairesinol possess a diversity of bioactivities. Here we investigated the cytotoxicity of arctigenin and matairesinol against a T-cell lymphoma cell line CCRF-CEM and the underlying mechanisms that have not been explored before. The cytotoxic activity was investigated using MTT assay. The cell cycle arrest and reactive oxygen species (ROS) accumulation were determined by flow cytometric analysis. The apoptosis induction was assessed using Annexin V/Propidium Iodide assay. The gene quantification analysis was measured through real-time polymerase chain reaction. Arctigenin and matairesinol exhibited significant antiproliferative activity against CCRF-CEM cells after 72 h treatment with IC50 values of 1.21 ± 0.15 μm and 4.27 ± 0.41 μm, respectively. In addition, both lignans arrest CCRF-CEM cells in the S phase. Furthermore, they could induce apoptosis in CCRF-CEM cells in a concentration- and time-dependent manner. Interestingly, the lignans differentially regulated the expression of several key genes involved in apoptosis pathways, including Bax, Bad and caspase-9. Moreover, both lignans could increase ROS levels in CCRF-CEM cells. Our study provides an insight into the potential of arctigenin and matairesinol as good candidates for the development of novel agents against T-cell lymphoma. © 2015 Royal Pharmaceutical Society.
[131I]FIAU labeling of genetically transduced, tumor-reactive lymphocytes: cell-level dosimetry and dose-dependent toxicity.

PubMed

Zanzonico, Pat; Koehne, Guenther; Gallardo, Humilidad F; Doubrovin, Mikhail; Doubrovina, Ekaterina; Finn, Ronald; Blasberg, Ronald G; Riviere, Isabelle; O'Reilly, Richard J; Sadelain, Michel; Larson, Steven M

2006-09-01

Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [(131)I]-2'-fluoro-2'-deoxy-1-beta-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular (131)I (even at tracer levels), the nucleus absorbed dose (D ( n )) and dose-dependent immune functionality were evaluated for NIT(+) T cells labeled ex vivo in [(131)I]FIAU-containing medium. Based on in vitro kinetic studies of [(131)I]FIAU uptake by NIT(+) T cells, D ( n ) was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [(131)I]FIAU-labeled cells was assayed against (51)Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. At median nuclear absorbed doses up to 830 cGy, a (51)Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.
Cytotoxic CD4 T Cells—Friend or Foe during Viral Infection?

PubMed Central

Juno, Jennifer A.; van Bockel, David; Kent, Stephen J.; Kelleher, Anthony D.; Zaunders, John J.; Munier, C. Mee Ling

2017-01-01

CD4 T cells with cytotoxic function were once thought to be an artifact due to long-term in vitro cultures but have in more recent years become accepted and reported in the literature in response to a number of viral infections. In this review, we focus on cytotoxic CD4 T cells in the context of human viral infections and in some infections that affect mice and non-human primates. We examine the effector mechanisms used by cytotoxic CD4 cells, the phenotypes that describe this population, and the transcription factors and pathways that lead to their induction following infection. We further consider the cells that are the predominant targets of this effector subset and describe the viral infections in which CD4 cytotoxic T lymphocytes have been shown to play a protective or pathologic role. Cytotoxic CD4 T cells are detected in the circulation at much higher levels than previously realized and are now recognized to have an important role in the immune response to viral infections. PMID:28167943
Depressed spontaneous cell-mediated cytotoxicity in Crohn's disease.

PubMed

Beeken, W L; Macpherson, B R; Gundel, R M; St Andre-Ukena, S; Wood, S G; Sylwester, D L

1983-02-01

Cytotoxicity of peripheral blood mononuclear cells of 30 patients with Crohn's disease (CD) and 30 matched controls was assayed by measuring isotope release from 75Se-L-methionine labelled RPMI 4788 human colon cancer cells. Effector populations were studied with and without monocyte depletion after 4 and 24 hr incubations in 10% fetal calf serum or autologous serum or plasma. Cytotoxicity was negligible at 4 hr. Twenty-four hour cytotoxicity was consistently lower in CD patients than in healthy controls, mean values ranging from 13.6 +/- 2.7% (s.e.m.) to 19.5 +/- 3.7% in patients and from 27.2 +/- 4.1% to 33.6 +/- 5.3% in controls. Cytotoxicity of disease controls was not significantly different from that of healthy subjects. Cytotoxicity was reduced by monocyte depletion, was weakly and inversely related to disease activity, was relatively stable for up to 24 months and was not HLA restricted. Cell lysis was attributable to spontaneous cell-mediated cytotoxicity. Antibody-dependent cellular cytotoxicity and antibody-complement-dependent cytotoxicity were not detected.
Depressed spontaneous cell-mediated cytotoxicity in Crohn's disease.

PubMed Central

Beeken, W L; Macpherson, B R; Gundel, R M; St Andre-Ukena, S; Wood, S G; Sylwester, D L

1983-01-01

Cytotoxicity of peripheral blood mononuclear cells of 30 patients with Crohn's disease (CD) and 30 matched controls was assayed by measuring isotope release from 75Se-L-methionine labelled RPMI 4788 human colon cancer cells. Effector populations were studied with and without monocyte depletion after 4 and 24 hr incubations in 10% fetal calf serum or autologous serum or plasma. Cytotoxicity was negligible at 4 hr. Twenty-four hour cytotoxicity was consistently lower in CD patients than in healthy controls, mean values ranging from 13.6 +/- 2.7% (s.e.m.) to 19.5 +/- 3.7% in patients and from 27.2 +/- 4.1% to 33.6 +/- 5.3% in controls. Cytotoxicity of disease controls was not significantly different from that of healthy subjects. Cytotoxicity was reduced by monocyte depletion, was weakly and inversely related to disease activity, was relatively stable for up to 24 months and was not HLA restricted. Cell lysis was attributable to spontaneous cell-mediated cytotoxicity. Antibody-dependent cellular cytotoxicity and antibody-complement-dependent cytotoxicity were not detected. PMID:6601555
Cytotoxic effect of artocarpin on T47D cells.

PubMed

Arung, Enos Tangke; Wicaksono, Britanto Dani; Handoko, Yohana Ayupriyanti; Kusuma, Irawan Wijaya; Shimizu, Kuniyoshi; Yulia, Dina; Sandra, Ferry

2010-10-01

In our screening projects for anticancer agents from natural resources, artocarpin [6-(3-methyl-1-butenyl)-5,2',4'-trihydroxy-3-isoprenyl-7-methoxyflavone] isolated from wood of jack fruit (Artocarpus heterophyllus) showed potent cytotoxic activity on human T47D breast cancer cells. The mode of action of artocarpin was evaluated by its effect on cell viability, nuclear morphology, cell cycle progression, expression of protein markers for apoptosis, and mitochondrial membrane potential (Delta psi m). These results showed that artocarpin caused a reduction of cell viability in a concentration-dependent manner and an alteration of cell and nuclear morphology. Moreover, the percentage of the sub-G1 phase formation was elevated dose-dependently. Artocarpin induced activation of caspase 8 and 10 as indicated by stronger signal intensity of cleaved-caspase 8 and weaker signal intensity of caspase 10 markers detected after artocarpin treatment. In addition, we also noticed the activation of caspase 3 by artocarpin. There were negligible changes in mitochondrial membrane potential (Delta psi m) due to artocarpin treatment. All together, these data indicated that artocarpin induced apoptosis in T47D cells possibly via an extrinsic pathway.
Protective effects of anethole dithiolethione against oxidative stress-induced cytotoxicity in human Jurkat T cells.

PubMed

Khanna, S; Sen, C K; Roy, S; Christen, M O; Packer, L

1998-07-01

The protective effects of anethole dithiolethione (ADT) against H2O2- or 4-hydroxynonenal (HNE)-induced cytotoxicity in human Jurkat T cells were investigated. Jurkat T cells were pretreated with ADT (10-50 microM) for 18 hr and then challenged with H202 or HNE for up to 4 hr. Cytotoxicity was assessed by measuring: 1) leakage of lactate dehydrogenase from cells to medium; and 2) exclusion of the DNA intercalating fluorescent probe propidium iodide by viable cells. Pretreatment of cells with ADT (10 or 25 microM) for 18 hr significantly protected cells against H202- or HNE-induced cytotoxicity. Treatment of cells with ADT (10-50 microM) for 72 hr significantly increased the activities of catalase and glutathione reductase. The maximum effect of ADT treatment on the activity of these enzymes was observed when cells were treated with 25 microM of ADT for 72 hr. A significant increase in cellular GSH was observed in cells that were treated with ADT for 72 hr. Using monobromobimane as a thiol probe, we consistently observed that cells pretreated for 18 hr with ADT (25 or 50 microM) had also increased total thiol content. Exposure of Jurkat T cells to H202 or HNE resulted in a time-dependent decrease in cellular GSH. ADT (10-50 microM, 18 hr) pretreatment circumvented H202-dependent lowering of cellular GSH. In conclusion, ADT proved to be a potent cytoprotective thiol antioxidant with multifaceted mechanisms of action, suggesting that the drug has a remarkable therapeutic potential.
HDAC6 regulates the dynamics of lytic granules in cytotoxic T lymphocytes

PubMed Central

Núñez-Andrade, Norman; Iborra, Salvador; Trullo, Antonio; Moreno-Gonzalo, Olga; Calvo, Enrique; Catalán, Elena; Menasche, Gaël; Sancho, David; Vázquez, Jesús; Yao, Tso-Pang

2016-01-01

HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4+ T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8+ T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6-/- CD8+ T cells to Rag1-/- mice demonstrated specific impairment in CD8+ T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin 1 – dynactin mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFNγ) production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs. PMID:26869226
Lactobacillus casei HY7213 ameliorates cyclophosphamide-induced immunosuppression in mice by activating NK, cytotoxic T cells and macrophages.

PubMed

Jang, Se-Eun; Joh, Eun-Ha; Ahn, Young-Tae; Huh, Chul-Sung; Han, Myung Joo; Kim, Dong-Hyun

2013-06-01

Lactic acid bacteria (LAB) have recently attracted considerable attention as treatment options for immune diseases, the incidence of which has been increasing worldwide. The ability of tumor necrosis factor-α producing LAB isolated from cheese to inhibit NF-κB activation in lipopolysaccharide (LPS)-stimulated peritoneal macrophages was investigated. Among the tested LAB, Lactobacillus casei HY7213 inhibited NF-κB activation most potently. Therefore, we measured its immunopotentiating effect in cyclophosphamide (CP)-immunosuppressed mice. When HY7213 was orally administered for 5 or 15 d, it reversed the CP immunosuppressant effect by increasing body and spleen weights, blood red and white blood cells levels, and splenocyte and bone marrow cells counts. Treatment with CP in mice markedly reduced concanavalin A (ConA)-induced T cell proliferation to 54% compared to the normal group. Oral administration of HY7213 in CP-immunosuppressed mice reversed that value to 95% of the normal group on day 15. Furthermore, oral administration of HY7213 to CP-treated mice significantly enhanced the expression of IL-2 and IFN-γ in ConA-induced splenic cytotoxic T cells, restored the CP-impaired phagocytosis of macrophage, and increased the cytotoxicity of natural killer (NK) and cytotoxic T cells derived from spleen and bone marrow against YAC-1. Based on these findings, we suggest that HY7213 may promote the recovery of immunosuppression caused by chemotherapeutic agents, such as CP, by activating NK cells, cytotoxic T cells and macrophages.
Ultra-fast stem cell labelling using cationised magnetoferritin

NASA Astrophysics Data System (ADS)

Correia Carreira, S.; Armstrong, J. P. K.; Seddon, A. M.; Perriman, A. W.; Hartley-Davies, R.; Schwarzacher, W.

2016-03-01

Magnetic cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) facilitates many important biotechnological applications, such as cell imaging and remote manipulation. However, to achieve adequate cellular loading of SPIONs, long incubation times (24 hours and more) or laborious surface functionalisation are often employed, which can adversely affect cell function. Here, we demonstrate that chemical cationisation of magnetoferritin produces a highly membrane-active nanoparticle that can magnetise human mesenchymal stem cells (hMSCs) using incubation times as short as one minute. Magnetisation persisted for several weeks in culture and provided significant T2* contrast enhancement during magnetic resonance imaging. Exposure to cationised magnetoferritin did not adversely affect the membrane integrity, proliferation and multi-lineage differentiation capacity of hMSCs, which provides the first detailed evidence for the biocompatibility of magnetoferritin. The combination of synthetic ease and flexibility, the rapidity of labelling and absence of cytotoxicity make this novel nanoparticle system an easily accessible and versatile platform for a range of cell-based therapies in regenerative medicine.Magnetic cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) facilitates many important biotechnological applications, such as cell imaging and remote manipulation. However, to achieve adequate cellular loading of SPIONs, long incubation times (24 hours and more) or laborious surface functionalisation are often employed, which can adversely affect cell function. Here, we demonstrate that chemical cationisation of magnetoferritin produces a highly membrane-active nanoparticle that can magnetise human mesenchymal stem cells (hMSCs) using incubation times as short as one minute. Magnetisation persisted for several weeks in culture and provided significant T2* contrast enhancement during magnetic resonance imaging. Exposure to cationised
Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes

PubMed Central

Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D.; Bullens, Dominique M.; Pinxteren, Jef; Verfaillie, Catherine M.; Van Gool, Stefaan W.

2016-01-01

MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8−CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Significance Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for
Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

PubMed

Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

2016-12-01

: MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8 + cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8 - CD69 + T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy
Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib.

PubMed

Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

2015-09-01

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8 + T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.
Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib

PubMed Central

Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

2015-01-01

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8+ T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses. PMID:26405606
Functional characterization of BK virus-specific CD4+ T cells with cytotoxic potential in seropositive adults.

PubMed

Zhou, Wendi; Sharma, Madeva; Martinez, Joy; Srivastava, Tumul; Diamond, Don J; Knowles, Wendy; Lacey, Simon F

2007-09-01

BK polyomavirus (BKV) reactivation is associated with a failure of T cell immunity in kidney transplant patients, and may lead to BKV-associated nephropathy (BKVN) and loss of the allograft. BKV reactivation in hematopoietic stem cell transplant recipients is associated with hemorrhagic cystitis. We have investigated T cell responses to overlapping peptide mixtures corresponding to the whole BKV major T antigen (TAg) and major capsid protein (VP1) in peripheral blood mononuclear cell samples from a cohort of healthy BKV-seropositive subjects. The majority of these individuals possessed populations of both CD8(+) and CD4(+) T cells specific for these BKV antigens. After expansion in culture, the majority of the BKV-specific CD4(+) T cells, in addition to expressing CD40L (CD154), secreted both interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, contained both granzyme A and granzyme B, and degranulated/mobilized CD107 in response to antigen-specific stimulation. These T cells thus represent potentially functional BKV-specific cytotoxic CD4(+) T lymphocytes. Secretion of both TNF-alpha and IFN-gamma by CD154(+)CD4(+) T cells on BKV-specific stimulation was associated with higher levels of granzyme B and a higher proportion of degranulating cells compared with CD154(+)CD4(+) T cells producing only IFN-gamma or neither cytokine. These healthy subjects also harbored populations of functional CD8(+) T cells specific for one or more of three newly defined HLA-A 02-restricted cytotoxic T lymphocyte epitopes within the BKV TAg as well as two HLA-A 02-restricted epitopes within the BKV VP1 we have previously described. The BKV-specific CD4(+) T cells characterized in this study may play a part in maintaining persistent memory T cell responses to the virus and thus contribute to the immune control of BKV in healthy individuals.
Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

PubMed

Oktem, Ozgur; Bildik, Gamze; Senbabaoglu, Filiz; Lack, Nathan A; Akin, Nazli; Yakar, Feridun; Urman, Defne; Guzel, Yilmaz; Balaban, Basak; Iwase, Akira; Urman, Bulent

2016-04-01

A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology. Copyright © 2015 Elsevier Inc. All rights reserved.
Epstein-Barr virus-positive cytotoxic T-cell lymphoma followed by chronic active Epstein-Barr virus infection-associated T/NK-cell lymphoproliferative disorder: a case report.

PubMed

Kato, Seiichi; Miyata, Tomoko; Takata, Katsuyoshi; Shimada, Satoko; Ito, Yoshinori; Tomita, Akihiro; Elsayed, Ahmed Ali; Takahashi, Emiko; Asano, Naoko; Kinoshita, Tomohiro; Kimura, Hiroshi; Nakamura, Shigeo

2013-12-01

A 30-year-old female patient presented with intestinal Epstein-Barr virus (EBV)-positive cytotoxic T-cell lymphoma (EBV+ CTL), which was surgically resected. Fourteen years later, she returned to our hospital with hypersensitivity to mosquito bites and was diagnosed with chronic active EBV infection-associated T/NK-cell lymphoproliferative disorder (CAEBV/TNK-LPD). She developed systemic EBV+ CTL at age 47 years during the 2.5-year clinical course of CAEBV/TNK-LPD, despite multiagent chemotherapy and allogeneic stem cell transplantation. Afterward, she had a rapidly deteriorating clinical course and died at age 48 years. The immunophenotype of the EBV+ CTL was consistently a CD3, CD8, and cytotoxic molecule-positive type with the same clonality in polymerase chain reaction analysis of T-cell receptor-γ chain gene rearrangement. This is the first reported case of EBV+ CTL preceding the clinical presentation of CAEBV/TNK-LPD. The present case was unique in suggesting a close relationship between EBV+ CTL and chronic active EBV infection. © 2013.

Exosomes derived from B16F0 melanoma cells alter the transcriptome of cytotoxic T cells that impacts mitochondrial respiration.

PubMed

Bland, Cassidy L; Byrne-Hoffman, Christina N; Fernandez, Audry; Rellick, Stephanie L; Deng, Wentao; Klinke, David J

2018-03-01

While recent clinical studies demonstrate the promise of cancer immunotherapy, a barrier for broadening the clinical benefit is identifying how tumors locally suppress cytotoxic immunity. As an emerging mode of intercellular communication, exosomes secreted by malignant cells can deliver a complex payload of coding and noncoding RNA to cells within the tumor microenvironment. Here, we quantified the RNA payload within tumor-derived exosomes and the resulting dynamic transcriptomic response to cytotoxic T cells upon exosome delivery to better understand how tumor-derived exosomes can alter immune cell function. Exosomes derived from B16F0 melanoma cells were enriched for a subset of coding and noncoding RNAs that did not reflect the abundance in the parental cell. Upon exosome delivery, RNAseq revealed the dynamic changes in the transcriptome of CTLL2 cytotoxic T cells. In analyzing transiently coexpressed gene clusters, pathway enrichment suggested that the B16F0 exosomal payload altered mitochondrial respiration, which was confirmed independently, and upregulated genes associated with the Notch signaling pathway. Interestingly, exosomal miRNA appeared to have no systematic effect on downregulating target mRNA levels. Gene expression data are available in the GEO database under the accession SuperSeries number GSE102951. © 2018 Federation of European Biochemical Societies.
A photosensitizer delivered by bispecific antibody redirected T lymphocytes enhances cytotoxicity against EpCAM-expressing carcinoma cells upon light irradiation.

PubMed

Blaudszun, André-René; Moldenhauer, Gerhard; Schneider, Marc; Philippi, Anja

2015-01-10

Recently conducted clinical trials have provided impressive evidence that chemotherapy resistant metastatic melanoma and several hematological malignancies can be cured using adoptive T cell therapy or T cell-recruiting bispecific antibodies. However, a significant fraction of patients did not benefit from these treatments. Here we have evaluated the feasibility of a novel combination therapy which aims to further enhance the killing potential of bispecific antibody-redirected T lymphocytes by using these cells as targeted delivery system for photosensitizing agents. For a first in vitro proof-of-concept study, ex vivo activated human donor T cells were loaded with a poly(styrene sulfonate) (PSS)-complex of the model photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). In the absence of light and when loading with the water-soluble PSS/mTHPP-complex occurred at a tolerable concentration, viability and cytotoxic function of loaded T lymphocytes were not impaired. When "drug-enhanced" T cells were co-cultivated with EpCAM-expressing human carcinoma cells, mTHPP was transferred to target cells. Notably, in the presence of a bispecific antibody, which cross-links effector and target cells thereby inducing the cytolytic activity of cytotoxic T lymphocytes, significantly more photosensitizer was transferred. Consequently, upon irradiation of co-cultures, redirected drug-loaded T cells were more effective in killing A549 lung and SKOV-3 ovarian carcinoma cells than retargeted unloaded T lymphocytes. Particularly, the additive approach using redirected unloaded T cells in combination with appropriate amounts of separately applied PSS/mTHPP was less efficient as well. Thus, by loading T lymphocytes with a stimulus-sensitive anti-cancer drug, we were able to enhance the cytotoxic capacity of carrier cells. Photosensitizer boosted T cells could open new perspectives for adoptive T cell therapy as well as targeted photodynamic therapy. Copyright © 2014
Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

PubMed

Ueda, Norihiro; Uemura, Yasushi; Zhang, Rong; Kitayama, Shuichi; Iriguchi, Shoichi; Kawai, Yohei; Yasui, Yutaka; Tatsumi, Minako; Ueda, Tatsuki; Liu, Tian-Yi; Mizoro, Yasutaka; Okada, Chihiro; Watanabe, Akira; Nakanishi, Mahito; Senju, Satoru; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Kiyoi, Hitoshi; Naoe, Tomoki; Kaneko, Shin

2018-06-05

CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2+ γδ T cell cytotoxicity in a perforin-dependent manner.

PubMed

Fowler, Daniel W; Copier, John; Dalgleish, Angus G; Bodman-Smith, Mark D

2017-09-01

Vδ2 + T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2 + T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2 + T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2 + T cell cytotoxicity. Vδ2 + T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2 + T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy.
Optimization of Dendritic Cell-Mediated Cytotoxic T-Cell Activation by Tracking of Dendritic Cell Migration Using Reporter Gene Imaging.

PubMed

Lee, Hongje; Lee, Ho Won; La Lee, You; Jeon, Yong Hyun; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

2018-06-01

The aim of this study is to optimize the dendritic cell (DC)-mediated T-cell activation using reporter gene imaging and flow cytometric analysis in living mice. A murine dendritic cell line (DC2.4) co-expressing effluc and Thy1.1 genes were established by transfection with retroviral vectors. Thy1.1 positive cells were sorted by magnetic bead separation system (DC2.4/effluc). Cell proliferation assay and phenotype analysis to determine the effects of gene transduction on the function of dendritic cells between parental DC2.4 and DC2.4/effluc were performed. To optimize the DC-mediated immune response by cell number or frequency, different cell numbers (5 × 10 5 , 1 × 10 6 , and 2 × 10 6 DC2.4/effluc) or different frequencies of DC2.4/effluc (first, second, and third injections) were injected in the right footpad of mice. The migration of the DC2.4/effluc into the draining popliteal lymph node of mice was monitored by bioluminescence imaging (BLI). Flow cytometric analysis was performed with splenocytes to determine the cytotoxic T-cell population after injection of DC2.4/effluc. Parental DC2.4 and DC2.4/effluc exhibit no significant differences in their proliferation and phenotype. BLI signals were observed in the draining popliteal lymph node at day 1 after injection of DC2.4/effluc in 1 × 10 6 and 2 × 10 6 cells-injected groups. The highest BLI signal intensity was detected in 2 × 10 6 cells-injected mice. On day 11, the BLI signal was detected in only 2 × 10 6 cell-injected group but not in other groups. Optimized cell numbers (2 × 10 6 ) were injected in three animal groups with a different frequency (first, second, and third injection groups). The BLI signal was detected at day 1 and maintained until day 7 in the first injection group, but there is low signal intensity in the second and the third injection groups. Although the expression levels of Thy1.1 gene in the first injection group were very high, there reveals no expression of
Virus-specific cytotoxic T cells in chronic active Epstein-Barr virus infection.

PubMed

Shibayama, Haruna; Imadome, Ken-Ichi; Onozawa, Erika; Tsuzura, Akiho; Miura, Osamu; Koyama, Takatoshi; Arai, Ayako

2017-01-01

Chronic active Epstein-Barr virus infection (CAEBV) is a disease characterized by clonally proliferating and activated EBV-infected T or NK cells accompanied by chronic inflammation and T- or NK-cell neoplasms. However, the mechanism for developing CAEBV has not been clarified to date. Because the decreased number or inactivation of EBV-specific cytotoxic T lymphocytes (CTLs) resulted in the development of EBV-positive B-cell neoplasms, we investigated the number of CTLs in CAEBV patients using the tetrameric complexes of HLA-restricted EBV-specific peptides. Among the seven patients examined, EBV-specific CTLs were detected in the peripheral blood mononuclear cells (PBMCs) of four cases but were not detected in three cases. The ratio of EBV-specific CTLs in PBMCs tended to be higher in the patients with active disease than in those with inactive disease. In two patients in whom EBV-specific CTLs had not been detected, CTLs appeared after the eradication of EBV-infected T cells by allogeneic bone marrow transplantation. These results suggested that the failure of CTLs had a role in developing CAEBV, although the induction number and function of EBV-specific CTLs might vary in each patient.
A NEW SENSITIVE ASSAY FOR ANTIBODY AGAINST CELL SURFACE ANTIGENS BASED ON INHIBITION OF CELL-DEPENDENT ANTIBODY-MEDIATED CYTOTOXICITY

PubMed Central

Halloran, Phil; Schirrmacher, Volker; Festenstein, Hilliard

1974-01-01

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells (61Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2–11 times more sensitive for anti-H-2 antisera and 20–780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man. PMID:4547657
In Vivo MR Imaging of Glioma Recruitment of Adoptive T-Cells Labeled with NaGdF4 -TAT Nanoprobes.

PubMed

Zhang, Hua; Wu, Yue; Wang, Jing; Tang, Zhongmin; Ren, Yan; Ni, Dalong; Gao, Hongbo; Song, Ruixue; Jin, Teng; Li, Qiao; Bu, Wenbo; Yao, Zhenwei

2018-01-01

Adoptive T lymphocyte immunotherapy is one of the most promising methods to treat residual lesions after glioma surgery. However, the fate of the adoptively transferred T-cells in vivo is unclear, hampering the understanding of this emerging therapy. Thus, it is highly desirable to develop noninvasive and quantitative in vivo tracking of these T-cells to glioma for better identification of the migratory fate and to provide objective evaluation of outcomes of adoptive T-cell immunotherapy targeting glioma. In this work, ultrasmall T 1 MR-based nanoprobes, NaGdF 4 -TAT, as molecular probes with high longitudinal relaxivity (8.93 mm -1 s -1 ) are designed. By means of HIV-1 transactivator (TAT) peptides, nearly 95% of the adoptive T-cells are labeled with the NaGdF 4 -TAT nanoprobes without any measurable side effects on the labeled T-cells, which is remarkably superior to that of the control fluorescein isothiocyanate-NaGdF 4 concerning labeling efficacy. Labeled adoptive T-cell clusters can be sensitively tracked in an orthotopic GL261-glioma model 24 h after intravenous infusion of 10 7 labeled T-cells by T 1 -weighted MR imaging. Both in vitro and in vivo experiments show that the NaGdF 4 -TAT nanoprobes labeling of T-cells may be a promising method to track adoptive T-cells to improve our understanding of the pathophysiology in adoptive immunotherapy for gliomas. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
HIV-TB coinfection impairs CD8(+) T-cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses.

PubMed

Suarez, Guadalupe V; Angerami, Matías T; Vecchione, María B; Laufer, Natalia; Turk, Gabriela; Ruiz, Maria J; Mesch, Viviana; Fabre, Bibiana; Maidana, Patricia; Ameri, Diego; Cahn, Pedro; Sued, Omar; Salomón, Horacio; Bottasso, Oscar A; Quiroga, María F

2015-09-01

Tuberculosis (TB) is the leading cause of death among HIV-positive patients. The decreasing frequencies of terminal effector (TTE ) CD8(+) T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV-TB patients. Here, we aimed to study Mtb-specific cytotoxicity, IFN-γ secretion, memory status of CD8(+) T cells, and their modulation by DHEA during HIV-TB coinfection. CD8(+) T cells from HIV-TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and TTE T-cell frequencies compared to healthy donors both in total and Mtb-specific CD8(+) T cells. Notably, CD8(+) T cells from HIV-TB patients displayed higher Terminal Effector (TTE ) CD45RA(dim) proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD-1 expression levels on CD8(+) T cells from HIV-TB patients increased although restricted to the CD27(+) population. Interestingly, DHEA plasma levels positively correlated with TTE in CD8(+) T cells and in vitro DHEA treatment enhanced Mtb-specific cytotoxic responses and terminal differentiation in CD8(+) T cells from HIV-TB patients. Our data suggest that HIV-TB coinfection promotes a deficient CD8(+) T-cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8(+) T-cell functions during HIV-TB coinfection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Inhibition of the activity of cytotoxic murine T lymphocytes by antibodies to idiotypic determinants.

PubMed Central

Rabinowitz, R; Schlesinger, M

1980-01-01

The nature of the receptors on the surface of cytotoxic T lymphocytes (CTL), which enable these cells to recognize antigens on allogeneic targets, is still a matter of controversy. In the present study various mouse alloantisera were tested for their capacity to inhibit, in the absence of complement, the cytotoxic activity of sensitized peritoneal T lymphocytes. The only antiserum which, even after heat inactivation, consistently inhibited cytotoxic T lymphocytes was an antiserum elicited in (C3H X C57B1/6)F1 mice by immunization with AKR/Cum thymus cells. The serum inhibited the cytotoxic reaction of either AKR/J or AKR/Cum CTL on EL-4 target cells but had no inhibitory activity on the cytotoxic reaction of AKR/J cells against P-815 target cells. Thus the inhibitory activity of the serum could not be attributed to antibodies against Ly-3 determinants present in the serum. This conclusion was strengthened by the finding that the inhibitory activity of the serum could be removed by absorption, not only with AKR/J thymus cells but also with AKR/J bone-marrow cells, a procedure which did not affect the titre of Ly-3 antibodies. The serum failed to exert any inhibition on cytotoxic T lymphocytes of BALB/c and C3H mice reacting against EL-4 target cells, indicating that the inhibitory activity of the antiserum did not result from contamination by antibodies against C57B1 antigenic determinants. It was concluded that the inhibitory activity of the antiserum resulted from the presence of antibodies against idiotypic determinants expressed on AKR/Cum thymus cells reacting against the hybrid hosts. It seems, therefore, that idiotypic determinants expressed on the surface of cytotoxic T lymphocytes may be directly involved in their cytotoxic activity. PMID:6155324
In vitro labelling and detection of mesenchymal stromal cells: a comparison between magnetic resonance imaging of iron-labelled cells and magnetic resonance spectroscopy of fluorine-labelled cells.

PubMed

Rizzo, Stefania; Petrella, Francesco; Zucca, Ileana; Rinaldi, Elena; Barbaglia, Andrea; Padelli, Francesco; Baggi, Fulvio; Spaggiari, Lorenzo; Bellomi, Massimo; Bruzzone, Maria Grazia

2017-01-01

Among the various stem cell populations used for cell therapy, adult mesenchymal stromal cells (MSCs) have emerged as a major new cell technology. These cells must be tracked after transplantation to monitor their migration within the body and quantify their accumulation at the target site. This study assessed whether rat bone marrow MSCs can be labelled with superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsion formulations without altering cell viability and compared magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) results from iron-labelled and fluorine-labelled MSCs, respectively. Of MSCs, 2 × 10 6 were labelled with Molday ION Rhodamine-B (MIRB) and 2 × 10 6 were labelled with Cell Sense. Cell viability was evaluated by trypan blue exclusion method. Labelled MSCs were divided into four samples containing increasing cell numbers (0.125 × 10 6 , 0.25 × 10 6 , 0.5 × 10 6 , 1 × 10 6 ) and scanned on a 7T MRI: for MIRB-labelled cells, phantoms and cells negative control, T1, T2 and T2* maps were acquired; for Cell Sense labelled cells, phantoms and unlabelled cells, a 19 F non-localised single-pulse MRS sequence was acquired. In total, 86.8% and 83.6% of MIRB-labelled cells and Cell Sense-labelled cells were viable, respectively. MIRB-labelled cells were visible in all samples with different cell numbers; pellets containing 0.5 × 10 6 and 1 × 10 6 of Cell Sense-labelled cells showed a detectable 19 F signal. Our data support the use of both types of contrast material (SPIO and PFC) for MSCs labelling, although further efforts should be dedicated to improve the efficiency of PFC labelling.
Unambiguous Identification of β-Tubulin as the Direct Cellular Target Responsible for the Cytotoxicity of Chalcone by Photoaffinity Labeling.

PubMed

Zhou, Bo; Yu, Xingxin; Zhuang, Chunlin; Villalta, Peter; Lin, Yong; Lu, Junxuan; Xing, Chengguo

2016-07-05

Chalcone is a simple and potentially privileged structure in medicinal chemistry with a diverse repertoire of biological activities, among which cytotoxicity is of particular interest. The sharp structure-activity relationship (SAR) for chalcone's cytotoxicity suggests structure-specific target interactions. Despite the numerous putative targets proposed, evidence for direct target interactions in cells is unavailable. In this study, guided by the sharp cytotoxic SAR, we developed a cytotoxic chalcone-based photoaffinity labeling (PAL) probe, (E)-3-(3-azidophenyl)-1-[3,5-dimethoxy-4-(prop-2-yn-1-yloxy)phenyl]-2-methylprop-2-en-1-one (C95; IC50 : 0.38±0.01 μm), along with two structurally similar non-cytotoxic probes. These probes were used to search for the direct cellular target responsible for chalcone's cytotoxicity through intact cell-based PAL experiments, in which β-tubulin was identified to specifically interact with the cytotoxic probe (i.e., C95) but not the non-cytotoxic probes. A set of phenotypical and biochemical assays further reinforced β-tubulin as the cytotoxic target of chalcones. Peptide mass quantitation by mass spectrometric analysis revealed one peptide potentially labeled by C95, providing information on chalcone's binding site on β-tubulin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
CXCR5+CD8+ T cells present elevated capacity in mediating cytotoxicity toward autologous tumor cells through interleukin 10 in diffuse large B-cell lymphoma.

PubMed

Tang, Jiahong; Zha, Jie; Guo, Xutao; Shi, Pengcheng; Xu, Bing

2017-09-01

Diffuse large B-cell lymphoma (DLBCL) is a common and aggressive subtype of non-Hodgkin's lymphomas, with limited treatment options in refractory and relapsed patients. Growing evidence supports the notion that CD8 + T cell immunity could be utilized to eliminate B cell lymphomas. CXCR5 + CD8 + T cell is a novel cell subtype and share CXCR5 expression with CD19 + tumor cells. In this study, we investigated the frequency and function of existing CXCR5 + CD8 + T cells in DLBCL patients. We found that DLBCL patients as a group demonstrated significantly higher level of CXCR5 + CD8 + T cells than healthy individuals, with huge variability in each patient. Using anti-CD3/CD28-stimulated CD8 + T cells as effector (E) cells and autologous CD19 + tumor cells as target (T) cells, at high E:T ratio, no difference between the intensities of CXCR5 + CD8 + T cell- and CXCR5 - CD8 + T cell-mediated cytotoxicity were observed. However, at intermediate and low E:T ratios, the CXCR5 + CD8 + T cells presented stronger cytotoxicity than CXCR5 - CD8 + T cells. The expressions of granzyme A, granzyme B, and perforin were significantly higher in CXCR5 + CD8 + T cells than in CXCR5 - CD8 + T cells, with no significant difference in the level of degranulation. Tumor cells in DLBCL were known to secrete high level of interleukin 10 (IL-10). We therefore blocked the IL-10/IL-10R pathway, and found that the expressions of granzyme A, granzyme B, and perforin by CXCR5 + CD8 + T cells were significantly elevated. Together, these results suggest that CXCR5 + CD8 + T cells are potential candidates of CD8 + T cell-based immunotherapies, could mediate elimination of autologous tumor cells in DLBCL patients, but are also susceptible to IL-10-mediated suppression. Copyright © 2017. Published by Elsevier B.V.
Synergism of Cytotoxic T Lymphocyte–Associated Antigen 4 Blockade and Depletion of Cd25+ Regulatory T Cells in Antitumor Therapy Reveals Alternative Pathways for Suppression of Autoreactive Cytotoxic T Lymphocyte Responses

PubMed Central

Sutmuller, Roger P.M.; van Duivenvoorde, Leonie M.; van Elsas, Andrea; Schumacher, Ton N.M.; Wildenberg, Manon E.; Allison, James P.; Toes, Rene E.M.; Offringa, Rienk; Melief, Cornelis J.M.

2001-01-01

Therapeutic efficacy of a tumor cell–based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte–associated antigen (CTLA)-4 pathway or the CD25+ regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25+ Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2180–188–specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8+ T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25+ Treg cells indicates that CD25+ Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity. PMID:11560997
Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice.

PubMed

Siveen, K S; Kuttan, Girija

2012-01-01

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn's neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.
The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

PubMed

Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

2016-10-17

Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.
Cytotoxic CD4+ T Cells Drive Multiple Sclerosis Progression.

PubMed

Peeters, Liesbet M; Vanheusden, Marjan; Somers, Veerle; Van Wijmeersch, Bart; Stinissen, Piet; Broux, Bieke; Hellings, Niels

2017-01-01

Multiple sclerosis (MS) is the leading cause of chronic neurological disability in young adults. The clinical disease course of MS varies greatly between individuals, with some patients progressing much more rapidly than others, making prognosis almost impossible. We previously discovered that cytotoxic CD4+ T cells (CD4+ CTL), identified by the loss of CD28, are able to migrate to sites of inflammation and that they contribute to tissue damage. Furthermore, in an animal model for MS, we showed that these cells are correlated with inflammation, demyelination, and disability. Therefore, we hypothesize that CD4+ CTL drive progression of MS and have prognostic value. To support this hypothesis, we investigated whether CD4+ CTL are correlated with worse clinical outcome and evaluated the prognostic value of these cells in MS. To this end, the percentage of CD4+CD28null T cells was measured in the blood of 176 patients with relapsing-remitting MS (=baseline). Multimodal evoked potentials (EP) combining information on motoric, visual, and somatosensoric EP, as well as Kurtzke expanded disability status scale (EDSS) were used as outcome measurements at baseline and after 3 and 5 years. The baseline CD4+CD28null T cell percentage is associated with EP ( P = 0.003, R 2 = 0.28), indicating a link between these cells and disease severity. In addition, the baseline CD4+CD28null T cell percentage has a prognostic value since it is associated with EP after 3 years ( P = 0.005, R 2 = 0.29) and with EP and EDSS after 5 years ( P = 0.008, R 2 = 0.42 and P = 0.003, R 2 = 0.27). To the best of our knowledge, this study provides the first direct link between the presence of CD4+ CTL and MS disease severity, as well as its prognostic value. Therefore, we further elaborate on two important research perspectives: 1° investigating strategies to block or reverse pathways in the formation of these cells resulting in new treatments that slow
GENERATION OF CYTOTOXIC LYMPHOCYTES IN MIXED LYMPHOCYTE REACTIONS

PubMed Central

Forman, James; Möller, Göran

1973-01-01

Generation of cytotoxic effector cells by a unidirectional mixed lymphocyte reaction (MLR) in the mouse H-2 system was studied using labeled YAC (H-2a) leukemia cells as targets. The responding effector cell displayed a specific cytotoxic effect against target cells of the same H-2 genotype as the stimulating cell population. Killing of syngeneic H-2 cells was not observed, even when the labeled target cells were "innocent bystanders" in cultures where specific target cells were reintroduced. Similar results were found with spleen cells taken from mice sensitized in vivo 7 days earlier. The effector cell was not an adherent cell and was not activated by supernatants from MLR. The supernatants were not cytotoxic by themselves. When concanavalin A or phytohemagglutinin was added to the cytotoxic test system, target and effector cells were agglutinated. Under these conditions, killing of H-2a target cells was observed in mixed cultures where H-2a lymphocytes were also the effector cells. These findings indicate that specifically activated, probably thymus-derived lymphocytes, can kill nonspecifically once they have been activated and providing there is close contact between effector and target cells. Thus, specificity of T cell killing appears to be restricted to recognition and subsequent binding to the targets, the actual effector phase being nonspecific. PMID:4269560
Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

PubMed Central

Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

2016-01-01

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139
Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Controlling Influenza by Cytotoxic T-Cells: Calling for Help from Destroyers

PubMed Central

Schotsaert, Michael; Ibañez, Lorena Itatí; Fiers, Walter; Saelens, Xavier

2010-01-01

Influenza is a vaccine preventable disease that causes severe illness and excess mortality in humans. Licensed influenza vaccines induce humoral immunity and protect against strains that antigenically match the major antigenic components of the vaccine, but much less against antigenically diverse influenza strains. A vaccine that protects against different influenza viruses belonging to the same subtype or even against viruses belonging to more than one subtype would be a major advance in our battle against influenza. Heterosubtypic immunity could be obtained by cytotoxic T-cell (CTL) responses against conserved influenza virus epitopes. The molecular mechanisms involved in inducing protective CTL responses are discussed here. We also focus on CTL vaccine design and point to the importance of immune-related databases and immunoinformatics tools in the quest for new vaccine candidates. Some techniques for analysis of T-cell responses are also highlighted, as they allow estimation of cellular immune responses induced by vaccine preparations and can provide correlates of protection. PMID:20508820
Cytotoxic cells induced after Chlamydia psittaci infection in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lammert, J.K.

1982-03-01

The ability of spleen cells from Chlamydia psittaci-infected mice to lyse C. psittaci-infected and uninfected target cell monolayers was studied. The cytotoxicity assay used was a terminal label method in which the number of adherent target cells surviving the interaction with effector cells was determined by measuring the uptake of (3H)uridine by such cells. It was observed that in the first few days postinfection (3 to 5), spleens contained cells that lysed infected and uninfected targets with equal efficiency. Subsequently, infected targets were killed primarily. The activity of effector spleen cells for infected targets continued, although at a reduced level,more » beyond 21 days postinfection. Intact effector cells were required since a disruption by sonication resulted in a loss of cytotoxicity. The enhanced killing observed with infected targets was also observed when target cells were sensitized with heat- or UV-inactivated C. psittaci. This study suggests that the induction of cytotoxic cells after C. psittaci infection may contribute to the ability of the host to control multiplication of the microorganism.« less
Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

PubMed

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke; Saini, Sunil Kumar; Ramskov, Sofie; Donia, Marco; Such, Lina; Furness, Andrew J S; McGranahan, Nicholas; Rosenthal, Rachel; Straten, Per Thor; Szallasi, Zoltan; Svane, Inge Marie; Swanton, Charles; Quezada, Sergio A; Jakobsen, Søren Nyboe; Eklund, Aron Charles; Hadrup, Sine Reker

2016-10-01

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.
Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12.

PubMed

Homma, Sadamu; Komita, Hideo; Sagawa, Yukiko; Ohno, Tsuneya; Toda, Gotaro

2005-08-01

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-gamma. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8(+) T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-gamma produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells.
Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12

PubMed Central

Homma, Sadamu; Komita, Hideo; Sagawa, Yukiko; Ohno, Tsuneya; Toda, Gotaro

2005-01-01

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-γ. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8+ T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-γ produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells. PMID:16011514
Abundant tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes.

PubMed

Hanon, E; Hall, S; Taylor, G P; Saito, M; Davis, R; Tanaka, Y; Usuku, K; Osame, M; Weber, J N; Bangham, C R

2000-02-15

The role of the cellular immune response in human T-cell leukemia virus type I (HTLV-I) infection is not fully understood. A persistently activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in the majority of infected individuals. However, it remains unclear whether this CTL response is protective or causes tissue damage. In addition, several observations paradoxically suggest that HTLV-I is transcriptionally silent in most infected cells and, therefore, not detectable by virus-specific CTLs. With the use of a new flow cytometric procedure, we show here that a high proportion of naturally infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10% and 80%) are capable of expressing Tax, the immunodominant target antigen recognized by virus-specific CTLs. Furthermore, we provide direct evidence that autologous CD8+ T cells rapidly kill CD4+ cells naturally infected with HTLV-I and expressing Tax in vitro by a perforin-dependent mechanism. Consistent with these observations, we observed a significant negative correlation between the frequency of Tax(11-19)-specific CD8+ T cells and the percentage of CD4+ T cells in peripheral blood of patients infected with HTLV-I. Those results are in accordance with the view that virus-specific CTLs participate in a highly efficient immune surveillance mechanism that persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo. (Blood. 2000;95:1386-1392)
Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

PubMed

Yan, Yuanwei; Sart, Sébastien; Calixto Bejarano, Fabian; Muroski, Megan E; Strouse, Geoffrey F; Grant, Samuel C; Li, Yan

2015-01-01

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers.
Withania somnifera Induces Cytotoxic and Cytostatic Effects on Human T Leukemia Cells

PubMed Central

Turrini, Eleonora; Calcabrini, Cinzia; Sestili, Piero; Catanzaro, Elena; de Gianni, Elena; Diaz, Anna Rita; Hrelia, Patrizia; Tacchini, Massimo; Guerrini, Alessandra; Canonico, Barbara; Papa, Stefano; Valdrè, Giovanni; Fimognari, Carmela

2016-01-01

Cancer chemotherapy is characterized by an elevated intrinsic toxicity and the development of drug resistance. Thus, there is a compelling need for new intervention strategies with an improved therapeutic profile. Immunogenic cell death (ICD) represents an innovative anticancer strategy where dying cancer cells release damage-associated molecular patterns promoting tumor-specific immune responses. The roots of Withania somnifera (W. somnifera) are used in the Indian traditional medicine for their anti-inflammatory, immunomodulating, neuroprotective, and anticancer activities. The present study is designed to explore the antileukemic activity of the dimethyl sulfoxide extract obtained from the roots of W. somnifera (WE). We studied its cytostatic and cytotoxic activity, its ability to induce ICD, and its genotoxic potential on a human T-lymphoblastoid cell line by using different flow cytometric assays. Our results show that WE has a significant cytotoxic and cytostatic potential, and induces ICD. Its proapoptotic mechanism involves intracellular Ca2+ accumulation and the generation of reactive oxygen species. In our experimental conditions, the extract possesses a genotoxic potential. Since the use of Withania is suggested in different contexts including anti-infertility and osteoarthritis care, its genotoxicity should be carefully considered for an accurate assessment of its risk–benefit profile. PMID:27187469
Involvement of tumour necrosis factor-alpha-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells.

PubMed

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-07-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257-264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand-tumour necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.
Induction of myeloma-specific cytotoxic T lymphocytes responses by natural killer cells stimulated-dendritic cells in patients with multiple myeloma.

PubMed

Nguyen-Pham, Thanh-Nhan; Im, Chang-Min; Nguyen, Truc-Anh Thi; Lim, Mi-Seon; Hong, Cheol Yi; Kim, Mi-Hyun; Lee, Hyun Ju; Lee, Youn-Kyung; Cho, Duck; Ahn, Jae-Sook; Yang, Deok-Hwan; Kim, Yeo-Kyeoung; Chung, Ik-Joo; Kim, Hyeoung-Joon; Lee, Je-Jung

2011-09-01

The interaction between dendritic cells (DCs) and natural killer (NK) cells plays a key role in inducing DC maturation for subsequent T-cell priming. We investigated to generate potent DCs by stimulated with NK cells to induce myeloma-specific cytotoxic T lymphocytes (CTLs). NK cells-stimulated-DCs exhibited high expression of costimulatory molecules and high production of IL-12p70. These DCs induce high potency of Th1 polarization and exhibit a high ability to generate myeloma-specific CTLs responses. These results suggest that functionally potent DCs can be generated by stimulation with NK cells and may provide an effective source of DC-based immunotherapy in multiple myeloma. Copyright © 2011 Elsevier Ltd. All rights reserved.
Increased Soluble CD226 in Sera of Patients with Cutaneous T-Cell Lymphoma Mediates Cytotoxic Activity against Tumor Cells via CD155.

PubMed

Takahashi, Naomi; Sugaya, Makoto; Suga, Hiraku; Oka, Tomonori; Kawaguchi, Makiko; Miyagaki, Tomomitsu; Fujita, Hideki; Inozume, Takashi; Sato, Shinichi

2017-08-01

Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances. CD155 is expressed in various types of cancer, and this surface molecule on tumor cells functions either as a co-stimulatory molecule or a co-inhibitory molecule, depending on its receptor. CD226, a CD155 ligand, is mainly expressed on natural killer cells and CD8 + T cells, playing important roles in natural killer cell-mediated cytotoxicity. In this study, we investigated the expression and function of CD155 and CD226 in cutaneous T-cell lymphoma (CTCL). CD155 was strongly expressed on tumor cells and CD155 mRNA expression levels were increased in CTCL lesional skin. CD226 expression on natural killer cells and CD8 + cells in peripheral blood of CTCL patients was decreased. On the other hand, serum CD226 levels were significantly elevated in CTCL patients, strongly reflecting disease activity, suggesting that soluble CD226 in sera was generated by shedding of its membrane form. Recombinant CD226 itself showed cytotoxic activity against CD155-expressing CTCL cells in vitro. These data suggest that soluble CD226 elevated in sera of CTCL patients would be important for tumor immunity by interacting with CD155 on tumor cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Cytotoxic and hemolytic effects of Tritrichomonas foetus on mammalian cells.

PubMed Central

Burgess, D E; Knoblock, K F; Daugherty, T; Robertson, N P

1990-01-01

Geographically distinct lines of Tritrichomonas foetus were assayed for their ability to cause cytotoxicity in nucleated mammalian cells and lysis of bovine erythrocytes. T. foetus was highly cytotoxic toward a human cervical cell line (HeLa) and early bovine lymphosarcoma (BL-3) but displayed low levels of cytotoxicity against African green monkey kidney (Vero) cells. In addition to variation in the extent of cytotoxicity toward different targets, differences in the levels of cytotoxicity in the same nucleated target occurred with different parasite lines. Whole T. foetus, unfractionated whole-cell extracts, and parasite-conditioned medium (RPMI 1640 without serum) all caused lysis of bovine erythrocytes. Lytic activity in the conditioned medium was substantially reduced by repeated freezing and thawing or heating to 90 degrees C for 30 min. Damage of mammalian target cells by live T. foetus could be reduced by the presence of protease inhibitors; however, such inhibitors did not diminish the lytic effects of conditioned medium. These results suggested that proteolytic enzymes were necessary for the lytic mechanism of the live parasites but were not required once lytic factors were released into the parasite-conditioned medium. They further suggested that the lytic molecules were either proteins or had proteinaceous components. Images PMID:2228233
Cervical cancer cells suppress effector functions of cytotoxic T cells through the adenosinergic pathway.

PubMed

Mora-García, M L; Ávila-Ibarra, L R; García-Rocha, R; Weiss-Steider, B; Hernández-Montes, J; Don-López, C A; Gutiérrez-Serrano, V; Titla-Vilchis, I J; Fuentes-Castañeda, M C; Monroy-Mora, A; Jave-Suárez, L F; Chacón-Salinas, R; Vallejo-Castillo, L; Pérez-Tapia, S M; Monroy-García, A

2017-10-01

The expression of CD73 in tumor cells plays a significant role in the production of adenosine (Ado) that suppresses antitumor effector cells. In this study we analyzed the capability of HPV-positive (HPV+) cervical cancer (CeCa) cell lines CaSki, SiHa, HeLa, and RoVa; and HPV-negative (HPV-) cell lines C33A and ViBo to produce Ado and inhibit effector functions of CD8+ T cells. HPV+ CeCa cells expressed significantly higher levels of CD73 in the membrane (p<0.01) than HPV- CeCa cells and this expression was associated with the production of larger amounts of Ado (>400μM) compared to HPV-CeCa cells (<200μM) in the presence of AMP, as well asa stronger inhibition of (>50%) proliferation, activation, and cytotoxic activity of CD8+ T cells via interaction with A2A adenosine receptor. We also provide evidence that silenced E6/E7 expression in CeCa cells, strongly reduced its CD73 expression level and its capability to generate Ado. This results suggest that HPV infection, which is associated with more than 99% of CeCa cases, may present an increased constitutive expression of CD73 in cervical neoplasia to contribute to the suppression of the immune response mediated by the production of large amounts of Ado. Copyright © 2017 Elsevier Inc. All rights reserved.
iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo

PubMed Central

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M.; Brennan, Patrick J.; Banerjee, Pinaki P.; Wiener, Susan J.; Orange, Jordan S.; Brenner, Michael B.; Grupp, Stephan A.; Nichols, Kim E.

2013-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we find that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially-induced by iNKT cell agonists of varying TCR affinities, such as OCH, α-galactosyl ceramide and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of T cell receptor (TCR) signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell-deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T-lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T-lymphoma. PMID:24563871
iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo .

PubMed

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M; Brennan, Patrick J; Banerjee, Pinaki P; Wiener, Susan J; Orange, Jordan S; Brenner, Michael B; Grupp, Stephan A; Nichols, Kim E

2014-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. ©2013 AACR.
Cytotoxic potential of decidual NK cells and CD8+ T cells awakened by infections.

PubMed

Crespo, Ângela C; van der Zwan, Anita; Ramalho-Santos, João; Strominger, Jack L; Tilburgs, Tamara

2017-02-01

To establish a healthy pregnancy the maternal immune system must tolerate fetal allo-antigens, yet remain competent to respond to infections. The ability of decidual NK cells (dNK) to promote migration of fetal extravillous trophoblasts (EVT) and placental growth as well as the capacity of EVT to promote immune tolerance are topics of high interest and extensive research. However, the problem of how dNK and decidual CD8+ T cells (CD8+ dT) provide immunity to infections of the placenta and the mechanisms that regulate their cytolytic function has thus far largely been ignored. Fetal EVT are the most invasive cells of the placenta and directly interact with maternal decidual immune cells at this maternal-fetal interface. Besides the expression of non-polymorphic HLA-E and HLA-G molecules that are associated with immune tolerance, EVT also express highly polymorphic HLA-C molecules that can serve as targets for maternal dNK and CD8+ dT responses. HLA-C expression by EVT has a dual role as the main molecule to which immune tolerance needs to be established and as the only molecule that can present pathogen-derived peptides and provide protective immunity when EVT are infected. The focus of this review is to address the regulation of cytotoxicity of dNK and CD8+ dT, which is essential for maternal-fetal immune tolerance as well as recent evidence that both cell types can provide immunity to infections at the maternal-fetal interface. A particular emphasis is given to the role of HLA-C expressed by EVT and its capacity to elicit dNK and CD8+ dT responses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Dynamic visualization the whole process of cytotoxic T lymphocytes killing the B16 tumor cells in vitro

NASA Astrophysics Data System (ADS)

Qi, Shuhong; Zhang, Zhihong

2016-03-01

Cytotoxic T lymphocytes (CTLs) played a key role in the immune system to destroy the tumor cells. Although some mechanisms of CTLs killing the tumor cells are revealed already, the dynamic information of CTLs interaction with tumor cells are still not known very clearly. Here we used confocal microscopy to visualize the whole process of CTLs killing the tumor cells in vitro. The imaging data showed that CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or some tumor cells and the average time for CTLs destroying one tumor cell is just a few minutes in vitro. The study displayed the temporal events of CTLs interacting with tumor cells at the beginning and finally killing them and directly presented the efficient tumor cell cytotoxicity of the CTLs. The results helped us to deeply understand the mechanism of the CTLs destroying the tumor cells and to develop the cancer immunotherapy.
Involvement of tumour necrosis factor-α-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells

PubMed Central

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-01-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257–264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand–tumour necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses. PMID:12100718
Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium

PubMed Central

Begum, Jusnara; Lal, Neeraj; Zuo, Jianmin; Beggs, Andrew; Moss, Paul

2016-01-01

Cytomegalovirus (CMV) infection elicits a very strong and sustained intravascular T cell immune response which may contribute towards development of accelerated immune senescence and vascular disease in older people. Virus-specific CD8+ T cell responses have been investigated extensively through the use of HLA-peptide tetramers but much less is known regarding CMV-specific CD4+ T cells. We used a range of HLA class II-peptide tetramers to investigate the phenotypic and transcriptional profile of CMV-specific CD4+ T cells within healthy donors. We show that such cells comprise an average of 0.45% of the CD4+ T cell pool and can reach up to 24% in some individuals (range 0.01–24%). CMV-specific CD4+ T cells display a highly differentiated effector memory phenotype and express a range of cytokines, dominated by dual TNF-α and IFN-γ expression, although substantial populations which express IL-4 were seen in some donors. Microarray analysis and phenotypic expression revealed a profile of unique features. These include the expression of CX3CR1, which would direct cells towards fractalkine on activated endothelium, and the β2-adrenergic receptor, which could permit rapid response to stress. CMV-specific CD4+ T cells display an intense cytotoxic profile with high level expression of granzyme B and perforin, a pattern which increases further during aging. In addition CMV-specific CD4+ T cells demonstrate strong cytotoxic activity against antigen-loaded target cells when isolated directly ex vivo. PD-1 expression is present on 47% of cells but both the intensity and distribution of the inhibitory receptor is reduced in older people. These findings reveal the marked accumulation and unique phenotype of CMV-specific CD4+ T cells and indicate how such T cells may contribute to the vascular complications associated with CMV in older people. PMID:27606804
Cytotoxic human peripheral blood-derived γδT cells kill glioblastoma cell lines: implications for cell-based immunotherapy for patients with glioblastoma.

PubMed

Nakazawa, Tsutomu; Nakamura, Mitsutoshi; Park, Young Soo; Motoyama, Yasushi; Hironaka, Yasuo; Nishimura, Fumihiko; Nakagawa, Ichiro; Yamada, Shuichi; Matsuda, Ryosuke; Tamura, Kentaro; Sugimoto, Tadashi; Takeshima, Yasuhiro; Marutani, Akiko; Tsujimura, Takahiro; Ouji, Noriko; Ouji, Yukiteru; Yoshikawa, Masahide; Nakase, Hiroyuki

2014-01-01

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell-cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αβT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32% U87MG, 15% U138MG, 1% A172, and 50% K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.

Enhanced Eradication of Lymphoma by Tumor-Specific Cytotoxic T-Cells Secreting an Engineered Tumor-Specific Immunotoxin

DTIC Science & Technology

2010-06-01

Boulad F, Carabasi M et al. Infusions of donor leukocytes to treat Epstein - Barr virus -associated lymphoproliferative disorders after allogeneic bone...Ng CY, Loftin SK, Sixbey JW, Gan Y et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein - Barr virus -induced lymphoma in... Epstein - Barr virus lymphoma after hemopoietic stem-cell transplantation. Blood 2000; 95: 1502-1505. 10. Davis TA, Czerwinski DK, Levy R. Therapy
T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses

PubMed Central

Tarasenko, Tatyana; Kole, Hemanta K.; Chi, Anthony W.; Mentink-Kane, Margaret M.; Wynn, Thomas A.; Bolland, Silvia

2007-01-01

The 5′-phosphoinositol phosphatase SHIP negatively regulates signaling pathways triggered by antigen, cytokine and Fc receptors in both lymphocytes and myeloid cells. Mice with germ-line (null) deletion of SHIP develop a myeloproliferative-like syndrome that causes early lethality. Lymphocyte anomalies have been observed in SHIP-null mice, but it is unclear whether they are due to an intrinsic requirement of SHIP in these cells or a consequence of the severe myeloid pathology. To precisely address the function of SHIP in T cells, we have generated mice with T cell-specific deletion of SHIP. In the absence of SHIP, we found no differences in thymic selection or in the activation state and numbers of regulatory T cells in the periphery. In contrast, SHIP-deficient T cells do not skew efficiently to Th2 in vitro. Mice with T cell-specific deletion of SHIP show poor antibody responses on Alum/NP-CGG immunization and diminished Th2 cytokine production when challenged with Schistosoma mansoni eggs. The failure to skew to Th2 responses may be the consequence of increased basal levels of the Th1-associated transcriptional factor T-bet, resulting from enhanced sensitivity to cytokine-mediated T-bet induction. SHIP-deficient CD8+ cells show enhanced cytotoxic responses, consistent with elevated T-bet levels in these cells. Overall our experiments indicate that in T cells SHIP negatively regulates cytokine-mediated activation in a way that allows effective Th2 responses and limits T cell cytotoxicity. PMID:17585010
Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes

PubMed Central

Posch, Wilfried; Cardinaud, Sylvain; Hamimi, Chiraz; Fletcher, Adam; Mühlbacher, Annelies; Loacker, Klaus; Eichberger, Paul; Dierich, Manfred P.; Pancino, Gianfranco; Lass-Flörl, Cornelia; Moris, Arnaud; Saez-Cirion, Asier; Wilflingseder, Doris

2014-01-01

Background Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8+ T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8+ T-cell responses. Objective We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)–mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. Methods Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8+ T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4+ T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. Results We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8+ T-cell response compared with the earlier described efficient CD8+ T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8+ T-cell clones. Conclusion Our ex vivo and in vitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs. PMID:23063584
Effect of lipoxygenase metabolites of arachidonic acid on proliferation of human T cells and T cell subsets.

PubMed

Gualde, N; Atluru, D; Goodwin, J S

1985-02-01

The lipoxygenase products LTB4 and 15 HPETE have been reported to stimulate T suppressor cell function and also to inhibit [3H]thymidine incorporation into mitogen-stimulated T cells. This present report documents that although these compounds do indeed inhibit [3H]thymidine incorporation into unfractionated T cells, they significantly enhance [3H]thymidine incorporation into T cell preparation enriched for cells bearing the cytotoxic suppressor cell phenotype identified by the OKT8 monoclonal antibody. The mitogen response of T cells enriched for OKT4+ helper-inducer cells is inhibited in manner similar to the response of unfractionated T cells. Thus, LTB4 and 15 HPETE stimulate both the function and the proliferation of the cytotoxic-suppressor T cell subset.
[Acquirement of autologous murine cytotoxic T lymphocytes via cryopreservation of lymphocytes].

PubMed

Wang, Lei; Peng, Na; Hu, Xiaoyan; Liang, Wentao; Liang, Kai; Peng, Guizhu

2016-11-01

Objective To evaluate the effects of cryopreservation on the proliferation and killing activity of lymphocytes, and explore a novel protocol of preparing autologous mouse cytotoxic T lymphocytes (CTLs). Methods Mononuclear cells derived from spleen (5.0×10 6 /mL) were cryopreserved in CELLBANKER2 for 6 days and recovered in water bath at 39DegreesCelsius. The fresh lymphocytes and post-cryopreservation lymphocytes were induced by CD3 mAb (100 ng/mL) and recombinant mouse interleukin 2 (rmIL-2, 100 ng/mL) to obtain cytokine-induced killer cells (CIKs). Dendritic cells (DCs) were co-cultured with fresh allogenic lymphocytes and post-cryopreservation autologous lymphocytes to obtain CTLs. The viable cells were counted by trypan blue staining; the percentages of CD3 + T cells and regulatory T cells (Tregs) were determined by flow cytometry; the levels of supernatant IFN-γ were detected through ELISA and the cytotoxicity was evaluated by lactate dehydrogenase (LDH) assay. Results The rate of viable lymphocytes declined to 78% after cryopreservation, and there were no significant differences in the percentages of CD3 + T cells and Tregs between pre-cryopreservation and post-cryopreservation. There were no significant differences in the proliferation of Tregs, the level of IFN-γ and the cytotoxicity between the fresh CIKs and cryopreservation CIKs, and the similar results were get between the autologous CTLs and allogenic CTLs. Conclusion The autologous CTLs acquired via cryopreservation of lymphocytes is equivalent to the allogenic CTLs with the similar proliferation and killing activity in vitro.
In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity

PubMed Central

Halle, Stephan; Keyser, Kirsten Anja; Stahl, Felix Rolf; Busche, Andreas; Marquardt, Anja; Zheng, Xiang; Galla, Melanie; Heissmeyer, Vigo; Heller, Katrin; Boelter, Jasmin; Wagner, Karen; Bischoff, Yvonne; Martens, Rieke; Braun, Asolina; Werth, Kathrin; Uvarovskii, Alexey; Kempf, Harald; Meyer-Hermann, Michael; Arens, Ramon; Kremer, Melanie; Sutter, Gerd; Messerle, Martin; Förster, Reinhold

2016-01-01

Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity. PMID:26872694
Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

PubMed Central

Uhlig, Katharina M.; Schülke, Stefan; Scheuplein, Vivian A. M.; Malczyk, Anna H.; Reusch, Johannes; Kugelmann, Stefanie; Muth, Anke; Koch, Vivian; Hutzler, Stefan; Bodmer, Bianca S.; Schambach, Axel; Buchholz, Christian J.; Waibler, Zoe; Scheurer, Stephan

2015-01-01

ABSTRACT To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8+ T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8+ T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8+ T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of
Possible role of PAPR-1 in protecting human HaCaT cells against cytotoxicity of SiO2 nanoparticles.

PubMed

Gong, Chunmei; Yang, Linqing; Zhou, Jichang; Guo, Xiang; Zhuang, Zhixiong

2017-10-05

Nano-SiO 2 materials play a significant role in the engineered nanomaterials (ENMs) field. The ease of their production as well as their relatively low cost has promoted the wide use of these products in many fields. Nano-SiO 2 exposure is known to cause severe DNA damage; however, the underlying mechanisms remain poorly understood. In a previous study, we found that nano-SiO 2 exposure regulate the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down PAPR-1 expression in HaCaT cells and explored the potential role of PARP-1 in nano-SiO 2 induced cytotoxicity. We found that nano-SiO 2 treatment of HaCaT cells causes decreased cell viability, increased apoptosis and DNA damage. Nano-SiO 2 -treated HaCaT cells were also found to have slightly changed cell cycle distribution. Lentivirus-mediated PAPR-1 knockdown partially aggravated cytotoxicity and increased apoptosis induced by nano-SiO 2 treatment. Nano-SiO 2 had significant toxicity to human HaCaT cells and causes DNA damage. PAPR-1 knock-down cell line appears more sensitive to nano-SiO 2 than the control cells in DNA damage. The results suggest that PAPR-1 is involved in protecting cells from damage caused by nano-SiO 2 . Copyright © 2017 Elsevier B.V. All rights reserved.
Identification of an HLA-A24-restricted OY-TES-1 epitope recognized by cytotoxic T-cells.

PubMed

Okumura, Hideo; Noguchi, Yuji; Uenaka, Akiko; Aji, Toshiki; Ono, Toshiro; Nakagawa, Kazuhiko; Aoe, Motoi; Shimizu, Nobuyoshi; Nakayama, Eiichi

2005-01-01

OY-TES-1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY-TES-1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY-TES-1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA-A24-binding OY-TES-1 peptide, TES(401-409) (KTPFVSPLL) recognized by CD8 T-cells. Purified CD8 T-cells from healthy donors stimulated in vitro with the peptide-pulsed autologous DC and PBMC produced IFNgamma in response to the peptide-pulsed PBMC and showed cytotoxicity against the peptide-pulsed autologous EBV-B specifically. Furthermore, cytotoxicity was also observed against an OY-TES-1 mRNA-expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV-B cells in recognition by CD8 CTL was the same as that of the fraction of the TES(401-409) peptide itself, suggesting that the TES(401-409) was a naturally processed peptide on LK79.
Cystatin F as a regulator of immune cell cytotoxicity.

PubMed

Kos, Janko; Nanut, Milica Perišić; Prunk, Mateja; Sabotič, Jerica; Dautović, Esmeralda; Jewett, Anahid

2018-05-10

Cysteine cathepsins are lysosomal peptidases involved in the regulation of innate and adaptive immune responses. Among the diverse processes, regulation of granule-dependent cytotoxicity of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells during cancer progression has recently gained significant attention. The function of cysteine cathepsins is regulated by endogenous cysteine protease inhibitors-cystatins. Whereas other cystatins are generally cytosolic or extracellular proteins, cystatin F is present in endosomes and lysosomes and is thus able to regulate the activity of its target directly. It is delivered to endosomal/lysosomal vesicles as an inactive, disulphide-linked dimer. Proteolytic cleavage of its N-terminal part leads to the monomer, the only form that is a potent inhibitor of cathepsins C, H and L, involved in the activation of granzymes and perforin. In NK cells and CTLs the levels of active cathepsin C and of granzyme B are dependent on the concentration of monomeric, active cystatin F. In tumour microenvironment, inactive dimeric cystatin F can be secreted from tumour cells or immune cells and further taken up by the cytotoxic cells. Subsequent monomerization and inhibition of cysteine cathepsins within the endosomal/lysosomal vesicles impairs granzyme and perforin activation, and provokes cell anergy. Further, the glycosylation pattern has been shown to be important in controlling secretion of cystatin F from target cells, as well as internalization by cytotoxic cells and trafficking to endosomal/lysosomal vesicles. Cystatin F is therefore an important mediator used by bystander cells to reduce NK and T-cell cytotoxicity.
Cytotoxic activities of CD8+ T cells collaborate with macrophages to protect against blood-stage murine malaria

PubMed Central

Imai, Takashi; Ishida, Hidekazu; Suzue, Kazutomo; Taniguchi, Tomoyo; Okada, Hiroko; Shimokawa, Chikako; Hisaeda, Hajime

2015-01-01

The protective immunity afforded by CD8+ T cells against blood-stage malaria remains controversial because no MHC class I molecules are displayed on parasite-infected human erythrocytes. We recently reported that rodent malaria parasites infect erythroblasts that express major histocompatibility complex (MHC) class I antigens, which are recognized by CD8+ T cells. In this study, we demonstrate that the cytotoxic activity of CD8+ T cells contributes to the protection of mice against blood-stage malaria in a Fas ligand (FasL)-dependent manner. Erythroblasts infected with malarial parasites express the death receptor Fas. CD8+ T cells induce the externalization of phosphatidylserine (PS) on the infected erythroblasts in a cell-to-cell contact-dependent manner. PS enhances the engulfment of the infected erythroid cells by phagocytes. As a PS receptor, T-cell immunoglobulin-domain and mucin-domain-containing molecule 4 (Tim-4) contributes to the phagocytosis of malaria-parasite-infected cells. Our findings provide insight into the molecular mechanisms underlying the protective immunity exerted by CD8+ T cells in collaboration with phagocytes. DOI: http://dx.doi.org/10.7554/eLife.04232.001 PMID:25760084
Peptide-independent Recognition by Alloreactive Cytotoxic T Lymphocytes (CTL)

PubMed Central

Smith, Pamela A.; Brunmark, Anders; Jackson, Michael R.; Potter, Terry A.

1997-01-01

We have isolated several H-2Kb–alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2Kb. These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2Kb molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2Kb on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2b) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2Kbm8 molecule. These findings suggested that the clones recognized determinants on H-2Kb that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2Kb produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2Kb molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2–incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent. PMID:9091576
Infiltration of γ⁢δ T cells, IL-17+ T cells and FoxP3+ T cells in human breast cancer

PubMed Central

Allaoui, Roni; Hagerling, Catharina; Desmond, Eva; Warfvinge, Carl-Fredrik; Jirström, Karin; Leandersson, Karin

2017-01-01

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) have a strong prognostic value in various forms of cancers. These data often refer to use of the pan-T cell marker CD3, or the cytotoxic T lymphocyte marker CD8α. However, T cells are a heterogeneous group of cells with a wide array of effector mechanisms ranging from immunosuppression to cytotoxicity. OBJECTIVE: In this study we have investigated the prognostic effects of some unconventional T cell subtypes in breast cancer; γ⁢δ T cells, IL-17+ T cells and FoxP3+ T cells (Tregs) in relation to the conventional CD3 and CD8α T cell markers. METHODS: This was done using immunohistochemistry on a human breast cancer tissue microarray consisting of 498 consecutive cases of primary breast cancer. RESULTS: Infiltration of γ⁢δ T cells and T cell infiltration in general (CD3), correlated with a good prognosis, while Treg infiltration with a worse. Infiltration of γ⁢δ T cells was associated with a significantly improved clinical outcome in all breast cancer subtypes except triple negative tumors. Only infiltration of either CD3+ or CD8α+ cells was independently associated with better prognosis for all breast cancer patients. CONCLUSIONS: This study sheds further light on the prognostic impact of various T cell subtypes in breast cancer. PMID:29060923
Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines.

PubMed

Zakrzewska, Karolina Ewa; Samluk, Anna; Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Sawosz, Ewa; Chwalibog, André; Pijanowska, Dorota Genowefa; Pluta, Krzysztof Dariusz

2015-01-01

Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests.
Fas/FasL interaction mediates imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells.

PubMed

Dou, Rui; Hong, Zhenya; Tan, Xiaosheng; Hu, Fenfen; Ding, Yajie; Wang, Wei; Liang, Zhihui; Zhong, Rongrong; Wu, Xiongwen; Weng, Xiufang

2018-07-01

The rapid antitumor cytokine production and direct cytotoxicity confer invariant NKT (iNKT) cells ideal candidates for cancer therapy. However, the therapeutic potential of iNKT cells in T-cell malignant diseases remains elusive, as antigen presentation by T cells (T-T presentation) has been suggested to induce hyporesponsiveness of iNKT cells. In this study, we found discrepancies in iNKT cell responses against two T cell-origin cell lines (Jurkat and Molt-4). Human iNKT cells exhibited more intensive cytotoxicity and less efficient cytokine production in response to Fas-bearing Jurkat cells than those to the Fas-negative tumor cells (Molt-4 and myeloid-derived K562). The imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells were CD1d-dependent and relied mostly on Fas/FasL interaction. The impairment in cytokine production could be overcome by Fas/FasL blocking antibodies and exogenous IL-2. Elevated CD1d levels as well as CD1d and Fas co-localization were found in T-cell lymphomas. However, defects in frequency and function of circulating iNKT cells were observed in the patients, which could be partly rescued by exogenous IL-2. Collectively, the Fas/FasL-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation in CD1d- and Fas-bearing T cell malignancies. Copyright © 2018. Published by Elsevier Ltd.
Adenosine, but not guanosine, protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity.

PubMed

Menezes, Camila Braz; Frasson, Amanda Piccoli; Meirelles, Lucia Collares; Tasca, Tiana

2017-02-01

Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5'-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
High-efficient and high-content cytotoxic recording via dynamic and continuous cell-based impedance biosensor technology.

PubMed

Hu, Ning; Fang, Jiaru; Zou, Ling; Wan, Hao; Pan, Yuxiang; Su, Kaiqi; Zhang, Xi; Wang, Ping

2016-10-01

Cell-based bioassays were effective method to assess the compound toxicity by cell viability, and the traditional label-based methods missed much information of cell growth due to endpoint detection, while the higher throughputs were demanded to obtain dynamic information. Cell-based biosensor methods can dynamically and continuously monitor with cell viability, however, the dynamic information was often ignored or seldom utilized in the toxin and drug assessment. Here, we reported a high-efficient and high-content cytotoxic recording method via dynamic and continuous cell-based impedance biosensor technology. The dynamic cell viability, inhibition ratio and growth rate were derived from the dynamic response curves from the cell-based impedance biosensor. The results showed that the biosensors has the dose-dependent manners to diarrhetic shellfish toxin, okadiac acid based on the analysis of the dynamic cell viability and cell growth status. Moreover, the throughputs of dynamic cytotoxicity were compared between cell-based biosensor methods and label-based endpoint methods. This cell-based impedance biosensor can provide a flexible, cost and label-efficient platform of cell viability assessment in the shellfish toxin screening fields.
Ionophore-A23187-induced cellular cytotoxicity: a cell fragment mediated process.

PubMed Central

Nash, G S; Niedt, G W; MacDermott, R P

1980-01-01

Calcium ionophore A23187 was found to induce human white blood cells to kill human red blood cells. Optimal conditions for ionophore-induced cellular cytotoxicity (IICC) included an 18 h time period, an incubation temperature of 25 degrees, a 25:1 or 50:1 killer:target cell ratio,and a final ionophore concentration of 2 . 5 microgram/ml. WBC or granulocytes which were either frozen and thawed three times or sonicated were capable of mediating IICC. As intact cells, granulocytes (67 . 2% cytotoxicity), monocytes (34 . 8%), B cells (22 . 0%) and Null cells (19 . 3%) were effector cells but T cells (7 . 4%) were not. After fragmenting these cells, all cell types including T cells were able to mediate IICC. When cell lines (K562, Chang, and NCTC) were used as effectors, none would mediate IICC when intact. After freezing and thawing, Chang and NCTC would not mediate IICC, whereas K562 cells did. These studies may be indicative of a calcium-dependent, membrane-localized mechanism in cellular cytotoxic processes, and may provide a useful indicator system for isolation of the enzyme systems involved in cellular cytotoxicity. PMID:6773881
Differential requirements of CD4(+) T-cell signals for effector cytotoxic T-lymphocyte (CTL) priming and functional memory CTL development at higher CD8(+) T-cell precursor frequency.

PubMed

Umeshappa, Channakeshava S; Nanjundappa, Roopa H; Xie, Yufeng; Freywald, Andrew; Xu, Qingyong; Xiang, Jim

2013-04-01

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions. © 2012 Blackwell Publishing Ltd.
Functional analysis of T cells expressing Ia antigens. I. Demonstration of helper T-cell heterogeneity.

PubMed

Swierkosz, J E; Marrack, P; Kappler, J W

1979-12-01

We have examined the expression of I-region antigens on functional subpopulations of murine T cells. A.TH anti-A.TL (anti-Ik, Sk, Gk) alloantiserum was raised by immunization of recipients with concanavalin A (Con A) stimulated thymic and peripheral T-cell blasts. In contrast to similar antisera made by conventional methods, the anti-Ia blast serum was highly cytotoxic for purified T lymphocytes. Moreover, it reacted in a specific fashion with T cells having particular functions. Treatment of keyhole limpet hemocyanin (KLH)-primed B10.A (H-2 alpha) T cells with this antiserum plus complement resulted in the elimination of helper activity for B-cell responses to trinitrophenyl-KLH. Inhibition was shown to be a result of the selective killing of one type of helper T cell whose activity could be replaced by a factor(s) found in the supernate of Con A-activated spleen cells. A second type of helper cell required for responses to protein-bound antigens appeared to be Ia-. By absorption and analysis on H-2 recombinants, at least two specificities were detectable on helper T cells; one mapping in the I-A subregion and a second in a region(s) to the right of I-J. In addition, the helper T cell(s) involved in the generation of alloreactive cytotoxic lymphocytes was shown to be Ia+, whereas cytotoxic effector cells and their precursors were Ia- with this antiserum. These results provide strong evidence for the selective expression of I-region determinants on T-cell subsets and suggest that T-cell-associated Ia antigens may play an important role in T-lymphocyte function.

Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy.

PubMed

Xu, Wei-li; Li, Suo-lin; Wen, Ming; Wen, Jun-ye; Han, Jie; Zhang, Hong-zhen; Gao, Fei; Cai, Jian-hui

2013-08-01

Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers, immunophenotype, tumor-specificity, and in vivo residence time, migration, and distribution. Therefore, tracing in vivo persistence, migration, and distribution of CTLs is important for cancer immunotherapy. Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared. Additionally, CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma, and their residence time, migration, and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values. Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed. Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity. No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P = 0.849) or interleukin-2 (P = 0.318) and interferon-γ (P = 0.201) levels. Distribution of CTLs in vivo varied with time. A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed. CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion. CTLs were observed up to three weeks later in the tumor, liver, kidneys, and spleen; this was related to the abundant blood supply or the nature of immune organs. CCK-8 assay is a novel method to select optimal CFSE staining concentrations. Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors. CFSE fluorescent markers can trace in vivo CTL persistence, migration, and distribution because of its stability
Analysis of the Cytotoxicity of Carbon-Based Nanoparticles, Diamond and Graphite, in Human Glioblastoma and Hepatoma Cell Lines

PubMed Central

Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Sawosz, Ewa; Chwalibog, André; Pijanowska, Dorota Genowefa; Pluta, Krzysztof Dariusz

2015-01-01

Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests. PMID:25816103
Effects of folic acid deficiency and MTHFRC677T polymorphisms on cytotoxicity in human peripheral blood lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu Xiayu; Liang Ziqing; Zou Tianning

2009-02-13

Apoptosis (APO) and necrosis (NEC) are two different types of cell death occurring in response to cellular stress factors. Cells with DNA damage may undergo APO or NEC. Folate is an essential micronutrient associated with DNA synthesis, repair and methylation. Methylenetetrahydrofolate reductase (MTHFR) regulates intracellular folate metabolism. Folate deficiency and MTHFR C677T polymorphisms have been shown to be related to DNA damage. To verify the cytotoxic effects of folate deficiency on cells with different MTHFR C677T genotypes, 15 human peripheral lymphocyte cases with different MTHFR C677T genotypes were cultured in folic acid (FA)-deficient and -sufficient media for 9 days. Cytotoxicitymore » was quantified using the frequencies of APO and NEC as endpoints, the nuclear division index (NDI), and the number of viable cells (NVC). These results showed that FA is an important factor in reducing cytotoxicity and increasing cell proliferation. Lymphocytes with the TT genotype proliferated easily under stress and exhibited different responses to FA deficiency than lymphocytes with the CC and CT genotypes. A TT individual may accumulate more cytotoxicity under cytotoxic stress, suggesting that the effects of FA deficiency on cytotoxicity are greater than the effects in individuals with the other MTHFR C677T variants.« less
Myeloid Cell COX-2 deletion reduces mammary tumor growth through enhanced cytotoxic T-lymphocyte function

PubMed Central

Chen, Edward P.; Markosyan, Nune; Connolly, Emma; Lawson, John A.; Li, Xuanwen; Grant, Gregory R.; Grosser, Tilo; FitzGerald, Garret A.; Smyth, Emer M.

2014-01-01

Cyclooxygenase-2 (COX-2) expression is associated with poor prognosis across a range of human cancers, including breast cancer. The contribution of tumor cell-derived COX-2 to tumorigenesis has been examined in numerous studies; however, the role of stromal-derived COX-2 is ill-defined. Here, we examined how COX-2 in myeloid cells, an immune cell subset that includes macrophages, influences mammary tumor progression. In mice engineered to selectively lack myeloid cell COX-2 [myeloid-COX-2 knockout (KO) mice], spontaneous neu oncogene-induced tumor onset was delayed, tumor burden reduced, and tumor growth slowed compared with wild-type (WT). Similarly, growth of neu-transformed mammary tumor cells as orthotopic tumors in immune competent syngeneic myeloid-COX-2 KO host mice was reduced compared with WT. By flow cytometric analysis, orthotopic myeloid-COX-2 KO tumors had lower tumor-associated macrophage (TAM) infiltration consistent with impaired colony stimulating factor-1-dependent chemotaxis by COX-2 deficient macrophages in vitro. Further, in both spontaneous and orthotopic tumors, COX-2-deficient TAM displayed lower immunosuppressive M2 markers and this was coincident with less suppression of CD8+ cytotoxic T lymphocytes (CTLs) in myeloid-COX-2 KO tumors. These studies suggest that reduced tumor growth in myeloid-COX-2 KO mice resulted from disruption of M2-like TAM function, thereby enhancing T-cell survival and immune surveillance. Antibody-mediated depletion of CD8+, but not CD4+ cells, restored tumor growth in myeloid-COX-2 KO to WT levels, indicating that CD8+ CTLs are dominant antitumor effectors in myeloid-COX-2 KO mice. Our studies suggest that inhibition of myeloid cell COX-2 can potentiate CTL-mediated tumor cytotoxicity and may provide a novel therapeutic approach in breast cancer therapy. PMID:24590894
Immunoglobulin-like transcript receptors on human dermal CD14+ dendritic cells act as a CD8-antagonist to control cytotoxic T cell priming

PubMed Central

Banchereau, Jacques; Zurawski, Sandra; Thompson-Snipes, LuAnn; Blanck, Jean-Philippe; Clayton, Sandra; Munk, Adiel; Cao, Yanying; Wang, Zhiqing; Khandelwal, Sunaina; Hu, Jiancheng; McCoy, William H.; Palucka, Karolina A.; Reiter, Yoram; Fremont, Daved H.; Zurawski, Gerard; Colonna, Marco; Shaw, Andrey S.; Klechevsky, Eynav

2012-01-01

Human Langerhans cells (LCs) are highly efficient at priming cytolytic CD8+ T cells compared with dermal CD14+ dendritic cells (DCs). Here we show that dermal CD14+ DCs instead prime a fraction of naïve CD8+ T cells into cells sharing the properties of type 2 cytokine-secreting CD8+ T cells (TC2). Differential expression of the CD8-antagonist receptors on dermal CD14+ DCs, the Ig-like transcript (ILT) inhibitory receptors, explains the difference between the two types of DCs. Inhibition of CD8 function on LCs inhibited cytotoxic T lymphocytes (CTLs) and enhanced TC2 generation. In addition, blocking ILT2 or ILT4 on dermal CD14+ DCs enhanced the generation of CTLs and inhibited TC2 cytokine production. Lastly, addition of soluble ILT2 and ILT4 receptors inhibited CTL priming by LCs. Thus, ILT receptor expression explains the polarization of CD8+ T-cell responses by LCs vs. dermal CD14+ DCs. PMID:23112154
CD8(+) T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types.

PubMed

Migueles, Stephen A; Mendoza, Daniel; Zimmerman, Matthew G; Martins, Kelly M; Toulmin, Sushila A; Kelly, Elizabeth P; Peterson, Bennett A; Johnson, Sarah A; Galson, Eric; Poropatich, Kate O; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A; Jones, Sara; Hallahan, Claire W; Follmann, Dean A; Connors, Mark

2015-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8(+) T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8(+) T-cell specificity and function of B*27/57(neg) LTNP/EC (n = 23), B*27/57(pos) LTNP/EC (n = 23) and B*27/57(neg) progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57(neg) LTNP/EC did not target more highly conserved epitopes, their CD8(+) T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57(pos) LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8(+) T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people.
Opposing Development of Cytotoxic and Follicular Helper CD4 T Cells Controlled by the TCF-1-Bcl6 Nexus.

PubMed

Donnarumma, Tiziano; Young, George R; Merkenschlager, Julia; Eksmond, Urszula; Bongard, Nadine; Nutt, Stephen L; Boyer, Claude; Dittmer, Ulf; Le-Trilling, Vu Thuy Khanh; Trilling, Mirko; Bayer, Wibke; Kassiotis, George

2016-11-01

CD4 + T cells develop distinct and often contrasting helper, regulatory, or cytotoxic activities. Typically a property of CD8 + T cells, granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4 + T cells. However, the conditions that induce CD4 + CTLs are not entirely understood. Using single-cell transcriptional profiling, we uncover a unique signature of Granzyme B (GzmB) + CD4 + CTLs, which distinguishes them from other CD4 + T helper (Th) cells, including Th1 cells, and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4 + CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4 + CTLs offers targets for their study, and its antagonism by the Tfh program separates CD4 + T cells with either helper or killer functions. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses.

PubMed

Sedlik, C; Dridi, A; Deriaud, E; Saron, M F; Rueda, P; Sarraseca, J; Casal, J I; Leclerc, C

1999-04-01

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.
Intranasal Delivery of Recombinant Parvovirus-Like Particles Elicits Cytotoxic T-Cell and Neutralizing Antibody Responses

PubMed Central

Sedlik, C.; Dridi, A.; Deriaud, E.; Saron, M. F.; Rueda, P.; Sarraseca, J.; Casal, J. I.; Leclerc, C.

1999-01-01

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4+ and CD8+ T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8+ T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8+ T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration. PMID:10074120
Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

PubMed

de Lourdes Mora-García, María; García-Rocha, Rosario; Morales-Ramírez, Omar; Montesinos, Juan José; Weiss-Steider, Benny; Hernández-Montes, Jorge; Ávila-Ibarra, Luis Roberto; Don-López, Christian Azucena; Velasco-Velázquez, Marco Antonio; Gutiérrez-Serrano, Vianey; Monroy-García, Alberto

2016-10-26

In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.
[gammadelta T cells stimulated by zoledronate kill osteosarcoma cells].

PubMed

Jiang, Hui; Xu, Qiang; Yang, Chao; Cao, Zhen-Guo; Li, Zhao-Xu; Ye, Zhao-Ming

2010-12-01

To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways. The peripheral blood mononuclear cells (PBMCs)of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks, analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit. Pre-treatment of γδT cells with anti-human γδTCR antibody, anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity. In vivo, BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors. And they were randomized into two groups: unteated group, γδT cell therapy group. Tumor volume and weight of the two groups were compared. After two weeks of culture, γδT cells from zoledronate-stimulated PBMCs could reach (95±3)%. When the E:T as 6:1, 12:1, 25:1, 50:1, the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%, 31.5%, 37.8%, 40.9%, respectively.When anti-huma γδTCR antibody, anti-human NKG2D antibody and concanamycin A blocked the relavent pathways, the percentage was 32.3%, 4.7%, 16.7% ( E:T as 25:1), respectively. In vivo, the tumor inhibition rate of the group of γδT cell therapy was 42.78%. γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells. And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.
LFA-1 Mediates Cytotoxicity and Tissue Migration of Specific CD8+ T Cells after Heterologous Prime-Boost Vaccination against Trypanosoma cruzi Infection

PubMed Central

Ferreira, Camila Pontes; Cariste, Leonardo Moro; Santos Virgílio, Fernando Dos; Moraschi, Barbara Ferri; Monteiro, Caroline Brandão; Vieira Machado, Alexandre M.; Gazzinelli, Ricardo Tostes; Bruna-Romero, Oscar; Menin Ruiz, Pedro Luiz; Ribeiro, Daniel Araki; Lannes-Vieira, Joseli; Lopes, Marcela de Freitas; Rodrigues, Mauricio Martins; de Vasconcelos, José Ronnie Carvalho

2017-01-01

Integrins mediate the lymphocyte migration into an infected tissue, and these cells are essential for controlling the multiplication of many intracellular parasites such as Trypanosoma cruzi, the causative agent of Chagas disease. Here, we explore LFA-1 and VLA-4 roles in the migration of specific CD8+ T cells generated by heterologous prime-boost immunization during experimental infection with T. cruzi. To this end, vaccinated mice were treated with monoclonal anti-LFA-1 and/or anti-VLA-4 to block these molecules. After anti-LFA-1, but not anti-VLA-4 treatment, all vaccinated mice displayed increased blood and tissue parasitemia, and quickly succumbed to infection. In addition, there was an accumulation of specific CD8+ T cells in the spleen and lymph nodes and a decrease in the number of those cells, especially in the heart, suggesting that LFA-1 is important for the output of specific CD8+ T cells from secondary lymphoid organs into infected organs such as the heart. The treatment did not alter CD8+ T cell effector functions such as the production of pro-inflammatory cytokines and granzyme B, and maintained the proliferative capacity after treatment. However, the specific CD8+ T cell direct cytotoxicity was impaired after LFA-1 blockade. Also, these cells expressed higher levels of Fas/CD95 on the surface, suggesting that they are susceptible to programmed cell death by the extrinsic pathway. We conclude that LFA-1 plays an important role in the migration of specific CD8+ T cells and in the direct cytotoxicity of these cells. PMID:29081775
Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy

PubMed Central

Chen, Can; Tan, Wee-Kiat; Chi, Zhixia; Xu, Xue-Hu; Wang, Shu

2016-01-01

Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer. PMID:27598655
Redirecting T cells to eradicate B-cell acute lymphoblastic leukemia: bispecific T-cell engagers and chimeric antigen receptors.

PubMed

Aldoss, I; Bargou, R C; Nagorsen, D; Friberg, G R; Baeuerle, P A; Forman, S J

2017-04-01

Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r
Effector CD8+ T cell IFN-γ production and cytotoxicity are enhanced by mild hyperthermia

PubMed Central

Mace, Thomas A.; Zhong, Lingwen; Kokolus, Kathleen M.; Repasky, Elizabeth A.

2012-01-01

Purpose Clinical trials combining hyperthermia with radiation and/or chemotherapy for cancer treatment have resulted in improved overall survival and control of local recurrences. The contribution of thermally enhanced anti-immune function in these effects is of considerable interest, but not understood; studies on the fundamental effects of elevated temperature on immune effector cells are needed. The goal of this study is to investigate the potential of mild hyperthermia to impact tumor antigen-specific (Ag) effector CD8+ T cell functions. Method Pmel-1 Ag-specific CD8+ T cells were exposed to mild hyperthermia and tested for changes in IFN-γ production and cytotoxicity. Additionally, overall plasma membrane organization and the phosphorylation of signaling proteins were also investigated following heat treatment. Results Exposing effector Pmel-1 specific CD8+ T cells to mild hyperthermia (39.5°C) resulted in significantly enhanced Ag-specific IFN-γ production and tumor target cell killing compared to that seen using lower temperatures (33 and 37°C). Further, inhibition of protein synthesis during hyperthermia did not reduce subsequent Ag-induced IFN-γ production by CD8+ T cells. Correlated with these effects, we observed a distinct clustering of GM1+ lipid microdomains at the plasma membrane and enhanced phosphorylation of LAT and PKCθ which may be related to an observed enhancement of Ag-specific effector CD8+ T cell IFN-γ gene transcription following mild hyperthermia. However, mitogen–mediated production of IFN-γ, which bypasses T cell receptor activation with antigen, was not enhanced. Conclusions Antigen-dependent effector T cell activity is enhanced following mild hyperthermia. These effects could potentially occur in patients being treated with thermal therapies. These data also provide support for the use of thermal therapy as an adjuvant for immunotherapies to improve CD8+ effector cell function. PMID:22235780
Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients

PubMed Central

Romano, Emanuela; Kusio-Kobialka, Monika; Foukas, Periklis G.; Baumgaertner, Petra; Meyer, Christiane; Ballabeni, Pierluigi; Michielin, Olivier; Weide, Benjamin; Romero, Pedro; Speiser, Daniel E.

2015-01-01

Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4–specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14++CD16− monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68+/CD163+ macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti–CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients. PMID:25918390
Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

PubMed

Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

2018-06-01

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.
Polyclonal proliferation of activated suppressor/cytotoxic T cells with transient depression of natural killer cell function in acute infectious mononucleosis.

PubMed Central

Williams, M L; Loughran, T P; Kidd, P G; Starkebaum, G A

1989-01-01

In acute infectious mononucleosis large numbers of atypical lymphocytes proliferate in response to B cells infected with Epstein-Barr virus, generally resulting in a self-limited illness. Although both T-cells and NK cells are known to be involved, the precise origin of the large granular lymphocytes in this disorder is incompletely understood. Using two-colour immunofluorescent flow cytometry, we sequentially examined the phenotype of selected T cell and NK cell subsets from nine patients with infectious mononucleosis. In parallel, we determined whether these lymphocytes utilized a restricted repertoire of the T cell receptor gene and also measured their NK activity. Our results show that in acute infectious mononucleosis there was a greater than three-fold increase in T lymphocytes with the phenotype CD2+, CD3+, CD8+ and DR+. A modest increase in Leu7(HNK1)+ and CD4+ T cells was also seen. In addition, there was a three-fold increase in cells coexpressing CD3- and CD16+, the phenotype reported to represent most NK cells. In spite of this latter finding, however, a marked decrease in NK function was found at the time of diagnosis, gradually returning to normal by day 28. Finally, Southern blot analysis of DNA from patient lymphocytes showed polyclonal rearrangements of the T cell receptor beta chain gene. These studies indicate that the proliferation of activated suppressor/cytotoxic T lymphocytes in acute infectious mononucleosis is polyclonal and is associated with transient depression of NK function. Images Fig. 2 PMID:2527653
Clinically Relevant Cytotoxic Immune Cell Signatures and Clonal Expansion of T Cell Receptors in High-risk MYCN-not-amplified Human Neuroblastoma.

PubMed

Wei, Jun S; Kuznetsov, Igor B; Zhang, Shile; Song, Young K; Asgharzadeh, Shahab; Sindiri, Sivasish; Wen, Xinyu; Patidar, Rajesh; Nagaraj, Sushma; Walton, Ashley; Guidry Auvil, Jaime M; Gerhard, Daniela S; Yuksel, Aysen; Catchpoole, Daniel R; Hewitt, Stephen M; Sondel, Paul M; Seeger, Robert C; Maris, John M; Khan, Javed

2018-05-21

High-risk neuroblastoma is an aggressive disease. DNA sequencing studies have revealed a paucity of actionable genomic alterations and a low mutation burden, posing challenges to develop effective novel therapies. We used RNA sequencing (RNA-seq) to investigate the biology of this disease including a focus on tumor-infiltrating lymphocytes (TILs). We performed deep RNA-seq on pre-treatment diagnostic tumors from 129 high-risk and 21 low- or intermediate-risk patients with neuroblastomas. We used single-sample gene set enrichment analysis to detect gene expression signatures of TILs in tumors and examined their association with clinical and molecular parameters including patient outcome. The expression profiles of 190 additional pre-treatment diagnostic neuroblastomas, a neuroblastoma tissue microarray, and T-cell receptor (TCR) sequencing were used to validate our findings. We found that MYCN -not-amplified ( MYCN -NA) tumors had significant higher cytotoxic TIL signatures compared to MYCN -amplified ( MYCN -A) tumors. A reported MYCN-activation-signature was significantly associated with poor outcome for high-risk patients with MYCN -NA tumors; however, a subgroup of these patients who had elevated activated NK cells, CD8+ T-cells, and cytolytic signatures showed improved outcome and expansion of infiltrating T-cell receptor (TCR) clones. Furthermore, we observed up-regulation of immune exhaustion marker genes, indicating an immune suppressive microenvironment in these neuroblastomas. Conclusions: This study provides evidence that RNA signatures of cytotoxic TIL are associated with the presence of activated NK-/T-cells and improved outcomes in high-risk neuroblastoma patients harboring MYCN -NA tumors. Our findings suggest that these high-risk patients with MYCN -NA neuroblastoma may benefit from additional immunotherapies incorporated into the current therapeutic strategies. Copyright ©2018, American Association for Cancer Research.
Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

PubMed

Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

1984-09-01

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

A hybrid of B and T lymphoblastic cell line could potentially substitute dendritic cells to efficiently expand out Her-2/neu-specific cytotoxic T lymphocytes from advanced breast cancer patients in vitro.

PubMed

Chen, Sheng; Gu, Feifei; Li, Kang; Zhang, Kai; Liu, Yangyang; Liang, Jinyan; Gao, Wei; Wu, Gang; Liu, Li

2017-02-28

Adoptive transfer of cytotoxic T lymphocytes (CTLs) holds promises to cure cancer. However, this treatment is hindered by lacking a robust way to specifically expand out CTLs. Here, we developed a hybrid of B lymphoblastic cell line and T lymphoblastic cell line (T2 cells) as a substitute of dendritic cells, together with irradiated autologous peripheral blood mononuclear cell (PBMC) as feeder cells and rhIL-2, to activate and expand Her-2/neu-specific CD8 + T cells from human epidermal growth factor receptor 2 (Her-2/neu) and human leukocyte antigen (HLA)-A2 double positive advanced breast cancer patients in vitro. These Her-2/neu-loaded T2 cells reproducibly activated and expanded out Her-2/neu-specific CD8 + T cells to 10 7 in 8 weeks. Furthermore, these Her-2/neu-specific CD8 + T cells had good sensitivity of recognition and killing Her-2/neu-overexpressed breast cancer cell line SK.BR.3. This technique gives us another insight on how to rapidly obtain sufficient CTLs for adoptive cancer immunotherapy.
A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Hui; Peng, Ji-Run, E-mail: pengjr@medmail.com.cn; Chen, Peng-Cheng

Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparationmore » of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.« less
iCELLigence real-time cell analysis system for examining the cytotoxicity of drugs to cancer cell lines

PubMed Central

Türker Şener, Leyla; Albeni̇z, Gürcan; Di̇nç, Bi̇rcan; Albeni̇z, Işil

2017-01-01

The recently developed iCELLigence™ real-time cell analyzer (RTCA) can be used for the label-free real-time monitoring of cancer cell proliferation, viability, invasion and cytotoxicity. The RTCA system uses 16-well microtiter plates with a gold microelectrode biosensor array that measures impedance when cells adhere to the microelectrodes causing an alternating current. By measuring the electric field generated in this process, the RTCA system can be used for the analysis of cell proliferation, viability, morphology and migration. The present review aimed to summarize the working method of the RTCA system, in addition to discussing the research performed using the system for various applications, including cancer drug discovery via measuring cytotoxicity. PMID:28962095
Cytotoxicity of ketamine, xylazine and Hellabrunn mixture in liver-, heart- and kidney-derived cells from fallow deer.

PubMed

Kovacova, Veronika; Abdelsalam, Ehdaa Eltayeb Eltigani; Bandouchova, Hana; Brichta, Jiri; Havelkova, Barbora; Piacek, Vladimir; Vitula, Frantisek; Pikula, Jiri

2016-12-18

Chemical restraint of wild animals is practiced to accomplish intended procedures such as capture, clinical examination, collection of diagnostic samples, treatment and/or transport. Extra-label use of animal medicinal drugs is often necessary in wildlife because most approved therapeutics do not list wild species on the labelling. Here, we used cellular in vitro models, a cutting-edge tool of biomedical research, to examine cytotoxicity of anaesthetic agents in fallow deer and extrapolate these data for anaesthetic risks in wildlife. We examined the cytotoxic effects of ketamine, xylazine, and ketamine-xylazine, i.e. the Hellabrunn mixture, on liver-, heart- and kidney-derived cell cultures prepared from a fallow deer (Dama dama) specimen. In line with preliminary studies we exposed cells to 10 µM, 50 µM, 100 µM, 1 mM, and 10 mM ketamine or xylazine. The combination of ketamine-xylazine was dosed at 0.025+0.02 mg/ml, 0.05+0.04 mg/ml, 0.75+0.06 mg/ml, 0.1+0.08 mg/ml, and 0.125+0.1 mg/ml per one well containing 10 000 cells. The quantification of cytotoxicity was based on lactate dehydrogenase activity released from damaged cells. Liver-derived cells show higher sensitivity to the cytotoxic effects of both ketamine and xylazine administered as single drugs when compared with cells cultured from the heart and kidney. The Hellabrunn mixture induced significantly higher cytotoxicity for kidney-derived cells ranging from 16.78% to 35.6%. Single and combined exposures to ketamine and xylazine resulted only in high-dose cytotoxicity in the heart-derived cells. Our results indicate that immobilization drugs significantly differ in their cytotoxic effects on cells derived from various organs of the fallow deer.
Cell Specific Cytotoxicity and Uptake of Graphene Nanoribbons

PubMed Central

Chowdhury, Sayan Mullick; Lalwani, Gaurav; Zhang, Kevin; Yang, Jeong Yun; Neville, Kayla; Sitharaman, Balaji

2012-01-01

The synthesis of oxidized graphene nanoribbons (O-GNR) via longitudinal unzipping of carbon nanotubes opens avenues for their further development for a variety of biomedical applications. Evaluation of the cyto- and bio-compatibility is necessary to develop any new material for in vivo biomedical applications. In this study, we report the cytotoxicity screening of O-GNRs water-solubilized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]), using six different assays, in four representative cell lines; Henrietta Lacks cells (HeLa) derived from cervical cancer tissue, National Institute of Health 3T3 mouse fibroblast cells (NIH-3T3), Sloan Kettering breast cancer cells (SKBR3) and Michigan cancer foundation-7 breast cancer cells (MCF7). These cell lines significantly differed in their response to O-GNR-PEG-DSPE formulations; assessed and evaluated using various endpoints (lactate dehydrogenase (LDH) release, cellular metabolism, lysosomal integrity and cell proliferation) for cytotoxicity. In general, all the cells showed a dose-dependent (10–400 μg/ml) and time-dependent (12–48 h) decrease in cell viability. However, the degree of cytotoxicity was significantly lower in MCF7 or SKBR3 cells compared to HeLa cells. These cells were 100% viable upto 48 hours, when incubated at 10μg/ml O-GNR-PEG-DSPE concentration, and showed decrease in cell viability above this concentration with ~78% of cells viable at the highest concentration (400 μg/ml). In contrast, significant cell death (5–25% cell death depending on the time point, and the assay) was observed for HeLa cells even at a low concentration of 10μg/ml. The decrease in cell viability was steep with increase in concentration with the CD50 values ≥ 100μg/ml depending on the assay, and time point. Transmission electron microscopy of the various cells treated with the O-GNR solutions show higher uptake of the O-GNR-PEG-DSPEs into HeLa cells compared to other cell
KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.

PubMed

Suck, Garnet; Branch, Donald R; Smyth, Mark J; Miller, Richard G; Vergidis, Joanna; Fahim, Soad; Keating, Armand

2005-10-01

To compare the cytotoxicity of KHYG-1 with other natural killer (NK)/NK T-cell lines and identify molecules that may be associated with enhanced cytotoxicity, thereby eventually leading to improved NK cell-mediated cancer immunotherapy. NK/NK T-cell lines KHYG-1, NK-92, YT, and SNT-8 were compared with a novel flow cytometric cytotoxicity assay under different culture conditions. Transcription, expression, and phosphorylation studies were performed using polymerase chain reaction sequence-specific primers, reverse transcription polymerase chain reaction, immunoblotting, and flow cytometry. KHYG-1 is a highly cytotoxic cell line, exceeding the cytolytic capacity of the other cell lines against K562. KHYG-1 is also highly cytotoxic against the leukemia cell lines EM2, EM3, and HL60. The novel activation receptor NKp44 and its adaptor, DAP12, NKG2D, and constitutively phosphorylated ERK2 may be associated with the enhanced cytotoxicity of KHYG-1. This cell line most likely mediates cytolysis by granzyme M (but not granzymes A and B) together with perforin, which is constitutively fully cleaved to the 60-kD form, in contrast to the other cell lines. KHYG-1 is a valuable model for the study of enhanced cytotoxicity by NK cells. In addition to the activation of NKp44, KHYG-1 may induce apoptosis of tumor cells by the newly described granzyme M/perforin pathway. Targeted modifications of effector molecules demonstrated in this model could generate NK cells with even greater killing ability that may be particularly attractive for clinical application. Moreover, our demonstration of greater cytotoxicity of KHYG-1 versus NK-92 cells, already in clinical trials, suggests a direct therapeutic role for KHYG-1.
CD8+ T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types

PubMed Central

Migueles, Stephen A.; Mendoza, Daniel; Zimmerman, Matthew G.; Martins, Kelly M.; Toulmin, Sushila A.; Kelly, Elizabeth P.; Peterson, Bennett A.; Johnson, Sarah A.; Galson, Eric; Poropatich, Kate O.; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A.; Jones, Sara; Hallahan, Claire W.; Follmann, Dean A.; Connors, Mark

2014-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8+ T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8+ T-cell specificity and function of B*27/57neg LTNP/EC (n = 23), B*27/57pos LTNP/EC (n = 23) and B*27/57neg progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57neg LTNP/EC did not target more highly conserved epitopes, their CD8+ T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57pos LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8+ T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people. PMID:26137533
Differential requirements for activation and growth of unprimed cytotoxic and helper T lymphocytes.

PubMed

Gullberg, M; Pobor, G; Bandeira, A; Larsson, E L; Coutinho, A

1983-09-01

The requirements for activation and growth of T lymphocytes capable of mediating either cytolytic activity or help to B lymphocytes were studied in unprimed splenic T cell populations. The selectivity of expression of Lyt-2 antigens, the reactivity to soluble concanavalin A (Con A), to partially purified interleukin 2 (IL 2, T cell growth factor[s]) and to lectin-pulsed macrophages (M phi) were used in this analysis. Lectin-dependent cytotoxicity assays and a novel method that allows for the detection of all effector helper cells, regardless of their clonal specificities, were used for the functional identification of the responding T cells. The results show a marked contrast between cytolytic and helper T cells in their growth and activation requirements. Thus, while Lyt-2+ cytotoxic T lymphocyte precursors grow exponentially in IL 2 after a short pulse with soluble Con A in the absence of accessory cells, Lyt-2- helper cell precursors completely fail to proliferate under the same conditions and require the continuous presence of lectin-pulsed M phi for significant growth. Furthermore, addition of IL 2 to M phi-stimulated cultures of Lyt-2- cells has no effect. T cells which produce IL 2 have the same growth characteristics as helper cells. In both cases, effector helper functions could be expanded more than 10-fold on a per cell basis by a 5-day-culture period under those growth supporting conditions. The development of effector helper functions, however, was strongly inhibited by the presence of Lyt-2+ T cells.
Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

NASA Technical Reports Server (NTRS)

Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

2000-01-01

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.
CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

PubMed

Yu, Shiguang; Fang, Yujiang; Sharav, Tumenjargal; Sharp, Gordon C; Braley-Mullen, Helen

2011-02-15

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.
Label-free optical biosensor for real-time monitoring the cytotoxicity of xenobiotics: A proof of principle study on glyphosate.

PubMed

Farkas, Eniko; Szekacs, Andras; Kovacs, Boglarka; Olah, Marianna; Horvath, Robert; Szekacs, Inna

2018-06-05

Rapid and inexpensive biosensor technologies allowing real-time analysis of biomolecular and cellular events have become the basis of next-generation cell-based screening techniques. Our work opens up novel opportunities in the application of the high-throughput label-free Epic BenchTop optical biosensor in cell toxicity studies. The Epic technology records integrated cellular responses about changes in cell morphology and dynamic mass redistribution of cellular contents at the 100-150 nm layer above the sensor surface. The aim of the present study was to apply this novel technology to identify the effect of the herbicide Roundup Classic, its co-formulant polyethoxylated tallow amine (POEA), and its active ingredient glyphosate, on MC3T3-E1 cells adhered on the biosensor surface. The half maximal inhibitory concentrations of Roundup Classic, POEA and glyphosate upon 1 h of exposure were found to be 0.024%, 0.021% and 0.163% in serum-containing medium and 0.028%, 0.019% and 0.538% in serum-free conditions, respectively (at concentrations equivalent to the diluted Roundup solution). These results showed a good correlation with parallel end-point assays, demonstrating the outstanding utility of the Epic technique in cytotoxicity screening, allowing not only high-throughput, real-time detection, but also reduced assay run time and cytotoxicity assessment at end-points far before cell death would occur. Copyright © 2018 Elsevier B.V. All rights reserved.
Working in "NK Mode": Natural Killer Group 2 Member D and Natural Cytotoxicity Receptors in Stress-Surveillance by γδ T Cells.

PubMed

Silva-Santos, Bruno; Strid, Jessica

2018-01-01

Natural killer cell receptors (NKRs) are germline-encoded transmembrane proteins that regulate the activation and homeostasis of NK cells as well as other lymphocytes. For γδ T cells, NKRs play critical roles in discriminating stressed (transformed or infected) cells from their healthy counterparts, as proposed in the "lymphoid stress-surveillance" theory. Whereas the main physiologic role is seemingly fulfilled by natural killer group 2 member D, constitutively expressed by γδ T cells, enhancement of their therapeutic potential may rely on natural cytotoxicity receptors (NCRs), like NKp30 or NKp44, that can be induced selectively on human Vδ1 + T cells. Here, we review the contributions of NCRs, NKG2D, and their multiple ligands, to γδ T cell biology in mouse and human.
Expression of activating natural killer-cell receptors is a hallmark of the innate-like T-cell neoplasm in peripheral T-cell lymphomas.

PubMed

Uemura, Yu; Isobe, Yasushi; Uchida, Akiko; Asano, Junko; Nishio, Yuji; Sakai, Hirotaka; Hoshikawa, Masahiro; Takagi, Masayuki; Nakamura, Naoya; Miura, Ikuo

2018-04-01

Peripheral T- or natural killer (NK)-cell lymphomas are rare and difficult-to-recognize diseases. It remains arduous to distinguish between NK cell- and cytotoxic T-lymphocyte-derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK-cell receptors and examined the expression using immunohistochemistry in 22 cases with T- and NK-cell neoplasms comprising angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive and -negative anaplastic large-cell lymphomas, extranodal NK/T-cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T-cell lymphoma, aggressive NK-cell leukemia, and other peripheral T-cell lymphomas. Inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK-cell receptors were expressed predominantly in TIA-1-positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK-cell neoplasms and cytotoxic T-lymphocyte-derived lymphomas including monomorphic epitheliotropic intestinal T-cell lymphoma. One Epstein-Barr virus-harboring cytotoxic T-lymphocyte-derived lymphoma mimicking extranodal NK/T-cell lymphoma, nasal type lacked these NK-cell receptors, indicating different cell origin from NK and innate-like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log-rank test, P = .024). We propose that the presence of activating NK-cell receptors may provide new insights into understanding peripheral T-cell lymphomas and characterizing them as innate-like T-cell neoplasm. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on
In vitro generation of viral-antigen dependent cytotoxic T-cells from ginbuna crucian carp, Carassius auratus langsdorfii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Somamoto, Tomonori, E-mail: somamoto@agr.kyushu-u.ac.j; Okamoto, Nobuaki; Nakanishi, Teruyuki

2009-06-20

Little is known about antigen-specific T-cell responses to viruses in teleosts due to a lack of a suitable experimental system using inbred or clonal animals. In the present study we have successfully induced an in vitro generation of virus-specific cytotoxic T-cells (CTLs) from isogeneic ginbuna crucian carp. Responder cells (primarily lymphocytes) from crucian carp haematopoietic necrosis virus (CHNV)-infected fish were capable of proliferating after stimulation in vitro with CHNV-infected syngeneic stimulator cells (primarily lymphocytes and macrophages). The effector cells collected 8 and 12 days after the in vitro stimulation efficiently lysed CHNV-infected syngeneic cells, but not CHNV-infected allogeneic cells ormore » different virus (EVA)-infected syngeneic cells. Furthermore, in situ hybridization analysis showed that some effector cells binding to a CHNV-infected target were TCRbeta or CD8alpha positive. These results provide evidence that the teleost effector cells generated in vitro correspond to virus-specific CTL and they recognize virus-infected target cells in a similar manner of mammalian counterparts.« less
FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

PubMed Central

Sellebjerg, F; Krakauer, M; Khademi, M; Olsson, T; Sørensen, P S

2012-01-01

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4+CD25high T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4+CD25high T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4+CD25high T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4+CD25high T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4+CD25high T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function. PMID:23039885
Cell specific cytotoxicity and uptake of graphene nanoribbons.

PubMed

Mullick Chowdhury, Sayan; Lalwani, Gaurav; Zhang, Kevin; Yang, Jeong Y; Neville, Kayla; Sitharaman, Balaji

2013-01-01

The synthesis of oxidized graphene nanoribbons (O-GNR) via longitudinal unzipping of carbon nanotubes opens avenues for their further development for a variety of biomedical applications. Evaluation of the cyto- and bio-compatibility is necessary to develop any new material for in vivo biomedical applications. In this study, we report the cytotoxicity screening of O-GNRs water-solubilized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]), using six different assays, in four representative cell lines; Henrietta Lacks cells (HeLa) derived from cervical cancer tissue, National Institute of Health 3T3 mouse fibroblast cells (NIH-3T3), Sloan Kettering breast cancer cells (SKBR3) and Michigan cancer foundation-7 breast cancer cells (MCF7). These cell lines significantly differed in their response to O-GNR-PEG-DSPE formulations; assessed and evaluated using various endpoints (lactate dehydrogenase (LDH) release, cellular metabolism, lysosomal integrity and cell proliferation) for cytotoxicity. In general, all the cells showed a dose-dependent (10-400 μg/ml) and time-dependent (12-48 h) decrease in cell viability. However, the degree of cytotoxicity was significantly lower in MCF7 or SKBR3 cells compared to HeLa cells. These cells were 100% viable upto 48 h, when incubated at 10 μg/ml O-GNR-PEG-DSPE concentration, and showed decrease in cell viability above this concentration with ~78% of cells viable at the highest concentration (400 μg/ml). In contrast, significant cell death (5-25% cell death depending on the time point, and the assay) was observed for HeLa cells even at a low concentration of 10 μg/ml. The decrease in cell viability was steep with increase in concentration with the CD(50) values ≥ 100 μg/ml depending on the assay, and time point. Transmission electron microscopy of the various cells treated with the O-GNR solutions show higher uptake of the O-GNR-PEG-DSPEs into HeLa cells compared to other cell types
Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

PubMed Central

Chen, Wanjun; Jin, Wenwen; Wahl, Sharon M.

1998-01-01

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4+ T cells. CD4+ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4+ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4+ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4+ T cell activation. PMID:9815262
The cytotoxic T lymphocyte immune synapse at a glance.

PubMed

Dieckmann, Nele M G; Frazer, Gordon L; Asano, Yukako; Stinchcombe, Jane C; Griffiths, Gillian M

2016-08-01

The immune synapse provides an important structure for communication with immune cells. Studies on immune synapses formed by cytotoxic T lymphocytes (CTLs) highlight the dynamic changes and specialised mechanisms required to facilitate focal signalling and polarised secretion in immune cells. In this Cell Science at a Glance article and the accompanying poster, we illustrate the different steps that reveal the specialised mechanisms used to focus secretion at the CTL immune synapse and allow CTLs to be such efficient and precise serial killers. © 2016. Published by The Company of Biologists Ltd.
IL-15 induces CD8+ T cells to acquire functional NK receptors capable of modulating cytotoxicity and cytokine secretion.

PubMed

Correia, Margareta P; Costa, Alexandra V; Uhrberg, Markus; Cardoso, Elsa M; Arosa, Fernando A

2011-05-01

During the last years several authors have described a small population of CD8+ T cells expressing NK receptors (NKRs). Although their origin remains largely unknown, we have recently demonstrated that IL-15 is capable of inducing NKR expression in purified human CD8+CD56- T cells. In this study we show that IL-15-driven NKR induction in CD8+ T cells was linked with CD56 de novo acquisition, consistent with an effector-memory phenotype, increased anti-apoptotic levels, high granzyme B/perforin expression and with the ability of displaying in vitro NK-like cytotoxicity. Interestingly, dissection of NKR functional outcome in IL-15-cultured CD8+ T cells revealed: (i) that NKG2D cross-linking was able per se to upregulate degranulation levels and (ii) that KIR and NKG2A cross-linking upregulated secretion of cytokines such as IFN-γ, TNF-α, IL-1β and IL-10. These results suggest that IL-15 is capable of differentiating CD8+ T cells into NK-like T cells displaying a regulatory phenotype. Copyright © 2010 Elsevier GmbH. All rights reserved.
In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

PubMed Central

Kustiawan, Paula M.; Puthong, Songchan; Arung, Enos T.; Chanchao, Chanpen

2014-01-01

Objective To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). Methods All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Results Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Conclusions Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s). PMID:25183275

In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines.

PubMed

Kustiawan, Paula M; Puthong, Songchan; Arung, Enos T; Chanchao, Chanpen

2014-07-01

To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s).
Thyroid Autoimmunity is Associated with Decreased Cytotoxicity T Cells in Women with Repeated Implantation Failure

PubMed Central

Huang, Chunyu; Liang, Peiyan; Diao, Lianghui; Liu, Cuicui; Chen, Xian; Li, Guangui; Chen, Cong; Zeng, Yong

2015-01-01

Thyroid autoimmunity (TAI), which is defined as the presence of autoantibodies against thyroid peroxidase (TPO) and/or thyroglobulin (TG), is related to repeated implantation failure (RIF). It is reported that TAI was involved in reproductive failure not only through leading thyroid function abnormality, but it can also be accompanied with immune imbalance. Therefore, this study was designed to investigate the association of thyroid function, immune status and TAI in women with RIF. Blood samples were drawn from 72 women with RIF to evaluate the prevalence of TAI, the thyroid function, the absolute numbers and percentages of lymphocytes. The prevalence of thyroid function abnormality in RIF women with TAI was not significantly different from that in RIF women without TAI (χ2 = 0.484, p > 0.05). The absolute number and percentage of T cells, T helper (Th) cells, B cells and natural killer (NK) cells were not significantly different in RIF women with TAI compared to those without TAI (all p > 0.05). The percentage of T cytotoxicity (Tc) cells was significantly decreased in RIF women with TAI compared to those without TAI (p < 0.05). Meanwhile, Th/Tc ratio was significantly increased (p < 0.05). These results indicated that the decreased Tc percentage and increased Th/Tc ratio may be another influential factor of adverse pregnancy outcomes in RIF women with TAI. PMID:26308040
Quantification of Superparamagnetic Iron Oxide (SPIO)-labeled Cells Using MRI

PubMed Central

Rad, Ali M; Arbab, Ali S; Iskander, ASM; Jiang, Quan; Soltanian-Zadeh, Hamid

2015-01-01

Purpose To show the feasibility of using magnetic resonance imaging (MRI) to quantify superparamagnetic iron oxide (SPIO)-labeled cells. Materials and Methods Lymphocytes and 9L rat gliosarcoma cells were labeled with Ferumoxides-Protamine Sulfate complex (FE-PRO). Cells were labeled efficiently (more than 95%) and iron concentration inside each cell was measured by spectrophotometry (4.77-30.21 picograms). Phantom tubes containing different number of labeled or unlabeled cells as well as different concentrations of FE-PRO were made. In addition, labeled and unlabeled cells were injected into fresh and fixed rat brains. Results Cellular viability and proliferation of labeled and unlabeled cells were shown to be similar. T2-weighted images were acquired using 7 T and 3 T MRI systems and R2 maps of the tubes containing cells, free FE-PRO, and brains were made. There was a strong linear correlation between R2 values and labeled cell numbers but the regression lines were different for the lymphocytes and gliosarcoma cells. Similarly, there was strong correlation between R2 values and free iron. However, free iron had higher R2 values than the labeled cells for the same concentration of iron. Conclusion Our data indicated that in vivo quantification of labeled cells can be done by careful consideration of different factors and specific control groups. PMID:17623892
A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

PubMed Central

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8–13% and reliably measures NK cell- or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. PMID:17617419
A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

PubMed

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.
Maximal exercise increases mucosal associated invariant T cell frequency and number in healthy young men.

PubMed

Hanson, Erik D; Danson, Eli; Nguyen-Robertson, Catriona V; Fyfe, Jackson J; Stepto, Nigel K; Bartlett, David B; Sakkal, Samy

2017-11-01

Mucosal associated invariant T (MAIT) cells have properties of the innate and acquired immune systems. While the response to vigorous exercise has been established for most leukocytes, MAIT cells have not been investigated. Therefore, the purpose was to determine if MAIT cell lymphocytosis occurs with acute maximal aerobic exercise and if this response is influenced by exercise duration, cardiovascular fitness, or body composition. Twenty healthy young males with moderate fitness levels performed an extended graded exercise test until volitional fatigue. Peripheral blood mononuclear cells were isolated from venous blood obtained prior and immediately after exercise and were labeled to identify specific T cell populations using flow cytometry. The percentage of MAIT cells relative to total T cells significantly increased from 3.0 to 3.8% and absolute MAIT cell counts increased by 2.2-fold following maximal exercise. MAIT cell subpopulation proportions were unchanged with exercise. Within cytotoxic T lymphocytes (CTL), MAIT cells consisted of 8% of these cells and this remained constant after exercise. MAIT cell counts and changes with exercise were not affected by body composition, VO 2peak , or exercise duration. Maximal exercise doubled MAIT cell numbers and showed preferential mobilization within total T cells but the response was not influenced by fitness levels, exercise duration, or body composition. These results suggest that acute exercise could be used to offset MAIT cell deficiencies observed with certain pathologies. MAIT cells also make up a substantial proportion of CTLs, which may have implications for cytotoxicity assays using these cells.
B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.

PubMed

Candolfi, Marianela; Curtin, James F; Yagiz, Kader; Assi, Hikmat; Wibowo, Mia K; Alzadeh, Gabrielle E; Foulad, David; Muhammad, A K M G; Salehi, Sofia; Keech, Naomi; Puntel, Mariana; Liu, Chunyan; Sanderson, Nicholas R; Kroeger, Kurt M; Dunn, Robert; Martins, Gislaine; Lowenstein, Pedro R; Castro, Maria G

2011-10-01

We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.
Tribody [(HER2)2xCD16] Is More Effective Than Trastuzumab in Enhancing γδ T Cell and Natural Killer Cell Cytotoxicity Against HER2-Expressing Cancer Cells.

PubMed

Oberg, Hans H; Kellner, Christian; Gonnermann, Daniel; Sebens, Susanne; Bauerschlag, Dirk; Gramatzki, Martin; Kabelitz, Dieter; Peipp, Matthias; Wesch, Daniela

2018-01-01

An enhanced expression of human epidermal growth factor receptor 2 (HER2, ErbB2) often occurs in an advanced stage of breast, ovarian, gastric or esophageal cancer, and pancreatic ductal adenocarcinoma (PDAC). Commonly, HER2 expression is associated with poor clinical outcome or chemoresistance in ovarian and breast cancer patients. Treatment with humanized anti-HER2 monoclonal antibodies, such as trastuzumab or pertuzumab, has improved the outcome of patients with HER2-positive metastatic gastric or breast cancer, but not all patients benefit. In this study, the bispecific antibody [(HER2) 2 xCD16] in the tribody format was employed to re-direct CD16-expressing γδ T lymphocytes as well as natural killer (NK) cells to the tumor-associated cell surface antigen HER2 to enhance their cytotoxic anti-tumor activity. Tribody [(HER2) 2 xCD16] comprises two HER2-specific single chain fragment variable fused to a fragment antigen binding directed to the CD16 (FcγRIII) antigen expressed on γδ T cells and NK cells. Our results revealed the superiority of tribody [(HER2) 2 xCD16] compared to trastuzumab in triggering γδ T cell and NK cell-mediated lysis of HER2-expressing tumor cells, such as PDAC, breast cancer, and autologous primary ovarian tumors. The increased efficacy of [(HER2) 2 xCD16] can be explained by an enhanced degranulation of immune cells. Although CD16 expression was decreased on γδ T cells in several PDAC patients and the number of tumor-infiltrating NK cells and γδ T cells was impaired in ovarian cancer patients, [(HER2) 2 xCD16] selectively enhanced cytotoxicity of cells from these patients. Here, unique anti-tumor properties of tribody [(HER2) 2 xCD16] are identified which beyond addressing HER2 overexpressing solid tumors may allow to treat with similar immunoconstructs combined with the adoptive transfer of γδ T cells and NK cells refractory hematological malignancies. A major advantage of γδ T cells and NK cells in the transplant
Tribody [(HER2)2xCD16] Is More Effective Than Trastuzumab in Enhancing γδ T Cell and Natural Killer Cell Cytotoxicity Against HER2-Expressing Cancer Cells

PubMed Central

Oberg, Hans H.; Kellner, Christian; Gonnermann, Daniel; Sebens, Susanne; Bauerschlag, Dirk; Gramatzki, Martin; Kabelitz, Dieter; Peipp, Matthias; Wesch, Daniela

2018-01-01

An enhanced expression of human epidermal growth factor receptor 2 (HER2, ErbB2) often occurs in an advanced stage of breast, ovarian, gastric or esophageal cancer, and pancreatic ductal adenocarcinoma (PDAC). Commonly, HER2 expression is associated with poor clinical outcome or chemoresistance in ovarian and breast cancer patients. Treatment with humanized anti-HER2 monoclonal antibodies, such as trastuzumab or pertuzumab, has improved the outcome of patients with HER2-positive metastatic gastric or breast cancer, but not all patients benefit. In this study, the bispecific antibody [(HER2)2xCD16] in the tribody format was employed to re-direct CD16-expressing γδ T lymphocytes as well as natural killer (NK) cells to the tumor-associated cell surface antigen HER2 to enhance their cytotoxic anti-tumor activity. Tribody [(HER2)2xCD16] comprises two HER2-specific single chain fragment variable fused to a fragment antigen binding directed to the CD16 (FcγRIII) antigen expressed on γδ T cells and NK cells. Our results revealed the superiority of tribody [(HER2)2xCD16] compared to trastuzumab in triggering γδ T cell and NK cell-mediated lysis of HER2-expressing tumor cells, such as PDAC, breast cancer, and autologous primary ovarian tumors. The increased efficacy of [(HER2)2xCD16] can be explained by an enhanced degranulation of immune cells. Although CD16 expression was decreased on γδ T cells in several PDAC patients and the number of tumor-infiltrating NK cells and γδ T cells was impaired in ovarian cancer patients, [(HER2)2xCD16] selectively enhanced cytotoxicity of cells from these patients. Here, unique anti-tumor properties of tribody [(HER2)2xCD16] are identified which beyond addressing HER2 overexpressing solid tumors may allow to treat with similar immunoconstructs combined with the adoptive transfer of γδ T cells and NK cells refractory hematological malignancies. A major advantage of γδ T cells and NK cells in the transplant situation of
Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model.

PubMed

Nomura, Machiko; Ohashi, Takashi; Nishikawa, Keiko; Nishitsuji, Hironori; Kurihara, Kiyoshi; Hasegawa, Atsuhiko; Furuta, Rika A; Fujisawa, Jun-ichi; Tanaka, Yuetsu; Hanabuchi, Shino; Harashima, Nanae; Masuda, Takao; Kannagi, Mari

2004-04-01

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.
A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

PubMed

Schaft, Niels; Dörrie, Jan; Müller, Ina; Beck, Verena; Baumann, Stefanie; Schunder, Tanja; Kämpgen, Eckhart; Schuler, Gerold

2006-09-01

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.
A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients

PubMed Central

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru

2017-01-01

ABSTRACT We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax301-309-specific CD8+ cytotoxic T cells (Tax301-309-CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02+) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+ CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax301-309-CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax301-309-CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax301-309-CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax301-309-CTLs, 1,458 Tax301-309-CTLs and 140 clones were identified in this cohort. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+ CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1
Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion*

PubMed Central

Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M.; Froelich, Christopher J.; Pardo, Julián

2015-01-01

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways. PMID:25605735
Preliminary analysis of murine cytotoxic T cell responses to the proteins of the flavivirus Kunjin using vaccinia virus expression.

PubMed

Parrish, C R; Coia, G; Hill, A; Müllbacher, A; Westaway, E G; Blanden, R V

1991-07-01

A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide.
Repeated cycles of 5-fluorouracil chemotherapy impaired anti-tumor functions of cytotoxic T cells in a CT26 tumor-bearing mouse model.

PubMed

Wu, Yanhong; Deng, Zhenling; Wang, Huiru; Ma, Wenbo; Zhou, Chunxia; Zhang, Shuren

2016-09-20

Recently, the immunostimulatory roles of chemotherapeutics have been increasingly revealed, although bone marrow suppression is still a common toxicity of chemotherapy. While the numbers and ratios of different immune subpopulations are analyzed after chemotherapy, changes to immune status after each cycle of treatment are less studied and remain unclear. To determine the tumor-specific immune status and functions after different cycles of chemotherapy, we treated CT26 tumor-bearing mice with one to four cycles of 5-fluorouracil (5-FU). Overall survival was not improved when more than one cycle of 5-FU was administered. Here we present data concerning the immune statuses after one and three cycles of chemotherapy. We analyzed the amount of spleen cells from mice treated with one and three cycles of 5-FU as well as assayed their proliferation and cytotoxicity against the CT26 tumor cell line. We found that the absolute numbers of CD8 T-cells and NK cells were not influenced significantly after either one or three cycles of chemotherapy. However, after three cycles of 5-FU, proliferated CD8 T-cells were decreased, and CT26-specific cytotoxicity and IFN-γ secretion of spleen cells were impaired in vitro. After one cycle of 5-FU, there was a greater percentage of tumor infiltrating CD8 T-cells. In addition, more proliferated CD8 T-cells, enhanced tumor-specific cytotoxicity as well as IFN-γ secretion of spleen cells against CT26 in vitro were observed. Given the increased expression of immunosuppressive factors, such as PD-L1 and TGF-β, we assessed the effect of early introduction of immunotherapy in combination with chemotherapy. We found that mice treated with cytokine induced killer cells and PD-L1 monoclonal antibodies after one cycle of 5-FU had a better anti-tumor performance than those treated with chemotherapy or immunotherapy alone. These data suggest that a single cycle of 5-FU treatment promoted an anti-tumor immune response, whereas repeated chemotherapy
Cytotoxic T Cell Adoptive Immunotherapy as a Treatment for Nasopharyngeal Carcinoma

PubMed Central

Crooks, Pauline; Morrison, Leanne; Stevens, Natasha; Davis, Joanne E.; Corban, Monika; Hall, David; Panizza, Benedict; Coman, William B.; Coman, Scott; Moss, Denis J.

2014-01-01

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress. PMID:24351754
Cytotoxic T cell adoptive immunotherapy as a treatment for nasopharyngeal carcinoma.

PubMed

Lutzky, Viviana P; Crooks, Pauline; Morrison, Leanne; Stevens, Natasha; Davis, Joanne E; Corban, Monika; Hall, David; Panizza, Benedict; Coman, William B; Coman, Scott; Moss, Denis J

2014-02-01

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress.
The cell labeling efficacy, cytotoxicity and relaxivity of copper-activated MRI/PET imaging contrast agents.

PubMed

Patel, Daksha; Kell, Arnold; Simard, Benoit; Xiang, Bo; Lin, Hung Yu; Tian, Ganghong

2011-02-01

A new class of nanoparticle-based dual-modality positron emission tomography/magnetic resonance imaging (PET/MRI) contrast agents has been developed. The probe consists of a superparamagnetic iron oxide (SPIO) or manganese oxide core coated with 3,4-dihydroxy-D,L-phenylalanine (DL-DOPA). The chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to DOPA termini. The DOTA modified nanoparticles allow chelation of copper for PET imaging. These surface functionalized nanoparticle-based probes have been characterized by various analytical techniques. The cell-labeling efficacy, cytotoxicity and relaxivity of these nanoparticles have been evaluated and compared with the same properties of one of the most commonly utilized MRI contrast agents, Feridex(®). Evidently, this new nanoparticle has a great potential for use in cell tracking with MRI and PET in the absence of transfecting agent. It is noteworthy that there is a sharp increase in r(2) relaxivity of these nanoparticles on coordination with Cu(2+) ions. Thus these iron oxide nanoparticles can also be explored as the smart magnetic resonance (MR) sensor for the detection of micromolar changes in copper concentration for neurodegenerative diseases such as Alzheimer's disease, Menkes and Wilson's diseases, amyotrophic lateral sclerosis and prion diseases. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.
Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

PubMed

Kahaleh, M B; Fan, P S; Otsuka, T

1999-05-01

In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc. Copyright 1999 Academic Press.
Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

PubMed

Utz, U; Banks, D; Jacobson, S; Biddison, W E

1996-02-01

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.

A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing.

PubMed

Pan, Deng; Kobayashi, Aya; Jiang, Peng; Ferrari de Andrade, Lucas; Tay, Rong En; Luoma, Adrienne M; Tsoucas, Daphne; Qiu, Xintao; Lim, Klothilda; Rao, Prakash; Long, Henry W; Yuan, Guo-Cheng; Doench, John; Brown, Myles; Liu, X Shirley; Wucherpfennig, Kai W

2018-02-16

Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of >100 genes-including Pbrm1 , Arid2 , and Brd7 , which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex-sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1 -deficient murine melanomas were more strongly infiltrated by cytotoxic T cells. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Expression of killer inhibitory receptors on cytotoxic cells from HIV-1-infected individuals

PubMed Central

Galiani, M D; Aguado, E; Tarazona, R; Romero, P; Molina, I; Santamaria, M; Solana, R; PeñA, J

1999-01-01

Dysfunction of cytotoxic activity of T and natural killer (NK) lymphocytes is a main immunological feature in patients with AIDS, but its basis are not well understood. It has been recently described that T and NK cell-mediated cytotoxicity can be regulated by HLA killer inhibitory receptors (KIR). In this work, we have determined on cytotoxic T cells and NK cells from HIV-1-infected individuals the expression of the following KIR molecules: p58, p70, and ILT2 (immunoglobulin-like family KIR) as well as CD94 and NKG2A (C-lectin-type family KIR). With some exceptions, no significant changes were found on the expression of immunoglobulin-like KIR in either CD8+ or CD56+ cells. Interestingly, the percentages of CD8+ and CD56+ cells expressing CD94 were significantly increased in these individuals. We also show that, in vitro, IL-10 up-regulates CD94 expression on CD8+ and CD56+ cells obtained from normal individuals, suggesting that the augmented expression observed in HIV-infected individuals could be related to the high levels of IL-10 previously described in HIV-1-infected individuals. PMID:10193420
Immunophenotypic characterization of the cutaneous exanthem of SIV-infected rhesus monkeys. Apposition of degenerative Langerhans cells and cytotoxic lymphocytes during the development of acquired immunodeficiency syndrome.

PubMed Central

Ringler, D. J.; Hancock, W. W.; King, N. W.; Letvin, N. L.; Daniel, M. D.; Desrosiers, R. C.; Murphy, G. F.

1987-01-01

A T-cell tropic retrovirus, simian immunodeficiency virus (SIV), has recently been isolated from immunodeficient rhesus monkeys. This virus has remarkable similarities to human immunodeficiency virus (HIV), the etiologic agent of acquired immunodeficiency syndrome. Subsequent studies of simian infection with SIV have shown it to be a relevant animal model for studying the pathogenesis of AIDS in man. In both HIV-infected humans and SIV-infected monkeys, a cutaneous maculopapular eruption has been described. To date, the pathogenesis and possible relationship of these exanthema to the evolution of systemic immunosuppression have remained obscure. In this study, the mononuclear cell infiltrates that characterize skin rashes of SIV-infected rhesus monkeys were found to be composed predominantly of cells with phenotypic characteristics of cytotoxic/suppressor (T8+) lymphocytes and natural killer cells. Many of these cells expressed membrane-bound interleukin-2 receptor molecules. Double labeling and immunoelectron microscopy revealed these cells in direct contact with degenerative Langerhans cells within the epidermis and dermis. These observations suggest that the cutaneous rash associated with SIV infection may be the consequence of target cell injury of Langerhans cells by effector cells with cytotoxic potential. Images Figure 1 Figure 2 Figure 3 PMID:3030113
Cytotoxic activity of ten algae from the Persian Gulf and Oman Sea on human breast cancer cell lines; MDA-MB-231, MCF-7, and T-47D

PubMed Central

Erfani, Nasrollah; Nazemosadat, Zahra; Moein, Mahmoodreza

2015-01-01

Background: Seaweeds have proven to be a promising natural source of bioactive metabolites for drug development. Objective: This study aimed to monitor the ethanol extract of ten algae from the Persian Gulf and Oman Sea, for their in vitro cytotoxic activity on three human breast cancer cell lines. Materials and Methods: Three human breast cancer cell lines including MDA-MB-231(ER−), MCF-7(ER+), and T-47D (ER+) were treated by different concentrations of total ethanol (90%) algae extracts and the cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Doxorubicin (Ebewe, Austria) was used as a positive control. After 72 h of incubation, the cytotoxic effect of the algae was calculated and presented as 50%-inhibitory concentration (IC50). Results: The results indicated Gracilaria foliifera and Cladophoropsis sp. to be the most active algae in terms of cytotoxic effects on the investigated cancer cell lines. The IC50 values against MDA-MB-231, MCF-7, and T-47D cells were, respectively, 74.89 ± 21.71, 207.81 ± 12.07, and 203.25 ± 30.98 µg/ml for G. foliifera and 66.48 ± 4.96, 150.86 ± 51.56 and >400 µg/ml for Cladophoropsis sp. The rest of the algal extracts were observed not to have significant cytotoxic effects in the concentration range from 6.25 µg/ml to 400 µg/ml. Conclusion: Our data conclusively suggest that G. foliifera and Cladophoropsis sp. may be good candidates for further fractionation to obtain novel anticancer substances. Moreover, stronger cytotoxic effects on estrogen negative breast cancer cell line (MDA-MB-231(ER−)) in comparison to estrogen positive cells (MCF-7 and T-47D) suggest that the extract of G. foliifera and Cladophoropsis sp. may have an estrogen receptor/progesterone receptor-independent mechanism for their cellular growth inhibition. PMID:25829786
Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes.

PubMed

Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2015-05-01

Identification and characterization of CD8(+) T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8(+) T cells have been only partially identified. In this study, we sought to identify CD8(+) T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8(+) T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10(-11)) and positively associated with CD4 count (P = 1.2 × 10(-11)), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8(+) T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52:01-restricted
Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes

PubMed Central

Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi

2015-01-01

ABSTRACT Identification and characterization of CD8+ T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8+ T cells have been only partially identified. In this study, we sought to identify CD8+ T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8+ T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10−11) and positively associated with CD4 count (P = 1.2 × 10−11), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8+ T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. IMPORTANCE HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52
Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

PubMed

Tanaka, Yuetsu; Takahashi, Yoshiaki; Tanaka, Reiko; Kodama, Akira; Fujii, Hideki; Hasegawa, Atsuhiko; Kannagi, Mari; Ansari, Aftab A; Saito, Mineki

2014-06-01

Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs.
Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors

NASA Astrophysics Data System (ADS)

Niederman, Thomas M. J.; Ghogawala, Zoher; Carter, Bob S.; Tompkins, Hillary S.; Russell, Margaret M.; Mulligan, Richard C.

2002-05-01

The demonstration that angiogenesis is required for the growth of solid tumors has fueled an intense interest in the development of new therapeutic strategies that target the tumor vasculature. Here we report the development of an immune-based antiangiogenic strategy that is based on the generation of T lymphocytes that possess a killing specificity for cells expressing vascular endothelial growth factor receptors (VEGFRs). To target VEGFR-expressing cells, recombinant retroviral vectors were generated that encoded a chimeric T cell receptor comprised of VEGF sequences linked to intracellular signaling sequences derived from the chain of the T cell receptor. After transduction of primary murine CD8 lymphocytes by such vectors, the transduced cells were shown to possess an efficient killing specificity for cells expressing the VEGF receptor, Flk-1, as measured by in vitro cytotoxicity assays. After adoptive transfer into tumor-bearing mice, the genetically modified cytotoxic T lymphocytes strongly inhibited the growth of a variety of syngeneic murine tumors and human tumor xenografts. An increased effect on in vivo tumor growth inhibition was seen when this therapy was combined with the systemic administration of TNP-470, a conventional angiogenesis inhibitor. The utilization of the immune system to target angiogenic markers expressed on tumor vasculature may prove to be a powerful means for controlling tumor growth.
Candidate mosaic proteins for a pan-filoviral cytotoxic T-Cell lymphocyte vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fenimore, Paul W; Fischer, William M; Kuiken, Carla

The extremely high fatality rates of many filovirus (FILV) strains the recurrent but rarely identified origin of human epidemics, the only partly identified viral reservoirs and the continuing non-human primate epizootics in Africa make a broadly-protective filovirus vaccine highly desirable. Cytotoxic T-cells (CTL) have been shown to be protective in mice, guinea pigs and non-human primates. In murine models the cytotoxic T-cell epitopes that are protective against Ebola virus have been mapped and in non-human primates CTL-mediated protection between viral strains (John Dye: specify) has been demonstrated using two filoviral proteins, nucleoprotein (NP) and glycoprotein (GP). These immunological results suggestmore » that the CTL avenue of immunity deserves consideration for a vaccine. The poorly-understood viral reservoirs means that it is difficult to predict what strains are likely to cause epidemics. Thus, there is a premium on developing a pan-filoviral vaccine. The genetic diversity of FILV is large, roughly the same scale as human immunodeficiency virus (HIV). This presents a serious challenge for the vaccine designer because a traditional vaccine aspiring to pan-filoviral coverage is likely to require the inclusion of many antigenic reagents. A recent method for optimizing cytotoxic T-cell lymphocyte epitope coverage with mosaic antigens was successful in improving potential CTL epitope coverage against HIV and may be useful in the context of very different viruses, such as the filoviruses discussed here. Mosaic proteins are recombinants composed of fragments of wild-type proteins joined at locations resulting in exclusively natural k-mers, 9 {le} k {le} 15, and having approximately the same length as the wild-type proteins. The use of mosaic antigens is motivated by three conjectures: (1) optimizing a mosaic protein to maximize coverage of k-mers found in a set of reference proteins will give better odds of including broadly-protective CTL epitopes in a
Cytotoxic T lymphocyte-dependent tumor growth inhibition by a vascular endothelial growth factor-superantigen conjugate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Qingwen; State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433; Jiang, Songmin

2012-11-02

Highlights: Black-Right-Pointing-Pointer We construct and purify a fusion protein VEGF-SEA. Black-Right-Pointing-Pointer VEGF-SEA strongly repressed the growth of murine solid sarcoma 180 (S180) tumors. Black-Right-Pointing-Pointer T cells driven by VEGF-SEA were accumulated around tumor cells bearing VEGFR by mice image model. Black-Right-Pointing-Pointer VEGF-SEA can serve as a tumor targeting agent and sequester CTLs into the tumor site. Black-Right-Pointing-Pointer The induced CTLs could release the cytokines, perforins and granzyme B to kill the tumor cells. -- Abstract: T cells are major lymphocytes in the blood and passengers across the tumor vasculature. If these T cells are retained in the tumor site, amore » therapeutic potential will be gained by turning them into tumor-reactive cytotoxic T lymphocytes (CTLs). A fusion protein composed of human vascular endothelial growth factor (VEGF) and staphylococcal enterotoxin A (SEA) with a D227A mutation strongly repressed the growth of murine solid sarcoma 180 (S180) tumors (control versus VEGF-SEA treated with 15 {mu}g, mean tumor weight: 1.128 g versus 0.252 g, difference = 0.876 g). CD4{sup +} and CD8{sup +} T cells driven by VEGF-SEA were accumulated around VEGFR expressing tumor cells and the induced CTLs could release the tumoricidal cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Meanwhile, intratumoral CTLs secreted cytolytic pore-forming perforin and granzyme B proteins around tumor cells, leading to the death of tumor cells. The labeled fusion proteins were gradually targeted to the tumor site in an imaging mice model. These results show that VEGF-SEA can serve as a tumor targeting agent and sequester active infiltrating CTLs into the tumor site to kill tumor cells, and could therefore be a potential therapeutical drug for a variety of cancers.« less
Dendritic cells induce specific cytotoxic T lymphocytes against prostate cancer TRAMP-C2 cells loaded with freeze- thaw antigen and PEP-3 peptide.

PubMed

Liu, Xiao-Qi; Jiang, Rong; Li, Si-Qi; Wang, Jing; Yi, Fa-Ping

2015-01-01

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-γ, TNF-β and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-γ, TNF-β and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.
In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.

PubMed

Boissonnas, Alexandre; Fetler, Luc; Zeelenberg, Ingrid S; Hugues, Stéphanie; Amigorena, Sebastian

2007-02-19

Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8(+) cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration.
In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor

PubMed Central

Boissonnas, Alexandre; Fetler, Luc; Zeelenberg, Ingrid S.; Hugues, Stéphanie; Amigorena, Sebastian

2007-01-01

Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8+ cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration. PMID:17261634
A mechanistic model for bromodeoxyuridine dilution naturally explains labelling data of self-renewing T cell populations

PubMed Central

Ganusov, Vitaly V.; De Boer, Rob J.

2013-01-01

Bromodeoxyuridine (BrdU) is widely used in immunology to detect cell division, and several mathematical models have been proposed to estimate proliferation and death rates of lymphocytes from BrdU labelling and de-labelling curves. One problem in interpreting BrdU data is explaining the de-labelling curves. Because shortly after label withdrawal, BrdU+ cells are expected to divide into BrdU+ daughter cells, one would expect a flat down-slope. As for many cell types, the fraction of BrdU+ cells decreases during de-labelling, previous mathematical models had to make debatable assumptions to be able to account for the data. We develop a mechanistic model tracking the number of divisions that each cell has undergone in the presence and absence of BrdU, and allow cells to accumulate and dilute their BrdU content. From the same mechanistic model, one can naturally derive expressions for the mean BrdU content (MBC) of all cells, or the MBC of the BrdU+ subset, which is related to the mean fluorescence intensity of BrdU that can be measured in experiments. The model is extended to include subpopulations with different rates of division and death (i.e. kinetic heterogeneity). We fit the extended model to previously published BrdU data from memory T lymphocytes in simian immunodeficiency virus-infected and uninfected macaques, and find that the model describes the data with at least the same quality as previous models. Because the same model predicts a modest decline in the MBC of BrdU+ cells, which is consistent with experimental observations, BrdU dilution seems a natural explanation for the observed down-slopes in self-renewing populations. PMID:23034350
Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

PubMed Central

Miltenburg, A M; Van Laar, J M; De Kuiper, P; Daha, M R; Breedveld, F C

1990-01-01

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function. PMID:2148285
Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity.

PubMed

Hwang, Kyung-Sun; Cho, Won-Kyung; Yoo, Jinsang; Seong, Young Rim; Kim, Bum-Kyeng; Kim, Samyong; Im, Dong-Soo

2004-06-01

We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5'-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32-), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32- produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32- compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32- contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18. Copyright 2004 Nature Publishing Group
The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

PubMed

Chieregato, Katia; Zanon, Cristina; Castegnaro, Silvia; Bernardi, Martina; Amati, Eliana; Sella, Sabrina; Rodeghiero, Francesco; Astori, Giuseppe

2017-01-01

Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.
Trypanosoma cruzi: sequence of phagocytosis and cytotoxicity by human polymorphonuclear leucocytes.

PubMed Central

Rimoldi, M T; Cardoni, R L; Olabuenaga, S E; de Bracco, M M

1981-01-01

We have studied the relationship between phagocytosis and cytotoxicity of human polymorphonuclear leucocytes (PMN) to sensitized Trypanosoma cruzi. Assays were done simultaneously using [3H]-uridine labelled epimastigotes as target cells. Phagocytosis was evaluated by the uptake and cytotoxicity by the release of parasite associated [3H]-uridine. Both reactions reached maximum levels at the same effector- to target-cell ratio and antibody concentration. Uptake of epimastigotes by PMN was highest at 30 min and intracellular disruption and release of parasite debris took place later. In conditions that precluded repeated uptake of sensitized radiolabelled T. cruzi, the release profile of [3H]-uridine from PMN that contained intracellular parasites was similar to that of the standard cytotoxic assay. However, as the ingestion phase was separated from the release step, no lag in the onset of the reaction was observed. Although we cannot rule out extracellular killing, the results of this study demonstrate that the bulk of damaged T. cruzi epimastigotes had been previously internalized by the PMN. PMID:7016743
A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients.

PubMed

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-Ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru; Kanda, Yoshinobu

2017-10-01

We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax 301-309 -specific CD8 + cytotoxic T cells (Tax 301-309 -CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02 + ) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR + CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax 301-309 -CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax 301-309 -CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax 301-309 -CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax 301-309 -CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax 301-309 -CTLs, 1,458 Tax 301-309 -CTLs and 140 clones were identified in this cohort. Tax 301-309 -CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR + CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02 + HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR + CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the
Induction of antigen-specific cytotoxic T-cell response by dendritic cells generated from ecto-mesenchymal stem cells infected with an adenovirus containing the MAGE-D4a gene.

PubMed

Hu, Shijie; Li, Bing; Shen, Xuefeng; Zhang, Rui; Gao, Dakuan; Guo, Qingdong; Jin, Yan; Fei, Zhou

2016-04-01

The present study aimed to investigate the feasibility of using ecto-mesenchymal stem cell (EMSC)-derived dendritic cells (DCs) for glioma immunotherapy following infection by a recombinant adenovirus containing the melanoma-associated antigen D4a (MAGE-D4a) gene. The ex vivo cultured EMSCs were infected by the adenoviral plasmid containing MAGE-D4a (pAd/MAGE-D4a). Efficiency of transfection was evaluated through the detection of green fluorescent protein-marked MAGE-D4a. The MAGE-EMSCs were induced to differentiate into DCs, termed as MAGE-EMSCs-DCs. The morphology was subsequently analyzed under a microscope, and methyl thiazolyl tetrazolium (MTT) and interferon-γ (IFN-γ) assays were performed to analyze the cytotoxicity of the MAGE-EMSC-DCs on the human glioma U251 cell line. Following purification by magnetic-activated cell sorting, the EMSCs grew into swirls, with a long spindle shape and were fibroblast-like. The gene transfected with recombinant adenovirus vectors maintained high and stable expression levels of MAGE-D4a, and its efficiency was increased in a multiplicity of infection-dependent manner. The results of the MTT assay indicated that the T cells, primed by the recombinant MAGE-D4a-infected EMSC-DCs in vitro , recognized MAGE-D4a-expressing tumor cell lines in a human leukocyte antigen class I-restricted manner, and evoked a higher cytotoxic T cell (CTL) response. The CTL response induced by the MAGE-EMSC-DCs, co-cultured with the U251 cells for 24 h, produced 765.0 pg/ml IFN-γ, which was significantly greater when compared to the control wells. T lymphocytes stimulated by MAGE-EMSC-DCs evoke a higher CTL response to human glioma cell lines, and may serve as a promising therapeutic modality for the treatment of MAGE-D4a-expressing glioma.

The Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell Development

PubMed Central

Hayes, Mark D.; Ovcinnikovs, Vitalijs; Smith, Andrew G.; Kimber, Ian; Dearman, Rebecca J.

2014-01-01

The aryl hydrocarbon receptor (AhR) has been shown to be required for optimal Thelper (Th) 17 cell activation. Th17 cells provide immunity against extracellular pathogens and are implicated in autoimmune diseases. Herein, the role of the AhR in cytokine production by Th17, and by the analogous population of T cytotoxic (Tc)17 cells, has been examined. Lymph node Tc (CD8+) and Th (CD4+) cells were isolated by negative selection from naive AhR+/− and AhR−/− mice and polarised to Tc1/Th1 or Tc17/Th17 phenotypes with appropriate cytokines. Cell differentiation was assessed as a function of mRNA and protein (ELISA and flow cytometry) expression for interferon (IFN)-γ and for key Th17 cytokines. In AhR+/− mice, Th17 cells displayed an exclusive IL-17 profile, which was markedly inhibited by a selective AhR antagonist to levels observed in AhR knockout mice. Addition of the natural AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) markedly enhanced Th17 cell activity in the heterozygotes. In contrast, Tc17 cells polarised into 3 distinct subsets: producing either IL-17 or IFN-γ alone, or both cytokines. Blocking AhR was also detrimental to Tc17 development, with reduced responses recorded in AhR−/− mice and antagonist-mediated reduction of IL-17 expression in the heterozygotes. However, Tc17 cells were largely refractory to exogenous FICZ, presumably because Tc17 cells express baseline AhR mRNA, but unlike Th17 cells, there is no marked up-regulation during polarisation. Thus, Th17 cell development is more dependent upon AhR activation than is Tc17 cell development, suggesting that endogenous AhR ligands play a much greater role in driving Th17 cell responses. PMID:25203682
Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

PubMed Central

Eberle, R; Russell, R G; Rouse, B T

1981-01-01

In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL. PMID:6277790
Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

PubMed Central

Sloan, Derek D.; Lam, Chia-Ying Kao; Irrinki, Alivelu; Liu, Liqin; Tsai, Angela; Pace, Craig S.; Kaur, Jasmine; Murry, Jeffrey P.; Balakrishnan, Mini; Moore, Paul A.; Johnson, Syd; Nordstrom, Jeffrey L.; Cihlar, Tomas; Koenig, Scott

2015-01-01

HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. PMID:26539983
In situ electrochemical assessment of cytotoxicity of chlorophenols in MCF-7 and HeLa cells.

PubMed

Qin, Hongwei; Liu, Jiguang; Zhang, Zeshi; Li, Jinlian; Gao, Guanggang; Yang, Yuxin; Yuan, Xing; Wu, Dongmei

2014-10-01

An in situ electrochemical method was used to assess the cytotoxicity of chlorophenols using human breast cancer (MCF-7) and cervical carcinoma (HeLa) cells as models. On treatment with different chlorophenols, the electrochemical responses of the selected cells, resulting from the oxidation of guanine and xanthine in the cytoplasm, indicated the cell viability. In addition, the in situ in vitro electrochemical method was further compared with the traditional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Although similar cytotoxicity data were obtained from both methods, the effective concentrations of chlorophenols that inhibited 50% cell growth (EC50 values) from the electrochemical method were only slightly lower than those from the MTT assay. These results indicate that the in situ in vitro electrochemical method paves a simple, rapid, strongly responsive, and label-free way to the cytotoxicity assessment of different chlorophenol pollutants. Copyright © 2014 Elsevier Inc. All rights reserved.
Cytotoxicity of fucosterol containing fraction of marine algae against breast and colon carcinoma cell line

PubMed Central

Khanavi, Mahnaz; Gheidarloo, Razieh; Sadati, Nargess; Ardekani, Mohammad Reza Shams; Nabavi, Seyed Mohammad Bagher; Tavajohi, Shohreh; Ostad, Seyed Nasser

2012-01-01

Context: Marine algae produce different secondary metabolites with a wide range of biological activities. Many studies have been achieved on the screening of biological effects of marine organisms and a lot of active compounds were isolated and characterized. Aims: In an attempt to find cytotoxic compound of hexane fraction, isolation, identification, and cytotoxicity of active compound of this fraction were performed. Materials and Methods: In this study, total methanolic (70%) extract and partition fractions of hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and MeOH–H2O of Sargassum angustifolium, Chondria dasyphylla, and Ulva flexuosa, collected from coastlines of the Persian Gulf in south of Iran, were studied against colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D), and Swiss mouse embryo fibroblast (NIH 3T3) cell lines by MTT assay. Statistical Analysis Used: IC50 (median growth inhibitory concentration) values were calculated by Sigmaplot (10) software. Results: Hexane fraction of Chondria dasyphylla (IC50 82.26 ± 4.09 μg/ml) and MeOH-H2O fraction of Ulva flexuosa (IC50 116.92 ± 8.58 μg/ml) showed cytotoxic activity against proliferation of T47D cells. Hexane fraction of Sargassum angustifolium was also observed for cytotoxicity against T47D and HT-29 cell lines (IC50 166.42 ± 26.7 and 190.24 ± 52.8 μg/ml), respectively. An investigation of a component from the hexane fraction of Sargassum angustifolium yielded a steroidal metabolite, fucosterol, with cytotoxicity in T47D and HT29 (IC50 27.94 ± 9.3 and 70.41 ± 7.5 μg/ml). Conclusions: These results indicated that fucosterol, the most abundant phytosterol in brown algae, is responsible for cytotoxic effect of this extract against breast and colon carcinoma cell lines. PMID:22438665
Adoptive transfer of Epstein-Barr virus-specific cytotoxic T-lymphocytes for the treatment of angiocentric lymphomas.

PubMed

Cho, Hyun-Il; Hong, Young Seon; Lee, Myung Ah; Kim, Eun-Kyung; Yoon, Sung-Hee; Kim, Chun-Choo; Kim, Tai-Gyu

2006-01-01

Angiocentric lymphoma, known as natural killer (NK)/T-cell non-Hodgkin's lymphoma, has been reported to be associated with the Epstein-Barr virus (EBV). We performed adoptive transfer of EBV-specific polyclonal T-cell lines in 3 patients with extranodal NK/T-cell lymphoma, nasal type, and evaluated the treatment for safety, immunologic reconstitution, and clinical outcomes. The tissue samples collected from the 3 patients were confirmed by polymerase chain reaction analysis to be EBV positive. In the cases of the first and second patients, EBV-transformed B-lymphoblastoid cell lines (LCLs) and T-cell lines were generated from peripheral lymphocytes of HLA-matched sibling donors. The third patient's T-cell lines were induced with autologous lymphocytes. Polyclonal T-cell infusion was carried out after high-dose radiotherapy because active relapsed disease remained in all of the patients. The first patient received 4 weekly infusions of 2 3 10(7) cells/m(2), and the second and third patients underwent treatment with 2 cycles of infusions of the same dosage. All T-cell lines showed >60% NK activity, cytotoxic T-lymphocyte (CTL) responses of >40% against autologous LCLs, and no CTL activity against patient-derived lymphoblasts. The level of cytotoxicity increased substantially in all patients after cell infusion. The 2 patients who received T-cell therapy twice had stabilized disease for more than 3 years. These safe treatments exhibited no severe inflammatory response, and no serious toxicity developed during T-cell therapy. Our findings demonstrate that adoptively transferred cells may provide reconstitution of EBV-specific CTL responses in patients with active relapsed angiocentric lymphoma. These results provide a rationale for the immunotherapy of angiocentric lymphoma.
Interleukins 12 and 15 induce cytotoxicity and early NK-cell differentiation in type 3 innate lymphoid cells.

PubMed

Raykova, Ana; Carrega, Paolo; Lehmann, Frank M; Ivanek, Robert; Landtwing, Vanessa; Quast, Isaak; Lünemann, Jan D; Finke, Daniela; Ferlazzo, Guido; Chijioke, Obinna; Münz, Christian

2017-12-26

Type 3 innate lymphoid cells (ILC3s) fulfill protective functions at mucosal surfaces via cytokine production. Although their plasticity to become ILC1s, the innate counterparts of type 1 helper T cells, has been described previously, we report that they can differentiate into cytotoxic lymphocytes with many characteristics of early differentiated natural killer (NK) cells. This transition is promoted by the proinflammatory cytokines interleukin 12 (IL-12) and IL-15, and correlates with expression of the master transcription factor of cytotoxicity, eomesodermin (Eomes). As revealed by transcriptome analysis and flow cytometric profiling, differentiated ILC3s express CD94, NKG2A, NKG2C, CD56, and CD16 among other NK-cell receptors, and possess all components of the cytotoxic machinery. These characteristics allow them to recognize and kill leukemic cells with perforin and granzymes. Therefore, ILC3s can be harnessed for cytotoxic responses via differentiation under the influence of proinflammatory cytokines.
Cytotoxicity Induced by a Redox-silent Analog of Tocotrienol in Human Mesothelioma H2452 Cell Line via Suppression of Cap-dependent Protein Translation.

PubMed

Sato, Ayami; Ueno, Haruka; Takase, Akari; Ando, Akira; Sekine, Yuko; Yano, Tomohiro

2016-04-01

De novo synthesis of proteins is regulated by cap-dependent protein translation. Aberrant activation of the translation is a hallmark of many cancer types including malignant mesothelioma (MM). We previously reported that a redox-silent analog of α-tocotrienol, 6-O-carboxypropyl-α-tocotrienol (T3E) induces potent cytotoxicity against human MM cells. However, the detailed mechanism of cytotoxicity of T3E remains unclear. In this study, we investigated if T3E induced potent cytotoxicity aganist MM cells. T3E reduced the formation of the cap-dependent translation complex and induced inactivation of oncogene from rat sarcoma virus (RAS). These events were associated with T3E cytotoxicity in MM cells. Furthermore, atorvastatin, an inhibitor of RAS function, had similar effects on MM cells. Moreover, 4EGI-1, a specific inhibitor of the cap-dependent translation complex, induced severe cytotoxicity in MM cells. Overall, T3E had a cytotoxic effect on MM cells via disruption of the activated cap-dependent translation complex through inactivation of RAS. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Modulation of cytotoxic T lymphocyte, natural killer cell, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by Vernonia cinerea L. and vernolide-A in BALB/c mice via enhanced production of cytokines IL-2 and IFN-γ.

PubMed

Pratheeshkumar, Poyil; Kuttan, Girija

2012-02-01

Effect of Vernonia cinerea L. and vernolide-A on cell-mediated immune (CMI) response was studied in normal as well as tumor-bearing BALB/c mice. Administration of V. cinerea and vernolide-A significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were also enhanced significantly in both normal as well as tumor-bearing animals after V. cinerea and vernolide-A administration compared with untreated control tumor-bearing animals. Extract and vernolide-A showed a significant increase in cytotoxic T lymphocyte (CTL) production in both the in vivo and in vitro models. The level of cytokines such as interleukin (IL)-2 and interferon (IFN)-γ were also enhanced by the treatment of V. cinerea and vernolide-A in both normal as well as tumor-bearing animals. This study demonstrated that V. cinerea extract and vernolide-A stimulate the CTL, NK cell, ADCC, and ADCC through enhanced secretion of IL-2 and IFN-γ.
Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death.

PubMed

Viola, K S; Rodrigues, E M; Tanomaru-Filho, M; Carlos, I Z; Ramos, S G; Guerreiro-Tanomaru, J M; Faria, G

2017-01-30

To evaluate the cytotoxicity and the mechanism of cell aggression of peracetic acid (PA) in comparison with sodium hypochlorite (NaOCl). L929 fibroblasts were exposed to 1% PA and 2.5% NaOCl, at several dilutions for 10 min. The following parameters were evaluated: cell metabolism by methylthiazol tetrazolium assay, external morphology by scanning electron microscopy, ultrastructure by transmission electron microscopy, the cytoskeleton by means of actin and α-tubulin labelling, and the type of cell death by flow cytometry (apoptosis/necrosis). The data were analysed by two-way anova and the Bonferroni post-test (α = 0.05). The PA group had lower cell viability and a higher percentage of necrotic cells than the NaOCl group (P < 0.05). Both solutions diminished cell metabolism, led to destructuring of the cytoskeleton, created changes in the external morphology, resulted in the accumulation of proteins in the rough endoplasmic reticulum and induced cell death predominantly by necrosis. However, these changes were observed in lower doses of PA when compared with NaOCl. Although they had the same mechanism of cytotoxicity, 1% PA had greater cytotoxic potential than 2.5% NaOCl. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.
A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

PubMed Central

Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Tamura, Kenji; Hamada, Akinobu

2016-01-01

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies. PMID:26813960
A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells.

PubMed

Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Tamura, Kenji; Hamada, Akinobu

2016-01-27

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The (51)Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.
Enhancement of T cell recruitment and infiltration into tumours

PubMed Central

Oelkrug, C; Ramage, J M

2014-01-01

Studies have documented that cancer patients with tumours which are highly infiltrated with cytotoxic T lymphocytes show enhanced survival rates. The ultimate goal of cancer immunotherapy is to elicit high-avidity tumour-specific T cells to migrate and kill malignant tumours. Novel antibody therapies such as ipilumimab (a cytotoxic T lymphocyte antigen-4 blocking antibody) show enhanced T cell infiltration into the tumour tissue and increased survival. More conventional therapies such as chemotherapy or anti-angiogenic therapy and recent therapies with oncolytic viruses have been shown to alter the tumour microenvironment and thereby lead to enhanced T cell infiltration. Understanding the mechanisms involved in the migration of high-avidity tumour-specific T cells into tumours will support and provide solutions for the optimization of therapeutic options in cancer immunotherapy. PMID:24828133
Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less
Optimization and validation of FePro cell labeling method.

PubMed

Janic, Branislava; Rad, Ali M; Jordan, Elaine K; Iskander, A S M; Ali, Md M; Varma, N Ravi S; Frank, Joseph A; Arbab, Ali S

2009-06-11

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17
Role of vitamin D in cytotoxic T lymphocyte immunity to pathogens and cancer.

PubMed

Sarkar, Surojit; Hewison, Martin; Studzinski, George P; Li, Yan Chun; Kalia, Vandana

2016-01-01

The discovery of vitamin D receptor (VDR) expression in immune cells has opened up a new area of research into immunoregulation by vitamin D, a niche that is distinct from its classical role in skeletal health. Today, about three decades since this discovery, numerous cellular and molecular targets of vitamin D in the immune system have been delineated. Moreover, strong clinical associations between vitamin D status and the incidence/severity of many immune-regulated disorders (e.g. infectious diseases, cancers and autoimmunity) have prompted the idea of using vitamin D supplementation to manipulate disease outcome. While much is known about the effects of vitamin D on innate immune responses and helper T (T(H)) cell immunity, there has been relatively limited progress on the frontier of cytotoxic T lymphocyte (CTL) immunity--an arm of host cellular adaptive immunity that is crucial for the control of such intracellular pathogens as human immunodeficiency virus (HIV), tuberculosis (TB), malaria, and hepatitis C virus (HCV). In this review, we discuss the strong historical and clinical link between vitamin D and infectious diseases that involves cytotoxic T lymphocyte (CTL) immunity, present our current understanding as well as critical knowledge gaps in the realm of vitamin D regulation of host CTL responses, and highlight potential regulatory connections between vitamin D and effector and memory CD8 T cell differentiation events during infections.
Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes.

PubMed Central

Matter, A

1979-01-01

A study was carried out to determine the sequence of events of T-cell mediated target cell lysis in microcinematography and electron microscopy. Highly efficient cytotoxic T lymphocytes (CTL) were generated in vivo and in vitro using preimmunized spleen cells and purification procedures. Such CTL were highly specific. This specificity correlated well with the number of adhesions formed between CTL and targets and this criterion was used to study killer-target cell interaction. Microcinematography showed that target cell lysis at the single cell level, despite time variations, could be clearly separated into three phases: (a) a recognition phase, visible by random crawling of CTL over the target cell surface until firm contact was established; (b) a post-recognition phase, during which firm contact between CTL and target was maintained without gross modification of either cell; (c) a phase of target cell disintegration, mainly characterized by vigorous blebbing of the cell membrane resulting in a motionless carcass of the target cell but not in its total dissolution. Only later this carcass decayed and formed a necrotic ghost. Electron microscopic observations were put into sequence according to microcinematography. Post-recognition phase was characterized by a tight apposition of the membranes of CTL and target cell. No gap junctions could be observed. During target cell disintegration, profound cytoplasmic and nuclear changes occurred simultaneous with surface blebbing. Most noticeable were extensive internal vacuolization, mitochondrial swelling, nuclear pycnosis and dissolution of the nucleolus. These observations suggested that target cell lysis does not start with a surface phenomenon similar to complement lysis, but a process involving practically the whole cell simultaneously. It is conceivable, therefore, that the signal from the CTL is transmitted across the target cell, and that the switch to sudden cell death is manipulated deep inside the cell. Images
PiggyBac-mediated Cancer Immunotherapy Using EBV-specific Cytotoxic T-cells Expressing HER2-specific Chimeric Antigen Receptor

PubMed Central

Nakazawa, Yozo; Huye, Leslie E; Salsman, Vita S; Leen, Ann M; Ahmed, Nabil; Rollins, Lisa; Dotti, Gianpietro; Gottschalk, Stephen M; Wilson, Matthew H; Rooney, Cliona M

2011-01-01

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy. PMID:21772253
PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor.

PubMed

Nakazawa, Yozo; Huye, Leslie E; Salsman, Vita S; Leen, Ann M; Ahmed, Nabil; Rollins, Lisa; Dotti, Gianpietro; Gottschalk, Stephen M; Wilson, Matthew H; Rooney, Cliona M

2011-12-01

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.
[Amplification of γδ T cells in PBMCs of healthy donors and osteosarcoma patients stimulated by zoledronate].

PubMed

Li, Zhao-xu; Sun, Ling-ling; Cheng, Rui-lin; Sun, Zheng-wang; Ye, Zhao-ming

2012-08-01

To investigate the amplification and cytotoxicity of γδ T cells in peripheral blood mononuclear cells (PBMCs) of healthy donors and osteosarcoma patients stimulated by zoledronate (Zol) and IL-2. PBMCs from healthy donors and osteosarcoma patients were stimulated with IL-2 and Zol+IL-2, respectively. After 14-day culture, the purity of γδ T cells was assessed by flow cytometry. The cytotoxicity of γδ T cells against target cells was analyzed using a standard lactate dehydrogenase release assay with γδ T lymphocyte-sensitive Daudi cells, γδ T lymphocyte-resistant Raji cells and human osteoblast cell line, hFOB, as the target cells. After 2-week culture ex vivo of PBMCs from healthy donors and osteosarcoma patients, compared with stimulation of IL-2, Zol+IL-2 significantly promoted the amplification of γδ T cells. In addition, γδ T cells showed the higher cytotoxicity against Daudi cells, but no cytotoxic effect on normal cells like hFOB. γδ T cells of high purity and high cytotoxicity can be obtained by the stimulation of Zol combined with IL-2 on PBMCs from healthy donors and osteosarcoma patients.

Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells.

PubMed

Medina, Daniel J; Sheay, Wendy; Osman, Mona; Goodell, Lauri; Martin, John; Rabson, Arnold B; Strair, Roger K

2005-11-01

Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin's lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k "antiapoptotic" gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells.
Protective effect of Edaravone against hypoxia-induced cytotoxicity in osteoblasts MC3T3-E1 cells.

PubMed

Cao, Bo; Chai, Chunxiang; Zhao, Sishun

2015-12-01

Edaravone is a newly developed clinical medicine for the treatment of acute cerebral infarction. Reduced blood supply to bones (hypoxia) has been involved in the pathological development of osteoporosis. In this study, we investigated the effect of Edaravone and its latent mechanism on hypoxia-induced cell toxicity in MC3T3-E1 cells. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were determined by the fluorescence dyes 2',7'-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), respectively. mRNA and proteins were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Edaravone significantly restored the hypoxia-induced reduction of MC3T3-E1 cell viability and inhibited lactate dehydrogenase release. In addition, we found that Edaravone inhibits the generation of ROS and NO. Hoechst staining results indicated that the nuclear condensation characteristic of apoptosis was increased in MC3T3-E1 cells after hypoxia exposure, which was significantly suppressed by Edaravone treatment. Mechanistically, we found that Edaravone markedly reduced the expression of cleaved caspase-3 and blunted the release of cytochrome c. These findings strongly suggested that Edaravone suppresses hypoxia-induced cytotoxicity in MC3T3-E1 cells. The pleiotropic effects of Edaravone on hypoxia exposure in osteoblasts suggest potential antiosteoporosis mechanisms of Edaravone. © 2015 International Union of Biochemistry and Molecular Biology.
Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway.

PubMed

Hodge, Greg; Mukaro, Violet; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2013-02-01

Natural killer (NK) and natural killer T (NKT)-like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro-inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin. We measured NK and NKT-like cells and their expression of CD94 in the blood of COPD patients (n = 71; 30 current and 41 ex-smokers), smokers (16) and healthy controls (25), and bronchoalveolar lavage fluid (BALF) from a cohort of subjects (19 controls, 12 smokers, 33 COPD). Activation was assessed by measuring CD69 in blood and the cytotoxic potential of NK cells by measuring granzymes A and B, and using a cytotoxicity assay in blood and BALF. In blood in COPD, there were no significant changes in the proportion of NK or NKT-like cells or expression of granzyme A or NK cytotoxic potential versus controls. There was, however, increased expression of granzyme B and decreased expression of CD94 by both cell types versus controls. The proportion of NK and NKT-like cells were increased in BALF in COPD, associated with increased NK cytotoxicity, increased expression of granzyme B and decreased expression of the inhibitory receptor CD94 by both cell types. Treatment strategies that target NK and NKT-like cells, their cytotoxicity and production of inflammatory mediators in the airway may improve COPD morbidity. © 2012 The Authors. Respirology © 2012 Asian Pacific Society of Respirology.
Labeling proteins on live mammalian cells using click chemistry.

PubMed

Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

2015-05-01

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.
RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma.

PubMed

Harrer, Dennis C; Simon, Bianca; Fujii, Shin-Ichiro; Shimizu, Kanako; Uslu, Ugur; Schuler, Gerold; Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

2017-08-17

Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA. PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8 + T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed. Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8 + MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8 + T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells. We
Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

PubMed

Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

1984-01-01

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.
Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells.

PubMed

Boonkaew, Benjawan; Kempf, Margit; Kimble, Roy; Cuttle, Leila

2014-12-01

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver(®) and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver(®) showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.
Epstein Barr virus–specific cytotoxic T lymphocytes expressing the anti-CD30ζ artificial chimeric T-cell receptor for immunotherapy of Hodgkin disease

PubMed Central

Rooney, Cliona M.; Di Stasi, Antonio; Abken, Hinrich; Hombach, Andreas; Foster, Aaron E.; Zhang, Lan; Heslop, Helen E.; Brenner, Malcolm K.; Dotti, Gianpietro

2007-01-01

Adoptive transfer of Epstein Barr virus (EBV)–specific cytotoxic T-lymphocytes (EBV-CTLs) has shown that these cells persist in patients with EBV+ Hodgkin lymphoma (HD) to produce complete tumor responses. Treatment failure, however, occurs if a subpopulation of malignant cells in the tumor lacks or loses expression of EBV antigens. We have therefore determined whether we could prepare EBV-CTLs that retained the antitumor activity conferred by their native receptor while expressing a chimeric antigen receptor (CAR) specific for CD30, a molecule highly and consistently expressed on malignant Hodgkin Reed-Sternberg cells. We made a CD30CAR and were able to express it on 26% (± 11%) and 22% (± 5%) of EBV-CTLs generated from healthy donors and HD patients, respectively. These CD30CAR+ CTLs killed both autologous EBV+ cells through their native receptor and EBV−/CD30+ targets through their major histocompatibility complex (MHC)–unrestricted CAR. A subpopulation of activated T cells also express CD30, but the CD30CAR+ CTLs did not impair cellular immune responses, probably because normal T cells express lower levels of the target antigen. In a xenograft model, CD30CAR+ EBV-CTLs could be costimulated by EBV-infected cells and produce antitumor effects even against EBV−/CD30+ tumors. EBV-CTLs expressing both a native and a chimeric antigen receptor may therefore have added value for treatment of HD. PMID:17507664
IL-7Rαlow memory CD8+ T cells are significantly elevated in patients with systemic lupus erythematosus.

PubMed

Kim, Jung-Sik; Cho, Bon-A; Sim, Ji Hyun; Shah, Kamini; Woo, Connie M; Lee, Eun Bong; Lee, Dong-Sup; Kang, Jae Seung; Lee, Wang Jae; Park, Chung-Gyu; Craft, Joe; Kang, Insoo; Kim, Hang-Rae

2012-09-01

Human effector memory (EM) CD8(+) T cells include IL-7Rα(high) and IL-7Rα(low) cells with distinct cellular characteristics, including the expression of cytotoxic molecules. Both NK cells and the NK cell-associated molecule 2B4 that is expressed on CD8(+) T cells promote cytotoxicity. Here we analysed the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells and its contribution to cytotoxicity. We also analysed the frequency of IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells in patients with SLE or lupus and in healthy individuals given the potential role of cytotoxic CD8(+) T cells in the pathogenesis of lupus. We used flow cytometry to measure the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells as well as the frequency of these cell populations in the peripheral blood of healthy individuals and patients with SLE. Also, 2B4-mediated cytotoxicity was quantitated in IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells using target cells with CD48 antigen. We found that IL-7Rα(high) EM CD8(+) T cells had higher levels of 2B4 expression compared with IL-7Rα(low) EM CD8(+) T cells. Triggering 2B4 enhanced the cytotoxic function of IL-7Rα(low) EM CD8(+) T cells against target cells. We also noticed that patients with SLE had an increased frequency of IL-7Rα(low) EM CD8(+) T cells that correlated with disease manifestation. Our findings show that SLE patients have increased IL-7Rα(low) EM CD8(+) T cells, possibly contributing to tissue damage through 2B4-mediated cytotoxicity.
Self-Assembled Superparamagnetic Iron Oxide Nanoclusters for Universal Cell Labeling and MRI

NASA Astrophysics Data System (ADS)

Chen, Shuzhen; Zhang, Jun; Jiang, Shengwei; Lin, Gan; Luo, Bing; Yao, Huan; Lin, Yuchun; He, Chengyong; Liu, Gang; Lin, Zhongning

2016-05-01

Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used in a variety of biomedical applications, especially as contrast agents for magnetic resonance imaging (MRI) and cell labeling. In this study, SPIO nanoparticles were stabilized with amphiphilic low molecular weight polyethylenimine (PEI) in an aqueous phase to form monodispersed nanocomposites with a controlled clustering structure. The iron-based nanoclusters with a size of 115.3 ± 40.23 nm showed excellent performance on cellular uptake and cell labeling in different types of cells, moreover, which could be tracked by MRI with high sensitivity. The SPIO nanoclusters presented negligible cytotoxicity in various types of cells as detected using MTS, LDH, and flow cytometry assays. Significantly, we found that ferritin protein played an essential role in protecting stress from SPIO nanoclusters. Taken together, the self-assembly of SPIO nanoclusters with good magnetic properties provides a safe and efficient method for universal cell labeling with noninvasive MRI monitoring capability.
Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage

PubMed Central

Huang, Ruili; Lin, Ja-An; Sedykh, Alexander; Zhao, Jinghua; Tice, Raymond R.; Paules, Richard S.; Xia, Menghang; Auerbach, Scott S.

2017-01-01

Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is
A cationic amphiphilic peptide ABP-CM4 exhibits selective cytotoxicity against leukemia cells.

PubMed

Chen, Yu Qing; Min, Cui; Sang, Ming; Han, Yang Yang; Ma, Xiao; Xue, Xiao Qing; Zhang, Shuang Quan

2010-08-01

Some cationic antibacterial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells, which could provide a new class of anticancer drugs. In the present study, the anticancer activity of ABP-CM4, an antibacterial peptide from Bombyx mori, against leukemic cell lines THP-1, K562 and U937 was evaluated, and the cytotoxicity compared with the effects on non-cancerous mammalian cells, including peripheral blood mononuclear cells (PBMCs), HEK-293 and erythrocytes. ABP-CM4 reduced the number of viable cells of the leukemic cell lines after exposure for 24h. The reduction was concentration dependent, and the IC50 values ranged from 14 to 18 microM. Conversely, ABP-CM4, even at 120 microM, exhibited no cytotoxicity toward HEK-293 or PBMCs, indicating that there was no significant effect on these two types of non-cancer cells. ABP-CM4 at a concentration of 200 microM had no hemolytic activity on mammalian erythrocytes. Together, these results suggested a selective cytotoxicity in leukemia cells. Flow cytometry demonstrated that the binding activity of ABP-CM4 to leukemia cells was much higher than that to HEK-293 or PBMCs, and there was almost no binding to erythrocytes. FITC-labeled ABP-CM4 molecules were examined under a confocal microscope and found to be concentrated at the surface of leukemia cells and changes of the cell membrane were determined by a cell permeability assay, which led us to the conclusion that ABP-CM4 could act at the cell membrane for its anticancer activity on leukemia cells. Collectively, our results indicated that ABP-CM4 has the potential for development as a novel antileukemic agent. Copyright 2010 Elsevier Inc. All rights reserved.
Activation of mouse liver natural killer cells and NK1.1(+) T cells by bacterial superantigen-primed Kupffer cells.

PubMed

Dobashi, H; Seki, S; Habu, Y; Ohkawa, T; Takeshita, S; Hiraide, H; Sekine, I

1999-08-01

Although bacterial superantigens have been well characterized as potent stimulators of T cells, their role in natural killer (NK)-type cells remains largely unknown. In the present study, we examined the effect of bacterial superantigens on mouse liver NK cells and NK1.1 Ag(+) (NK1(+)) T cells. C57BL/6 mice were intravenously injected with staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A), and mononuclear cells (MNC) of various organs were obtained from mice 4 hours after being injected with superantigen. MNC were cultured for 48 hours, and interferon gamma (IFN-gamma) levels of supernatants were measured. The antitumor cytotoxicities of the liver and spleen MNC were also evaluated 24 hours after the mice were injected with superantigen. Liver MNC produced more IFN-gamma than did splenocytes, and peripheral blood and lung MNC did not produce any detectable IFN-gamma. In addition, liver MNC acquired a potent antitumor cytotoxicity by the SEB injection, and both NK cells and NK1(+)T cells but not cluster of differentiation (CD)8(+) T cells were responsible for the cytotoxicity as demonstrated by either in vivo or in vitro cell depletion experiments, and the NK-type cells were partly responsible for the increased serum IFN-gamma. Activation of liver NK-type cells was also supported by the fact that liver NK cells proportionally increased and NK1(+) T cells augmented their CD11a expressions after SEB injection. The pretreatment of mice with anti-IFN-gamma Ab and/or with anti-interleukin-12 (IL-12) Ab diminished the SEB-induced cytotoxicity of liver MNC. Furthermore, the in vivo depletion of Kupffer cells decreased the SEB-induced cytotoxicity of liver MNC. Consistent with these results, liver MNC stimulated with superantigens in the presence of Kupffer cells in vitro produced a greater amount of IFN-gamma than did the liver MNC without Kupffer cells or splenocytes. Our results suggest that bacterial superantigen-primed Kupffer cells
Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

PubMed Central

Graf, Erin H.; Pace, Matthew J.; Peterson, Bennett A.; Lynch, Lindsay J.; Chukwulebe, Steve B.; Mexas, Angela M.; Shaheen, Farida; Martin, Jeffrey N.; Deeks, Steven G.; Connors, Mark; Migueles, Stephen A.; O’Doherty, Una

2013-01-01

Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy. PMID:23951263
In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity.

PubMed

Sedlik, C; Dadaglio, G; Saron, M F; Deriaud, E; Rojas, M; Casal, S I; Leclerc, C

2000-07-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.
In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity†

PubMed Central

Sedlik, C.; Dadaglio, G.; Saron, M. F.; Deriaud, E.; Rojas, M.; Casal, S. I.; Leclerc, C.

2000-01-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+ cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+ class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+ T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+ T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies. PMID:10846055
Fucoidan cytotoxicity against human breast cancer T47D cell line increases with higher level of sulfate ester group

NASA Astrophysics Data System (ADS)

Saepudin, Endang; Alfita Qosthalani, Fildzah; Sinurat, Ellya

2018-01-01

The anticancer activity of different sulfate ester group content in different molecular weight was examined. The anticancer activity was achieved in vitro on human breast cancer T47D cell line. Fucoidan with lower molecular weight (5.79 kDa) tends to have lower sulfate ester group content (8.69%) and resulted in higher IC50 value (184.22 μg/mL). While fucoidan with higher molecular weight (785.12 kDa) tends to have higher sulfate level (18.63%) and achieved lower IC50 value (75.69 μg/mL). The result showed that in order to maintain fucoidan cytotoxic activity against human breast cancer T47D cell line, the sulfate content should be remain high. Keywords: fucoidan, sulfate ester group, human breast cancer
Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability.

PubMed

Birch, Ditlev; Christensen, Malene Vinther; Staerk, Dan; Franzyk, Henrik; Nielsen, Hanne Mørck

2017-12-01

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC 50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical. Copyright © 2017 Elsevier B.V. All rights reserved.
Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

PubMed

Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

2013-01-01

To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.
Highly conserved CDR3 region in circulating CD4+Vβ5+ T cells may be associated with cytotoxic activity in Chagas disease

PubMed Central

Menezes, C A S; Sullivan, A K; Falta, M T; Mack, D G; Freed, B M; Rocha, M O C; Gollob, K J; Fontenot, A P; Dutra, W O

2012-01-01

Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4+ T cells from chagasic patients preferentially express T cell receptor (TCR) β-chain variable region (Vβ) 5. The aim of this work was to determine whether T cells expressing this particular Vβ region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vβ5·1 expressing CD4+ T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jβ region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4+Vβ5·1+ cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy. PMID:22774985

Circulating cytotoxic T cells and natural killer cells as potential predictors for antidepressant response in melancholic depression. Restoration of T regulatory cell populations after antidepressant therapy.

PubMed

Grosse, Laura; Carvalho, Livia A; Birkenhager, Tom K; Hoogendijk, Witte J; Kushner, Steven A; Drexhage, Hemmo A; Bergink, Veerle

2016-05-01

There is a substantial unmet need for biomarkers to predict treatment response in major depressive disorder (MDD). Evidence has converged on activation of the inflammatory response system as a fundamental mechanism underlying MDD. By investigating circulating leukocyte subsets quantified by fluorescence-activated cell sorting (FACS) analysis before treatment, we aim to predict antidepressant response. Forty medication-free inpatients with melancholic, non-psychotic depression before treatment with either venlafaxine or imipramine and 40 age- and gender-matched healthy controls were included. Leukocyte subsets were quantified by FACS analysis using frozen peripheral blood mononuclear cells (PBMC) collected prior to and after 7 weeks of treatment with either venlafaxine (375 mg/day) or imipramine (blood level 200-300 ng/ml). Response was defined as at least 50 % reduction of the baseline Hamilton Rating Scale for Depression (HAM-D) score. Prior to treatment, MDD patients showed reduced percentages of CD4(+)CD25(high)Foxp3(+) T regulatory (Treg) cells when compared with controls (1.5 ± 0.6 vs. 1.8 ± 0.6, p = .037). After treatment, robust rises in Treg cells were observed in patients (1.8 ± 0.7, p < .001), yet Treg cells were not predictors of the clinical outcome of treatment. Antidepressant non-responders showed increased CD8(+) cytotoxic T cell percentages (24.0 ± 8.6 vs. 15.9 ± 5.9, p = .004) and decreased natural killer (NK) cell percentages (14.0 ± 6.9 vs. 21.4 ± 11.9, p = .020) compared with responders before treatment. Both lymphocyte levels were not significantly modulated by treatment. In melancholic MDD, FACS analysis of circulating leukocyte subpopulations might help to discriminate between patients with high or low responsiveness to antidepressant treatment.
Low Dose Decitabine Treatment Induces CD80 Expression in Cancer Cells and Stimulates Tumor Specific Cytotoxic T Lymphocyte Responses

PubMed Central

Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

2013-01-01

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8+, but not CD4+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy. PMID:23671644
Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

PubMed

Wang, Li-Xin; Mei, Zhen-Yang; Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

2013-01-01

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.
Steroid Resistant CD8+CD28null NKT-Like Pro-inflammatory Cytotoxic Cells in Chronic Obstructive Pulmonary Disease.

PubMed

Hodge, Greg; Hodge, Sandra

2016-01-01

Corticosteroid resistance is a major barrier to effective treatment in chronic obstructive pulmonary disease (COPD), and failure to suppress systemic inflammation in these patients may result in increased comorbidity. Although much of the research to date has focused on the role of macrophages and neutrophils involved in inflammation in the airways in COPD, recent evidence suggests that CD8 + T cells may be central regulators of the inflammatory network in this disease. CD8 + cytotoxic pro-inflammatory T cells have been shown to be increased in the peripheral blood and airways in patients with COPD, whereas smokers that have not progressed to COPD only show an increase in the lungs. Although the mechanisms underlying steroid resistance in these lymphocytes is largely unknown, new research has identified a role for cytotoxic pro-inflammatory CD8 + T-cells and CD8 + natural killer T-like (NKT-like) cells. Increased numbers of these cells and their significant loss of the co-stimulatory molecule CD28 have been shown in COPD, consistent with findings in the elderly and in clinical conditions involving chronic activation of the immune system. In COPD, these senescent cells expressed increased levels of the cytotoxic mediators, perforin and granzyme b, and the pro-inflammatory cytokines, IFNγ and TNFα. They also demonstrated increased cytotoxicity toward lung epithelial cells and importantly were resistant to immunosuppression by corticosteroids compared with their CD28 + counterparts. Further research has shown these cells evade the immunosuppressive effects of steroids via multiple mechanisms. This mini review will focus on cytotoxic pro-inflammatory CD8 + CD28 null NKT-like cells involved in COPD and novel approaches to reverse steroid resistance in these cells.
Steroid Resistant CD8+CD28null NKT-Like Pro-inflammatory Cytotoxic Cells in Chronic Obstructive Pulmonary Disease

PubMed Central

Hodge, Greg; Hodge, Sandra

2016-01-01

Corticosteroid resistance is a major barrier to effective treatment in chronic obstructive pulmonary disease (COPD), and failure to suppress systemic inflammation in these patients may result in increased comorbidity. Although much of the research to date has focused on the role of macrophages and neutrophils involved in inflammation in the airways in COPD, recent evidence suggests that CD8+ T cells may be central regulators of the inflammatory network in this disease. CD8+ cytotoxic pro-inflammatory T cells have been shown to be increased in the peripheral blood and airways in patients with COPD, whereas smokers that have not progressed to COPD only show an increase in the lungs. Although the mechanisms underlying steroid resistance in these lymphocytes is largely unknown, new research has identified a role for cytotoxic pro-inflammatory CD8+ T-cells and CD8+ natural killer T-like (NKT-like) cells. Increased numbers of these cells and their significant loss of the co-stimulatory molecule CD28 have been shown in COPD, consistent with findings in the elderly and in clinical conditions involving chronic activation of the immune system. In COPD, these senescent cells expressed increased levels of the cytotoxic mediators, perforin and granzyme b, and the pro-inflammatory cytokines, IFNγ and TNFα. They also demonstrated increased cytotoxicity toward lung epithelial cells and importantly were resistant to immunosuppression by corticosteroids compared with their CD28+ counterparts. Further research has shown these cells evade the immunosuppressive effects of steroids via multiple mechanisms. This mini review will focus on cytotoxic pro-inflammatory CD8+CD28null NKT-like cells involved in COPD and novel approaches to reverse steroid resistance in these cells. PMID:28066427
Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

PubMed Central

Cartellieri, Marc; Bachmann, Michael; Feldmann, Anja; Bippes, Claudia; Stamova, Slava; Wehner, Rebekka; Temme, Achim; Schmitz, Marc

2010-01-01

CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs). First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells. PMID:20467460
Localized Irradiation of Cell Membrane by Auger Electrons Is Cytotoxic Through Oxidative Stress-Mediated Nontargeted Effects.

PubMed

Paillas, Salomé; Ladjohounlou, Riad; Lozza, Catherine; Pichard, Alexandre; Boudousq, Vincent; Jarlier, Marta; Sevestre, Samuel; Le Blay, Marion; Deshayes, Emmanuel; Sosabowski, Jane; Chardès, Thierry; Navarro-Teulon, Isabelle; Mairs, Robert J; Pouget, Jean-Pierre

2016-09-10

We investigated whether radiation-induced nontargeted effects are involved in the cytotoxic effects of anticell surface monoclonal antibodies labeled with Auger electron emitters, such as iodine 125 (monoclonal antibodies labeled with (125)I [(125)I-mAbs]). We showed that the cytotoxicity of (125)I-mAbs targeting the cell membrane of p53(+/+) HCT116 colon cancer cells is mainly due to nontargeted effects. Targeted and nontargeted cytotoxicities were inhibited in vitro following lipid raft disruption with Methyl-β-cyclodextrin (MBCD) or filipin or use of radical oxygen species scavengers. (125)I-mAb efficacy was associated with acid sphingomyelinase activation and modulated through activation of the AKT, extracellular signal-related kinase ½ (ERK1/2), p38 kinase, c-Jun N-terminal kinase (JNK) signaling pathways, and also of phospholipase C-γ (PLC-γ), proline-rich tyrosine kinase 2 (PYK-2), and paxillin, involved in Ca(2+) fluxes. Moreover, the nontargeted response induced by directing 5-[(125)I]iodo-2'-deoxyuridine to the nucleus was comparable to that of (125)I-mAb against cell surface receptors. In vivo, we found that the statistical significance of tumor growth delay induced by (125)I-mAb was removed after MBCD treatment and observed oxidative DNA damage beyond the expected Auger electron range. These results suggest the involvement of nontargeted effects in vivo also. Low-energy Auger electrons, such as those emitted by (125)I, have a short tissue range and are usually targeted to the nucleus to maximize their cytotoxicity. In this study, we show that targeting the cancer cell surface with (125)I-mAbs produces a lipid raft-mediated nontargeted response that compensates for the inferior efficacy of non-nuclear targeting. Our findings describe the mechanisms involved in the efficacy of (125)I-mAbs targeting the cancer cell surface. Antioxid. Redox Signal. 25, 467-484.
Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

PubMed

Wakuri, S; Yamakage, K; Kazuki, Y; Kazuki, K; Oshimura, M; Aburatani, S; Yasunaga, M; Nakajima, Y

2017-04-01

The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay. Copyright © 2017 Elsevier Inc. All rights reserved.
SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

PubMed

Shen, Wei-Bin; Vaccaro, Dennis E; Fishman, Paul S; Groman, Ernest V; Yarowsky, Paul

2016-05-01

This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Cytotoxicity of citral against melanoma cells: The involvement of oxidative stress generation and cell growth protein reduction.

PubMed

Sanches, Larissa Juliani; Marinello, Poliana Camila; Panis, Carolina; Fagundes, Tatiane Renata; Morgado-Díaz, José Andrés; de-Freitas-Junior, Julio Cesar Madureira; Cecchini, Rubens; Cecchini, Alessandra Lourenço; Luiz, Rodrigo Cabral

2017-03-01

Citral is a natural compound that has shown cytotoxic and antiproliferative effects on breast and hematopoietic cancer cells; however, there are few studies on melanoma cells. Oxidative stress is known to be involved in all stages of melanoma development and is able to modulate intracellular pathways related to cellular proliferation and death. In this study, we hypothesize that citral exerts its cytotoxic effect on melanoma cells by the modulation of cellular oxidative status and/or intracellular signaling. To test this hypothesis, we investigated the antiproliferative and cytotoxic effects of citral on B16F10 murine melanoma cells evaluating its effects on cellular oxidative stress, DNA damage, cell death, and important signaling pathways, as these pathways, namely, extracellular signal-regulated kinases 1/2 (ERK1/2), AKT, and phosphatidylinositol-3 kinase, are involved in cell proliferation and differentiation. The p53 and nuclear factor kappa B were also investigated due to their ability to respond to intracellular stress. We observed that citral exerted antiproliferative and cytotoxic effects in B16F10; induced oxidative stress, DNA lesions, and p53 nuclear translocation; and reduced nitric oxide levels and nuclear factor kappa B, ERK1/2, and AKT. To investigate citral specificity, we used non-neoplastic human and murine cells, HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts), and observed that although citral effects were not specific for cancer cells, non-neoplastic cells were more resistant to citral than B16F10. These findings highlight the potential clinical utility of citral in melanoma, with a mechanism of action involving the oxidative stress generation, nitric oxide depletion, and interference in signaling pathways related to cell proliferation.
Assessment of Pb, Cd and Hg soil contamination and its potential to cause cytotoxic and genotoxic effects in human cell lines (CaCo-2 and HaCaT).

PubMed

Sljivic Husejnovic, Maida; Bergant, Martina; Jankovic, Sasa; Zizek, Suzana; Smajlovic, Aida; Softic, Adaleta; Music, Omer; Antonijevic, Biljana

2018-01-23

Soil contamination by heavy metals is a serious global environmental problem, especially for developing countries. A large number of industrial plants, which continually pollute the environment, characterize Tuzla Canton, Bosnia and Herzegovina. The aim of this study was to assess the level of soil pollution by heavy metals and to estimate cytotoxicity and genotoxicity of soil leachates from this area. Lead (Pb), cadmium (Cd) and mercury (Hg) were analyzed by ICP-AES and AAS. Soil contamination was assessed using contamination factor, degree of contamination, geoaccumulation index and pollution load index. To determine the connection of variables and understanding their origin in soils, principal component analysis (PCA) and cluster analysis (CA) were used. The results indicate that Cd and Hg originated from natural and anthropogenic activities, while Pb is of anthropogenic origin. For toxicity evaluation, CaCo-2 and HaCaT cells were used. PrestoBlue assay was used for cytotoxicity testing, and γH2A.X for genotoxicity evaluation. Concerning cytotoxicity, Cd and Hg had a positive correlation with cytotoxicity in HaCaT cells, but only Hg induced cytotoxicity in CaCo-2 cells. We also demonstrate that soil leachates contaminated by heavy metals can induce genotoxicity in both used cell lines. According to these results, combining bioassays with standard physicochemical analysis can be useful for evaluating environmental and health risks more accurately. These results are important for developing proper management strategies to decrease pollution. This is one of the first studies from this area and an important indication of soil quality in Southeast Europe.
Selective in vivo metabolic cell-labeling-mediated cancer targeting

PubMed Central

Wang, Hua; Wang, Ruibo; Cai, Kaimin; He, Hua; Liu, Yang; Yen, Jonathan; Wang, Zhiyu; Xu, Ming; Sun, Yiwen; Zhou, Xin; Yin, Qian; Tang, Li; Dobrucki, Iwona T; Dobrucki, Lawrence W; Chaney, Eric J; Boppart, Stephen A; Fan, Timothy M; Lezmi, Stéphane; Chen, Xuesi; Yin, Lichen; Cheng, Jianjun

2017-01-01

Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo. Specifically, we inhibit the cell-labeling activity of tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac4ManAz analog developed, mediated cancer-selective labeling in vivo, which enhanced tumor accumulation of a dibenzocyclooctyne–doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice. PMID:28192414
A Multiepitope of XBP1, CD138 and CS1 Peptides Induces Myeloma-Specific Cytotoxic T lymphocytes in T cells of Smoldering Myeloma Patients

PubMed Central

Bae, Jooeun; Prabhala, Rao; Voskertchian, Annie; Brown, Andrew; Maguire, Craig; Richardson, Paul; Dranoff, Glen; Anderson, Kenneth C.; Munshi, Nikhil C.

2014-01-01

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL), for their ability to elicit multipeptide specific cytotoxic T lymphocytes (MP-CTL) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTL generated from SMM patients’ T cells show effective anti-MM responses including CD137 (4-1BB) upregulation, CTL proliferation, IFN-γ production, and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically, we observed increased total CD3+CD8+ T cells (>80%) and cellular activation (CD69+) within the memory SMM MP-CTL (CD45RO+/CD3+CD8+) subset after repeated multipeptide stimulation. Importantly, SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and anti-tumor activity. In high responders, the effector memory (CCR7-CD45RO+/CD3+CD8+) T cell subset was enriched, while the remaining responders’ CTL contained a higher frequency of the terminal effector (CCR7-CD45RO-/CD3+CD8+) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore, our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease. PMID:24935722
High Rate of Induction of Human Autologous Cytotoxic T Lymphocytes against Renal Carcinoma Cells Cultured with an Interleukin Cocktail

PubMed Central

Liu, Shu Qin; Kawai, Koji; Shiraiwa, Hiroshi; Hayashi, Hitoshi; Akaza, Hideyuki; Hashizaki, Kazuko; Shiba, Reiko; Saijo, Kaoru

1998-01-01

A high rate of induction (9 of 10 cases) of human autologous cytotoxic T lymphocytes (CTL) was achieved in vitro from peripheral blood mononuclear cells of renal carcinoma patients by applying an interleukin (IL)‐cocktail consisting of IL‐1, ‐2, ‐4, and ‐6. The CTL specifically lysed their own target carcinoma cells within 24 h but did not kill neighboring autologous normal kidney cells or allogeneic renal cancer cell lines. In the case of TUHR4TKB, for which autologous CTL were not induced, no expression of MHC class‐I molecules was observed on the surface of these carcinoma cells, although they were sensitive to autologous natural killer cells. The results imply that adoptive immunotherapy for metastasized renal carcinoma will be feasible with autologous CTL in combination with natural killer cells. PMID:9914789
Design of polymeric immunomicrospheres for cell labelling and cell separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Margel, S.

1978-01-01

Synthesis of several classes of hydrophylic microspheres applied to cell labeling and cell separation is described. Five classes of cross-linked microspheres with functional groups such as carboxyl, hydroxyl, amide and/or pyridine groups were synthesized. These functional groups were used to bind covalently antibodies and other proteins to the surface of the microspheres. To optimize the derivatisation technique, polyglutaraldehyde immunomicrospheres were prepared and utilized. Specific populations of human and murine lymphocytes were labelled with microspheres synthesized by the emulsion of the ionizing radiation technique. The labelling of the cells by means of microspheres containing an iron core produced successful separation of B from T lymphocytes by means of a magnetic field.
CYTOTOXICITY AND CELL SIGNALING IN MH-S CELLS: RELATIVE POTENCY OF DIESEL AND COAL COMBUSTION PARTICLES

EPA Science Inventory

Cytotoxicity and Cell Signaling in MH-S Cells: Relative Potency of Diesel and Coal Combustion Particles P. Singh1, Y. Kostetski2, M. Daniels1, T. Stevens3 and MI Gilmour 1USEPA, RTP, NC, 2National University of Singapore, Singapore, 3University of North Carolina, Chapel Hill, NC<...
Iodination of Escherichia coli with chloramine T: selective labeling of the outer membrane lipoprotein.

PubMed Central

Munford, R S; Gotschlich, E C

1977-01-01

Iodination of Escherichia coli cells with chloramine T preferentially labels the free and murein-bound forms of the outer membrane lipoprotein. Iodination for 15 s at 15 degrees C labels the two forms of the lipoprotein almost exclusively, whereas iodination for 60 s at 25 degrees C also labels the other major outer membrane proteins. Chloramine T iodination is a rapid, simple technique for labeling the outer membrane lipoprotein. PMID:400793
Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stępnik, Maciej, E-mail: mstep@imp.lodz.pl; Arkusz, Joanna; Smok-Pieniążek, Anna

The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but notmore » to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (γ-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ► Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ► Ludox CL-X silica NPs are cytotoxic to both cell lines. ► In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ► Cell cycle analysis
In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.
Priming of Anti-Human Immunodeficiency Virus (HIV) CD8^+ Cytotoxic T Cells in vivo by Carrier-Free HIV Synthetic Peptides

NASA Astrophysics Data System (ADS)

Hart, Mary Kate; Weinhold, Kent J.; Scearce, Richard M.; Washburn, Eileen M.; Clark, Cynthia A.; Palker, Thomas J.; Haynes, Barton F.

1991-11-01

The generation of antiviral cytotoxic T lymphocytes (CTLs) is a critical component of the immune response to viral infections. A safe and nontoxic vaccine for AIDS would optimally use a carrier-free synthetic peptide immunogen containing only components of HIV necessary for induction of protective immune responses. We report that hybrid synthetic peptides containing either a HIV envelope gp120 T-cell determinant (T1) or the envelope gp41 fusion domain (F) N-terminal to HIV CTL determinants are capable of priming murine CD8^+, major histocompatibility complex class I-restricted anti-HIV CTLs in vivo. These data demonstrate that carrier-free, nonderivatized synthetic peptides can be used in vivo to induce anti-HIV CTL responses.

IL-2 activation of STAT5 enhances production of IL-10 from human cytotoxic regulatory T cells, HOZOT.

PubMed

Tsuji-Takayama, Kazue; Suzuki, Motoyuki; Yamamoto, Mayuko; Harashima, Akira; Okochi, Ayumi; Otani, Takeshi; Inoue, Toshiya; Sugimoto, Akira; Motoda, Ryuichi; Yamasaki, Fumiyuki; Nakamura, Shuji; Kibata, Masayoshi

2008-02-01

Interleukin (IL)-10 is an immunosuppressive cytokine produced by many cell types, including T cells. We previously reported that a novel type of regulatory T (Treg) cells, termed HOZOT, which possesses a FOXP3+CD4+CD8+CD25+ phenotype and dual suppressor/cytotoxic activities, produced high levels of IL-10. In this study, we examined the mechanisms of high IL-10 production by HOZOT, focusing on Janus activating kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway. We prepared five different types of T cells, including HOZOT from human umbilical cord blood. Cytokine productions of IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were compared among these T cells after anti-CD3/CD28 antibody stimulation in the presence or absence of IL-2. Specific inhibitors for JAK/STAT, nuclear factor-kappaB (NF-kappaB), and nuclear factor for activated T cell (NFAT) were used to analyze signal transduction mechanisms. IL-10 production by HOZOTs was greatly enhanced by the addition of IL-2. Little or no enhancement of IFN-gamma and TNF-alpha production was observed under the same conditions. The enhancing effect of IL-2 was specific for both HOZOT and IL-10-secreting Treg cells. T helper type 2 cells, whose IL-10 production mechanisms involve GATA-3, failed to show IL-2-mediated enhancement of IL-10. Similar enhancing effects of IL-15 and IFN-alpha suggested a major role of JAK/STAT activation pathway for high IL-10 production. Further inhibitor experiments demonstrated that STAT5 rather than STAT3 was critically involved in this mechanism. Our results demonstrated that IL-2 selectively enhanced production of IL-10 in HOZOT primarily through activation of STAT5, which synergistically acts with NF-kappaB/NFAT activation, implying a novel regulatory mechanism of IL-10 production in Treg cells.
Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells

PubMed Central

Liu, Chunming; Sherpa, Tshering

2013-01-01

Purpose To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. Methods Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3′,5′-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3′,5′-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. Results Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. Conclusions These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels. PMID:23805033
Identification of HLA-A2–restricted CD8+ Cytotoxic T Cell Responses in Primary Biliary Cirrhosis

PubMed Central

Kita, Hiroto; Lian, Zhe-Xiong; Van de Water, Judy; He, Xiao-Song; Matsumura, Shuji; Kaplan, Marshall; Luketic, Velimir; Coppel, Ross L.; Ansari, Aftab A.; Gershwin, M. Eric

2002-01-01

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4+ and CD8+ T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4+ T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2–restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159–167 of PDC-E2, induces specific MHC class I–restricted CD8+ CTL lines from 10/12 HLA-A2+ PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2–specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2–specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2–specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen–immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease. PMID:11781370
The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

PubMed

Eliassen, Liv Tone; Berge, Gerd; Leknessund, Arild; Wikman, Mari; Lindin, Inger; Løkke, Cecilie; Ponthan, Frida; Johnsen, John Inge; Sveinbjørnsson, Baldur; Kogner, Per; Flaegstad, Trond; Rekdal, Øystein

2006-08-01

Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN-amplified and non-MYCN-amplified cell lines. Non-transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno- and fluorescence-labeled LfcinB revealed that the peptide co-localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase-6, -7 and -9 followed by cell death. However, neither addition of the pan-caspase inhibitor, zVAD-fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH-SY-5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic- and the mitochondria membrane. Copyright (c) 2006 Wiley-Liss, Inc.
A Comparison of Exogenous Labels for the Histological Identification of Transplanted Neural Stem Cells

PubMed Central

Nicholls, Francesca J.; Liu, Jessie R.; Modo, Michel

2017-01-01

The interpretation of cell transplantation experiments is often dependent on the presence of an exogenous label for the identification of implanted cells. The exogenous labels Hoechst 33342, 5-bromo-2′-deoxyuridine (BrdU), PKH26, and Qtracker were compared for their labeling efficiency, cellular effects, and reliability to identify a human neural stem cell (hNSC) line implanted intracerebrally into the rat brain. Hoechst 33342 (2 mg/ml) exhibited a delayed cytotoxicity that killed all cells within 7 days. This label was hence not progressed to in vivo studies. PKH26 (5 μM), Qtracker (15 nM), and BrdU (0.2 μM) labeled 100% of the cell population at day 1, although BrdU labeling declined by day 7. BrdU and Qtracker exerted effects on proliferation and differentiation. PKH26 reduced viability and proliferation at day 1, but this normalized by day 7. In an in vitro coculture assay, all labels transferred to unlabeled cells. After transplantation, the reliability of exogenous labels was assessed against the gold standard of a human-specific nuclear antigen (HNA) antibody. BrdU, PKH26, and Qtracker resulted in a very small proportion (<2%) of false positives, but a significant amount of false negatives (~30%), with little change between 1 and 7 days. Exogenous labels can therefore be reliable to identify transplanted cells without exerting major cellular effects, but validation is required. The interpretation of cell transplantation experiments should be presented in the context of the label's limitations. PMID:27938486
Cytotoxic activity of four Mexican medicinal plants.

PubMed

Vega-Avila, Elisa; Espejo-Serna, Adolfo; Alarcón-Aguilar, Francisco; Velasco-Lezama, Rodolfo

2009-01-01

Ibervillea sonorae Greene, Cucurbita ficifolia Bouché, Tagetes lucida Cav and Justicia spicigera Scheltdd are Mexican native plants used in the treatment of different illnesses. The ethanolic extract of J. spicigera and T. lucida as well as aqueous extracts from I. sonorae, C. ficifolia, T. lucida and J. spicigera were investigated using sulforhodamine B assay. These extracts were assessed using two cell line: T47D (Human Breast cancer) and HeLa (Human cervix cancer). Colchicine was used as the positive control. Data are presented as the dose that inhibited 50% control growth (ED50). All of the assessed extracts were cytotoxic (ED50 < 20 microg/ml) against T47D cell line, meanwhile only the aqueous extract from T. lucida and the ethanolic extract from J. spicigera were cytotoxic to HeLa cell line. Ethanolic extract from J. spicigera presented the best cytotoxic effect. The cytotoxic activity of J. spicigera correlated with one of the popular uses, the treatment of cancer.
Determination of the reactivity of cytotoxic immune cells with preimplantation mouse embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ewoldsen, M.A.

1987-01-01

Cytotoxic immune cells were used in an assay, MELIA (mixed embryo leukocyte interaction assay) to test the ability of the cells to kill blastocyst stage embryos. The cytotoxic immune cells generated for use in this study, cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and lymphokine activated killer (LAK) cells were shown to have phenotypic and cytolytic characteristics similar to those reported by other investigators. The lysis of the blastocysts in the MELIA was determined by measuring the inhibition of blastocoel retention and/or by the inhibition of incorporation of tritiated thymidine (/sup 3/H-TdR) into embryonic DNA. Blastocysts which possess ormore » lack their zonae pellucidae were tested to determine whether the zona pellucida plays an immunoprotective role in preimplantation development. The results indicated that CTLs only lysed embryonic cells when the zona pellucida was absent, but NK and LAK cells lysed embryonic cells whether the zona pellucida was present or absent. The results suggest that the zona pellucida may protect the preimplantation mouse embryo from lysis by CTLs but what protects the embryo from lysis by NK and LAK cells is unclear.« less
PEROXIDASE-MEDIATED MAMMALIAN CELL CYTOTOXICITY

PubMed Central

Edelson, Paul J.; Cohn, Zanvil A.

1973-01-01

Lactoperoxidase, in the presence of hydrogen peroxide and iodide is cytotoxic for human and mouse lymphoid cells, and human erythrocytes. Myeloperoxidase, in amounts equivalent to 1.5 x 106 neutrophils, readily replaces lactoperoxidase, and allows the substitution of the iodide ion by chloride. The myeloperoxidase-mediated reaction is rapid, and highly efficient, leading to 85–90% cell death in 90 min, as measured by 51chromium release and dye exclusion. The mixture of granulocytes. monocytes, and lymphocytes present in an inflammatory exudate, and the intimate cell-to-cell association characteristic of cytotoxic phenomena may provide the in vivo requirements for such a system. PMID:4717124
Cytotoxic T Lymphocyte Trafficking and Survival in an Augmented Fibrin Matrix Carrier

PubMed Central

Zou, Zhaoxia; Denny, Erin; Brown, Christine E.; Jensen, Michael C.; Li, Gang; Fujii, Tatsuhiro; Neman, Josh; Jandial, Rahul; Chen, Mike

2012-01-01

Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment. PMID:22496835
Cytotoxic T lymphocyte trafficking and survival in an augmented fibrin matrix carrier.

PubMed

Zou, Zhaoxia; Denny, Erin; Brown, Christine E; Jensen, Michael C; Li, Gang; Fujii, Tatsuhiro; Neman, Josh; Jandial, Rahul; Chen, Mike

2012-01-01

Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment.
The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

PubMed

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

2013-04-25

The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.
Evaluation of the In Vitro Cytotoxicity of Crosslinked Biomaterials

PubMed Central

Wang, Martha O.; Etheridge, Julie M.; Thompson, Joshua A.; Vorwald, Charlotte E.; Dean, David; Fisher, John P.

2013-01-01

This study evaluated the in vitro cytotoxicity of poly(propylene fumarate) (PPF). PPF is an aliphatic biodegradable polymer that has been well characterized for use in bone tissue engineering scaffolds. Four different cell types, human mesenchymal stem cells (hMSC), fibroblasts (L929), pre-osteoblasts (MC3T3), and canine mesenchymal stem cells (cMSC), were used to evaluate the cytotoxicity of PPF. These cell types represent the tissues that PPF would interact with in vivo as a bone tissue scaffold. The sol fraction of the PPF films was measured and then utilized to estimate crosslinking density. Cytotoxicity was evaluated using XTT assay and fluorescence imaging. Results showed that PPF supported similar cell metabolic activities of hMSC, L929, MC3T3 and cMSC compared to the non-cytotoxic control, high density polyethylene (HDPE) and were statistically different than those cultured with the cytotoxic control, a polyurethane film containing 0.1% zinc diethyldithiocarbamate (ZCF). Results showed differing cellular responses to ZCF, the cytotoxic control. The L929 cells had the lowest cell metabolic activity levels after exposure to ZCF compared to the cell metabolic activity levels of the MC3T3, hMSC or cMSC cells. Qualitative verification of the results using fluorescence imaging demonstrated no change in cell morphology, vacuolization, or detachment when cultured with PPF compared to HDPE or blank media cultures. Overall the cytotoxicity response of the cells to PPF was demonstrated to be similar to the cytotoxic response of cells to known non-cytotoxic materials (HDPE). PMID:23627804
T cell priming versus T cell tolerance induced by synthetic peptides

PubMed Central

1995-01-01

It is well known that synthetic peptides are able to both induce and tolerize T cells. We have examined the parameters leading either to priming or tolerance of CD8+ cytotoxic T lymphocytes (CTL) in vivo with a major histocompatibility complex class I (H-2 Db) binding peptide derived from the glycoprotein (GP aa33-41) of lymphocytic choriomeningitis virus (LCMV). By varying dose, route, and frequency of LCMV GP peptide application, we found that a single local subcutaneous injection of 50-500 micrograms peptide emulsified in incomplete Freund's adjuvant protected mice against LCMV infection, whereas repetitive and systemic intraperitoneal application of the same dose caused tolerance of LCMV-specific CTL. The peptide-induced tolerance was transient in euthymic mice but permanent in thymectomized mice. These findings are relevant for a selective use of peptides as a therapeutic approach: peptide-induced priming of T cells for vaccination and peptide-mediated T cell tolerance for intervention in immunopathologies and autoimmune diseases. PMID:7540654
The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation

PubMed Central

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P.; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S.

2013-01-01

The adaptor molecule signaling lymphocytic activation molecule–associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase–mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)–induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell–mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA–mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS. PMID:23430111
Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

PubMed

Krakowiak, Agnieszka; Pęcherzewska, Róża; Kaczmarek, Renata; Tomaszewska, Agnieszka; Nawrot, Barbara; Stec, Wojciech J

2011-08-15

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 μM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.
Fish oil feeding enhances lymphocyte proliferation but impairs virus-specific T lymphocyte cytotoxicity in mice following challenge with influenza virus

PubMed Central

Byleveld, M; Pang, G T; Clancy, R L; Roberts, D C K

2000-01-01

The effect of a fish oil diet on virus-specific cytotoxicity and lymphocyte proliferation was investigated. Mice were fed fish oil (17 g fish oil and 3 g sunflower/100 g) or beef tallow (17 g tallow and 3 g sunflower/100 g) diets for 14 days before intranasal challenge with influenza virus. At day 5 after infection, lung virus-specific T lymphocyte, but not macrophage or natural killer (NK) cell, cytotoxicity was significantly lower in mice fed fish oil, while bronchial lymph node cell proliferation to virus was significantly higher. In mice fed fish oil, spleen cell proliferation to virus was also significantly higher following immunization. The results showed that, despite improved lymphocyte proliferation, fish oil impairs primary virus-specific T lymphocyte cytotoxicity. This impairment may explain the delayed virus clearance that we have previously reported in infected mice fed the fish oil diet. PMID:10632664
Fish oil feeding enhances lymphocyte proliferation but impairs virus-specific T lymphocyte cytotoxicity in mice following challenge with influenza virus.

PubMed

Byleveld, M; Pang, G T; Clancy, R L; Roberts, D C

2000-02-01

The effect of a fish oil diet on virus-specific cytotoxicity and lymphocyte proliferation was investigated. Mice were fed fish oil (17 g fish oil and 3 g sunflower/100 g) or beef tallow (17 g tallow and 3 g sunflower/100 g) diets for 14 days before intranasal challenge with influenza virus. At day 5 after infection, lung virus-specific T lymphocyte, but not macrophage or natural killer (NK) cell, cytotoxicity was significantly lower in mice fed fish oil, while bronchial lymph node cell proliferation to virus was significantly higher. In mice fed fish oil, spleen cell proliferation to virus was also significantly higher following immunization. The results showed that, despite improved lymphocyte proliferation, fish oil impairs primary virus-specific T lymphocyte cytotoxicity. This impairment may explain the delayed virus clearance that we have previously reported in infected mice fed the fish oil diet.
Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy

PubMed Central

Maus, Marcela V.; June, Carl H.

2016-01-01

Chimeric antigen receptors (CARs) are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that can be expressed in cytotoxic T cells with the purpose of reprograming the T cells to specifically target tumor cells. CAR T-cell therapy uses gene transfer technology to reprogram a patient's own T cells to stably express CARs, thereby combining the specificity of an antibody with the potent cytotoxic and memory functions of a T cell. In early phase clinical trials, CAR T cells targeting CD19 have resulted in sustained complete responses within a population of otherwise refractory patients with B-cell malignancies and, more specifically, have shown complete response rates of ≈90% in patients with relapsed or refractory acute lymphoblastic leukemia. Given this clinical efficacy, preclinical development of CAR T-cell therapy for a number of cancer indications has been actively investigated, and the future of the CAR T-cell field is extensive and dynamic. Several approaches to increase the feasibility and safety of CAR T cells are currently being explored, including investigation into mechanisms regulating the persistence of CAR T cells. Additionally, numerous early-phase clinical trials are now investigating CAR T-cell therapy beyond targeting CD19, especially in solid tumors. Trials investigating combinations of CAR T cells with immune checkpoint blockade therapies are now beginning and results are eagerly awaited. This review evaluates several of the ongoing and future directions of CAR T-cell therapy. PMID:27084741
Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

PubMed

Bhatia, Sujata K; Yetter, Ann B

2008-08-01

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.
Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells.

PubMed

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine Kl; Berman, Jason N; Rupasinghe, Hp Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo . We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway.

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells

PubMed Central

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine KL; Berman, Jason N; Rupasinghe, HP Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo. We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway. PMID:29312799
Casein expression in cytotoxic T lymphocytes.

PubMed Central

Grusby, M J; Mitchell, S C; Nabavi, N; Glimcher, L H

1990-01-01

A cDNA that expresses a mRNA restricted to cytotoxic T lymphocytes (CTL) and mammary tissue has been isolated and characterized. The deduced amino acid sequence from this cDNA shows extensive homology with the previously reported amino acid sequence for rat alpha-casein. Indeed, the presence of a six-residue-repeated motif that is specific for rodent alpha-caseins strongly supports the identification of this cDNA as mouse alpha-casein. Northern (RNA) blot analysis of many hematopoietic cell types revealed that this gene is restricted to CTL, being expressed in four of six CTL lines examined. Furthermore, CTL that express this gene were also found to express other members of the casein gene family, such as beta- and kappa-casein. These results suggest that caseins may be important in CTL function, and their potential role in CTL-mediated lysis is discussed. Images PMID:2395885
TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

PubMed Central

Catakovic, Kemal; Gassner, Franz Josef; Ratswohl, Christoph; Zaborsky, Nadja; Rebhandl, Stefan; Schubert, Maria; Steiner, Markus; Gutjahr, Julia Christine; Pleyer, Lisa; Egle, Alexander; Hartmann, Tanja Nicole; Greil, Richard; Geisberger, Roland

2018-01-01

ABSTRACT While research on T cell exhaustion in context of cancer particularly focuses on CD8+ cytotoxic T cells, the role of inhibitory receptors on CD4+ T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8+ T cells of healthy controls and CLL cells, we found an enrichment of TIGIT+ T cells in the CD4+ T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4+ TIGIT+ T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4+ TIGIT+ expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGIT+CD4+ T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGIT+CD4+T cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment. PMID:29296521
Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8(+) T cell-mediated cell death.

PubMed

Kim, Jin Hee; Kang, Tae Heung; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Seok-Ho; Yoo, Young Do; Seong, Seung-Yong; Kim, Tae Woo

2009-01-29

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8(+) T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8(+) T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.
Longevity of T-cell memory following acute viral infection.

PubMed

Walker, Joshua M; Slifka, Mark K

2010-01-01

Investigation of T-cell-mediated immunity following acute viral infection represents an area of research with broad implications for both fundamental immunology research as well as vaccine development. Here, we review techniques that are used to assess T-cell memory including limiting dilution analysis, enzyme-linked immunospot (ELISPOT) assays, intracellular cytokine staining (ICCS) and peptide-MHC Class I tetramer staining. The durability of T-cell memory is explored in the context of several acute viral infections including vaccinia virus (VV), measles virus (MV) and yellow fever virus (YFV). Following acute infection, different virus-specific T-cell subpopulations exhibit distinct cytokine profiles and these profiles change over the course of infection. Differential regulation of the cytotoxic proteins, granzyme A, granzyme B and perforin are also observed in virus-specific T cells following infection. As a result of this work, we have gained a broader understanding of the kinetics and magnitude of antiviral T-cell immunity as well as new insight into the patterns of immunodominance and differential regulation of cytokines and cytotoxicity-associated molecules. This information may eventually lead to the generation of more effective vaccines that elicit T-cell memory with the optimal combination of functional characteristics required for providing protective immunity against infectious disease.
Localized Irradiation of Cell Membrane by Auger Electrons Is Cytotoxic Through Oxidative Stress-Mediated Nontargeted Effects

PubMed Central

Paillas, Salomé; Ladjohounlou, Riad; Lozza, Catherine; Pichard, Alexandre; Boudousq, Vincent; Jarlier, Marta; Sevestre, Samuel; Le Blay, Marion; Deshayes, Emmanuel; Sosabowski, Jane; Chardès, Thierry; Navarro-Teulon, Isabelle; Mairs, Robert J.

2016-01-01

Abstract Aims: We investigated whether radiation-induced nontargeted effects are involved in the cytotoxic effects of anticell surface monoclonal antibodies labeled with Auger electron emitters, such as iodine 125 (monoclonal antibodies labeled with 125I [125I-mAbs]). Results: We showed that the cytotoxicity of 125I-mAbs targeting the cell membrane of p53+/+ HCT116 colon cancer cells is mainly due to nontargeted effects. Targeted and nontargeted cytotoxicities were inhibited in vitro following lipid raft disruption with Methyl-β-cyclodextrin (MBCD) or filipin or use of radical oxygen species scavengers. 125I-mAb efficacy was associated with acid sphingomyelinase activation and modulated through activation of the AKT, extracellular signal-related kinase ½ (ERK1/2), p38 kinase, c-Jun N-terminal kinase (JNK) signaling pathways, and also of phospholipase C-γ (PLC-γ), proline-rich tyrosine kinase 2 (PYK-2), and paxillin, involved in Ca2+ fluxes. Moreover, the nontargeted response induced by directing 5-[(125)I]iodo-2′-deoxyuridine to the nucleus was comparable to that of 125I-mAb against cell surface receptors. In vivo, we found that the statistical significance of tumor growth delay induced by 125I-mAb was removed after MBCD treatment and observed oxidative DNA damage beyond the expected Auger electron range. These results suggest the involvement of nontargeted effects in vivo also. Innovation: Low-energy Auger electrons, such as those emitted by 125I, have a short tissue range and are usually targeted to the nucleus to maximize their cytotoxicity. In this study, we show that targeting the cancer cell surface with 125I-mAbs produces a lipid raft-mediated nontargeted response that compensates for the inferior efficacy of non-nuclear targeting. Conclusion: Our findings describe the mechanisms involved in the efficacy of 125I-mAbs targeting the cancer cell surface. Antioxid. Redox Signal. 25, 467–484. PMID:27224059
Labeling Human Mesenchymal Stem Cells with Gold Nanocages for in vitro and in vivo Tracking by Two-Photon Microscopy and Photoacoustic Microscopy

PubMed Central

Zhang, Yu Shrike; Wang, Yu; Wang, Lidai; Wang, Yucai; Cai, Xin; Zhang, Chi; Wang, Lihong V.; Xia, Younan

2013-01-01

Stem cell tracking is a highly important subject. Current techniques based on nanoparticle-labeling, such as magnetic resonance imaging, fluorescence microscopy, and micro-computed tomography, are plagued by limitations including relatively low sensitivity or penetration depth, involvement of ionizing irradiation, and potential cytotoxicity of the nanoparticles. Here we introduce a new class of contrast agents based on gold nanocages (AuNCs) with hollow interiors and porous walls to label human mesenchymal stem cells (hMSCs) for both in vitro and in vivo tracking using two-photon microscopy and photoacoustic microscopy. As demonstrated by the viability assay, the AuNCs showed negligible cytotoxicity under a reasonable dose, and did not alter the differentiation potential of the hMSCs into desired lineages. We were able to image the cells labeled with AuNCs in vitro for at least 28 days in culture, as well as to track the cells that homed to the tumor region in nude mice in vivo. PMID:23946820
Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

PubMed Central

Chen, Jiang; Li, Hong-Yu; Wang, Di; Shao, Xiao-Dong

2015-01-01

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination. PMID:25736302
Empowering gamma delta T cells with antitumor immunity by dendritic cell-based immunotherapy

PubMed Central

Van Acker, Heleen H; Anguille, Sébastien; Van Tendeloo, Viggo F; Lion, Eva

2015-01-01

Gamma delta (γδ) T cells are the all-rounders of our immune-system with their major histocompatibility complex-unrestricted cytotoxicity, capacity to secrete immunosti-mulatory cytokines and ability to promote the generation of tumor antigen-specific CD8+ and CD4+ T cell responses. Dendritic cell (DC)-based vaccine therapy has the prospective to harness these unique features of the γδ T cells in the fight against cancer. In this review, we will discuss our current knowledge on DC-mediated γδ T cell activation and related opportunities for tumor immunologists. PMID:26405575
Analysis of cytotoxic activity of the CD4+ T lymphocytes generated by local immunotherapy.

PubMed Central

Katsumoto, Y.; Monden, T.; Takeda, T.; Haba, A.; Ito, Y.; Wakasugi, E.; Wakasugi, T.; Sekimoto, M.; Kobayashi, T.; Shiozaki, H.; Shimano, T.; Monden, M.

1996-01-01

We previously reported that the anti-tumour effect of OK-432 is considerably enhanced by its intratumoral injection together with fibrinogen. In the present study, we generated killer T cells by culturing tumour-infiltrating lymphocytes from thyroid cancer patients who had received this local immunotherapy. Phenotypic analysis revealed that the T cells were positive for CD3+, CD4+, Leu8-, CD45RO+ and T-cell receptor (TCR)alpha beta+, as well as showing strong surface expression of HLA-DR, CD25, LFA-1 and ICAM-1. The generated CD4+ T cells secreted interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, TNF-beta, and interleukin (IL)-6 (but not IL-4), and exhibited a high level of cytolytic activity against several tumour cell lines. The cytolytic activity of these T cells for Daudi cells was inhibited by preincubation with an anti-intercellular adhesion molecule (ICAM)-1 antibody, but not by preincubation with anti-TCR alpha beta, anti-CD2, or anti-LFA-1 antibodies. Pretreatment with anti-ICAM-1 antibody inhibited T-cell cytolytic activity, but not conjugation with target cells. In addition, incubation with immobilised anti-ICAM-1 enhanced the secretion of IFN-gamma by T cells. We conclude that ICAM-1 expressed on the effector cytotoxic CD4+ T lymphocytes delivers regulatory signals that enhance IFN-gamma secretion. PMID:8554971
Bortezomib Improves Adoptive T-cell Therapy by Sensitizing Cancer Cells to FasL Cytotoxicity.

PubMed

Shanker, Anil; Pellom, Samuel T; Dudimah, Duafalia F; Thounaojam, Menaka C; de Kluyver, Rachel L; Brooks, Alan D; Yagita, Hideo; McVicar, Daniel W; Murphy, William J; Longo, Dan L; Sayers, Thomas J

2015-12-15

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(-/-) mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy. ©2015 American Association for Cancer Research.
The Protein Corona around Nanoparticles Facilitates Stem Cell Labeling for Clinical MR Imaging.

PubMed

Nejadnik, Hossein; Taghavi-Garmestani, Seyed-Meghdad; Madsen, Steven J; Li, Kai; Zanganeh, Saeid; Yang, Phillip; Mahmoudi, Morteza; Daldrup-Link, Heike E

2018-03-01

Purpose To evaluate if the formation of a protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for in vivo tracking with magnetic resonance (MR) imaging. Materials and Methods Ferumoxytol was incubated in media containing human serum (group 1), fetal bovine serum (group 2), StemPro medium (group 3), protamine (group 4), and protamine plus heparin (group 5). Formation of a protein corona was characterized by means of dynamic light scattering, ζ potential, and liquid chromatography-mass spectrometry. Iron uptake was evaluated with 3,3'-diaminobenzidine-Prussian blue staining, lysosomal staining, and inductively coupled plasma spectrometry. To evaluate the effect of a protein corona on stem cell labeling, human mesenchymal stem cells (hMSCs) were labeled with the above formulations, implanted into pig knee specimens, and investigated with T2-weighted fast spin-echo and multiecho spin-echo sequences on a 3.0-T MR imaging unit. Data in different groups were compared by using a Kruskal-Wallis test. Results Compared with bare nanoparticles, all experimental groups showed significantly increased negative ζ values (from -37 to less than -10; P = .008). Nanoparticles in groups 1-3 showed an increased size because of the formation of a protein corona. hMSCs labeled with group 1-5 media showed significantly shortened T2 relaxation times compared with unlabeled control cells (P = .0012). hMSCs labeled with group 3 and 5 media had the highest iron uptake after cells labeled with group 1 medium. After implantation into pig knees, hMSCs labeled with group 1 medium showed significantly shorter T2 relaxation times than hMSCs labeled with group 2-5 media (P = .0022). Conclusion The protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for clinical cell tracking with MR imaging. © RSNA, 2017 Online supplemental material is available for this article.
Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dannemann, B.R.; Morris, V.A.; Araujo, F.G.

1989-10-15

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in themore » absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant. In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively, as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites.« less
Cytotoxic effect of butein on human colon adenocarcinoma cell proliferation.

PubMed

Yit, C C; Das, N P

1994-07-15

Several classes of plant polyphenols namely, flavonoids, chalcones and coumarins exhibited varying degrees of inhibition on the cell proliferation of human colon adenocarcinoma cell line 220.1. At the highest concentration tested (100 microM), many of the chalcones showed > 100% growth inhibition and their order of potency was butein > 2'-hydroxychalcone > 2-hydroxychalcone > 2',6'-dihydroxy-4'-methoxychalcone > 2',4-dihydroxychalcone with IC50 values of 1.75, 6.2, 7.5, 17, 23 microM, respectively. Butein (the most potent chalcone) at 2 microM concentration inhibited the incorporation of 14C-labelled thymidine, uridine and leucine into the colon cancer cells whilst 5-fluorouracil (5-FU, a chemotherapeutic drug) at 50 microM concentration could significantly inhibit only the uridine incorporation. The mode of cytotoxic action of butein was different from 5-FU but may be similar to colchicine, a known HeLa cell inhibitor.
Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists

PubMed Central

Petersen, Christopher T.; Hassan, Mojibade; Morris, Anna B.; Jeffery, Jasmin; Lee, Kunhee; Jagirdar, Neera; Staton, Ashley D.; Raikar, Sunil S.; Spencer, Harold T.; Sulchek, Todd; Flowers, Christopher R.

2018-01-01

Adoptive therapy with ex vivo–expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27+CD28+ T cells vs 27.4% in control cultures; P < .05). This strategy results in a mean of 83.7% more T cells cultured from lymphoma patients in the presence of PI3Kδ and VIP antagonists, increased survival of human T cells from a lymphoma patient in a murine xenograft model, enhanced cytotoxic activity of antigen-specific human CAR T cells and murine T cells against lymphoma, and increased transduction and expansion of anti-CD5 human CAR T cells. PI3Kδ and VIP antagonist-expanded T cells from lymphoma patients show reduced terminal differentiation, enhanced polyfunctional cytokine expression, and preservation of costimulatory molecule expression. Taken together, synergistic blockade of these pathways is an attractive strategy to enhance the expansion and functional capacity of ex vivo–expanded cancer-specific T cells. PMID:29386194
In vitro generation of Epstein-Barr virus-specific cytotoxic T cells in patients receiving haplo-identical allogeneic stem cell transplantation.

PubMed

Musk, P; Szmania, S; Galloway, A T; Johnson, K; Scott, A; Guttman, S; Bridges, K; Bruorton, M; Gatlin, J; Garcia, J V; Lamb, L; Chiang, K Y; Spencer, T; Henslee-Downey, J; van Rhee, F

2001-01-01

Use of a partially mismatched related donor (PMRD) is an option for patients who require allogeneic transplantation but do not have a matched sibling or unrelated donor. Epstein-Barr virus (EBV)-induced lymphoma is a major cause of mortality after PMRD transplantation. In this study, we present a clinical grade culture system for donor-derived EBV-specific cytotoxic T cells (CTLs) that do not recognize haplo-identical recipient cells. The EBV-specific CTLs were tested for cytolytic specificity and other functional properties, including ability to transgress into tissues, propensity for apoptosis, degree of clonality, stability of dominant T-cell clones, and Tc and Th phenotypes. The EBV-specific CTLs were routinely expanded to greater than 80 x 10(6) over a period of 5 weeks, which is sufficient for clinical application. A CD8+ phenotype predominated, and the CTLs were highly specific for donor lymphoblastoid cell lines (LCLs) without killing of recipient targets or K562. Vbeta spectratyping showed an oligoclonal population that was stable on prolonged culture. The EBV-specific CTLs were activated (D-related human leukocyte antigen [HLA-DR+], L-selectin+/-) and of memory phenotype (CD45RO+). Expression of the integrin VLA-4 suggested that these CTLs could adhere to endothelium and migrate into tissues. The Bcl-2 message was upregulated, which may protect the CTLs from the apoptosis. The first demonstration of overexpression of bcl-2 in human memory CTLs. In addition, we show that lymphoblastoid cell lines used to generate CTLs are readily genetically modified with recombinant lentivirus, indicating that genetically engineered antigen presentation is feasible.
Differential cytotoxic properties of Helleborus niger L. on tumour and immunocompetent cells.

PubMed

Schink, Michael; Garcia-Käufer, Manuel; Bertrams, Julia; Duckstein, Sarina M; Müller, Margit B; Huber, Roman; Stintzing, Florian C; Gründemann, Carsten

2015-01-15

In Romanian folk medicine, Helleborus niger L. is used for the treatment of rheumatoid arthritis or viral infections and in complementary therapy, especially in anthroposophic medicine (AM), where the plant is administered as an adjuvant to treat malignant diseases. In the present study, we investigated the differential cytotoxic effects of H. niger on human tumour and healthy cells of the human immune system in vitro. Protoanemonin and saponins, as significant constituents of H. niger extracts, were quantified in five individual batches using validated HPLC methods. Further, the impact of H. niger on proliferation capacity (MTT assay) as well as on apoptosis and necrosis induction in a panel of tumour cell lines and human lymphocytes (combined annexin V and propidium iodide staining) was monitored. In addition, NK cell function (degranulation-CD107a assay and IFN-gamma secretion) was also investigated since these immunocompetent cells are important for the control of malignancies within the human body. Extracts of H. niger induced proliferation inhibition not only of lymphoblastic leukaemia cells (MOLT4; IC50: 171 µg/mL) but also of myosarcoma (SK-UT-1b; IC50: 304 µg/mL) and melanoma cells (HT-144; IC50: 569 µg/mL) due to the induction of apoptosis. Purified T cells or NK cells were significantly affected through the presence of high H. niger concentrations while bulk lymphocytes were not affected. NK cells' anti-tumour functions expressed by degranulation capacity as well as IFN-y production were unaffected by the presence of the H. niger extract. Since protoanemonin and saponins have been reported in the literature to exert cytotoxic effects, their content was also determined. H. niger extracts exhibit differential cytotoxicity towards tumour cell lines and healthy human T- and NK-cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

PubMed

Yang, Andrew C; du Bois, Haley; Olsson, Niclas; Gate, David; Lehallier, Benoit; Berdnik, Daniela; Brewer, Kyle D; Bertozzi, Carolyn R; Elias, Joshua E; Wyss-Coray, Tony

2018-06-13

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr Y43G ) and a phenylalanyl ( MmPhe T413G ) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr Y43G and MmPhe T413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr Y43G and MmPhe T413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.
Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumour, leading to generation of endogenous memory responses to non-targeted tumour epitopes.

PubMed

Li, Kunyu; Donaldson, Braeden; Young, Vivienne; Ward, Vernon; Jackson, Christopher; Baird, Margaret; Young, Sarah

2017-10-01

The results of adoptive T-cell therapies (ACTs) are very encouraging and show clinical evidence that ACT can provide a cure for patients with metastatic disease. However, various response rates and long-term cancer remission have been observed in different ACT trials. The types of T cells, prior treatment with chemotherapy and co-administration of other immune-target therapies have been found to influence the efficacy of ACT. In this study, we investigate the ability of ACT using CD4 + T helper 1 (Th1) cells and CD8 + cytotoxic T lymphocytes (CTLs) to reject the growth of established B16-ovalbumin (OVA) melanoma. CD8 + CTLs were found to be the main effector T cells that mediated tumour regression. However, low tumour-free survival rates were observed in ACT with CD8 + CTLs only. Co-transferring CD4 + Th1 cells and CD8 + CTLs has been observed to induce a synergistic antitumour response, resulting in complete regression in 80% of the tumour-bearing mice. We also examined a prior Dacarbazine (DTIC) and after virus-like particle (VLP)-OVA vaccine treatment to enhance ACT, but no therapeutic benefit was observed during primary B16-OVA tumour growth. Nevertheless, the ACT-mediated antitumour response was able to generate memory responses to both B16-OVA and B16-gp33 tumours. VLP-OVA vaccination following ACT enhances the memory responses to tumours that express a heterogenic population of both B16-OVA and B16-gp33 cells; however, it abolished the memory response to tumours consisting of only gp33-expressing cells. These findings provide important information for designing therapeutic treatments for patients with metastatic disease and cancer relapse to achieve durable cancer remission.
Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumour, leading to generation of endogenous memory responses to non-targeted tumour epitopes

PubMed Central

Li, Kunyu; Donaldson, Braeden; Young, Vivienne; Ward, Vernon; Jackson, Christopher; Baird, Margaret; Young, Sarah

2017-01-01

The results of adoptive T-cell therapies (ACTs) are very encouraging and show clinical evidence that ACT can provide a cure for patients with metastatic disease. However, various response rates and long-term cancer remission have been observed in different ACT trials. The types of T cells, prior treatment with chemotherapy and co-administration of other immune-target therapies have been found to influence the efficacy of ACT. In this study, we investigate the ability of ACT using CD4+ T helper 1 (Th1) cells and CD8+ cytotoxic T lymphocytes (CTLs) to reject the growth of established B16-ovalbumin (OVA) melanoma. CD8+ CTLs were found to be the main effector T cells that mediated tumour regression. However, low tumour-free survival rates were observed in ACT with CD8+ CTLs only. Co-transferring CD4+ Th1 cells and CD8+ CTLs has been observed to induce a synergistic antitumour response, resulting in complete regression in 80% of the tumour-bearing mice. We also examined a prior Dacarbazine (DTIC) and after virus-like particle (VLP)-OVA vaccine treatment to enhance ACT, but no therapeutic benefit was observed during primary B16-OVA tumour growth. Nevertheless, the ACT-mediated antitumour response was able to generate memory responses to both B16-OVA and B16-gp33 tumours. VLP-OVA vaccination following ACT enhances the memory responses to tumours that express a heterogenic population of both B16-OVA and B16-gp33 cells; however, it abolished the memory response to tumours consisting of only gp33-expressing cells. These findings provide important information for designing therapeutic treatments for patients with metastatic disease and cancer relapse to achieve durable cancer remission. PMID:29114389

T cell-B cell interactions in primary immunodeficiencies.

PubMed

Tangye, Stuart G; Deenick, Elissa K; Palendira, Umaimainthan; Ma, Cindy S

2012-02-01

Regulated interactions between cells of the immune system facilitate the generation of successful immune responses, thereby enabling efficient neutralization and clearance of pathogens and the establishment of both cell- and humoral-mediated immunological memory. The corollary of this is that impediments to efficient cell-cell interactions, normally necessary for differentiation and effector functions of immune cells, underly the clinical features and disease pathogenesis of primary immunodeficiencies. In affected individuals, these defects manifest as impaired long-term humoral immunity and susceptibility to infection by specific pathogens. In this review, we discuss the importance of, and requirements for, effective interactions between B cells and T cells during the formation of CD4(+) T follicular helper cells and the elicitation of cytotoxic function of virus-specific CD8(+) T cells, as well as how these processes are abrogated in primary immunodeficiencies due to loss-of-function mutations in defined genes. © 2012 New York Academy of Sciences.
Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

PubMed Central

Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

2014-01-01

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells. PMID:24830447
Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

PubMed

Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

2014-05-16

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.
Phospholipase A2 activation regulates cytotoxicity of methylmercury in vascular endothelial cells.

PubMed

Mazerik, Jessica N; Hagele, Thomas; Sherwani, Shariq; Ciapala, Valorie; Butler, Susan; Kuppusamy, M Lakshmi; Hunter, Melissa; Kuppusamy, Periannan; Marsh, Clay B; Parinandi, Narasimham L

2007-01-01

Mercury has been identified as a risk factor for cardiovascular disease among humans. Through diet, mainly fish consumption, humans are exposed to methylmercury, the biomethylated organic form of environmental mercury. As the endothelium is an important player in homeostasis of the cardiovascular system, here, the authors tested their hypothesis that methylmercury activates the lipid signaling enzyme phospholipase A(2) (PLA(2)) in vascular endothelial cells (ECs), causing upstream regulation of cytotoxicity. To test this hypothesis, the authors used bovine pulmonary artery ECs (BPAECs) cultured in monolayers, following labeling of their membrane phospholipids with [(3)H]arachidonic acid (AA). The cells were exposed to methylmercury chloride (MMC) and then the release of free AA (index of PLA(2) activity) and lactate dehydrogenase (LDH; index of cytotoxicity) were determined by liquid scintillation counting and spectrophotometry, respectively. MMC significantly activated PLA(2) in a dose-dependent (5 to 15 microM) and time-dependent (0 to 60 min) fashion. Sulfhydryl (thiol-protective) agents, calcium chelators, antioxidants, and PLA(2)-specific inhibitors attenuated the MMC-induced PLA(2) activation, suggesting the role of thiols, reactive oxygen species (ROS), and calcium in the activation of PLA(2) in BPAECs. MMC also induced the loss of thiols and increase of lipid peroxidation in BPAECs. MMC induced cytotoxicity in BPAECs as observed by the altered cell morphology and LDH leak, which was significantly attenuated by PLA(2) inhibitors. This study established that PLA(2) activation through thiols, calcium, and oxidative stress was associated with the cytotoxicity of MMC in BPAECs, drawing attention to the involvement of PLA(2) signaling in the methylmercury-induced vascular endothelial dysfunctions.
Cancer cell specific cytotoxic effect of Rhoeo discolor extracts and solvent fractions.

PubMed

García-Varela, Rebeca; Fajardo Ramírez, Oscar Raúl; Serna-Saldivar, Sergio O; Altamirano, Julio; Cardineau, Guy A

2016-08-22

Traditional or folk medicine has led to the discovery of important bioactive substances used in several health-related areas. Phytochemicals in Rhoeo discolor (R. discolor) extracts have proven to have important cancer cell specific cytotoxic activity. In the present research, we determined the cytotoxic effect of extracts of R. discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the cytotoxic effects against three representative human cancer cell lines: HT-29 colon cancer, Hep-G2 liver cancer and PC-3 prostate cancer cell lines, as well as a control fibroblast cell line NIH 3T3. Ten different crude extracts were tested along with fractions derived from the five most bioactive crude extracts. Analytical data, HPLC-MS-TOF, revealed a high content of phenolic compounds such as anthocyanins, ferulic, vanillic, chlorogenic and p-coumaric acid in the extracts. Phenolic compounds have previously been reported as health beneficial with antioxidant and potential cancer specific cytotoxic effects. Studies revealed that low concentrations of these crude bioactive extracts (10µg/ml) and their fractions (50µg/ml) were effective as cancer specific cytotoxic agents, since they caused a significant proliferation inhibition on cancer cell lines (up to 94.2% in HT-29, 92.9% in Hep-G2 and 61.8% in PC-3 of apoptosis induction) with little harm to the control cell line (no higher than 28.3% apoptosis induction), and, importantly, the most effective extracts were mainly water, methanol and ethanol based. These results suggest that a diet containing these compounds may function as a medical aid or chemoprotective. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing.

PubMed

Akhmetzyanova, Ilseyar; Drabczyk, Malgorzata; Neff, C Preston; Gibbert, Kathrin; Dietze, Kirsten K; Werner, Tanja; Liu, Jia; Chen, Lieping; Lang, Karl S; Palmer, Brent E; Dittmer, Ulf; Zelinskyy, Gennadiy

2015-10-01

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.
Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; McGuinness, Dagmara; Faley, Shannon; Kolch, Walter; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines. PMID:19572560
Real-time, label-free monitoring of cell viability based on cell adhesion measurements with an atomic force microscope.

PubMed

Yang, Fang; Riedel, René; Del Pino, Pablo; Pelaz, Beatriz; Said, Alaa Hassan; Soliman, Mahmoud; Pinnapireddy, Shashank R; Feliu, Neus; Parak, Wolfgang J; Bakowsky, Udo; Hampp, Norbert

2017-03-22

The adhesion of cells to an oscillating cantilever sensitively influences the oscillation amplitude at a given frequency. Even early stages of cytotoxicity cause a change in the viscosity of the cell membrane and morphology, both affecting their adhesion to the cantilever. We present a generally applicable method for real-time, label free monitoring and fast-screening technique to assess early stages of cytotoxicity recorded in terms of loss of cell adhesion. We present data taken from gold nanoparticles of different sizes and surface coatings as well as some reference substances like ethanol, cadmium chloride, and staurosporine. Measurements were recorded with two different cell lines, HeLa and MCF7 cells. The results obtained from gold nanoparticles confirm earlier findings and attest the easiness and effectiveness of the method. The reported method allows to easily adapt virtually every AFM to screen and assess toxicity of compounds in terms of cell adhesion with little modifications as long as a flow cell is available. The sensitivity of the method is good enough indicating that even single cell analysis seems possible.
Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line: a potential improvement of intravesical chemotherapy in bladder cancer.

PubMed

Vásquez, Juan L; Gehl, Julie; Hermann, Gregers G

2012-12-01

Intravesical mitomycin instillation combined with electric pulses is being used experimentally for the treatment of T1 bladder tumors, in patients unfit for surgery. Electroporation may enhance the uptake of chemotherapeutics by permeabilization of cell membranes. We investigated if electroporation improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01-2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability was assessed by colorimetric assay (MTT). For both cell lines, mitomycin's IC_50 was approximately 1000μM in both pulsed and unpulsed cells. On T24 cells, electroporation and mitomycin caused (relative reduction) RR of survival of: 25%, 31% and 29%, by concentrations 0μM, 500μM and 1000μM respectively. For DC3F cells, the RRs of survival were: 28%, 29%, and 33%, by concentrations 0μM, 500μM and 1000μM respectively. In conclusion, electroporation and mitomycin together are about 30% more effective than mitomycin alone. The results help to elucidate the additive effect of mitomycin and electric pulses and support the use of this combination in the treatment of bladder cancer. Copyright © 2012 Elsevier B.V. All rights reserved.
Micro-Computed Tomography Detection of Gold Nanoparticle-Labelled Mesenchymal Stem Cells in the Rat Subretinal Layer.

PubMed

Mok, Pooi Ling; Leow, Sue Ngein; Koh, Avin Ee-Hwan; Mohd Nizam, Hairul Harun; Ding, Suet Lee Shirley; Luu, Chi; Ruhaslizan, Raduan; Wong, Hon Seng; Halim, Wan Haslina Wan Abdul; Ng, Min Hwei; Idrus, Ruszymah Binti Hj; Chowdhury, Shiplu Roy; Bastion, Catherine Mae-Lynn; Subbiah, Suresh Kumar; Higuchi, Akon; Alarfaj, Abdullah A; Then, Kong Yong

2017-02-08

Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases.
Micro-Computed Tomography Detection of Gold Nanoparticle-Labelled Mesenchymal Stem Cells in the Rat Subretinal Layer

PubMed Central

Mok, Pooi Ling; Leow, Sue Ngein; Koh, Avin Ee-Hwan; Mohd Nizam, Hairul Harun; Ding, Suet Lee Shirley; Luu, Chi; Ruhaslizan, Raduan; Wong, Hon Seng; Halim, Wan Haslina Wan Abdul; Ng, Min Hwei; Idrus, Ruszymah Binti Hj.; Chowdhury, Shiplu Roy; Bastion, Catherine Mae-Lynn; Subbiah, Suresh Kumar; Higuchi, Akon; Alarfaj, Abdullah A.; Then, Kong Yong

2017-01-01

Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases. PMID:28208719
Cell-mediated immunity in herpes simplex virus-infected mice: functional analysis of lymph node cells during periods of acute and latent infection, with reference to cytotoxic and memory cells.

PubMed

Nash, A A; Quartey-Papafio, R; Wildy, P

1980-08-01

The functional characteristics of lymphoid cells were investigated during acute and latent infection of mice with herpes simplex virus (HSV). Cytotoxic T cells were found in the draining lymph node (DLN) 4 days p.i. and had reached maximum activity between 6 and 9 days. After the 12th day and during the period of latent infection (> 20 days) no cytotoxic cell activity was observed. Cytotoxic activity could only be detected when the lymphoid cells had been cultured for a period of 3 days. In general, the cell killing was specific for syngeneic infected target cells, although some killing of uninfected targets was observed. In contrast to the cytotoxic response, DLN cells responding to HSV in a proliferation assay were detected towards the end of the acute phase and at lease up to 9 months thereafter. The significance of these observations for the pathogenesis of HSV is discussed.
CD62L− memory T cells enhance T-cell regeneration after allogeneic stem cell transplantation by eliminating host resistance in mice

PubMed Central

Zhang, Jifeng; Barefoot, Brice E.; Mo, Wenjian; Deoliveira, Divino; Son, Jessica; Cui, Xiuyu; Ramsburg, Elizabeth

2012-01-01

A major challenge in allogeneic hematopoietic cell transplantation is how to transfer T-cell immunity without causing graft-versus-host disease (GVHD). Effector memory T cells (CD62L−) are a cell subset that can potentially address this challenge because they do not induce GVHD. Here, we investigated how CD62L− T cells contributed to phenotypic and functional T-cell reconstitution after transplantation. On transfer into allogeneic recipients, CD62L− T cells were activated and expressed multiple cytokines and cytotoxic molecules. CD62L− T cells were able to deplete host radioresistant T cells and facilitate hematopoietic engraftment, resulting in enhanced de novo T-cell regeneration. Enhanced functional immune reconstitution was demonstrated in CD62L− T-cell recipients using a tumor and an influenza virus challenge model. Even though CD62L− T cells are able to respond to alloantigens and deplete host radioresistant immune cells in GVHD recipients, alloreactive CD62L− T cells lost the reactivity over time and were eventually tolerant to alloantigens as a result of prolonged antigen exposure, suggesting a mechanism by which CD62L− T cells were able to eliminate host resistance without causing GVHD. These data further highlight the unique characteristics of CD62L− T cells and their potential applications in clinical hematopoietic cell transplantation. PMID:22596261
Direct evidence for a chronic CD8+-T-cell-mediated immune reaction to tax within the muscle of a human T-cell leukemia/lymphoma virus type 1-infected patient with sporadic inclusion body myositis.

PubMed

Ozden, Simona; Cochet, Madeleine; Mikol, Jacqueline; Teixeira, Antonio; Gessain, Antoine; Pique, Claudine

2004-10-01

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection can lead to the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), concomitantly with or without other inflammatory disorders such as myositis. These pathologies are considered immune-mediated diseases, and it is assumed that migration within tissues of both HTLV-1-infected CD4(+) T cells and anti-HTLV-1 cytotoxic T cells represents a pivotal event. However, although HTLV-1-infected T cells were found in inflamed lesions, the antigenic specificity of coinfiltrated CD8(+) T cells remains to be determined. In this study, we performed both ex vivo and in situ analyses using muscle biopsies obtained from an HTLV-1-infected patient with HAM/TSP and sporadic inclusion body myositis. We found that both HTLV-1-infected CD4(+) T cells and CD8(+) T cells directed to the dominant Tax antigen can be amplified from muscle cell cultures. Moreover, we were able to detect in two successive muscle biopsies both tax mRNA-positive mononuclear cells and T cells recognized by the Tax11-19/HLA-A*02 tetramer and positive for perforin. These findings provide the first direct demonstration that anti-Tax cytotoxic T cells are chronically recruited within inflamed tissues of an HTLV-1 infected patient, which validates the cytotoxic immune reaction model for the pathogenesis of HTLV-1-associated inflammatory disease.
A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

PubMed Central

Matsuda, Shigeaki; Okada, Natsumi; Kodama, Toshio; Honda, Takeshi; Iida, Tetsuya

2012-01-01

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H+-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death. PMID:22829766
Dendritic cell internalization of α-galactosylceramide from CD8 T cells induces potent antitumor CD8 T-cell responses.

PubMed

Choi, Dong Hoon; Kim, Kwang Soon; Yang, Se Hwan; Chung, Doo Hyun; Song, Boyeong; Sprent, Jonathan; Cho, Jae Ho; Sung, Young Chul

2011-12-15

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.
In Vitro Evidence of the Presence of Mesenchymal Stromal Cells in Cervical Cancer and Their Role in Protecting Cancer Cells from Cytotoxic T Cell Activity

PubMed Central

Montesinos, Juan J.; Mora-García, María de L.; Mayani, Héctor; Flores-Figueroa, Eugenia; García-Rocha, Rosario; Fajardo-Orduña, Guadalupe R.; Castro-Manrreza, Marta E.; Weiss-Steider, Benny

2013-01-01

Mesenchymal stromal cells (MSCs) have been isolated from different tumors and it has been suggested that they support tumor growth through immunosuppression processes that favor tumor cell evasion from the immune system. To date, however, the presence of MSCs in cervical cancer (CeCa) and their possible role in tumor growth remains unknown. Herein we report on the presence of MSCs in cervical tissue, both in normal conditions (NCx-MSCs) and in CeCa (CeCa-MSCs), and described several biological properties of such cells. Our study showed similar patterns of cell surface antigen expression, but distinct differentiation potentials, when we compared both cervical MSC populations to MSCs from normal bone marrow (BM-MSCs, the gold standard). Interestingly, CeCa-MSCs were negative for the presence of human papiloma virus, indicating that these cells are not infected by such a viral agent. Also, interestingly, and in contrast to NCx-MSCs, CeCa-MSCs induced significant downregulation of surface HLA class I molecules (HLA-A*0201) on CaSki cells and other CeCa cell lines. We further observed that CeCa-MSCs inhibited antigen-specific T cell recognition of CaSki cells by cytotoxic T lymphocytes (CTLs). HLA class I downregulation on CeCa cells correlated with the production of IL-10 in cell cocultures. Importantly, this cytokine strongly suppressed recognition of CeCa cells by CTLs. In summary, this study demonstrates the presence of MSCs in CeCa and suggests that tumor-derived MSCs may provide immune protection to tumor cells by inducing downregulation of HLA class I molecules. This mechanism may have important implications in tumor growth. PMID:23656504
HIV-1 dynamics revisited: biphasic decay by cytotoxic T lymphocyte killing?

PubMed Central

Arnaout, R A; Nowak, M A; Wodarz, D

2000-01-01

The biphasic decay of blood viraemia in patients being treated for human immunodeficiency virus type 1 (HIV-1) infection has been explained as the decay of two distinct populations of cells: the rapid death of productively infected cells followed by the much slower elimination of a second population the identity of which remains unknown. Here we advance an alternative explanation based on the immune response against a single population of infected cells. We show that the biphasic decay can be explained simply, without invoking multiple compartments: viral load falls quickly while cytotoxic T lymphocytes (CTL) are still abundant, and more slowly as CTL disappear. We propose a method to test this idea, and develop a framework that is readily applicable to treatment of other infections. PMID:10972131
Designing bovine T-cell vaccines via reverse immunology

USDA-ARS?s Scientific Manuscript database

T-cell responses contribute to immunity against many intra-cellular infections. There is, for example, strong evidence that major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTLs) play an essential role in mediating immunity to East Coast fever (ECF), a fatal lymphop...
Receptor-mediated cytotoxicity of alpha-MSH fragments containing melphalan in a human melanoma cell line.

PubMed

Morandini, R; Süli-Vargha, H; Libert, A; Loir, B; Botyánszki, J; Medzihradszky, K; Ghanem, G

1994-01-02

Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human

HIV-specific CD8+ T cells: serial killers condemned to die?

PubMed

Petrovas, Constantinos; Mueller, Yvonne M; Katsikis, Peter D

2004-04-01

An increasing body of evidence supports a key role for cytotoxic CD8+ T cells (CTL) in controlling HIV infection. Although a vigorous HIV-specific CD8+ T cell response is raised during the primary infection, these cells ultimately fail to control virus and prevent disease progression. The failure of CTL to control HIV infection has been attributed to a number of strategies HIV employs to evade the immune system. Recently, intrinsic defects in the CTL themselves have been proposed to contribute to the failure of CTL to control HIV. HIV-specific CD8+ T cells differ in their effector/memory phenotype from other virus-specific CD8+ T cells indicating that their differentiation status differs. This altered differentiation may affect effector functions as well as homing properties of these cells. Other studies have indicated that activation of HIV-specific CTL may be impaired and this contributes to their dysfunction. The effector function of these CTL may also be affected. There are conflicting reports about their ability to kill, whereas IFNgamma production does not appear to be impaired in these cells. In this review we focus on recent work indicating that apoptosis may be an important mechanism through which HIV evades the CTL response. In particular, HIV-specific CD8+ T cells are highly susceptible to CD95/Fas-induced apoptosis. This leads to the hypothesis that virus-specific cytotoxic T cells can be eliminated upon binding CD95L/FasL on HIV-infected cells. Understanding the intrinsic defects of CTL in HIV infection could lead to new therapeutic strategies and optimized vaccination protocols that enhance the HIV-specific cytotoxic response.
Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes.

PubMed

Moritoyo, T; Reinhart, T A; Moritoyo, H; Sato, E; Izumo, S; Osame, M; Haase, A T

1996-07-01

Infection by human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia and a slowly progressive disease of the central nervous system (CNS), HTLV-I-associated myelopathy/tropical spastic paraparesis, characterized pathologically by inflammation and white matter degeneration in the spinal cord. One of the explanations for the tissue destruction is that HTLV-I infects cells in the CNS, or HTLV-I-infected CD4+ T lymphocytes enter the CNS, and this drives local expansion of virus-specific CD8+ cytotoxic T lymphocytes, which along with cytokines cause the pathological changes. Because both in the circulation and in the cerebrospinal fluid, CD8+ cytotoxic T lymphocytes are primarily reactive to the product of the HTLV-I tax gene, we sought evidence of expression of this gene within cells in the inflammatory lesions. After using double-label in situ hybridization techniques, we now report definitive localization of HTLV-I tax gene expression in CD4+ T lymphocytes in areas of inflammation and white matter destruction. These findings lend support to a hypothetical scheme of neuropathogenesis in which HTLV-I tax gene expression provokes and sustains an immunopathological process that progressively destroys myelin and axons in the spinal cord.
Natural killer cells regulate T cell immune responses in primary biliary cirrhosis.

PubMed

Shimoda, Shinji; Hisamoto, Satomi; Harada, Kenichi; Iwasaka, Sho; Chong, Yong; Nakamura, Minoru; Bekki, Yuki; Yoshizumi, Tomoharu; Shirabe, Ken; Ikegami, Toru; Maehara, Yoshihiko; He, Xiao-Song; Gershwin, M Eric; Akashi, Koichi

2015-12-01

The hallmark of primary biliary cirrhosis (PBC) is the presence of autoreactive T- and B-cell responses that target biliary epithelial cells (BECs). Biliary cell cytotoxicity is dependent upon initiation of innate immune responses followed by chronic adaptive, as well as bystander, mechanisms. Critical to these mechanisms are interactions between natural killer (NK) cells and BECs. We have taken advantage of the ability to isolate relatively pure viable preparations of liver-derived NK cells, BECs, and endothelial cells, and studied interactions between NK cells and BECs and focused on the mechanisms that activate autoreactive T cells, their dependence on interferon (IFN)-γ, and expression of BEC major histocompatibility complex (MHC) class I and II molecules. Here we show that at a high NK/BEC ratio, NK cells are cytotoxic for autologous BECs, but are not dependent on autoantigen, yet still activate autoreactive CD4(+) T cells in the presence of antigen presenting cells. In contrast, at a low NK/BEC ratio, BECs are not lysed, but IFN-γ production is induced, which facilitates expression of MHC class I and II molecules on BEC and protects them from lysis upon subsequent exposure to autoreactive NK cells. Furthermore, IFN-γ secreted from NK cells after exposure to autologous BECs is essential for this protective function and enables autoreactive CD4(+) T cells to become cytopathic. NK cell-mediated innate immune responses are likely critical at the initial stage of PBC, but also facilitate and maintain the chronic cytopathic effect of autoantigen-specific T cells, essential for progression of disease. © 2015 by the American Association for the Study of Liver Diseases.
FasL Mediates T-Cell Eradication of Tumor Cells Presenting Low Levels of Antigens | Center for Cancer Research

Cancer.gov

One approach to cancer immunotherapy, as opposed to therapeutic vaccination, is the transfusion of large numbers of tumor-specific killer T cells (cytotoxic T cells or CTLs) into patients. The body’s own defense killer T cells are a subgroup of T lymphocytes (a type of white blood cells) that are capable of inducing death in tumor cells. CTLs can cause the death of target
Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia.

PubMed

Lee, Woo-Yong; Sanz, Maria-Jesus; Wong, Connie H Y; Hardy, Pierre-Olivier; Salman-Dilgimen, Aydan; Moriarty, Tara J; Chaconas, George; Marques, Adriana; Krawetz, Roman; Mody, Christopher H; Kubes, Paul

2014-09-23

CXCR6-GFP(+) cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60-70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi, which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints.
Selective cytotoxicity of transformed cells but not normal cells by a sialoglycopeptide growth regulator in the presence of tumor necrosis factor

NASA Technical Reports Server (NTRS)

Woods, K. M.; Fattaey, H.; Johnson, T. C.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.
T cells raised against allogeneic HLA-A2/CD20 kill primary follicular lymphoma and acute lymphoblastic leukemia cells.

PubMed

Abrahamsen, Ingerid Weum; Kjellevoll, Synneva; Greve-Isdahl, Margrethe; Mensali, Nadia; Wälchli, Sébastien; Kumari, Shraddha; Loland, Beate Fossum; Egeland, Torstein; Kolstad, Arne; Olweus, Johanna

2012-04-15

T cells mediating a graft-versus-leukemia/lymphoma effects without causing graft-versus-host disease would greatly improve the safety and applicability of hematopoietic stem cell transplantation. We recently demonstrated that highly peptide- and HLA-specific T cells can readily be generated against allogeneic HLA-A*02:01 in complex with a peptide from the B cell-restricted protein CD20. Here, we show that such CD20-specific T cells can easily be induced from naïve precursors in cord blood, demonstrating that they do not represent cross-reactive memory cells. The cells displayed high avidity and mediated potent cytotoxic effects on cells from patients with the CD20(pos) B cell malignancies follicular lymphoma (FL) and acute lymphoblastic leukemia (ALL). However, the cytotoxicity was consistently lower for cells from two of the ALL patients. The ALL cells that were less efficiently killed did not display lower surface expression of CD20 or HLA-A*02:01, or mutations in the CD20 sequence. Peptide pulsing fully restored the levels of cytotoxicity, indicating that they are indeed susceptible to T cell-mediated killing. Adoptive transfer of CD20-specific T cells to an HLA-A*02:01(pos) patient requires an HLA-A*02:01(neg) , but otherwise HLA identical, donor. A search clarified that donors meeting these criteria can be readily identified even for patients with rare haplotypes. The results bear further promise for the clinical utility of CD20-specific T cells in B cell malignancies. Copyright © 2011 UICC.
IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro.

PubMed

Alves, Nuno L; Hooibrink, Berend; Arosa, Fernando A; van Lier, René A W

2003-10-01

Recent studies in mice have shown that although interleukin 15 (IL-15) plays an important role in regulating homeostasis of memory CD8+ T cells, it has no apparent function in controlling homeostatic proliferation of naive T cells. We here assessed the influence of IL-15 on antigen-independent expansion and differentiation of human CD8+ T cells. Both naive and primed human T cells divided in response to IL-15. In this process, naive CD8+ T cells successively down-regulated CD45RA and CD28 but maintained CD27 expression. Concomitant with these phenotypic changes, naive cells acquired the ability to produce interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), expressed perforin and granzyme B, and acquired cytotoxic properties. Primed CD8+ T cells, from both noncytotoxic (CD45RA-CD27+) and cytotoxic (CD45RA+CD27-) subsets, responded to IL-15 and yielded ample numbers of cytokine-secreting and cytotoxic effector cells. In summary, all human CD8+ T-cell subsets had the ability to respond to IL-15, which suggests a generic influence of this cytokine on CD8+ T-cell homeostasis in man.
Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells, an Extrathymic αβT-cell Progenitor.

PubMed

Welker, Christian; Handgretinger, Rupert; Schilbach, Karin

2015-12-07

The thymus, the primary organ for the generation of αβ T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of αβT cells. Yet, thymic involution begins early in life leading to a drastically reduced output of naïve αβT cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic αβT cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. γδ T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a γδ T cell subset by showing that the scarce entity of CD4(+) Vδ1(+)γδ T cells can transdifferentiate into αβT cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. Vδ1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4(+) cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4(+) Vδ1(+)γδ T cells. This knowledge is prerequisite for studies that relate to this αβT cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.
Viability and proliferation potential of adipose-derived stem cells following labeling with a positron-emitting radiotracer.

PubMed

Elhami, Esmat; Goertzen, Andrew L; Xiang, Bo; Deng, Jixian; Stillwell, Chris; Mzengeza, Shadreck; Arora, Rakesh C; Freed, Darren; Tian, Ganghong

2011-07-01

Adipose-derived stem cells (ASCs) have promising potential in regenerative medicine and cell therapy. Our objective is to examine the biological function of the labeled stem cells following labeling with a readily available positron emission tomography (PET) tracer, (18)F-fluoro-2-deoxy-D: -glucose (FDG). In this work we characterize labeling efficiency through assessment of FDG uptake and retention by the ASCs and the effect of FDG on cell viability, proliferation, transdifferentiation, and cell function in vitro using rat ASCs. Samples of 10(5) ASCs (from visceral fat tissue) were labeled with concentrations of FDG (1-55 Bq/cell) in 0.75 ml culture medium. Label uptake and retention, as a function of labeling time, FDG concentration, and efflux period were measured to determine optimum cell labeling conditions. Cell viability, proliferation, DNA structure damage, cell differentiation, and other cell functions were examined. Non-labeled ASC samples were used as a control for all experimental groups. Labeled ASCs were injected via tail vein in several healthy rats and initial cell biodistribution was assessed. Our results showed that FDG uptake and retention by the stem cells did not depend on FDG concentration but on labeling and efflux periods and glucose content of the labeling and efflux media. Cell viability, transdifferentiation, and cell function were not greatly affected. DNA damage due to FDG radioactivity was acute, but reversible; cells managed to repair the damage and continue with cell cycles. Over all, FDG (up to 25 Bq/cell) did not impose severe cytotoxicity in rat ASCs. Initial biodistribution of the FDG-labeled ASCs was 80% + retention in the lungs. In the delayed whole-body images (2-3 h postinjection) there was some activity distribution resembling typical FDG uptake patterns. For in vivo cell tracking studies with PET tracers, the parameter of interest is the amount of radiotracer that is present in the cells being labeled and consequent
Discorhabdins S, T, and U, new cytotoxic pyrroloiminoquinones from a deep-water Caribbean sponge of the genus Batzella.

PubMed

Gunasekera, Sarath P; Zuleta, Ignacio A; Longley, Ross E; Wright, Amy E; Pomponi, Shirley A

2003-12-01

Discorhabdins S, T, and U (1-3), three new discorhabdin analogues, have been isolated from a deep-water marine sponge of the genus Batzella. These discorhabdin analogues showed in vitro cytotoxicity against PANC-1, P-388, and A-549 cell lines. The isolation and structure elucidation of discorhabdins S, T, and U are described.
Regulation of NK92-MI cell cytotoxicity by substance P.

PubMed

Fu, W X; Qin, B; Zhou, A P; Yu, Q Y; Huang, Q J; Liang, Z F

2011-08-01

The neuropeptide substance P (SP) can regulate a number of immunological functions in vitro and in vivo and may regulate natural killer (NK) cell activity. Here, we investigated whether SP has a role in regulating NK92-MI cell function in vitro, and how it influences NK cell activity. We found that SP dose dependently increased the cytotoxicity of NK92-MI cells and had a maximal effect at a concentration of 10(-12) and 10(-10) m. Furthermore, the expression of cytotoxic-associated molecules (perforin, granzyme) and activating receptor NKp46 [a member of natural cytotoxicity receptors (NCRs)] was observed to be upregulated by SP at optimal concentration, at which SP enhanced the cytotoxicity of NK92-MI cells. Neurokinin-1 receptor (NK-1R), a functional receptor of SP, was found on NK92-MI cells, and the observed effects of SP on NK92-MI cells could be more partially blocked by an NK-1R antagonist. Our data suggest that SP induces NK92-MI cell cytotoxicity by directly increasing the expression of cytotoxic granules and upregulates NK92-MI cell receptor-mediated functions indirectly. Thus, SP may regulate NK cell function mainly through NK-1R. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.
Hemophagocytic lymphohistiocytosis secondary to T-cell/histiocyte-rich large B-cell lymphoma

PubMed Central

Devitt, Katherine; Cerny, Jan; Switzer, Bradley; Ramanathan, Muthalagu; Nath, Rajneesh; Yu, Hongbo; Woda, Bruce A.; Chen, Benjamin J.

2014-01-01

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening clinical syndrome characterized by dysregulation of the immune system. Impaired function of cytotoxic T cells and natural killer cells is often seen, and T-cell malignancies represent most cases of lymphoma-associated HLH. HLH associated with B-cell lymphoma is rare. We describe a case of a 30-year-old man who presented with fever, splenomegaly, and hyperferritinemia. Bone marrow biopsy revealed T-cell/histiocyte-rich large B-cell lymphoma, a rare, aggressive B-cell malignancy. This case highlights the interplay between a pro-inflammatory cytokine microenvironment and tumor-mediated immune suppression, and addresses the importance of accurately diagnosing these entities for appropriate clinical management. PMID:24955327
Hemophagocytic lymphohistiocytosis secondary to T-cell/histiocyte-rich large B-cell lymphoma.

PubMed

Devitt, Katherine; Cerny, Jan; Switzer, Bradley; Ramanathan, Muthalagu; Nath, Rajneesh; Yu, Hongbo; Woda, Bruce A; Chen, Benjamin J

2014-01-01

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening clinical syndrome characterized by dysregulation of the immune system. Impaired function of cytotoxic T cells and natural killer cells is often seen, and T-cell malignancies represent most cases of lymphoma-associated HLH. HLH associated with B-cell lymphoma is rare. We describe a case of a 30-year-old man who presented with fever, splenomegaly, and hyperferritinemia. Bone marrow biopsy revealed T-cell/histiocyte-rich large B-cell lymphoma, a rare, aggressive B-cell malignancy. This case highlights the interplay between a pro-inflammatory cytokine microenvironment and tumor-mediated immune suppression, and addresses the importance of accurately diagnosing these entities for appropriate clinical management.
Investigation of T-2 Mycotoxin-Induced Cytotoxicity in vitro and Protective Effects of Flavonoid Compounds

DTIC Science & Technology

1986-01-01

Quercetin , a flavonoid compound was able to decrease the effect of T-2 toxin when the drug was added within an hour of mixing the T-2 toxin with the...were examined microscopically using a Neubauer hemocytometer and viability of at least 200 cells was deter- mined. Quercetin or other flavonold... quercetin and additional OMSO had a cytotoxic effect on the thymocytes. RESULTS Figure 1 shows the results of 8 separate experiments performed at 2 week
Radionuclide therapy using ¹³¹I-labeled anti-epidermal growth factor receptor-targeted nanoparticles suppresses cancer cell growth caused by EGFR overexpression.

PubMed

Li, Wei; Liu, Zhongyun; Li, Chengxia; Li, Ning; Fang, Lei; Chang, Jin; Tan, Jian

2016-03-01

Anti-epidermal growth factor receptor (EGFR)-targeted nanoparticles can be used to deliver a therapeutic and imaging agent to EGFR-overexpressing tumor cells. (131)I-labeled anti-EGFR nanoparticles derived from cetuximab were used as a tumor-targeting vehicle in radionuclide therapy. This paper describes the construction of the anti-EGFR nanoparticle EGFR-BSA-PCL. This nanoparticle was characterized for EGFR-targeted binding and cellular uptake in EGFR-overexpressing cancer cells by using flow cytometry and confocal microscopy. Anti-EGFR and non-targeted nanoparticles were labeled with (131)I using the chloramine-T method. Analyses of cytotoxicity and targeted cell killing with (131)I were performed using the MTT assay. The time-dependent cellular uptake of (131)I-labeled anti-EGFR nanoparticles proved the slow-release effects of nanoparticles. A radioiodine therapy study was also performed in mice. The EGFR-targeted nanoparticle EGFR-BSA-PCL and the non-targeted nanoparticle BSA-PCL were constructed; the effective diameters were approximately 100 nm. The results from flow cytometry and confocal microscopy revealed significant uptake of EGFR-BSA-PCL in EGFR-overexpressing tumor cells. Compared with EGFR-BSA-PCL, BSA-PCL could also bind to cells, but tumor cell retention was minimal and weak. In MTT assays, the EGFR-targeted radioactive nanoparticle (131)I-EGFR-BSA-PCL showed greater cytotoxicity and targeted cell killing than the non-targeted nanoparticle (131)I-BSA-PCL. The radioiodine uptake of both (131)I-labeled nanoparticles, (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL, was rapid and reached maximal levels 4 h after incubation, but the (131)I uptake of (131)I-EGFR-BSA-PCL was higher than that of (131)I-BSA-PCL. On day 15, the average tumor volumes of the (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL groups showed a slow growth relationship compared with that of the control group. The EGFR-targeted nanoparticle EGFR-BSA-PCL demonstrated superior cellular binding and uptake
Chemotherapeutic Effect of CD147 Antibody-labeled Micelles Encapsulating Doxorubicin Conjugate Targeting CD147-Expressing Carcinoma Cells.

PubMed

Asakura, Tadashi; Yokoyama, Masayuki; Shiraishi, Koichi; Aoki, Katsuhiko; Ohkawa, Kiyoshi

2018-03-01

CD147 (basigin/emmprin) is expressed on the surface of carcinoma cells. For studying the efficacy of CD147-targeting medicine on CD147-expressing cells, we studied the effect of anti-CD147-labeled polymeric micelles (CD147ab micelles) that encapsulated a conjugate of doxorubicin with glutathione (GSH-DXR), with specific accumulation and cytotoxicity against CD147-expressing A431 human epidermoid carcinoma cells, Ishikawa human endometrial adenocarcinoma cells, and PC3 human prostate carcinoma cells. By treatment of each cell type with CD147ab micelles for 1 h, a specific accumulation of CD147ab micelles in CD147-expressing cells was observed. In addition, the cytotoxicity of GSH-DXR-encapsulated micelles against each cell type was measured by treatment of the micelles for 1 h. The cytotoxic effect of CD147ab micelles carrying GSH-DXR was 3- to 10-fold higher for these cells than that of micelles without GSH-DXR. These results suggest that GSH-DXR-encapsulated CD147ab micelles could serve as an effective drug delivery system to CD147-expressing carcinoma cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3).

PubMed

Bisht, Gunjan; Rayamajhi, Sagar; Kc, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G

2016-12-01

Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe 3 O 4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe 3 O 4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe 3 O 4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe 3 O 4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles. Schematic representation of the conjugation, characterization and cytotoxicity analysis of Fe 3 O 4 -ZnO magnetic composite particles (MCPs).
Cystatin F Affects Natural Killer Cell Cytotoxicity

PubMed Central

Perišić Nanut, Milica; Sabotič, Jerica; Švajger, Urban; Jewett, Anahid; Kos, Janko

2017-01-01

Cystatin F is a cysteine peptidase inhibitor which, unlike other cystatin family members, is targeted to endosomal/lysosomal compartments. It is synthesized as an inactive disulfide-linked dimer which is then converted to an active monomer by proteolytic cleavage of 15 N-terminal residues. Cystatin F has been suggested to regulate the cytotoxicity of natural killer (NK) cells by inhibiting the major granzyme convertases, cathepsins C and H. To test this hypothesis, we prepared variants of cystatin F and analyzed their uptake, subcellular trafficking, and peptidase inhibition, as well as their impact on the cytotoxicity of NK-92 cells and primary NK cells. The N-glycosylation pattern is responsible for the secretion, uptake, and subcellular sorting of cystatin F in HeLa and Hek293 cells, whereas the legumain binding site had no effect on these processes. Active, N-terminally truncated, monomeric cystatin F can also be internalized by recipient cells and targeted to endo/lysosomes, affecting also cells lacking the activating peptidase. Cystatin F mutants capable of cell internalization and trafficking through the endo/lysosomal pathway significantly decreased cathepsin C and H activities, both in situ, following transfection and in trans, using conditioned media. Further, incubation of IL-2 stimulated NK-92 and primary NK cells with full-length and N-terminally truncated cystatin F mutants led to suppression of their granule-mediated cytotoxicity. This effect was most significant with the N-terminally truncated mutants. These results suggest that cystatin F can be an important mediator within tumor microenvironment affecting the cytotoxicity of NK cells and consequently antitumor immune response. PMID:29180998
Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Do Youn; Institute of Genetic Engineering, Kyungpook National University, Daegu; Kim, Jun Seok

2007-07-15

To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 {mu}M) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressingmore » Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.« less

Fish Scale Collagen Peptides Protect against CoCl2/TNF-α-Induced Cytotoxicity and Inflammation via Inhibition of ROS, MAPK, and NF-κB Pathways in HaCaT Cells

PubMed Central

Subhan, Fazli; Kang, Hae Yeong; Lim, Yeseon; Ikram, Muhammad; Baek, Sun-Yong; Jin, Songwan; Jeong, Young Hun; Kwak, Jong Young

2017-01-01

Skin diseases associated with inflammation or oxidative stress represent the most common problem in dermatology. The present study demonstrates that fish scale collagen peptides (FSCP) protect against CoCl2-induced cytotoxicity and TNF-α-induced inflammatory responses in human HaCaT keratinocyte cells. Our study is the first to report that FSCP increase cell viability and ameliorate oxidative injury in HaCaT cells through mechanisms mediated by the downregulation of key proinflammatory cytokines, namely, TNF-α, IL-1β, IL-8, and iNOS. FSCP also prevent cell apoptosis by repressing Bax expression, caspase-3 activity, and cytochrome c release and by upregulating Bcl-2 protein levels in CoCl2- or TNF-α-stimulated HaCaT cells. In addition, the inhibitory effects of FSCP on cytotoxicity and the induction of proinflammatory cytokine expression were found to be associated with suppression of the ROS, MAPK (p38/MAPK, ERK, and JNK), and NF-κB signaling pathways. Taken together, our data suggest that FSCP are useful as immunomodulatory agents in inflammatory or immune-mediated skin diseases. Furthermore, our results provide new insights into the potential therapeutic use of FSCP in the prevention and treatment of various oxidative- or inflammatory stress-related inflammation and injuries. PMID:28717410
CMV drives the expansion of highly functional memory T cells expressing NK-cell receptors in renal transplant recipients.

PubMed

Makwana, Nandini; Foley, Bree; Fernandez, Sonia; Lee, Silvia; Irish, Ashley; Pircher, Hanspeter; Price, Patricia

2017-08-01

Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post-transplant. We describe how active and latent CMV affect T-cell subsets in RTRs who are stable on maintenance therapy. T-cell responses to CMV were assessed in RTRs (n = 54) >2 years post-transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8 + T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T-cell (T EM ), terminally differentiated T-cell (T EMRA ) and CD57 + T EMRA cell populations. Expression of NK-cell receptors, LIR-1 and KLRG1 on CD4 + and CD8 + CD57 + T EM and T EMRA cells correlated with elevated interferon-γ and cytotoxic responses to anti-CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8 + T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) -1 peptides. The data show that latent and active CMV infection can alter T-cell subsets in RTRs many years after transplantation, and up-regulate T-cell expression of NK-cell receptors. This may enhance effector responses of CD4 + and CD8 + T cells against CMV. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Deep Learning in Label-free Cell Classification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less
Deep Learning in Label-free Cell Classification

PubMed Central

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-01-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219
Deep Learning in Label-free Cell Classification

DOE PAGES

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; ...

2016-03-15

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less
Deep Learning in Label-free Cell Classification

NASA Astrophysics Data System (ADS)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-03-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.
Nrf2 suppresses the function of dendritic cells to facilitate the immune escape of glioma cells.

PubMed

Wang, Jialiang; Liu, Peng; Xin, Shaoyan; Wang, Zongbao; Li, Jun

2017-11-15

Nrf2 is presented in dendritic cells (DCs) and contributes to the maintenance of redox homeostasis. However, the expression pattern and function of Nrf2 in the maturation of DCs in the glioma-infiltrated microenvironment remain unrevealed. Our study aims to investigate the roles of Nrf2 in glioma cell-tamed DCs and their impact on the downstream T cell proliferation and cytotoxicity to glioma cells. It was showed that the inducible maturation of DCs was significantly suppressed after stimulation with tumor-conditioned medium (TCM) prepared from glioma cells (LN-18 and U118MG), as suggested by the decreased CD80, CD86 and IL-12 p70 expression and higher levels of IL-10 than the normal astrocyte medium treated DCs. Moreover, the TCM-exposed DCs had significantly increased expression and transcriptional activity of Nrf2 compared to the negative control. Nrf2 inhibition in DC cells substantially antagonized the inhibitory effects of TCM on the maturation and activation of DC cells, reflected by the elevated maturation markers and IL-12 p70. We further confirmed that Nrf2 inhibition in TCM-exposed DC cells promoted the proliferation of T cells as evaluated by the CFSE-labeled assay and Th1 response shown by the elevated production of IFN-γ. The cytotoxic T lymphocyte assay revealed that Nrf2 genetic suppression in DC cells greatly enhanced the capacity of T cells in the cytotoxicity to glioma cells dependent on the E:T ratio. Collectively, our study demonstrated that Nrf2 inhibition in DCs in glioma-exposed microenvironment could enhance the maturation of DCs and the subsequent activation of T cells and their cytotoxicity on glioma cells. Copyright © 2017. Published by Elsevier Inc.
Dacarbazine-mediated upregulation of NKG2D ligands on tumor cells activates NK and CD8 T cells and restrains melanoma growth.

PubMed

Hervieu, Alice; Rébé, Cédric; Végran, Frédérique; Chalmin, Fanny; Bruchard, Mélanie; Vabres, Pierre; Apetoh, Lionel; Ghiringhelli, François; Mignot, Grégoire

2013-02-01

Dacarbazine (DTIC) is a cytotoxic drug widely used for melanoma treatment. However, the putative contribution of anticancer immune responses in the efficacy of DTIC has not been evaluated. By testing how DTIC affects host immune responses to cancer in a mouse model of melanoma, we unexpectedly found that both natural killer (NK) and CD8(+) T cells were indispensable for DTIC therapeutic effect. Although DTIC did not directly affect immune cells, it triggered the upregulation of NKG2D ligands on tumor cells, leading to NK cell activation and IFNγ secretion in mice and humans. NK cell-derived IFNγ subsequently favored upregulation of major histocompatibility complex class I molecules on tumor cells, rendering them sensitive to cytotoxic CD8(+) T cells. Accordingly, DTIC markedly enhanced cytotoxic T lymphocyte antigen 4 inhibition efficacy in vivo in an NK-dependent manner. These results underscore the immunogenic properties of DTIC and provide a rationale to combine DTIC with immunotherapeutic agents that relieve immunosuppression in vivo.
Comparison of cytotoxicity, genotoxicity and immunological inflammatory biomarker activity of several endodontic sealers against immortalized human pulp cells.

PubMed

Martinho, F C; Camargo, S E A; Fernandes, A M M; Campos, M S; Prado, R F; Camargo, C H R; Valera, M C

2018-01-01

To establish an SV40 T-Ag-transfected cell line of human pulp-derived cells in order to compare the cytotoxicity, genotoxicity and to investigate the activities of immunological biomarkers of several endodontic sealers. Primary human pulp cells and transfected cells were cultured. Cell morphology and proliferation were analysed, and the expression of cell-specific gene transcripts and proteins was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining phenotypic characteristics from the primarily cells tested. The SV40 T-Ag-transfected cells were cultured and stimulated by sealers (Apexit Plus, Real Seal, AH Plus, and EndoREZ) to evaluate the cytotoxicity and genotoxicity by MTT and MTN assays, respectively. Immunological inflammatory biomarkers (IL6, IL8 and TNF-α) were determined by ELISA assay. The differences between median values were statistically analysed using Kruskal-Wallis and Dunn's tests at 5% significance level. The cytotoxicity assay revealed that multimethacrylate (Real Seal) was the most cytotoxic sealer (P < 0.05) and exhibited the highest inflammatory potential against the SV40 T-Ag-transfected cells (P < 0.05). All root canal sealers tested were able to stimulate the immortalized pulp cells to produce IL-6, IL-8 and TNF-α, with differences in relation to the control group (P < 0.05). Higher levels of IL-6, IL-8 and TNF-α were found in cell supernatant after stimulation with multimethacrylate (Real Seal) compared to all other sealers tested (P < 0.05). No differences were found comparing epoxy resin-based sealer (AHPlus), single-methacrylate sealer (EndoREZ) and calcium hydroxide-based sealer (Apexit Plus), regardless of the cytokine investigated (all P > 0.05). A SV40 T-Ag-transfected cell line of human pulp-derived cells was established. The methacrylate resin-based sealer (Real Seal) exhibited the greatest cytoxicity and inflammatory potential against
Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells

PubMed Central

Wells, Alexandria C; Daniels, Keith A; Angelou, Constance C; Fagerberg, Eric; Burnside, Amy S; Markstein, Michele; Alfandari, Dominique; Welsh, Raymond M; Pobezinskaya, Elena L; Pobezinsky, Leonid A

2017-01-01

The differentiation of naive CD8 T cells into effector cytotoxic T lymphocytes upon antigen stimulation is necessary for successful antiviral, and antitumor immune responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs in the regulation of CD8 T cell responses, where maintenance of the naive phenotype in CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T lymphocytes depends upon T cell receptor-mediated let-7 downregulation. Decrease of let-7 expression in activated T cells enhances clonal expansion and the acquisition of effector function through derepression of the let-7 targets, including Myc and Eomesodermin. Ultimately, we have identified a novel let-7-mediated mechanism, which acts as a molecular brake controlling the magnitude of CD8 T cell responses. DOI: http://dx.doi.org/10.7554/eLife.26398.001 PMID:28737488
Studies on the immunopotentiating effects of a streptococcal preparation, OK-432. I. Enhancement of T cell-mediated immune responses of mice.

PubMed Central

Kai, S; Tanaka, J; Nomoto, K; Torisu, M

1979-01-01

The effects of the anti-tumour agent OK-432 on the immune response to hamster erythrocytes (HRBC) and nucleated chicken erythrocytes (CRBC) were studied in inbred SL mice. Mice were treated repeatedly with OK-432 before immunization with erythrocytes in saline. The cytotoxicity of CRBC-primed spleen cells, as demonstrated by 51Cr release from labelled CRBC, was markedly increased by treatment with OD-432. The delayed footpad reaction to CRBC was significantly augmented by treatment with OK-432. These results in mice indicate that OK-432 can enhance the cellular immune responses which require the contribution of T cells. Such an activation of T cells by OK-432 was observed in the humoral immune response to a trinitrophenyl group. Augmentation of anti-hapten antibody production, suggesting the enhancement of helper T cell activity by OK-432, was noticed after immunization with trinitrophenyl conjugated to erythrocytes. Furthermore, this enhancement of helper T cell activity by OK-432 was confirmed by utilizing an adoptive transfer system. These results support the possibility that T cell activation may be one of the important effects of OK-432 as an immunopotentiator. PMID:314874
A genetically encoded and gate for cell-targeted metabolic labeling of proteins.

PubMed

Mahdavi, Alborz; Segall-Shapiro, Thomas H; Kou, Songzi; Jindal, Granton A; Hoff, Kevin G; Liu, Shirley; Chitsaz, Mohsen; Ismagilov, Rustem F; Silberg, Jonathan J; Tirrell, David A

2013-02-27

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNA(Met). Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.
A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

PubMed Central

Mahdavi, Alborz; Segall-Shapiro, Thomas H.; Kou, Songzi; Jindal, Granton A.; Hoff, Kevin G.; Liu, Shirley; Chitsaz, Mohsen; Ismagilov, Rustem F.; Silberg, Jonathan J.; Tirrell, David A.

2013-01-01

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within five minutes after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals. PMID:23406315
Gamma delta T cell responses associated with the development of tuberculosis in health care workers.

PubMed

Ordway, Diane J; Pinto, Luisa; Costa, Leonor; Martins, Marta; Leandro, Clara; Viveiros, Miguel; Amaral, Leonard; Arroz, Maria J; Ventura, Fernando A; Dockrell, Hazel M

2005-03-01

This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.
Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7

PubMed Central

Tsang, Kwong Y.; Fantini, Massimo; Fernando, Romaine I.; Palena, Claudia; David, Justin M.; Hodge, James W.; Gabitzsch, Elizabeth S.; Jones, Frank R.; Schlom, Jeffrey

2017-01-01

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen–A2 allele (HLA-A2)–Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer–cell platforms. PMID:28389098
Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

PubMed

Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

2016-09-15

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process. Copyright © 2016 by The American Association of Immunologists, Inc.
Astrocyte-specific expression of human T-cell lymphotropic virus type 1 (HTLV-1) Tax: induction of tumor necrosis factor alpha and susceptibility to lysis by CD8+ HTLV-1-specific cytotoxic T cells.

PubMed

Méndez, E; Kawanishi, T; Clemens, K; Siomi, H; Soldan, S S; Calabresi, P; Brady, J; Jacobson, S

1997-12-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with a chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraperesis (HAM/TSP). Although the pathogenesis of this disease remains to be elucidated, the evidence suggests that immunopathological mechanisms are involved. Since HTLV-1 tax mRNA was colocalized with glial acidic fibrillary protein, a marker for astrocytes, we developed an in vitro model to assess whether HTLV-1 infection activates astrocytes to secrete cytokines or present viral immunodominant epitopes to virus-specific T cells. Two human astrocytic glioma cell lines, U251 and U373, were transfected with the 3' portion of the HTLV-1 genome and with the HTLV-1 tax gene under astrocyte-specific promoter control. In this study, we report that Tax-expressing astrocytic glioma transfectants activate the expression of tumor necrosis factor alpha mRNA in vitro. Furthermore, these Tax-expressing glioma transfectants can serve as immunological targets for HTLV-1-specific cytotoxic T lymphocytes (CTL). We propose that these events could contribute to the neuropathology of HAM/TSP, since infected astrocytes can become a source for inflammatory cytokines upon HTLV-1 infection and serve as targets for HTLV-1-specific CTL, resulting in parenchymal damage by direct lysis and/or cytokine release.
Successful Cord Blood Stem Cell Transplantation for Primary Cutaneous CD8-positive Aggressive Epidermotropic Cytotoxic T-cell Lymphoma Complicated with Cerebral Infiltration.

PubMed

Ichikawa, Satoshi; Fukuhara, Noriko; Hatta, Shunsuke; Himuro, Masahito; Katsushima, Hiroki; Nasu, Kentaro; Ono, Koya; Inokura, Kyoko; Kobayashi, Masahiro; Onishi, Yasushi; Fujii, Hiroshi; Ishizawa, Kenichi; Ichinohasama, Ryo; Harigae, Hideo

2018-03-09

A 16-year-old boy, who had been initially examined for bilateral blepharedema and slight eruption, presented with rapidly deteriorating symptoms in associating with headache and consciousness disturbance. He was diagnosed to have primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma (PCAE-CTL) by a biopsy of the skin and brain. After whole-brain radiation and some courses of chemotherapy, cord blood transplantation was performed with myeloablative conditioning. After transplantation, the cerebral dysfunction gradually improved. Disease remission was confirmed by the disappearance of any abnormal findings on electroencephalogram and magnetic resonance imaging. PCAE-CTL is reported to be an extremely aggressive disease with a poor prognosis, but the timely performance of cord blood transplantation is considered to be a promising treatment strategy.
Innovative T Cell-Targeted Therapy for Ovarian Cancer

DTIC Science & Technology

2012-10-01

from co-culture with EL4 -ROR1neg and EL4 -ROR1+ tumor targets. Ovarian cancer cell lines (A2780, EFO21, EFO27, IGROV1, OC314, and UPN251) were...profiled for ROR1 expression in normoxia (20% O2) and hypoxia (1% O2). Four-hour CRA was used to evaluate cytotoxicity against the OvCa and EL4 tumor...loaded aAPC for negative controls. EL4 is a murine T cell lymphoma cell line used to test specificity of CAR+ T cells with limited allo-reactivity
An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.

PubMed

Anticoli, Simona; Manfredi, Francesco; Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Ferrantelli, Flavia; Capocefalo, Antonio; Falcone, Emiliana; Ruggieri, Anna; Federico, Maurizio

2018-04-01

Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, that is, Nef mut , the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen-specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nef mut , and are uploaded in exosomes at levels comparable to Nef mut . When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen-specific CD8 + T cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

PubMed

Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

2010-07-01

Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.
Phenotypic and functional characterization of T cells from patients with myasthenia gravis.

PubMed Central

Mokhtarian, F; Pino, M; Ofosu-Appiah, W; Grob, D

1990-01-01

A study of cell surface phenotypes of PBL of myasthenia gravis (MG) patients showed that their T cells had a significantly higher percentage of 4B4+ T cells (the helper/inducer subset) than age- and sex-matched controls. The PBL of MG patients proliferated significantly higher than those of normal subjects (NS) in response to the purified alpha chain of the acetylcholine receptor (AChR). Anti-AChR antibody was present in sera of 88% of MG and none of the NS. The PBL B cells from MG only, when cultured with autologous T cells and stimulated with either pokeweed mitogen (69%), or AChR-alpha chain (38%), secreted antibody to AChR-alpha chain, whereas T and B cells alone secreted no antibody. T cells from PBL of MG patients were more readily cloned than T cells of NS, by limiting dilution, in the presence of recombinant IL-2 and in the absence of AChR-alpha chain. About 50% of T cell clones from MG patients, compared to none from NS, proliferated to AChR-alpha chain. This response was HLA-DR restricted. MG T cell clones did not display significant cytotoxic activity, as compared to control T cell clones. Our results indicate that in MG, 4B4+ regulatory T cells play their role in the pathogenesis of MG, not by cytotoxicity, but more likely by their ability to stimulate specific antibody production by B cells. Images PMID:1979338
Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds.

PubMed

Lema, Carolina; Varela-Ramirez, Armando; Aguilera, Renato J

As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z' factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC 50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity.
Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds

PubMed Central

LEMA, Carolina; VARELA-RAMIREZ, Armando; AGUILERA, Renato J.

2016-01-01

As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z′ factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity. PMID:27042697
Epstein-Barr virus-transformed B-cells as efficient antigen presenting cells to propagate Aspergillus-specific cytotoxic T-lymphocytes.

PubMed

Ramadan, Gamal

2008-01-01

To overcome the cytotoxic T-lymphocytes (CTL) expansion limitations imposed by the lack of sufficient dendritic cells (DC) alternative sources of autologous antigen presenting cells (APC) such as Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BLCL), which are easy to establish in vitro, have been considered and studied in the present work. Non-adherent peripheral blood mononuclear cells of three healthy donors were repeatedly primed with autologous Aspergillus fumigatus commercial culture-filtrate antigen-pulsed fast monocyte-derived DC (Aspf-CFA-DC) alone, Aspf-CFA-pulsed BLCL (Aspf-CFA-BLCL) alone or Aspf-CFA-BLCL after one, two, or three primings with Aspf-CFA-DC (1DC/BLCL, 2DC/BLCL or 3DCIBLCL; respectively). After 5th priming, lines generated by Aspf-CFA-BLCL only showed strong/weak lytic activity for EBV/Aspf; respectively. Aspf-specific lytic activity in all donors was increased by increasing the number of primings with Aspf-CFA-DC before switching to Aspf-CFA-BLCL (18.20 +/- 1.65% versus 35.67 +/- 1.02% and 40.03 +/- 1.41% in bulk cultures generated by 1DC/BLCL versus 2DC/BLCL and 3DC/BLCL, respectively). Bulk cultures generated by Aspf-CFA-BLCL after at least two primings with Aspf-CFA-DC showed approximately the same Aspf-specific lytic activity, effector cell phenotype, expansion level and percentage expression of IFN-gamma, CD69 and CD107a without any significant differences (p > 0.05) as standard bulk cultures generated by only Aspf-CFA-DC. Thus, this study explored the use of a combined DC/BLCL protocol to establish/propagate Aspf-specific CTL for adoptive immunotherapy to prevent or treat invasive pulmonary aspergillosis.
Small RNA Enhances Antitumor T-cell Therapy | Center for Cancer Research

Cancer.gov

Adoptive T-cell transfer is an effective form of anticancer immunotherapy in which patients receive infusions of cytotoxic T cells that seek out and destroy targeted cancer cells. This type of therapy is usually preceded by a lymphodepleting chemotherapy regimen and combined with high doses of the cytokine interleukin-2 (IL-2) to eliminate immunosuppressive and other immune
Dengue virus-specific human T cell clones. Serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity

PubMed Central

1989-01-01

The severe complications of dengue virus infections, hemorrhagic manifestation and shock, are much more commonly observed during secondary infections caused by a different serotype of dengue virus than that which caused the primary infections. It has been speculated, therefore, that dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are caused by serotype crossreactive immunopathological mechanisms. We analyzed clones of dengue serotype crossreactive T lymphocytes derived from the PBMC of a donor who had been infected with dengue 3 virus. These PBMC responded best to dengue 3 antigen, but also responded to dengue 1, 2, and 4 antigens, in bulk culture proliferation assays. 12 dengue antigen-specific clones were established using a limiting dilution technique. All of the clones had CD3+ CD4+ CD8 phenotypes. Eight clones responded to dengue 1, 2, 3, and 4 antigens and are crossreactive, while four other clones responded predominantly to dengue 3 antigen. These results indicate that the serotype crossreactive dengue-specific T lymphocyte proliferation observed in bulk cultures reflects the crossreactive responses detected at the clonal level. Serotype crossreactive clones produced high titers of IFN- gamma after stimulation with dengue 3 antigens, and also produced IFN- gamma to lower levels after stimulation with dengue 1, 2, and 4 antigens. The crossreactive clones lysed autologous lymphoblastoid cell line (LCL) pulsed with dengue antigens, and the crossreactivity of CTL lysis by T cell clones was consistent with the crossreactivity observed in proliferation assays. Epidemiological studies have shown that secondary infections with dengue 2 virus cause DHF/DSS at a higher rate than the other serotypes. We hypothesized that the lysis of dengue virus-infected cells by CTL may lead to DHF/DSS; therefore, the clones were examined for cytotoxic activity against dengue 2 virus-infected LCL. All but one of the serotype crossreactive clones lysed dengue 2 virus
[Cytotonic and cytotoxic effect of cholera toxin on Vero cells and its relation to PCR].

PubMed

Rodríguez-Angeles, M G; Giono-Cerezo, S; Valdespino-Gómez, J L

1994-01-01

We studied 40 Vibrio cholerae strains: 16 from stool, 16 from sewage and 8 from food. The serotypes were Inaba in 21 strains, 8 Ogawa strains and 11 V. cholerae non-O1. PCR was made with ctx2 and ctx3 primers with 25 cycles of temperature: 1 min at 94 degrees C, 1 min at 60 degrees C and 1 min at 72 degrees C. 24 V. cholerae strains were positive: 18/24 Inaba y 6/24 Ogawa. PCR was negative for 16 strains: 3 Inaba serotype, 2 Ogawa y 11 V. cholerae non-O1. In Vero culture cells 18 strains were cytotonic, 21 cytotoxic and 1 strain was negative. ELISA was positive for 11 strains with PCR positive. The PCR sensitivity was 95.83% compared with culture cells. V. cholerae O1 produced cytotoxic effect on Vero culture cells, maybe related to ACE factor. Colony blot was made with a specific probe labeled with digoxigenin and it could detect 4 Vibrio cholerae toxigenic strains with PCR negative. All V. cholerae Non O1 strains were PCR negative.
Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

PubMed

Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

1996-02-15

Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast.
Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells.

PubMed

Keller, Eric J; Porter, Meghan; Garrett, Joy E; Varie, Meredith; Wang, Haiyan; Pollok, Karen E; Turchi, John J; Zaleski, Jeffrey M; Dynlacht, Joseph R

2018-05-15

Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of Py
Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells.

PubMed

Bennett Saidu, Nathaniel Edward; Bretagne, Marie; Mansuet, Audrey Lupo; Just, Pierre-Alexandre; Leroy, Karen; Cerles, Olivier; Chouzenoux, Sandrine; Nicco, Carole; Damotte, Diane; Alifano, Marco; Borghese, Bruno; Goldwasser, François; Batteux, Frédéric; Alexandre, Jérôme

2018-02-06

KRAS mutation, one of the most common molecular alterations observed in adult carcinomas, was reported to activate the anti-oxidant program driven by the transcription factor NRF2 (Nuclear factor-erythroid 2-related factor 2). We previously observed that the antitumoral effect of Dimethyl fumarate (DMF) is dependent of NRF2 pathway inhibition. We used in vitro methods to examine the effect of DMF on cell death and the activation of the NRF2/DJ-1 antioxidant pathway. We report here that DMF is preferentially cytotoxic against KRAS mutated cancer cells. This effect was observed in patient-derived cancer cell lines harbouring a G12V KRAS mutation, compared with cell lines without such a mutation. In addition, KRAS*G12V over-expression in the human Caco-2 colon cancer cell line significantly promoted DMF-induced cell death, as well as DMF-induced- reactive oxygen species (ROS) formation and -glutathione (GSH) depletion. Moreover, in contrast to malignant cells, our data confirms that the same concentration of DMF has no significant cytotoxic effects on non-tumorigenic human ARPE-19 retinal epithelial, murine 3T3 fibroblasts and primary mice bone marrow cells; but is rather associated with NRF2 activation, decreased ROS and increased GSH levels. Furthermore, DJ-1 down-regulation experiments showed that this protein does not play a protective role against NRF2 in non-tumorigenic cells, as it does in malignant ones. This, interestingly, could be at the root of the differential effect of DMF observed between malignant and non-tumorigenic cells. Our results suggest for the first time that the dependence on NRF2 observed in mutated KRAS malignant cells makes them more sensitive to the cytotoxic effect of DMF, which thus opens up new prospects for the therapeutic applications of DMF.
A monoclonal antibody against SV40 large T antigen (PAb416) does not label Merkel cell carcinoma.

PubMed

Pelletier, Daniel J; Czeczok, Thomas W; Bellizzi, Andrew M

2018-07-01

Merkel cell carcinoma represents poorly differentiated neuroendocrine carcinoma of cutaneous origin. In most studies, the vast majority of Merkel cell carcinomas are Merkel cell polyomavirus (MCPyV)-associated. SV40 polyomavirus immunohistochemistry is typically used in the diagnosis of other polyomavirus-associated diseases, including tubulointerstitial nephritis and progressive multifocal leukoencephalopathy, given cross-reactivity with BK and JC polyomaviruses. MCPyV-specific immunohistochemistry is commercially available, but, if antibodies against SV40 also cross-reacted with MCPyV, that would be advantageous from a resource-utilisation perspective. Tissue microarrays were constructed from 39 Merkel cell carcinomas, 24 small-cell lung carcinomas, and 18 extrapulmonary visceral small-cell carcinomas. SV40 large T antigen immunohistochemistry (clone PAb416) was performed; MCPyV large T antigen immunohistochemistry (clone CM2B4) had been previously performed. UniProt was used to compare the amino acid sequences of the SV40, BK, JC and MCPyV large T antigens, focusing on areas recognised by the PAb416 and CM2B4 clones. SV40 immunohistochemistry was negative in all tumours; MCPyV immunohistochemistry was positive in 38% of Merkel cell carcinomas and in 0% of non-cutaneous poorly differentiated neuroendocrine carcinomas. UniProt analysis revealed a high degree of similarity between SV40, BK, and JC viruses in the region recognised by PAb416. There was less homology between SV40 and MCPyV in this region, which was also interrupted by two long stretches of amino acids unique to MCPyV. The CM2B4 clone recognises a unique epitope in one of these stretches. The PAb416 antibody against the SV40 large T antigen does not cross-react with MCPyV large T antigen, and thus does not label Merkel cell carcinoma. © 2018 John Wiley & Sons Ltd.
Comparative cytotoxicity of chlorophenols to cultured fish cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Hotaka; Shigeoka, Tadayoshi

1994-10-01

In vitro cytotoxicity of chlorophenols to Cyprinus carpio brain (CCB) cells derived from carp and Oryzias latipes fin (OLF-136) cells derived from medaka was examined with the neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) incorporation assay. Results were compared with previous cytotoxicity data of chlorophenols to goldfish (Carassius auratus) scale (GFS) cells derived from the goldfish. There were excellent correlations between the 24-h NR50 values of chlorophenols toward the three kinds of cells (r[sup 2] > 0.94).
Cytotoxic lymphocytes in Hashimoto thyroiditis: an in vitro assay system using 51Cr-labelled chicken red blood cells coated with thyroglobulin

PubMed Central

Calder, Elizabeth A.; Penhale, W. J.; Barnes, E. W.; Irvine, W. J.

1973-01-01

An in vitro method is described to detect lymphocytes in patients with Hashimoto thyroiditis that are cytotoxic to thyroglobulin-coated chicken red blood cells. Using this technique, the cytotoxic index of lymphocytes from patients with Hashimoto thyroiditis was 25·46±3·81 (SEM), which is significantly different from that obtained with lymphocytes from control subjects, 6·28±0·80. PMID:4740396
Do cancer cells in human and meristematic cells in plant exhibit similar responses toward plant extracts with cytotoxic activities?

PubMed

Khalifa, Noha S; Barakat, Hoda S; Elhallouty, Salwa; Salem, Dina

2015-01-01

We examined the effect of water extracts of Persea americana fruit, and of the leaves of Tabernamontana divericata, Nerium oleander and Annona cherimolia (positive control) on Vicia faba root cells. We had confirmed in our previously published data the cytotoxicity of these plant extracts on four human cancer cell lines: liver (HepG-2), lung (A549), colon (HT-29) and breast (MCF-7). Vicia faba roots were soaked in plant extracts at dilutions of 100, 1,250, 2,500, 5,000, 10,000, 20,000 ppm for 4 and 24 h. All treatments resulted in a significant reduction in the mitotic index in a dose dependant manner. Root cells treated with T. divericata, N. oleander and A. cherimolia exhibited a decrease in prophase cell percentage, increase in micronuclei and chromosomal abnormalities as concentration increased. The P. americana treatment showed the highest cytotoxic effect on cancer cells, prophase cell percentage increased linearly with the applied concentration and no micronuclei were detected. This study shows that root tip assay of beans can be used in initial screening for new plant extracts to validate their use as candidates for containing active cytotoxic agents against malignant cells. This will greatly help in exploring new plant extracts as drugs for cancer treatment.
Infection of human T lymphotropic virus-I-specific immune T cell clones by human T lymphotropic virus-I.

PubMed Central

Mitsuya, H; Jarrett, R F; Cossman, J; Cohen, O J; Kao, C S; Guo, H G; Reitz, M S; Broder, S

1986-01-01

Human T lymphotropic virus-I (HTLV-I)-specific T cell lines were established and cloned. K5, an OKT8+ clone bearing multiple proviral integration sites, retained its HTLV-I-specific cytotoxicity and a normal dependence on interleukin 2 (IL-2), indicating that there is a finite number of transforming integration sites. R2, an OKT4+ HTLV-I-infected clone, initially mounted a proliferative response to HTLV-I; but then its IL-2-independent proliferation increased and the antigen specificity was lost. All HTLV-I-infected clones tested including K7, another OKT8+ transformed cytotoxic clone that had lost its reactivity, expressed comparable levels of T cell receptor beta-chain (TCR-beta) messenger (m)RNA. Although clones K5 and K7 had different functional properties, they had the same rearrangement of the TCR-beta gene, suggesting that they had the same clonal origin. These data indicate that HTLV-I-specific T cells retain their immune reactivity for variable periods of time following infection, but then usually lose it; in some cases, however, no alteration in function can be detected. The data also suggest that different consequences can take place in the same clone depending on the pattern of retroviral infection. Images PMID:2877011
Poly(ADP-ribose) polymerase-independent potentiation of nitrosourea cytotoxicity by 3-aminobenzamide in human malignant glioma cells.

PubMed

Winter, S; Weller, M

2000-06-16

Poly(ADP-ribose) polymerase is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents and is thought to be involved in DNA repair. Here, we examined the effects of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, on the chemosensitivity of human malignant glioma cells. 3-Aminobenzamide selectively potentiated the cytotoxicity of the nitrosoureas, nimustine, carmustine and lomustine in 10 of 12 human malignant glioma cell lines. In contrast, 3-aminobenzamide did not modulate the cytotoxic effects of doxorubicine, teniposide, vincristine, camptothecin or cytarabine. The nitrosoureas did not induce poly(ADP-ribose) polymerase activity in the glioma cells. Ectopic expression of truncated poly(ADP-ribose) polymerase containing the poly(ADP-ribose) polymerase DNA-binding domain, which acts as a dominant-negative mutant, in LN-18 or LN-229 cells did not alter the 3-aminobenzamide effect on nitrosourea-mediated cytotoxicity. Thus, 3-aminobenzamide may target another nicotinamide adenine dinucleotide (NAD)-requiring enzyme, but not poly(ADP-ribose) polymerase, when enhancing nitrosourea cytotoxicity in human malignant glioma cells. Carmustine cytotoxicity was associated with a G2/M arrest. Coexposure to carmustine and 3-aminobenzamide overcame this G2/M arrest in T98G cells, which are sensitized to carmustine by 3-aminobenzamide, but not in U251MG cells, which are refractory to 3-aminobenzamide-mediated sensitization to carmustine. Thus, 3-aminobenzamide-mediated sensitization to carmustine cytotoxicity may result from interference with the stable G2/M arrest response to carmustine in human glioma cells.
T cell function in tuatara (Sphenodon punctatus).

PubMed

Burnham, D Kim; Keall, Susan N; Nelson, Nicola J; Daugherty, Charles H

2005-05-01

Tuatara are the sole survivors of an entire order of reptiles that thrived during the age of the dinosaurs. Therefore, knowledge of their physiology is critical to understanding the phylogeny of reptiles. Previous studies of the immune system of the tuatara did not assess T cell function. We analyzed T cell function among six captive tuatara by assessing concanavalin A (Con A), phytohemagglutinin (PHA) and mixed lymphocyte reaction (MLR) induced T cell proliferation. Peripheral blood mononuclear cells from six out of six and four out of four tuatara tested exhibited significant proliferative responses to Con A and PHA, respectively, as measured by an MTT reduction assay. A lower level of proliferation was detected in an MLR. However, Con A activated lymphocytes were not cytotoxic for a xenogeneic murine mastocytoma cell line (P815).
Trypanosoma cruzi Subverts Host Cell Sialylation and May Compromise Antigen-specific CD8+ T Cell Responses*

PubMed Central

Freire-de-Lima, Leonardo; Alisson-Silva, Frederico; Carvalho, Sebastião T.; Takiya, Christina M.; Rodrigues, Maurício M.; DosReis, George A.; Mendonça-Previato, Lucia; Previato, José O.; Todeschini, Adriane R.

2010-01-01

Upon activation, cytotoxic CD8+ T lymphocytes are desialylated exposing β-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8+ T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8+ T cell surface, thereby dampening antigen-specific CD8+ T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8+ T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8+ T cell surface. The cytotoxic activity of antigen-experienced CD8+ T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase- mediated resialylation in vitro and in vivo. Inhibition of the parasite's native trans-sialidase activity during infection strongly decreased CD8+ T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8+ T cell interactions with peptide-major histocompatibility complex class I complexes. CD8+ T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism. PMID:20106975
Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection

PubMed Central

Tario, Joseph D.; Conway, Alexis N.; Muirhead, Katharine A.; Wallace, Paul K.

2018-01-01

In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells. The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: Assessment of the dye’s spectral profile on the laboratory’s flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems;Evaluating the effect of labeling on cell growth rate;Testing the fidelity with which dye dilution reports cell division;Determining the maximum number of generations to be included when using dye dilution profiles to estimate fold population expansion or frequency of responder cells; andVerifying that relevant cell functions (e.g., effector activity) remain unaltered by tracking dye labeling. PMID:29071683

TCRγ4δ1-Engineered αβT Cells Exhibit Effective Antitumor Activity

PubMed Central

He, Kangxia; You, Hongqin; Li, Yuxia; Cui, Lianxian; Zhang, Jianmin; He, Wei

2016-01-01

T cell engineering with T cell receptors (TCRs) specific for tumors plays an important role in adoptive T cell transfer (ATC) therapy for cancer. Here, we present a novel strategy to redirect peripheral blood-derived αβT cells against tumors via TCRγ4δ1 gene transduction. The broad-spectrum antitumor activity of TCRδ1 cells in innate immunity is dependent on CDR3δ1. TCRγ4δ1-engineered αβT cells were prepared by lentiviral transduction and characterized by analyzing in vitro and in vivo cytotoxicity to tumors, ability of proliferation and cytokine production, and potential role in autoimmunity. Results show that TCRγ4δ1 genes were transduced to approximately 36% of polyclonal αβT cells. TCRγ4δ1-engineered αβT cells exhibited effective in vitro TCRγδ-dependent cytotoxicity against various tumor cells via the perforin-granzyme pathway. They also showed a strong proliferative capacity and robust cytokine production. TCRγ4δ1-engineered αβT cells neither expressed mixed TCR dimers nor bound/killed normal cells in vitro. More important, adoptive transfer of TCRγ4δ1-engineered αβT cells into nude mice bearing a human HepG2 cell line significantly suppressed tumor growth. Our results demonstrate a novel role for TCRγ4δ1 in gene therapy and ATC for cancer. PMID:27463149
A novel subset of helper T cells promotes immune responses by secreting GM-CSF

PubMed Central

Zhang, J; Roberts, A I; Liu, C; Ren, G; Xu, G; Zhang, L; Devadas, S; Shi, Yufang

2013-01-01

Helper T cells are crucial for maintaining proper immune responses. Yet, they have an undefined relationship with one of the most potent immune stimulatory cytokines, granulocyte macrophage-colony-stimulating factor (GM-CSF). By depleting major cytokines during the differentiation of CD4+ T cells in vitro, we derived cells that were found to produce large amounts of GM-CSF, but little of the cytokines produced by other helper T subsets. By their secretion of GM-CSF, this novel subset of helper T cells (which we have termed ThGM cells) promoted the production of cytokines by other T-cell subtypes, including type 1 helper T cell (Th1), type 2 helper T cell (Th2), type 1 cytotoxic T cell (Tc1), type 2 cytotoxic T cell (Tc2), and naive T cells, as evidenced by the fact that antibody neutralization of GM-CSF abolished this effect. ThGM cells were found to be highly prone to activation-induced cell death (AICD). Inhibitors of TRAIL or granzymes could not block AICD in ThGM cells, whereas inhibition of FasL/Fas interaction partially rescued ThGM cells from AICD. Thus, ThGM cells are a novel subpopulation of T helper cells that produce abundant GM-CSF, exhibit exquisite susceptibility to apoptosis, and therefore play a pivotal role in the regulation of the early stages of immune responses. PMID:24076588
An MSI Tumor Specific Frameshift Mutation in a Coding Microsatellite of MSH3 Encodes for HLA-A0201-Restricted CD8+ Cytotoxic T Cell Epitopes

PubMed Central

Garbe, Yvette; Maletzki, Claudia; Linnebacher, Michael

2011-01-01

Background Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI+ colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. Methodology/Principal Findings FSP-specific CD8+ T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI+ colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. Conclusions We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were 386-FLLALWECSL (FSP18) and 387-LLALWECSL (FSP19) as well as 403-IVSRTLLLV (FSP23) and 402-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI+-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or –even more important– preventive purposes. PMID:22110587
An MSI tumor specific frameshift mutation in a coding microsatellite of MSH3 encodes for HLA-A0201-restricted CD8+ cytotoxic T cell epitopes.

PubMed

Garbe, Yvette; Maletzki, Claudia; Linnebacher, Michael

2011-01-01

Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI(+) colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. FSP-specific CD8(+) T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI(+) colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were (386)-FLLALWECSL (FSP18) and (387)-LLALWECSL (FSP19) as well as (403)-IVSRTLLLV (FSP23) and (402)-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI(+)-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or--even more important--preventive purposes.
Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28- T cells.

PubMed

Arosa, F A; Oliveira, L; Porto, G; da Silva, B M; Kruijer, W; Veltman, J; de Sousa, M

1997-03-01

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8+ CD28- T cells with a corresponding reduction in the percentage of CD8+ CD28+ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4+ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8+ CD28+ T cell population 'to expand', coinciding with an 'expansion' of CD8+ CD28- T cells in peripheral blood of HLA-A3+ but not HLA-A3- HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR+ but not CD45RO+ cells was also found within the peripheral CD8+ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8+ CD28+ T cells both in controls and HH were able to expand in vitro; (ii) that CD8+ CD28- T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8+ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard 51Cr-release assays when compared with CD8+ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8+ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.
A carbon nanotube-polymer composite for T-cell therapy

NASA Astrophysics Data System (ADS)

Fadel, Tarek R.; Sharp, Fiona A.; Vudattu, Nalini; Ragheb, Ragy; Garyu, Justin; Kim, Dongin; Hong, Enping; Li, Nan; Haller, Gary L.; Pfefferle, Lisa D.; Justesen, Sune; Harold, Kevin C.; Fahmy, Tarek M.

2014-08-01

Clinical translation of cell therapies requires strategies that can manufacture cells efficiently and economically. One promising way to reproducibly expand T cells for cancer therapy is by attaching the stimuli for T cells onto artificial substrates with high surface area. Here, we show that a carbon nanotube-polymer composite can act as an artificial antigen-presenting cell to efficiently expand the number of T cells isolated from mice. We attach antigens onto bundled carbon nanotubes and combined this complex with polymer nanoparticles containing magnetite and the T-cell growth factor interleukin-2 (IL-2). The number of T cells obtained was comparable to clinical standards using a thousand-fold less soluble IL-2. T cells obtained from this expansion were able to delay tumour growth in a murine model for melanoma. Our results show that this composite is a useful platform for generating large numbers of cytotoxic T cells for cancer immunotherapy.
Memory CD8+ T Cells Protect Dendritic Cells from CTL Killing1

PubMed Central

Watchmaker, Payal B.; Urban, Julie A.; Berk, Erik; Nakamura, Yutaro; Mailliard, Robbie B.; Watkins, Simon C.; van Ham, S. Marieke; Kalinski, Pawel

2010-01-01

CD8+ T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8+ T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8+ T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4+ and CD8+ T cell populations. Moreover, memory CD8+ T cells that release the DC-activating factor TNF-α before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4+ Th cells. The currently identified DC-protective function of memory CD8+ T cells helps to explain the phenomenon of CD8+ T cell memory, reduced dependence of recall responses on CD4+ T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization. PMID:18322193
Preparation, characterization and pharmacokinetics of fluorescence labeled propylene glycol alginate sodium sulfate

NASA Astrophysics Data System (ADS)

Li, Pengli; Li, Chunxia; Xue, Yiting; Zhang, Yang; Liu, Hongbing; Zhao, Xia; Yu, Guangli; Guan, Huashi

2014-08-01

A rapid and sensitive fluorescence labeling method was developed and validated for the microanalysis of a sulfated polysaccharide drug,namely propylene glycol alginate sodium sulfate (PSS), in rat plasma. Fluorescein isothiocyanate (FITC) was selected to label PSS, and 1, 6-diaminohexane was used to link PSS and FITC in order to prepare FITC-labeled PSS (F-PSS) through a reductive amination reaction. F-PSS was identified by UV-Vis, FT-IR and 1H-NMR spectrum. The cell stability and cytotoxicity of F-PSS were tested in Madin-Darby canine kidney (MDCK) cells. The results indicated that the labeling efficiency of F-PSS was 0.522% ± 0.0248% and the absolute bioavailability was 8.39%. F-PSS was stable in MDCK cells without obvious cytotoxicity. The method was sensitive and reliable; it showed a good linearity, precision, recovery and stability. The FITC labeling method can be applied to investigating the absorption and metabolism of PSS and other polysaccharides in biological samples.
T cell-recruiting triplebody 19-3-19 mediates serial lysis of malignant B-lymphoid cells by a single T cell

PubMed Central

Roskopf, Claudia C.; Schiller, Christian B.; Braciak, Todd A.; Kobold, Sebastian; Schubert, Ingo A.; Fey, Georg H.; Hopfner, Karl-Peter; Oduncu, Fuat S.

2014-01-01

Triplebody 19-3-19, an antibody-derived protein, carries three single chain fragment variable domains in tandem in a single polypeptide chain. 19-3-19 binds CD19-bearing lymphoid cells via its two distal domains and primary T cells via its CD3-targeting central domain in an antigen-specific manner. Here, malignant B-lymphoid cell lines and primary cells from patients with B cell malignancies were used as targets in cytotoxicity tests with pre-stimulated allogeneic T cells as effectors. 19-3-19 mediated up to 95% specific lysis of CD19-positive tumor cells and, at picomolar EC50 doses, had similar cytolytic potency as the clinically successful agent BlinatumomabTM. 19-3-19 activated resting T cells from healthy unrelated donors and mediated specific lysis of both autologous and allogeneic CD19-positive cells. 19-3-19 led to the elimination of 70% of CD19-positive target cells even with resting T cells as effectors at an effector-to-target cell ratio of 1 : 10. The molecule is therefore capable of mediating serial lysis of target cells by a single T cell. These results highlight that central domains capable of engaging different immune effectors can be incorporated into the triplebody format to provide more individualized therapy tailored to a patient’s specific immune status. PMID:25115385
Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

PubMed Central

Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

2013-01-01

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112
Labeling single cell for in-vivo study of cell fate mapping and lineage tracing

NASA Astrophysics Data System (ADS)

He, Sicong; Xu, Jin; Wu, Yi; Tian, Ye; Sun, Qiqi; Wen, Zilong; Qu, Jianan Y.

2018-02-01

Cell fate mapping and lineage tracing are significant ways to understanding the developmental origins of biological tissues. It requires labeling individual cells and tracing the development of their progeny. We develop an infrared laser-evoked gene operator heat-shock microscope system to achieve single-cell labeling in zebrafish. With a fluorescent thermometry technique, we measure the temperature increase in zebrafish tissues induced by infrared laser and identify the optimal heat shock conditions for single-cell gene induction in different types of zebrafish cells. We use this technique to study the fate mapping of T lymphocytes and discover the distinct waves of lymphopoiesis during the zebrafish development.
A semi high-throughput method for screening small bispecific antibodies with high cytotoxicity.

PubMed

Sugiyama, Aruto; Umetsu, Mitsuo; Nakazawa, Hikaru; Niide, Teppei; Onodera, Tomoko; Hosokawa, Katsuhiro; Hattori, Shuhei; Asano, Ryutaro; Kumagai, Izumi

2017-06-06

Small bispecific antibodies that induce T-cell-mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell-recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 10 3 -times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.
Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy.

PubMed

Yang, Degang; Shui, Tiejun; Miranda, Jake W; Gilson, Danny J; Song, Zhengyu; Chen, Jia; Shi, Chao; Zhu, Jianyu; Yang, Jun; Jing, Zhichun

2016-01-01

The persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions. Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings. Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity.
Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.
The Effects of TLR Activation on T-Cell Development and Differentiation

PubMed Central

Jin, Bo; Sun, Tao; Yu, Xiao-Hong; Yang, Ying-Xiang; Yeo, Anthony E. T.

2012-01-01

Invading pathogens have unique molecular signatures that are recognized by Toll-like receptors (TLRs) resulting in either activation of antigen-presenting cells (APCs) and/or costimulation of T cells inducing both innate and adaptive immunity. TLRs are also involved in T-cell development and can reprogram Treg cells to become helper cells. T cells consist of various subsets, that is, Th1, Th2, Th17, T follicular helper (Tfh), cytotoxic T lymphocytes (CTLs), regulatory T cells (Treg) and these originate from thymic progenitor thymocytes. T-cell receptor (TCR) activation in distinct T-cell subsets with different TLRs results in differing outcomes, for example, activation of TLR4 expressed in T cells promotes suppressive function of regulatory T cells (Treg), while activation of TLR6 expressed in T cells abrogates Treg function. The current state of knowledge of regarding TLR-mediated T-cell development and differentiation is reviewed. PMID:22737174
Improving T Cell Expansion with a Soft Touch

PubMed Central

Lambert, Lester H.; Goebrecht, Geraldine K.E.; De Leo, Sarah E.; O’Connor, Roddy S.; Nunez-Cruz, Selene; Li, Tai-De; Yuan, Jinglun; Milone, Michael C.; Kam, Lance C.

2017-01-01

Protein-coated microbeads provide a consistent approach for activating and expanding populations of T cells for immunotherapy, but don’t fully capture the properties of antigen presenting cells. In this report, we enhance T cell expansion by replacing the conventional, rigid bead with a mechanically soft elastomer. Polydimethylsiloxane (PDMS) was prepared in a microbead format and modified with activating antibodies to CD3 and CD28. Three different formulations of PDMS provided an extended proliferative phase in both CD4+-only and mixed CD4+/CD8+ T cell preparations. CD8+ T cells retained cytotoxic function, as measured by a set of biomarkers (perforin production, LAMP2 mobilization, and IFN-γ secretion) and an in vivo assay of targeted cell killing. Notably, PDMS beads presented a nanoscale polymer structure and higher rigidity than associated with conventional bulk material. These data suggest T cells respond to this higher rigidity, indicating an unexpected effect of curing conditions. Together, these studies demonstrate that adopting mechanobiology ideas into the bead platform can provide new tools for T cell-based immunotherapy. PMID:28122453
Treatment of mcf-7 breast cancer cells with a red grape wine polyphenol fraction results in disruption of calcium homeostasis and cell cycle arrest causing selective cytotoxicity.

PubMed

Hakimuddin, Fatima; Paliyath, Gopinadhan; Meckling, Kelly

2006-10-04

Food components influence the physiology by modulating gene expression and biochemical pathways within the human body. The disease-preventive roles of several fruit and vegetable components have been related to such properties. Polyphenolic components such as flavonoids are strong antioxidants and induce the expression of several xenobiotic-detoxifying enzymes. The mechanism of selective cytotoxicity induced by red grape wine polyphenols against MCF-7 breast cancer cells was investigated in relation to their interference with calcium homeostasis. MCF-7 cells showed an increase in cytosolic calcium levels within 10 min of treatment with the polyphenols. Immunohistochemical localization of calmodulin with secondary gold-labeled antibodies showed similar levels of gold labeling in both MCF-7 cells and the spontaneously immortalized, normal MCF-10A cell line. MCF-7 cells treated with the red wine polyphenol fraction (RWPF) showed swelling of endoplasmic reticulum, dissolution of the nucleus, and loss of plasma membrane integrity as well as reduced mitochondrial membrane potential. These cells were arrested at the G2/M interphase. By contrast, MCF-10A cells did not show such changes after RWPF treatment. The results suggest that polyphenol-induced calcium release may disrupt mitochondrial function and cause membrane damage, resulting in selective cytotoxicity toward MCF-7 cells. This property could further be developed toward breast cancer prevention strategies either independently or in conjunction with conventional prevention therapies where a positive drug-nutrient interaction can be demonstrated.
T-cell tracking using Cerenkov and Radioluminescence imaging.

PubMed

Boschi, F; De Sanctis, F; Ugel, S; Spinelli, A E

2018-05-16

Cancer immunotherapy is a promising strategy based on the ability of the immune system to kill selected cells. In the development of an effective T-cell therapy the non-invasive cell tracking methods play a crucial role. Here we investigate the potentialities of T-cell marked with radionuclides in order to detect their localization with imaging techniques in small animal rodents. A protocol to label T-cells with 32 P-ATP was tested and evaluated. The homing of 32 P-ATP labeled T lymphocytes was investigated by Cerenkov luminescence imaging and radioluminescence imaging The first approach relies on the acquisition of Cerenkov photons produced by the beta particles emitted by the 32 P internalized by lymphocytes; the second one on the detection of photons coming from the conversion of radioactive energy in light done by scintillator crystals layered on the animals. The results show that T-cell biodistribution can be optically observed by both Cerenkov and radioluminescence imaging in small animal rodents in in-vivo and ex-vivo acquisitions. T-cell localization in the tumor mass was definitively confirmed by flow cytometry. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Cytotoxicity Testing of Temporary Luting Cements with Two- and Three-Dimensional Cultures of Bovine Dental Pulp-Derived Cells

PubMed Central

Ülker, Hayriye Esra; Ülker, Mustafa; Gümüş, Hasan Önder; Yalçın, Muhammet; Şengün, Abdulkadir

2013-01-01

This study evaluated the cytotoxicity of eugenol-containing and eugenol-free temporary luting cements. For cytotoxicity testing, bovine pulp-derived cells transfected with Simian virus 40 Large T antigen were exposed to extracts of eugenol-containing (Rely X Temp E) and eugenol-free (Provicol, PreVISION CEM, and Rely X Temp NE) temporary luting cements for 24 h. The cytotoxicity of the same materials was also evaluated in a dentin barrier test device using three-dimensional cell cultures of bovine pulp-derived cells. The results of the cytotoxicity studies with two-dimensional cultures of bovine dental pulp-derived cells revealed that cell survival with the extracts of Rely X Temp E, Provicol, PreVISION CEM, and Rely X Temp NE was 89.1%, 84.9%, 92.3%, and 66.8%, respectively. Rely X Temp NE and Provicol showed cytotoxic effects on bovine dental pulp-derived cells (P < 0.05). The results of the dentin barrier test revealed that cell survival with the above-mentioned temporary cement was 101.5%, 91.9%, 93.5%, and 90.6%, respectively. None of the temporary luting cements significantly reduced cell survival compared with the negative control in the dentin barrier test (P > 0.05). Biologically active materials released from temporary luting cements may not influence the dentine-pulp complex if the residual dentine layer is at least 0.5 mm thick. PMID:23984419
The Role of T Lymphocytes in Cancer Patients Undergoing Immunotherapy with Autologous Dendritic Cells

PubMed Central

Rodrigues, Cláudia M.; Matias, Bruna F.; Murta, Eddie F.C.; Michelin, Márcia A.

2011-01-01

Introduction: Cancer stems from mutations in specific genes that induce uncontrolled cell proliferation. Dendritic cells (DCs) are important immunologic cells and play a crucial role in the induction of an antitumour response. Patients and methods: We examined the immune response mediated by T lymphocytes, helper T cells, cytotoxic T cells, and regulatory T cells, as well as the cytokines [interleukin (IL)-2, IL-12, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-10], produced by these cell populations, in cancer patients (N = 7) undergoing immunotheraphy with autologous DCs. Results: We observed an initial increase in T helper cells (CD4+) expressing IL-2, IFN-γ, IL-12, TNF-α, and IL-10 after initiation of treatment, with statistically significant for the cytokines IL-2, TNF-α and IL-10. A similar significant effect was observed for IL-2-expressing cytotoxic T cells (CD8+). The percentage of total T cells (CD3+) remained elevated throughout immunotherapy. Regulatory T cells (CD25+/FOXP3+) only showed high percentage of their maximum value when analyzed the pretreatment levels, with statistically significant. Conclusion: Immunotherapy with DCs stimulated the immune response, as evidenced by an increase in percent fluorescence of most cell populations investigated during the specified treatment period. PMID:21603246

The role of T lymphocytes in cancer patients undergoing immunotherapy with autologous dendritic cells.

PubMed

Rodrigues, Cláudia M; Matias, Bruna F; Murta, Eddie F C; Michelin, Márcia A

2011-01-01

Cancer stems from mutations in specific genes that induce uncontrolled cell proliferation. Dendritic cells (DCs) are important immunologic cells and play a crucial role in the induction of an antitumour response. We examined the immune response mediated by T lymphocytes, helper T cells, cytotoxic T cells, and regulatory T cells, as well as the cytokines [interleukin (IL)-2, IL-12, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-10], produced by these cell populations, in cancer patients (N = 7) undergoing immunotheraphy with autologous DCs. We observed an initial increase in T helper cells (CD4+) expressing IL-2, IFN-γ, IL-12, TNF-α, and IL-10 after initiation of treatment, with statistically significant for the cytokines IL-2, TNF-α and IL-10. A similar significant effect was observed for IL-2-expressing cytotoxic T cells (CD8+). The percentage of total T cells (CD3+) remained elevated throughout immunotherapy. Regulatory T cells (CD25+/FOXP3+) only showed high percentage of their maximum value when analyzed the pretreatment levels, with statistically significant. Immunotherapy with DCs stimulated the immune response, as evidenced by an increase in percent fluorescence of most cell populations investigated during the specified treatment period.
The Role of B Cells for in Vivo T Cell Responses to a Friend Virus-Induced Leukemia

NASA Astrophysics Data System (ADS)

Schultz, Kirk R.; Klarnet, Jay P.; Gieni, Randall S.; Hayglass, Kent T.; Greenberg, Philip D.

1990-08-01

B cells can function as antigen-presenting cells and accessory cells for T cell responses. This study evaluated the role of B cells in the induction of protective T cell immunity to a Friend murine leukemia virus (F-MuLV)-induced leukemia (FBL). B cell-deficient mice exhibited significantly reduced tumor-specific CD4^+ helper and CD8^+ cytotoxic T cell responses after priming with FBL or a recombinant vaccinia virus containing F-MuLV antigens. Moreover, these mice had diminished T cell responses to the vaccinia viral antigens. Tumor-primed T cells transferred into B cell-deficient mice effectively eradicated disseminated FBL. Thus, B cells appear necessary for efficient priming but not expression of tumor and viral T cell immunity.
The new numerology of immunity mediated by virus-specific CD8(+) T cells.

PubMed

Doherty, P C

1998-08-01

Our understanding of virus-specific CD8(+) T cell responses is currently being revolutionized by peptide-based assay systems that allow flow cytometric analysis of effector and memory cytotoxic T lymphocyte populations. These techniques are, for the first time, putting the analysis of T-cell-mediated immunity on a quantitative basis.
Colorectal cancer cells suppress CD4+ T cells immunity through canonical Wnt signaling.

PubMed

Sun, Xuan; Liu, Suoning; Wang, Daguang; Zhang, Yang; Li, Wei; Guo, Yuchen; Zhang, Hua; Suo, Jian

2017-02-28

Understanding how colorectal cancer escapes from immunosurveillance and immune attack is important for developing novel immunotherapies for colorectal cancer. In this study we evaluated the role of canonical Wnt signaling in the regulation of T cell function in a mouse colorectal cancer model. We found that colorectal cancer cells expressed abundant Wnt ligands, and intratumoral T cells expressed various Frizzled proteins. Meanwhile, both active β-catenin and total β-catenin were elevated in intratumoral T cells. In vitro study indicated that colorectal cancer cells suppressed IFN-γ expression and increased IL-17a expression in activated CD4+ T cells. However, the cytotoxic activity of CD8+ T cells was not altered by colorectal cancer cells. To further evaluate the importance of Wnt signaling for CD4+ T cell-mediated cancer immunity, β-catenin expression was enforced in CD4+ T cells using lentiviral transduction. In an adoptive transfer model, enforced expression of β-catenin in intratumoral CD4+ T cells increased IL-17a expression, enhanced proliferation and inhibited apoptosis of colorectal cancer cells. Taken together, our study disclosed a new mechanism by which colorectal cancer impairs T cell immunity.
Role of Proteus mirabilis MR/P fimbriae and flagella in adhesion, cytotoxicity and genotoxicity induction in T24 and Vero cells.

PubMed

Scavone, Paola; Villar, Silvia; Umpiérrez, Ana; Zunino, Pablo

2015-06-01

Proteus mirabilis is frequently associated with complicated urinary tract infections (UTI). It is proposed that several virulence factors are associated with P. mirabilis uropathogenicity. The aim of this work was to elucidate genotoxic and cytotoxic effects mediated by MR/P fimbriae and flagella in eukaryotic cells in vitro. Two cell lines (kidney- and bladder-derived) were infected with a clinical wild-type P. mirabilis strain and an MR/P and a flagellar mutant. We evaluated adhesion, genotoxicity and cytotoxicity by microscopy, comet assay and triple staining technique, respectively. Mutant strains displayed lower adhesion rates than the P. mirabilis wild-type strain and were significantly less effective to induce genotoxic and cytotoxic effects compared to the wild type. We report for the first time that P. mirabilis MR/P fimbriae and flagella mediate genotoxic and cytotoxic effects on eukaryotic cells, at least in in vitro conditions. These results could contribute to design new strategies for the control of UTI. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology

PubMed Central

Rauch, Jessica; Papp, Stefanie; Kuehl, Svenja; Richardt, Ulricke; Fleischer, Bernhard; Osterloh, Anke

2017-01-01

Endemic typhus caused by Rickettsia (R.) typhi is an emerging febrile disease that can be fatal due to multiple organ pathology. Here we analyzed the requirements for protection against R. typhi by T cells in the CB17 SCID model of infection. BALB/c wild-type mice generate CD4+ TH1 and cytotoxic CD8+ T cells both of which are sporadically reactivated in persistent infection. Either adoptively transferred CD8+ or CD4+ T cells protected R. typhi-infected CB17 SCID mice from death and provided long-term control. CD8+ T cells lacking either IFNγ or Perforin were still protective, demonstrating that the cytotoxic function of CD8+ T cells is not essential for protection. Immune wild-type CD4+ T cells produced high amounts of IFNγ, induced the release of nitric oxide in R. typhi-infected macrophages and inhibited bacterial growth in vitro via IFNγ and TNFα. However, adoptive transfer of CD4+IFNγ-/- T cells still protected 30–90% of R. typhi-infected CB17 SCID mice. These cells acquired a TH17 phenotype, producing high amounts of IL-17A and IL-22 in addition to TNFα, and inhibited bacterial growth in vitro. Surprisingly, the neutralization of either TNFα or IL-17A in CD4+IFNγ-/- T cell recipient mice did not alter bacterial elimination by these cells in vivo, led to faster recovery and enhanced survival compared to isotype-treated animals. Thus, collectively these data show that although CD4+ TH1 cells are clearly efficient in protection against R. typhi, CD4+ TH17 cells are similarly protective if the harmful effects of combined production of TNFα and IL-17A can be inhibited. PMID:28222146
Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

PubMed

Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

2016-01-01

This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.
Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma

PubMed Central

Quezada, Sergio A.; Peggs, Karl S.; Simpson, Tyler R.; Shen, Yuelei; Littman, Dan R.; Allison, James P.

2008-01-01

Interference with inhibitory immunological checkpoints controlling T cell activation provides new opportunities to augment cancer immunotherapies. Whereas cytotoxic T lymphocyte–associated antigen-4 blockade has shown promising preclinical and clinical results, therapeutic CD4+CD25+ T reg cell depletion has failed to consistently enhance immune-based therapies. Using B16/BL6, a transplantable murine melanoma model, we show a dichotomy between the effects of T reg cell depletion on tumor rejection dependent on whether depletion occurs before (prophylactic) or after (therapeutic) tumor engraftment. Failure to promote rejection with therapeutic depletion is not related to lack of T reg cell depletion, to elimination of CD25+ effector T cells, or to a failure to enhance systemic antitumor T cell responses, but correlates with failure of effector cells to infiltrate the tumor and increase the intratumor ratio of effector T cell/T reg cell. Finally, systemic antitumor responses generated upon therapeutic T reg cell depletion are significantly stronger than those generated in the presence of T reg cells, and are capable of eliciting rejection of established tumors after transfer into immunoablated recipients receiving combination immunotherapy. The data demonstrate a dissociation between measurable systemic responses and tumor rejection during CD25-directed T reg cell depletion, and suggest an alternative, clinically applicable strategy for the treatment of established tumors. PMID:18725522
Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4

PubMed Central

Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M.; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L.; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A.; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L.; Burgdorf, Sven

2016-01-01

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8+ T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte–associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality. PMID:27601670
Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

PubMed

Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L; Burgdorf, Sven

2016-09-20

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.
A complex of serine protease genes expressed preferentially in cytotoxic T-lymphocytes is closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14.

PubMed

Crosby, J L; Bleackley, R C; Nadeau, J H

1990-02-01

A complex of genes encoding serine proteases that are preferentially expressed in cytotoxic T-cells was shown to be closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14. A striking difference in recombination frequencies among linkage crosses was reported. Two genes, Np-1 and Tcra, which fail to recombine in crosses involving conventional strains of mice, were shown to recombine readily in interspecific crosses involving Mus spretus. This difference in recombination frequency suggests chromosomal rearrangements that suppress recombination in conventional crosses, recombination hot spots in interspecific crosses, or selection against recombinant haplotypes during development of recombinant inbred strains. Finally, a mutation called disorganization, which is located near the serine protease complex, is of considerable interest because it causes an extraordinarily wide variety of congenital defects. Because of the involvement of serine protease loci in several homeotic mutations in Drosophila, disorganization must be considered a candidate for a mutation in a serine protease-encoding gene.
Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28− T cells

PubMed Central

AROSA, F A; OLIVEIRA, L; PORTO, G; DA SILVA, B M; KRUIJER, W; VELTMAN, J; DE SOUSA, M

1997-01-01

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8+ CD28− T cells with a corresponding reduction in the percentage of CD8+ CD28+ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4+ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8+ CD28+ T cell population ‘to expand’, coinciding with an ‘expansion’ of CD8+ CD28− T cells in peripheral blood of HLA-A3+ but not HLA-A3− HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR+ but not CD45RO+ cells was also found within the peripheral CD8+ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8+ CD28+ T cells both in controls and HH were able to expand in vitro; (ii) that CD8+ CD28− T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8+ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard 51Cr-release assays when compared with CD8+ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8+ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH. PMID:9067531
Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043
Anti-inflammatory effects of Artemisia princeps in antigen-stimulated T cells and regulatory T cells.

PubMed

Chang, Sung Ho; Jung, Eun Jung; Park, Youn Hee; Lim, Dong Gyun; Ko, Na Young; Choi, Wahn Soo; Her, Erk; Kim, Soo Hyun; Choi, Kang Duk; Bae, Jae Ho; Kim, Sun Hee; Kang, Chi Dug; Han, Duck Jong; Kim, Song Cheol

2009-08-01

The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells and antigen-expanded regulatory T cells. CD4(+)CD25(-) T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [(3)H]thymidine and, after harvesting the cells, [(3)H]thymidine incorporation was measured. For analysis of interleukin-2 and interferon-gamma secreted from CD4(+)CD25(-) T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4(+)CD25(-) T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography-mass spectrometry. A. princeps (30 microg/ml) effectively suppressed proliferation of CD4(+)CD25(-) T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-gamma in anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3. The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4(+)CD25(-) T cells and increasing activity of expanded regulatory T cells.
Inhibition of phosphoantigen-mediated gammadelta T-cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells.

PubMed

Kunzmann, Volker; Kimmel, Brigitte; Herrmann, Thomas; Einsele, Hermann; Wilhelm, Martin

2009-02-01

Tumour growth promotes the expansion of CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) which suppress various arms of immune responses and might therefore contribute to tumour immunosurveillance. In this study, we found an inverse correlation between circulating Treg frequencies and phosphoantigen-induced gammadelta T-cell proliferation in cancer patients, which prompted us to address the role of Tregs in controlling the gammadelta T-cell arm of innate immune responses. In vitro, human Treg-peripheral blood mononuclear cell (PBMC) co-cultures strongly inhibited phosphoantigen-induced proliferation of gammadelta T cells and depletion of Tregs restored the impaired phosphoantigen-induced gammadelta T-cell proliferation of cancer patients. Tregs did not suppress other effector functions of gammadelta T cells such as cytokine production or cytotoxicity. Our experiments indicate that Tregs do not mediate their suppressive activity via a cell-cell contact-dependent mechanism, but rather secrete a soluble non-proteinaceous factor, which is independent of known soluble factors interacting with amino acid depletion (e.g. arginase-diminished arginine and indolamine 2,3-dioxygenase-diminished tryptophan) or nitric oxide (NO) production. However, the proliferative activity of alphabeta T cells was not affected by this cell-cell contact-independent suppressive activity induced by Tregs. In conclusion, these findings indicate a potential new mechanism by which Tregs can specifically suppress gammadelta T cells and highlight the strategy of combining Treg inhibition with subsequent gammadelta T-cell activation to enhance gammadelta T cell-mediated immunotherapy.
Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection

PubMed Central

Knuschke, Torben; Rotan, Olga; Bayer, Wibke; Kollenda, Sebastian; Dickow, Julia; Sutter, Kathrin; Hansen, Wiebke; Dittmer, Ulf; Lang, Karl S.; Epple, Matthias; Buer, Jan; Westendorf, Astrid M.

2018-01-01

T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR−/−) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections. PMID:29740425
Endosulfan decreases cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus.

PubMed

Téllez-Bañuelos, Martha Cecilia; Ortiz-Lazareno, Pablo Cesar; Jave-Suárez, Luis Felipe; Siordia-Sánchez, Victor Hugo; Bravo-Cuellar, Alejandro; Santerre, Anne; Zaitseva, Galina P

2014-05-01

The effect of the organochlorinated insecticide endosulfan, on the cytotoxic activity of Nile tilapia nonspecific cytotoxic cells (NCC) was assessed. Juvenile Nile tilapia were exposed to endosulfan (7 ppb) for 96 h and splenic NCC were isolated. Flow cytometric phenotyping of NCC was based on the detection of the NCC specific membrane signaling protein NCCRP-1 by using the monoclonal antibody Mab 5C6; granzyme expression was evaluated by quantitative RT-PCR. The cytotoxic activity of sorted NCC on HL-60 tumoral cells was assessed using propidium iodide (PI) staining of DNA in HL-60 nuclei, indicating dead cells. Nile tilapia splenic NCC had the ability to kill HL-60 tumoral cells, however, the exposure to endosulfan significantly reduced, by a 65%, their cytotoxic activity when using the effector:target ratio of 40:1. Additionally, the exposure to endosulfan tended to increase the expression of NCCRP-1, which is involved in NCC antigen recognition and signaling. Moreover, it decreased the expression of the granzyme gene in exposed group as compared with non-exposed group; however significant differences between groups were not detected. In summary, the acute exposure of Nile tilapia to sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species. Copyright © 2014 Elsevier Ltd. All rights reserved.
Analysis of the Effects of Cell Stress and Cytotoxicity on In ...

EPA Pesticide Factsheets

Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, concentration-dependent responses of 1063 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a diverse battery of 821 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to better distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress / cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least two viability/cytotoxicity assays within the concentration range tested (typically up to 100 M) activated a median of 12% of assay endpoints while those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (e.g., receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering of specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a g
FasL Mediates T-Cell Eradication of Tumor Cells Presenting Low Levels of Antigens | Center for Cancer Research

Cancer.gov

One approach to cancer immunotherapy, as opposed to therapeutic vaccination, is the transfusion of large numbers of tumor-specific killer T cells (cytotoxic T cells or CTLs) into patients. The body’s own defense killer T cells are a subgroup of T lymphocytes (a type of white blood cells) that are capable of inducing death in tumor cells. CTLs can cause the death of target cells either by releasing granules containing toxic molecules including perforin, or by producing a membrane protein called Fas ligand (FasL) which on interaction with the tumor cell results in cell death.
The effect of phloretin on human γδ T cells killing colon cancer SW-1116 cells.

PubMed

Zhu, Sheng-Ping; Liu, Gang; Wu, Xiao-Ting; Chen, Fu-Xing; Liu, Jun-Quan; Zhou, Zhong-Hai; Zhang, Jian-Fu; Fei, Su-Juan

2013-01-01

To explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells. γδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells. After cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31±3.00% to 78.40±10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35μg/ml to 18.75μg/ml, it could significantly proliferate the γδ T cell growth (P<0.05) and inhibit the growth of SW-1116 cells in dose-response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P<0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P<0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group. Phloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity. Copyright © 2012 Elsevier B.V. All rights reserved.

Adoptive transfer of natural killer cells promotes the anti-tumor efficacy of T cells.

PubMed

Goding, Stephen R; Yu, Shaohong; Bailey, Lisa M; Lotze, Michael T; Basse, Per H

2017-04-01

The density of NK cells in tumors correlates positively with prognosis in many types of cancers. The average number of infiltrating NK cells is, however, quite modest (approximately 30 NK cells/sq.mm), even in tumors deemed to have a "high" density of infiltrating NK cells. It is unclear how such low numbers of tumor-infiltrating NK cells can influence outcome. Here, we used ovalbumin-expressing tumor cell lines and TCR transgenic, OVA-specific cytotoxic T lymphocytes (OT-I-CTLs) to determine whether the simultaneous attack by anti-tumor CTLs and IL-2-activated NK (A-NK) cells synergistically increases the overall tumor cell kill and whether upregulation of tumor MHC class-I by NK cell-derived interferon-gamma (IFNγ) improves tumor-recognition and kill by anti-tumor CTLs. At equal E:T ratios, A-NK cells killed OVA-expressing tumor cells better than OT-I-CTLs. The cytotoxicity against OVA-expressing tumor cells increased by combining OT-I-CTLs and A-NK cells, but the increase was additive rather than synergistic. A-NK cells adenovirally-transduced to produce IL-12 (A-NK IL-12 ) produced high amounts of IFNγ. The addition of a low number of A-NK IL-12 cells to OT-I-CTLs resulted in a synergistic, albeit modest, increase in overall cytotoxicity. Pre-treatment of tumor cells with NK cell-conditioned medium increased tumor MHC expression and sensitivity to CTL-mediated killing. Pre-treatment of CTLs with NK cell-conditioned medium had no effect on CTL cytotoxicity. In vivo, MHC class-I expression by OVA-expressing B16 melanoma lung metastases increased significantly within 24-48h after adoptive transfer of A-NK IL-12 cells. OT-I-CTLs and A-NK IL-12 cells localized selectively and equally well into OVA-expressing B16 lung metastases and treatment of mice bearing 7-days-old OVA-B16 lung metastases with both A-NK IL-12 cells and OT-I-CTLs lead to a significant prolongation of survival. Thus, an important function of tumor-infiltrating NK cells may be to increase tumor
Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy

PubMed Central

Miranda, Jake W.; Gilson, Danny J.; Song, Zhengyu; Chen, Jia; Shi, Chao; Zhu, Jianyu; Yang, Jun; Jing, Zhichun

2016-01-01

Background The persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions. Methodology/Principal Findings Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings. Conclusions/Significance Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity. PMID:26751388
CD8+ T-cell immunosurveillance constrains lymphoid pre-metastatic myeloid cell accumulation

PubMed Central

Li, Wenzhao; Deng, Jiehui; Herrmann, Andreas; Priceman, Saul J.; Liang, Wei; Shen, Shudan; Pal, Sumanta K.; Hoon, Dave S.B.; Yu, Hua

2014-01-01

Increasing evidence suggests that pre-metastatic niches, consisting mainly of myeloid cells, provide microenvironment critical for cancer cell recruitment and survival to facilitate metastasis. While CD8+ T cells exert immunosurveillance in primary human tumors, whether they can exert similar effects on myeloid cells in the pre-metastatic environment is unknown. Here, we show that CD8+ T cells are capable of constraining pre-metastatic myeloid cell accumulation by inducing myeloid cell apoptosis in C57BL/6 mice. Antigen-specific CD8+ T-cell cytotoxicity against myeloid cells in pre-metastatic lymph nodes is compromised by Stat3. We demonstrate here that Stat3 ablation in myeloid cells leads to CD8+ T-cell activation and increased levels of IFN-γ and granzyme B in the pre-metastatic environment. Furthermore, Stat3 negatively regulates soluble antigen cross-presentation by myeloid cells to CD8+ T cells in the pre-metastatic niche. Importantly, in tumor-free lymph nodes of melanoma patients, infiltration of activated CD8+ T cells inversely correlates with STAT3 activity, which is associated with a decrease in number of myeloid cells. Our study suggested a novel role for CD8+ T cells in constraining myeloid cell activity through direct killing in the pre-metastatic environment, and the therapeutic potential by targeting Stat3 in myeloid cells to improve CD8+ T-cell immunosurveillance against metastasis. PMID:25310972
Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

PubMed Central

Dressel, Ralf; Guan, Kaomei; Nolte, Jessica; Elsner, Leslie; Monecke, Sebastian; Nayernia, Karim; Hasenfuss, Gerd; Engel, Wolfgang

2009-01-01

Background Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). Results We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. Conclusion Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific
Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.
T- and B-cell subpopulations in infectious mononucleosis

PubMed Central

Papamichail, M.; Sheldon, P. J.; Holborow, E. J.

1974-01-01

Mononuclear cells separated from the blood in fourteen cases of infectious mononucleosis at various intervals from the onset were tested for the presence of surface immunoglobulin and for ability to form spontaneous rosettes with washed sheep red blood cells. The mononucleosis during the acute phase of the illness consisted largely of a T lymphocytosis. The absolute count of T lymphocytes returned to the normal range approximately 2 months after the onset of the illness. B cells (bearing surface immunoglobulin) were only slightly increased in the acute phase. In four cases appreciable numbers of fluorescent rosetting cells were also present, and investigation suggested that these were T cells coated with anti-T-cell autoantibody. During the first 2 weeks of the illness responsiveness to phytohaemagglutinin was severely depressed, but thereafter returned towards normal. It is thought likely that in infectious mononucleosis the vast majority of atypical mononuclear cells are T cells proliferating in response to E-B virus-infected B cells, and cytotoxic towards them. ImagesFig. 3 PMID:4549622
Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

PubMed

Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R

2018-02-27

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.
Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity

PubMed Central

Davenport, A. J.; Cross, R. S.; Watson, K. A.; Liao, Y.; Shi, W.; Prince, H. M.; Beavis, P. A.; Trapani, J. A.; Kershaw, M. H.; Ritchie, D. S.; Darcy, P. K.; Jenkins, M. R.

2018-01-01

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. PMID:29440406
Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4+ Vα24 natural killer T cells expressing CD40L

PubMed Central

Nieda, M; Kikuchi, A; Nicol, A; Koezuka, Y; Ando, Y; Ishihara, S; Lapteva, N; Yabe, T; Tokunaga, K; Tadokoro, K; Juji, T

2001-01-01

Human Vα24 natural killer T (Vα24NKT) cells are activated by α-glycosylceramide-pulsed dendritic cells (DCs) in a CD1d-dependent and T-cell receptor-mediated manner. There are two major subpopulations of Vα24NKT cells, CD4– CD8– Vα24NKT and CD4+ Vα24NKT cells. We have recently shown that activated CD4– CD8– Vα24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of Vα24NKT cells is currently limited. We aimed to investigate whether CD4+ Vα24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4+ Vα24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4+ Vα24NKT cells, but not with resting CD4+ Vα24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb. Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40–CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Vα24NKT cells. The apoptosis of DCs from normal donors, triggered by the CD40–CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4+ Vα24NKT cells by virtue of apoptosis of DCs. PMID:11260318
Human Epidermal Langerhans Cells Maintain Immune Homeostasis in Skin by Activating Skin Resident Regulatory T Cells

PubMed Central

Seneschal, Julien; Clark, Rachael A.; Gehad, Ahmed; Baecher-Allan, Clare M.; Kupper, Thomas S.

2013-01-01

Recent discoveries indicate that the skin of a normal individual contains 10-20 billion resident memory T cells ( which include various T helper, T cytotoxic, and T regulatory subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both in vitro and in vivo, resting epidermal Langerhan cells (LC) selectively and specifically induced the activation and proliferation of skin resident regulatory T cells (Treg), a minor subset of skin resident memory T cells. In the presence of foreign pathogen, however, the same LC activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells activation. These underappreciated properties of LC: namely maintenance of tolerance in normal skin, and activation of protective skin resident memory T cells upon infectious challenge, help clarify the role of LC in skin. PMID:22560445
Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms.

PubMed

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-07-01

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (alpha-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and alpha-GalCer in the treatment of mice engrafted with CD1d(+) lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence alpha-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and alpha-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d(+) masses. In addition, CD1d-restricted T-cell treatment plus alpha-GalCer eradicated small C1R-CD1d(+) nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and alpha-GalCer may represent a new immunotherapeutic tool for treatment of CD1d(+) hematologic malignancies.
Heterogeneity of Human CD4(+) T Cells Against Microbes.

PubMed

Sallusto, Federica

2016-05-20

CD4(+) T helper (Th) cells play a central role in the adaptive immune response by providing help to B cells and cytotoxic T cells and by releasing different types of cytokines in tissues to mediate protection against a wide range of pathogenic microorganisms. These functions are performed by different types of Th cells endowed with distinct migratory capacities and effector functions. Here we discuss how studies of the human T cell response to microbes have advanced our understanding of Th cell functional heterogeneity, in particular with the discovery of a distinct Th1 subset involved in the response to Mycobacteria and the characterization of two types of Th17 cells specific for extracellular bacteria or fungi. We also review new approaches to dissect at the clonal level the human CD4(+) T cell response induced by pathogens or vaccines that have revealed an unexpected degree of intraclonal diversification and propose a progressive and selective model of CD4(+) T cell differentiation.
High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these
Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-06-05

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.
Reappraisal of nodal Epstein-Barr Virus-negative cytotoxic T-cell lymphoma: identification of indolent CD5+ diseases.

PubMed

Yamashita, Daisuke; Shimada, Kazuyuki; Takata, Katsuyoshi; Miyata-Takata, Tomoko; Kohno, Kei; Satou, Akira; Sakakibara, Ayako; Nakamura, Shigeo; Asano, Naoko; Kato, Seiichi

2018-05-29

Nodal cytotoxic molecule (CM)-positive peripheral T-cell lymphoma (CTL) has recently been recognized as a clinicopathologically distinct disease. To further characterize this disease, here we compared 58 patients with Epstein-Barr virus (EBV)-negative CTL to 48 patients with EBV-positive CTL. The two groups did not differ in histopathology, T-cell receptor (TCR) expression or rearrangement incidences, or survival curves. However, patients with EBV-negative CTL less frequently showed hepatic involvement (P = 0.007), B symptoms (P = 0.020), hemophagocytosis (P = 0.024), and detectable CD4 (P = 0.002) and CD5 (P = 0.009). Univariate and multivariate analyses identified three factors that independently predicted favorable survival, onset age <60 years (P = 0.002), CD5 expression (P = 0.002), and mixed morphology (P = 0.013), TCRαβ was not an independent predictor (P = 0.30), but was strongly linked with long survivorship among patients younger than 60 years old. A prognostic model incorporating these factors worked well for prognostic delineation, independently of the International Prognostic Index (P = 0.007 vs. P = 0.082) and Prognostic Index for PTCL (P = 0.020 vs. P = 0.15). Moreover, this constellation of findings indicated two nodal indolent diseases: CD5 + TCRαβ (n = 13), and CD5 + NK-cell type lacking TCR expression or clonal TCRγ rearrangement (n = 4). The survival curves for these two groups were significantly superior to others (n = 29, P < 0.001). These diseases appear to be unique in their indolent clinical behavior, and should be managed differently from other diseases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Cytotoxic potential of lung CD8(+) T cells increases with chronic obstructive pulmonary disease severity and with in vitro stimulation by IL-18 or IL-15.

PubMed

Freeman, Christine M; Han, MeiLan K; Martinez, Fernando J; Murray, Susan; Liu, Lyrica X; Chensue, Stephen W; Polak, Timothy J; Sonstein, Joanne; Todt, Jill C; Ames, Theresa M; Arenberg, Douglas A; Meldrum, Catherine A; Getty, Christi; McCloskey, Lisa; Curtis, Jeffrey L

2010-06-01

Lung CD8(+) T cells might contribute to progression of chronic obstructive pulmonary disease (COPD) indirectly via IFN-gamma production or directly via cytolysis, but evidence for either mechanism is largely circumstantial. To gain insights into these potential mechanisms, we analyzed clinically indicated lung resections from three human cohorts, correlating findings with spirometrically defined disease severity. Expression by lung CD8(+) T cells of IL-18R and CD69 correlated with severity, as did mRNA transcripts for perforin and granzyme B, but not Fas ligand. These correlations persisted after correction for age, smoking history, presence of lung cancer, recent respiratory infection, or inhaled corticosteroid use. Analysis of transcripts for killer cell lectin-like receptor G1, IL-7R, and CD57 implied that lung CD8(+) T cells in COPD do not belong to the terminally differentiated effector populations associated with chronic infections or extreme age. In vitro stimulation of lung CD8(+) T cells with IL-18 plus IL-12 markedly increased production of IFN-gamma and TNF-alpha, whereas IL-15 stimulation induced increased intracellular perforin expression. Both IL-15 and IL-18 protein expression could be measured in whole lung tissue homogenates, but neither correlated in concentration with spirometric severity. Although lung CD8(+) T cell expression of mRNA for both T-box transcription factor expressed in T cells and GATA-binding protein 3 (but not retinoic acid receptor-related orphan receptor gamma or alpha) increased with spirometric severity, stimulation of lung CD8(+) T cells via CD3epsilon-induced secretion of IFN-gamma, TNF-alpha, and GM-CSF, but not IL-5, IL-13, and IL-17A. These findings suggest that the production of proinflammatory cytokines and cytotoxic molecules by lung-resident CD8(+) T cells contributes to COPD pathogenesis.
Human germinal center CD4+CD57+ T cells act differently on B cells than do classical T-helper cells.

PubMed

Bouzahzah, F; Bosseloir, A; Heinen, E; Simar, L J

1995-01-01

We have isolated two subtypes of helper T cells from human tonsils: CD4+CD57+ cells, mostly located in the germinal center (GC), and CD4+CD57- cells, distributed through the interfollicular areas but also present in the GC. In a functional study, we have compared the capacities of these T-cell subtypes to stimulate B cells in cocultures. In order to block T-cell proliferation while maintaining their activation level, we pretreated isolated T cells with mitomycin C prior to culture in the presence of B cells and added polyclonal activators such as PHA and Con A, combined or not with IL-2. Contrary to CD4+ CD57- cells, CD4+CD57+ cells did not markedly enhance B-cell proliferation. Even when sIgD.B cells typical of germinal center cells were tested, the CD4+CD57+ cells had no significant effect. This is in accordance with the location of these cells: They mainly occupy the light zones of the GC where few B cells divide. Even when added to preactivated, actively proliferating cells, CD4+CD57 cells failed to modulate B-cell multiplication. On the supernatants of B-cell-T-cell cocultures, we examined by the ELISA technique the effect of T cells on Ig synthesis. Contrary to CD57+ T cells, whose effect was strong, CD57- T cells weakly stimulated Ig synthesis. More IgM than IgG was generally found. Because CD57 antigen is a typical marker of natural killer cells, we tested the cytolytic activity of tonsillar CD4+CD57+ cells on K562 target cells. Unlike NK cells, neither CD4+CD57+ nor CD4+CD57- cells exhibit any cytotoxicity. Thus, germinal center CD4+CD57+ cells are not cytolytic and do not strongly stimulate either B-cell proliferation or Ig secretion. CD4+CD57- cells, however, enhance B-cell proliferation and differentiation, thus acting like the classical helper cells of the T-dependent areas.
4-1BB regulates NKG2D costimulation in human cord blood CD8+ T cells.

PubMed

Kim, Young-June; Han, Myung-Kwan; Broxmeyer, Hal E

2008-02-01

Ligation of NKG2D, a potent costimulatory receptor, can be either beneficial or detrimental to CD8(+) cytotoxic T cell (CTL) responses. Factors for these diverse NKG2D effects remain elusive. In this study, we demonstrate that 4-1BB, another costimulatory receptor, is an essential regulator of NKG2D in CD8(+) T cells. Costimulation of NKG2D caused down-modulation of NKG2D, but induced 4-1BB expression on the cell surface, even in the presence of TGF-beta1, which inhibits 4-1BB expression. Resulting NKG2D(-)4-1BB(+) cells were activated but still in an immature state with low cytotoxic activity. However, subsequent 4-1BB costimulation induced cytotoxic activity and restored down-modulated NKG2D. The cytotoxic activity and NKG2D expression induced by 4-1BB on NKG2D(+)4-1BB(+) cells were refractory to TGF-beta1 down-modulation. Such 4-1BB effects were enhanced by IL-12. In contrast, in the presence of IL-4, 4-1BB effects were abolished because IL-4 down-modulated NKG2D and 4-1BB expression in cooperation with TGF-beta1, generating another CD8(+) T-cell type lacking both NKG2D and 4-1BB. These NKG2D(-)4-1BB(-) cells were inert and unable to gain cytotoxic activity. Our results suggest that 4-1BB plays a critical role in protecting NKG2D from TGF-beta1-mediated down-modulation. Co-expression of NKG2D and 4-1BB may represent an important biomarker for defining competency of tumor infiltrating CD8(+) T cells.
(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

PubMed

Sato, Noriko; Wu, Haitao; Asiedu, Kingsley O; Szajek, Lawrence P; Griffiths, Gary L; Choyke, Peter L

2015-05-01

To develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy. This study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses. (89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7). We have developed a (89)Zr-oxine complex cell tracking technique
89Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies

PubMed Central

Wu, Haitao; Asiedu, Kingsley O.; Szajek, Lawrence P.; Griffiths, Gary L.; Choyke, Peter L.

2015-01-01

Purpose To develop a clinically translatable method of cell labeling with zirconium 89 (89Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy. Materials and Methods This study was approved by the institutional animal care committee. 89Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of 89Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444–555 kBq/[5 × 106] cells, n = 5) and CTLs (185 kBq/[5 × 106] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 106] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses. Results 89Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%–43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. 89Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7). Conclusion We have developed a 89Zr

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A{sub 2}-induced degranulation in mast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishikawa, Hirofumi; Kitani, Seiichi, E-mail: drkitani@kaiyodai.ac.jp

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of {beta}-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation andmore » cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G{sub M1}), di-sialoganglioside (G{sub D1a}) and tri-sialoganglioside (G{sub T1b}). In contrast, honeybee venom-derived phospholipase A{sub 2} induced the net degranulation directly without cytotoxicity, which was not inhibited by G{sub M1}, G{sub D1a} and G{sub T1b}. For analysis of distribution of G{alpha}{sub q} and G{alpha}{sub i} protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of G{alpha}{sub q} and G{alpha}{sub i} at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A{sub 2}-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A{sub 2}-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.« less
Chemical composition and cytotoxic activity of Garcinia atroviridis Griff. ex T. Anders. essential oils in combination with tamoxifen.

PubMed

Tan, Wen-Nee; Lim, Jia-Qin; Afiqah, Fatin; Nik Mohamed Kamal, Nik Nur Syazni; Abdul Aziz, Fatin Athirah; Tong, Woei-Yenn; Leong, Chean-Ring; Lim, Jun-Wei

2018-04-01

Garcinia atroviridis Griff. ex T. Anders. is used as a medication agent in folkloric medicine. The present study was to examine the chemical composition of the stem bark and leaf of G. atroviridis as well as their cytotoxic effects against MCF-7 cells. The constituents obtained by hydrodistillation were identified using GC-MS. The stem bark oil (EO-SB) composed mainly the palmitoleic acid (51.9%) and palmitic acid (21.9%), while the leaf oil (EO-L) was dominated by (E)-β-farnesene (58.5%) and β-caryophyllene (16.9%). Treatment of MCF-7 cells using EO-L (100 μg/mL) caused more than 50% cell death while EO-SB did not induce cytotoxic effect. EO-L has stimulated the growth of BEAS-2B normal cells, but not in MCF-7 cancerous cells. The IC 50 of EO-L in MCF-7 and BEAS-2B cells were 71 and 95 μg/mL, respectively. A combination treatment of EO-L and tamoxifen induced more cell death than the treatment with drug alone at lower doses.
Cytotoxicity of protein corona-graphene oxide nanoribbons on human epithelial cells

NASA Astrophysics Data System (ADS)

Mbeh, Doris A.; Akhavan, Omid; Javanbakht, Taraneh; Mahmoudi, Morteza; Yahia, L.'Hocine

2014-11-01

Graphene oxide nanoribbons (GONRs) were synthesized using an oxidative unzipping of multi-walled carbon nanotubes. The interactions of the GONRs with various concentrations of fetal bovine serum or human plasma serum indicated that the GONRs were functionalized substantially by the albumin originated from the two different protein sources. Then, concentration-dependent cytotoxicity of the protein-functionalized GONRs on human epithelial cells was studied. Although the GONRs with concentrations ≤50 μg/mL did not exhibit significant cytotoxicity on the cells (with the cell viability >85%), the concentration of 100 μg/mL exhibited significant cytotoxicity including prevention of cell proliferation and induction of cell apoptosis. These results can provide more in-depth understanding about cytotoxic effects of graphene nanostructures which can be functionalized by the proteins of media.
Clinical methods of cryopreservation for donor lymphocyte infusions vary in their ability to preserve functional T-cell subpopulations.

PubMed

Worsham, D Nicole; Reems, Jo-Anna; Szczepiorkowski, Zbigniew M; McKenna, David H; Leemhuis, Thomas; Mathew, Aby J; Cancelas, Jose A

2017-06-01

Cryopreserved donor lymphocyte infusion (DLI) products are manufactured and administered to treat relapse after allogeneic hematopoietic stem cell transplantation. Reported clinical responses to DLIs vary broadly, even within the same group of patients. While there is an implicit recognition of the fact that different manufacturing protocols may have specific effects on different cell types, cryopreservation protocols are frequently derived from our experience in the cryopreservation of stem cell products and do not account for the heterogeneous functional nature of DLI T-cell populations. Here, we report the results of a prospective, multicenter trial on the effect of four different cryopreservation solutions that were used to freeze DLIs compared to control DLIs that were refrigerated overnight. Cryopreserved postthawed and refrigerated specimens were analyzed side by side for their T-cell subpopulation content and viability, as well as T-cell proliferation, cytokine secretion, and cytotoxic activities. This study indicates that "homemade" 10% dimethyl sulfoxide (DMSO) results in reduced viability of different CD4+ T-cell populations, including T-helper, T-cytotoxic, and T-regulatory populations, and a decrease in their proliferative and cytotoxic response to immunologically relevant stimuli, while the use of solutions containing 5% DMSO with intracellular-like cryoprotectant stabilizers maintains T-cell function at levels similar to refrigerated control samples. This study has important implications in determining the best cryoprotectant solution for specific clinical applications in allogeneic immunotherapy. © 2017 AABB.
Cytotoxicity of extracts of spices to cultured cells.

PubMed

Unnikrishnan, M C; Kuttan, R

1988-01-01

The cytotoxicity of the extracts from eight different spices used in the Indian diet was determined using Dalton's lymphoma ascites tumor cells and human lymphocytes in vitro and Chinese Hamster Ovary cells and Vero cells in tissue culture. Alcoholic extracts of the spices were found to be more cytotoxic to these cells than their aqueous extracts. Alcoholic extracts of several spices inhibited cell growth at concentrations of 0.2-1 mg/ml in vitro and 0.12-0.3 mg/ml in tissue culture. Ginger, pippali (native to India; also called dried catkins), pepper, and garlic showed the highest activity followed by asafetida, mustard, and horse-gram (native to India). These extracts also inhibited the thymidine uptake into DNA.
Divergent effects of norepinephrine, dopamine and substance P on the activation, differentiation and effector functions of human cytotoxic T lymphocytes

PubMed Central

2009-01-01

Background Neurotransmitters are important regulators of the immune system, with very distinct and varying effects on different leukocyte subsets. So far little is known about the impact of signals mediated by neurotransmitters on the function of CD8+ T lymphocytes. Therefore, we investigated the influence of norepinephrine, dopamine and substance P on the key tasks of CD8+ T lymphocytes: activation, migration, extravasation and cytotoxicity. Results The activation of naïve CD8+ T lymphocytes by CD3/CD28 cross-linking was inhibited by norepinephrine and dopamine, which was caused by a downregulation of interleukin (IL)-2 expression via Erk1/2 and NF-κB inhibition. Furthermore, all of the investigated neurotransmitters increased the spontaneous migratory activity of naïve CD8+ T lymphocytes with dopamine being the strongest inducer. In contrast, activated CD8+ T lymphocytes showed a reduced migratory activity in the presence of norepinephrine and substance P. With regard to extravasation we found norepinephrine to induce adhesion of activated CD8+ T cells: norepinephrine increased the interleukin-8 release from endothelium, which in turn had effect on the activated CXCR1+ CD8+ T cells. At last, release of cytotoxic granules from activated cells in response to CD3 cross-linking was not influenced by any of the investigated neurotransmitters, as we have analyzed by measuring the β-hexosamidase release. Conclusion Neurotransmitters are specific modulators of CD8+ T lymphocytes not by inducing any new functions, but by fine-tuning their key tasks. The effect can be either stimulatory or suppressive depending on the activation status of the cells. PMID:19968887
Cytotoxic activity of natural killer cells in vitro under microgravity

NASA Astrophysics Data System (ADS)

Grigorieva, O. V.; Buravkova, L. B.; Rykova, M. P.

2005-08-01

Changes in the immune response during space flight are close relation to functions of NK lymphocytes and their ability to interact with target cells. The aim of this research was to study NK cells cytotoxic activity and their ability to produce cytokines under microgravity in vitro. The modification of the method to study NK cells cytotoxic activity with the use of human peripheral blood mononuclear cells and myeloblasts K-562 (as target cells) proved highly effective (Buravkova et al., 2004). The flight experiment "Cell-to-cell interaction" with the use of the special device "Fibroblast-1" was carried out by Russian cosmonauts within the first two days after the docking when a new crew was taking over on International Space Station (ISS 8 - 10). The data collected on board ISS revealed that NK lymphocytes cytotoxic activity in vitro can increase under microgravity. The ground-based simulation experiments showed that long-term changes in gravity vector direction clinorotation resulted in a smaller increase of NK cells cytotoxic activity than it did in microgravity. As lymphocytes produce cytokines while interacting with target cells, the levels of TNF-α, IL-1α, IL- 2, IL-6 in cell-conditioned medium were assessed. The data showed that microgravity has varied effects on cytokines production level.
Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-08-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.
T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells

PubMed Central

Witkover, Aviva; Tanaka, Yuetsu; Fields, Paul; Bangham, Charles R. M.

2016-01-01

There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease. PMID:27893842
Improve T Cell Therapy in Neuroblastoma

DTIC Science & Technology

2015-09-01

bioluminescence was then measured overtime. The graph is representative of one of 4 experiments using CMV-CTLs from 4 donors. Panel E. Kaplan-Meier...whole-cell vaccine expressing the iC9 gene and labeled with an enhanced firefly luciferase. Tumor growth was measured by in vivo imaging. Panel E...down regulation in LTE -T cells is not caused by specific culture conditions. T lymphocytes were activated with immobilized OKT3 (1 μg ml) and
Expansion of natural killer cell receptor (CD94/NKG2A)-expressing cytolytic CD8 T cells and CD4+CD25+ regulatory T cells from the same cord blood unit.

PubMed

Tanaka, Junji; Sugita, Junichi; Kato, Naoko; Toubai, Tomomi; Ibata, Makoto; Shono, Yusuke; Ota, Shuichi; Kondo, Takeshi; Kobayashi, Takahiko; Kobayashi, Masanobu; Asaka, Masahiro; Imamura, Masahiro

2007-10-01

Cord blood contains a significant number of precursor cells that differentiate to cytotoxic effector cells and immunoregulatory cells. We tried to expand inhibitory natural killer cell receptor CD94-expressing CD8 T cells with cytolytic activity and CD4(+)CD25(+) regulatory T cells from the same cord cell unit. Cytotoxic CD94-expressing CD8 T cells were expanded from CD4-depleted cord blood using an immobilized anti-CD3 monoclonal antibody and a cytokine and also CD4(+)CD25(+) regulatory T cells were expanded from a CD4-enriched fraction derived from the same cord blood unit using anti-CD3/CD28 monoclonal antibody-coated Dynabeads and cytokines. We were able to obtain a more than 1000-fold expansion of CD94-expressing CD8 T cells and a more than 50-fold expansion of CD4(+)CD25(+) cells from the same cord blood unit. These expanded CD4(+)CD25(+) cells expressed FoxP3 mRNA at a level about 100-fold higher than that in isolated CD25(-) cells and could suppress allogeneic mixed lymphocyte culture by >80% (effector cells: CD4(+)CD25(+) cells = 2:1). Cytolytic activities of purified CD94-expressing cells detected by a 4-hour (51)Cr release assay against K562 were >60%. Coculture of CD94-expressing cells with expanded CD4(+)CD25(+) cells did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells. These expanded cytolytic CD94-expressing CD8 cells might be able to induce a graft-vs-leukemia effect without enhancing graft-vs-host disease, and CD4(+)CD25(+) cells might be able to suppress allogeneic responses, including graft-vs-host disease and graft rejection after cord blood transplantation.
Cytotoxicity of Etch-and-Rinse, Self-Etch, and Universal Dental Adhesive Systems in Fibroblast Cell Line 3T3

PubMed Central

Bernardo, Cintia Fernanda de Freitas; de Souza, Francielly Fernanda de Freitas A.; Michél, Milton Domingos; Ribeiro, Camila Nunes de Morais; Germano, Sandro; Maluf, Daniela Florencio

2017-01-01

The aim of this study was to evaluate in fibroblast cultures the direct cytotoxic effects of etch-and-rinse, self-etch, and universal adhesive systems. The sterile glass cover slips (n = 3) were then immersed in culture medium to obtain the eluates for the experimental groups: (1) Adper™ Single Bond 2; (2) Ambar; (3) Adper™ Scotchbond™ Multi-Purpose; (4) Scotchbond™ Universal; (5) Ambar Universal; and (6) OptiBond All-In-One. As a negative control, sterile glass cover slips were immersed in culture medium only. After 24 h, the eluate obtained was applied on fibroblast culture. Cell viability and cell morphology were evaluated by MTT assay and SEM, respectively. Data were analyzed by Kruskal–Wallis and Mann–Whitney tests (α = 0.05). All adhesive systems except universal reduced cell viability in 3T3 cells to between 26.04% and 56.57%, and Scotchbond Universal and Ambar Universal reduced cell viability to 2.13% and 3.57%, respectively, when compared to the negative control. Cytoplasmic membrane shrinkage and cell-free areas with residual membrane fragments from dead cells were observed. In conclusion, improvements in universal adhesive system formulations and their mechanisms of action are not accompanied by increased toxicity compared with those in other systems, warranting commitment to the use of these dentin-pulp complexes. PMID:29109829
Peripheral blood TIM-3 positive NK and CD8+ T cells throughout pregnancy: TIM-3/galectin-9 interaction and its possible role during pregnancy.

PubMed

Meggyes, Matyas; Miko, Eva; Polgar, Beata; Bogar, Barbara; Farkas, Balint; Illes, Zsolt; Szereday, Laszlo

2014-01-01

The T-cell immunoglobulin and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important negative regulator of Th1 immunity and tolerance induction. Data about the TIM-3/Gal-9 pathway in the pathogenesis of human diseases is emerging, but their possible role during human pregnancy is not precisely known. The aim of our study was to investigate the number, phenotype and functional activity of TIM-3+ peripheral blood mononuclear cells during healthy human pregnancy. 57 healthy pregnant women [first trimester (n = 16); second trimester (n = 19); third trimester (n = 22)] and 30 non-pregnant controls were enrolled in the study. We measured the surface expression of TIM-3 by cytotoxic T cells, NK cells and NK cell subsets as well as Galectin-9 expression by regulatory T cells by flow cytometry. We analyzed the cytokine production and cytotoxicity of TIM3+ and TIM3- CD8 T and NK cells obtained from non-pregnant and healthy pregnant women at different stages of pregnancy by flow cytometry. Serum Galectin-9 levels were measured by ELISA. Our results show that the numbers of peripheral NK and cytotoxic T cells and their TIM-3 expression do not change between the first, second and third trimesters of pregnancy. Compared to non-pregnant individuals, regulatory T cells show higher level of Galectin-9 expression as pregnancy proceeds, which is in line with the level of Galectin-9 in the patients sera. Cytotoxic T cells, NK cells and NK cell subsets expressing TIM-3 molecule show altered cytokine production and cytotoxicity during pregnancy compared to non-pregnant individuals. Our results indicate that Galectin-9 expressing regulatory T cells, TIM-3+ cytotoxic T cells and NK cells could play an important role in the maintenance of healthy pregnancy.
Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.
How Do CD4+ T Cells Detect and Eliminate Tumor Cells That Either Lack or Express MHC Class II Molecules?

PubMed Central

Haabeth, Ole Audun Werner; Tveita, Anders Aune; Fauskanger, Marte; Schjesvold, Fredrik; Lorvik, Kristina Berg; Hofgaard, Peter O.; Omholt, Hilde; Munthe, Ludvig A.; Dembic, Zlatko; Corthay, Alexandre; Bogen, Bjarne

2014-01-01

CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR) transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor-specific antigen by host antigen-presenting cells (APCs) appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315), where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR-transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-γ stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed. PMID:24782871
Practical cell labeling with magnetite cationic liposomes for cell manipulation.

PubMed

Ito, Hiroshi; Nonogaki, Yurika; Kato, Ryuji; Honda, Hiroyuki

2010-07-01

Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10(3)-10(5) cells for personalized cell processing, we determined that 10 microg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method. 2010. Published by Elsevier B.V.
Qualitative features of the HIV-specific CD8+ T-cell response associated with immunologic control.

PubMed

Hersperger, Adam R; Migueles, Stephen A; Betts, Michael R; Connors, Mark

2011-05-01

Over the past 2 years, a clearer picture has emerged regarding the properties of HIV-specific CD8+ T cells associated with immunologic control of HIV replication. These properties represent a potential mechanism by which rare patients might control HIV replication in the absence of antiretroviral therapy. This review addresses the background and recent findings that have lead to our current understanding of these mechanism(s). Patients with immunologic control of HIV are not distinguished by targeted specificities, or greater numbers or breadth of their HIV-specific CD8+ T-cell response. For this reason, recent work has focused greater attention on qualitative features of this response. The qualitative features most closely associated with immunologic control of HIV are related to the granule-exocytosis-mediated elimination of HIV-infected CD4 T cells. The ability of HIV-specific CD8+ T cells to increase their contents of proteins known to mediate cytotoxicity, such as granzyme B and perforin, appears to be a critical means by which HIV-specific cytotoxic capacity is regulated. Investigation from multiple groups has now focused upon HIV-specific CD8+ T-cell granule-exocytosis-mediated cytotoxicity as a correlate of immunologic control of HIV. In the near future, a more detailed understanding of the qualities associated with immunologic control may provide critical insights regarding the necessary features of a response that should be stimulated by immunotherapies or T-cell-based vaccines.
A phase I-II trial to examine the toxicity of CMV- and EBV-specific cytotoxic T lymphocytes when used for prophylaxis against EBV and CMV disease in recipients of CD34-selected/T cell-depleted stem cell transplants.

PubMed

Lucas, K G; Sun, Q; Burton, R L; Tilden, A; Vaughan, W P; Carabasi, M; Salzman, D; Ship, A

2000-07-01

Epstein-Barr virus (EBV)-induced lymphoproliferative disease and cytomegalovirus (CMV) infection are major causes of morbidity and mortality in individuals with compromised cellular immunity. Although anti-viral pharmacological agents exist, severe side effects such as myelosuppression often limit the application of these medications. Infusion of ex vivo-expanded, virus-specific cytotoxic T-lymphocytes (CTL) has been proven to be safe and efficacious for the prophylaxis and treatment of EBV and CMV complications. While EBV-specific CTL can be readily and reliably produced with EBV-immortalized B-lymphoblastoid cell lines (BLCL) as stimulators, current protocols for CMV-specific CTL, which use CMV-infected fibroblasts as stimulators, may be associated with alloreactivity and the need for cloning, as well as the potential for exposure to human blood-born infectious agents. Our laboratory has developed a novel system to generate EBV/CMV-bi-specific CTL by co-culturing PBMC with autologous BLCL expressing a CMV protein pp65 (BLCLpp65) (Sun et al., 1999). pp65, an immunodominant CMV antigen, is transduced into BLCL by a recombinant retrovirus MSCVpp65. While low in alloreactivity, BLCLpp65-stimulated CTL are cytolytic to autologous cells infected with EBV or CMV, and this cytotoxicity is mediated by polyclonal, CD8+, MHC Class I-restricted T-cells. Further experiments revealed that retroviral transduction and expression of pp65 do not compromise the capacity of presenting EBV antigens, and T cells stimulated by BLCLpp65 recognize clinical strains of CMV (Sun et al., 2000). These data indicated that BLCLpp65 could substitute for BLCL as antigen presenting cells in adoptive immunotherapy against EBV-LPD, with the benefit of providing protection against CMV reactivation. This protocol is a Phase I/II study to examine the toxicity associated with and the immunologic effects of ex vivo simultaneously expanded EBV- and CMV-specific CTL for prophylaxis against EBV and CMV
Endowing carbon nanotubes with superparamagnetic properties: applications for cell labeling, MRI cell tracking and magnetic manipulations.

PubMed

Lamanna, Giuseppe; Garofalo, Antonio; Popa, Gabriela; Wilhelm, Claire; Bégin-Colin, Sylvie; Felder-Flesch, Delphine; Bianco, Alberto; Gazeau, Florence; Ménard-Moyon, Cécilia

2013-05-21

Coating of carbon nanotubes (CNTs) with magnetic nanoparticles (NPs) imparts novel magnetic, optical, and thermal properties with potential applications in the biomedical domain. Multi-walled CNTs have been decorated with iron oxide superparamagnetic NPs. Two different approaches have been investigated based on ligand exchange or "click chemistry". The presence of the NPs on the nanotube surface allows conferring magnetic properties to CNTs. We have evaluated the potential of the NP/CNT hybrids as a contrast agent for magnetic resonance imaging (MRI) and their interactions with cells. The capacity of the hybrids to magnetically monitor and manipulate cells has also been investigated. The NP/CNTs can be manipulated by a remote magnetic field with enhanced contrast in MRI. They are internalized into tumor cells without showing cytotoxicity. The labeled cells can be magnetically manipulated as they display magnetic mobility and are detected at a single cell level through high resolution MRI.
Human Umbilical Cord Mesenchymal Stem Cells Inhibit the Function of Allogeneic Activated Vγ9Vδ2 T Lymphocytes In Vitro

PubMed Central

Liu, Xiaohuan; Feng, Ting; Gong, Tianxiang; Shen, Chongyang; Zhu, Tingting; Wu, Qihong; Li, Qiang; Li, Hong

2015-01-01

Background. Human umbilical cord mesenchymal stem cells (UC-MSCs) can regulate the function of immune cells. However, whether and how UC-MSCs can modulate the function of Vγ9Vδ2 T cells has not been fully understood. Methods. The PBMCs or Vγ9Vδ2 T cells were activated and expanded with pamidronate (PAM) and interleukin-2 (IL-2) with or without the presence UC-MSCs. The effects of UC-MSCs on the proliferation, cytokine expression, and cytotoxicity of Vγ9Vδ2 T cells were determined by flow cytometry. The effects of UC-MSCs on Fas-L, TRAIL-expressing Vγ9Vδ2 T cells, and Vγ9Vδ2 T cell apoptosis were determined by flow cytometry. Results. UC-MSCs inhibited Vγ9Vδ2 T cell proliferation in a dose-dependent but cell-contact independent manner. Coculture with UC-MSCs reduced the frequency of IFNγ+ but increased granzyme B+ Vγ9Vδ2 T cells. UC-MSCs inhibited the cytotoxicity of Vγ9Vδ2 T cells against influenza virus H1N1 infected A549 cells and also reduced the frequency of Fas-L+, TRAIL+ Vγ9Vδ2 T cells but failed to modulate the apoptosis of Vγ9Vδ2 T cells. Conclusions. These results indicated that UC-MSCs efficiently suppressed the proliferation and cytotoxicity of Vγ9Vδ2 T cells and modulated their cytokine production. Fas-L and TRAIL were involved in the regulation. Cell contact and apoptosis of Vγ9Vδ2 T cells were not necessary for the inhibition. PMID:25984529

T-cell receptor transfer for boosting HIV-1-specific T-cell immunity in HIV-1-infected patients.

PubMed

Mummert, Christiane; Hofmann, Christian; Hückelhoven, Angela G; Bergmann, Silke; Mueller-Schmucker, Sandra M; Harrer, Ellen G; Dörrie, Jan; Schaft, Niels; Harrer, Thomas

2016-09-10

Strategies to cure HIV-1 infection require the eradication of viral reservoirs. An innovative approach for boosting the cytotoxic T-lymphocyte response is the transfer of T-cell receptors (TCRs). Previously, we have shown that electroporation of TCR-encoding mRNA is able to reprogram CD8 T cells derived from healthy donors. So far, it is unknown whether the transfer of HIV-1-specific TCRs is capable to reprogram CD8 T cells of HIV-1-infected patients. To assess the efficiency of TCR-transfer by mRNA electroporation and the functionality of reprogramed T cells in HIV-1-infected patients, we performed an in-vitro analysis of TCR-transfer into T cells from HIV-1-infected patients in various stages of disease and from healthy controls. Peripheral blood mononuclear cells from 16 HIV-1-infected patients (nine HLA-A02-positive, seven HLA-A02-negative) and from five healthy controls were electroporated with mRNA-constructs encoding TCRs specific for the HLA-A02/HIV-1-gag p17 epitope SLYNTVATL (SL9). Functionality of the TCRs was measured by γIFN-ELISpot assays. SL9/TCR transfection into peripheral blood mononuclear cells from both HLA-A02-positive and HLA-A02-negative HIV-1-infected patients and from healthy blood donors reprogramed T cells for recognition of SL9-presenting HLA-A02-positive cells in γIFN-ELISpot assays. SL9/TCR-transfer into T cells from an immunodeficient AIDS patient could induce recognition of SL9-expressing target cells only after reversion of T-cell dysfunction by antiretroviral therapy. The transfer of HIV-1-p17-specific TCRs into T cells is functional both in HIV-1-infected patients as well as in healthy blood donors. TCR-transfer is a promising method to boost the immune system against HIV-1.
Weak vaccinia virus-induced NK cell regulation of CD4 T cells is associated with reduced NK cell differentiation and cytolytic activity.

PubMed

Hatfield, Steven D; Daniels, Keith A; O'Donnell, Carey L; Waggoner, Stephen N; Welsh, Raymond M

2018-06-01

Natural killer (NK) cells control antiviral adaptive immune responses in mice during some virus infections, but the universality of this phenomenon remains unknown. Lymphocytic choriomeningitis virus (LCMV) infection of mice triggered potent cytotoxic activity of NK cells (NK LCMV ) against activated CD4 T cells, tumor cells, and allogeneic lymphocytes. In contrast, NK cells activated by vaccinia virus (VACV) infection (NK VACV ) exhibited weaker cytolytic activity against each of these target cells. Relative to NK LCMV cells, NK VACV cells exhibited a more immature (CD11b - CD27 + ) phenotype, and lower expression levels of the activation marker CD69, cytotoxic effector molecules (perforin, granzyme B), and the transcription factor IRF4. NK VACV cells expressed higher levels of the inhibitory molecule NKG2A than NK LCMV cells. Consistent with this apparent lethargy, NK VACV cells only weakly constrained VACV-specific CD4 T-cell responses. This suggests that NK cell regulation of adaptive immunity, while universal, may be limited with viruses that poorly activate NK cells. Published by Elsevier Inc.
δ- and γ-tocotrienols induce classical ultrastructural apoptotic changes in human T lymphoblastic leukemic cells.

PubMed

Wong, Rebecca S Y; Radhakrishnan, Ammu K; Ibrahim, Tengku Azmi Tengku; Cheong, Soon-Keng

2012-06-01

Tocotrienols are isomers of the vitamin E family, which have been reported to exert cytotoxic effects in various cancer cells. Although there have been some reports on the effects of tocotrienols in leukemic cells, ultrastructural evidence of tocotrienol-induced apoptotic cell death in leukemic cells is lacking. The present study investigated the effects of three isomers of tocotrienols (alpha, delta, and gamma) on a human T lymphoblastic leukemic cell line (CEM-SS). Cell viability assays showed that all three isomers had cytotoxic effects (p < 0.05) on CEM-SS cells with delta-tocotrienol being the most potent. Transmission electron microscopy showed that the cytotoxic effects by delta- and gamma-tocotrienols were through the induction of an apoptotic pathway as demonstrated by the classical ultrastructural apoptotic changes characterized by peripheral nuclear chromatin condensation and nuclear fragmentation. These findings were confirmed biochemically by the demonstration of phosphatidylserine externalization via flow cytometry analysis. This is the first study showing classical ultrastructural apoptotic changes induced by delta- and gamma-tocotrienols in human T lymphoblastic leukemic cells.
Implication of Highly Cytotoxic Natural Killer Cells for Esophageal Squamous Cell Carcinoma Treatment.

PubMed

Lim, Kee Siang; Mimura, Kosaku; Kua, Ley-Fang; Shiraishi, Kensuke; Kono, Koji

2018-04-20

Esophageal squamous cell carcinoma (ESCC) is an aggressive upper gastrointestinal cancer and effective treatments are limited. Previous studies reported that natural killer (NK) cells expanded by coculturing with K562-mb15-41BBL feeder cells, a genetically modified K562 leukemia cell line that expresses membrane-bound interleukin (IL)-15 and 41BBL ligand, were highly proliferative and highly cytotoxic. Here, we investigated the potential of expanded NK cells for ESCC treatment. We analyzed both genetic and surface expression levels of NKG2D ligands (NKG2DLs) in ESCC using publicly available microarray data sets and ESCC cell lines. The cytotoxicity of resting and of IL-2-activated NK cells against ESCC cell lines was compared with that of expanded NK cells. We then also investigated the effect of epithelial mesenchymal transition (EMT) inducers, GSK3β inhibitor and epidermal growth factor, on NKG2DLs expressions. As a result, MICA and MICB were significantly overexpressed in ESCC compared with adjacent normal tissues and surface NKG2DLs were expressed in ESCC cell lines. Expanded NK cells were much potent than IL-2-activated and resting NK cells against ESCC cell lines. Blocking of NKG2D with anti-NKG2D monoclonal antibody dampened expanded NK cell cytotoxicity, suggesting that the NKG2DLs-NKG2D interaction is crucial for NK cells to eliminate ESCC cells. EMT inducers concurrently induced EMT and NKG2DLs expression in ESCC cell lines rendering transitioned cells more sensitive to expanded NK cells. In conclusion, expanded NK cells were highly cytotoxic against NKG2DLs-expressing ESCC cells, particularly the EMT phenotype. These results provide a strong rationale for clinical use of these NK cells in ESCC patients.
Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

PubMed Central

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-01-01

Background CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. Design and Methods We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d+ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. Results The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d+ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d+ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Conclusions Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d+ hematologic malignancies. PMID:19454494
Micrometer-sized iron oxide particle labeling of mesenchymal stem cells for magnetic resonance imaging-based monitoring of cartilage tissue engineering.

PubMed

Saldanha, Karl J; Doan, Ryan P; Ainslie, Kristy M; Desai, Tejal A; Majumdar, Sharmila

2011-01-01

To examine mesenchymal stem cell (MSC) labeling with micrometer-sized iron oxide particles (MPIOs) for magnetic resonance imaging (MRI)-based tracking and its application to monitoring articular cartilage regeneration. Rabbit MSCs were labeled using commercial MPIOs. In vitro MRI was performed with gradient echo (GRE) and spin echo (SE) sequences at 3T and quantitatively characterized using line profile and region of interest analysis. Ex vivo MRI of hydrogel-encapsulated labeled MSCs implanted within a bovine knee was performed with spoiled GRE (SPGR) and T(1ρ) sequences. Fluorescence microscopy, labeling efficiency, and chondrogenesis of MPIO-labeled cells were also examined. MPIO labeling results in efficient contrast uptake and signal loss that can be visualized and quantitatively characterized via MRI. SPGR imaging of implanted cells results in ex vivo detection within native tissue, and T(1ρ) imaging is unaffected by the presence of labeled cells immediately following implantation. MPIO labeling does not affect quantitative glycosaminoglycan production during chondrogenesis, but iron aggregation hinders extracellular matrix visualization. This aggregation may result from excess unincorporated particles following labeling and is an issue that necessitates further investigation. This study demonstrates the promise of MPIO labeling for monitoring cartilage regeneration and highlights its potential in the development of cell-based tissue engineering strategies. Published by Elsevier Inc.
Identification and characterization of polyclonal αβ T cells with dendritic cell properties

PubMed Central

Kuka, Mirela; Munitic, Ivana; Ashwell, Jonathan D.

2012-01-01

An efficient immune response requires coordination between innate and adaptive immunity, which act through cells different in origin and function. Here we report the identification of thymus-derived αβ TCR+ cells that express CD11c and MHC class II, and require FLT3L for development (TDC). TDC express genes heretofore found uniquely in T cells or DC, as well as a distinctive signature of cytotoxicity-related genes. Unlike other innate T cell subsets, TDC have a polyclonal TCR repertoire andrespond to cognate antigens. However, they differ from conventional T cells in that they do not require help from antigen-presenting cells, respond to TLR-mediated stimulation by producing IL-12 and process and present antigen. The physiologic relevance of TDC, found in mice and humans, is still under investigation, but the fact that they combine key features of T and DC cells suggests that they provide a bridge between the innate and adaptive immune systems. PMID:23187623
In Vivo Cytometry of Antigen-Specific T Cells Using 19F MRI

PubMed Central

Srinivas, Mangala; Turner, Michael S.; Janjic, Jelena M.; Morel, Penelope A.; Laidlaw, David H.; Ahrens, Eric T.

2009-01-01

Noninvasive methods to image the trafficking of phenotypically defined immune cells are paramount as we attempt to understand adaptive immunity. A 19F MRI-based methodology for tracking and quantifying cells of a defined phenotype is presented. These methods were applied to a murine inflammation model using antigen-specific T cells. The T cells that were intracellularly labeled ex vivo with a perfluoropolyether (PFPE) nanoemulsion and cells were transferred to a host receiving a localized inoculation of antigen. Longitudinal 19F MRI over 21 days revealed a dynamic accumulation and clearance of T cells in the lymph node (LN) draining the antigen. The apparent T-cell numbers were calculated in the LN from the time-lapse 19F MRI data. The effect of in vivo T-cell division on the 19F MRI cell quantification accuracy was investigated using fluorescence assays. Overall, in vivo cytometry using PFPE labeling and 19F MRI is broadly applicable to studies of whole-body cell biodistribution. PMID:19585593
One-pot synthesis of fluorescent nitrogen-doped carbon dots with good biocompatibility for cell labeling.

PubMed

Zhang, Zhengwei; Yan, Kun; Yang, Qiulian; Liu, Yanhua; Yan, Zhengyu; Chen, Jianqiu

2017-12-01

Here we report an easy and economical hydrothermal carbonization approach to synthesize the fluorescent nitrogen-doped carbon dots (N-CDs) that was developed using citric acid and triethanolamine as the precursors. The synthesis conditions were optimized to obtain the N-CDs with superior fluorescence performances. The as-prepared N-CDs are monodispersed sphere nanoparticles with good water solubility, and exhibited strong fluorescence, favourable photostability and excitation wavelength-dependent behavior. Furthermore, the in vitro cytotoxicity and cellular labeling of N-CDs were investigated using the rat glomerular mesangial cells. The results showed the N-CDs have more inconspicuous cytotoxicity and better biosafety in comparison with ZnSe quantum dots, although both targeted the cells successfully. Considering their admirable photostability, low toxicity and good compatibility, the as-obtained N-CDs could have potential applications in biosensors, cellular imaging, and other fields. Copyright © 2017 John Wiley & Sons, Ltd.
Regulatory T Cells in Autoimmune and Viral Chronic Hepatitis

PubMed Central

Lapierre, Pascal; Lamarre, Alain

2015-01-01

In both autoimmune liver disease and chronic viral hepatitis, the injury results from an immune-mediated cytotoxic T cell response to liver cells. As such, it is not surprising that CD4+ regulatory T cells, a key regulatory population of T cells able to curb immune responses, could be involved in both autoimmune hepatitis and chronic viral hepatitis. The liver can induce the conversion of naïve CD4+ T cells to CD4+ regulatory T cells and induce tolerance to locally expressed antigens. This tolerance mechanism is carefully regulated in physiological conditions but any imbalance could be pathological. An overly tolerant immune response can lead to chronic infections while an overreactive and unbridled immune response can lead to autoimmune hepatitis. With the recent advent of monoclonal antibodies able to target regulatory T cells (daclizumab) and improve immune responses and several ongoing clinical trials analysing the impact of regulatory T cell infusion on autoimmune liver disease or liver transplant tolerance, modulation of immunological tolerance through CD4+ regulatory T cells could be a key element of future immunotherapies for several liver diseases allowing restoring the balance between proper immune responses and tolerance. PMID:26106627
The Herpes Simplex Virus Type 1 Latency-Associated Transcript Can Protect Neuron-Derived C1300 and Neuro2A Cells from Granzyme B-Induced Apoptosis and CD8 T-Cell Killing▿

PubMed Central

Jiang, Xianzhi; Alami Chentoufi, Aziz; Hsiang, Chinhui; Carpenter, Dale; Osorio, Nelson; BenMohamed, Lbachir; Fraser, Nigel W.; Jones, Clinton; Wechsler, Steven L.

2011-01-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LAT's antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LAT's antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LAT's ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LAT's antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo. PMID:21177822
Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

PubMed Central

Poiret, Thomas; Axelsson-Robertson, Rebecca; Remberger, Mats; Luo, Xiao-Hua; Rao, Martin; Nagchowdhury, Anurupa; Von Landenberg, Anna; Ernberg, Ingemar; Ringden, Olle; Maeurer, Markus

2018-01-01

Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMV-specific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. PMID:29692783
Memory CD4 T cell subsets are kinetically heterogeneous and replenished from naive T cells at high levels.

PubMed

Gossel, Graeme; Hogan, Thea; Cownden, Daniel; Seddon, Benedict; Yates, Andrew J

2017-03-10

Characterising the longevity of immunological memory requires establishing the rules underlying the renewal and death of peripheral T cells. However, we lack knowledge of the population structure and how self-renewal and de novo influx contribute to the maintenance of memory compartments. Here, we characterise the kinetics and structure of murine CD4 T cell memory subsets by measuring the rates of influx of new cells and using detailed timecourses of DNA labelling that also distinguish the behaviour of recently divided and quiescent cells. We find that both effector and central memory CD4 T cells comprise subpopulations with highly divergent rates of turnover, and show that inflows of new cells sourced from the naive pool strongly impact estimates of memory cell lifetimes and division rates. We also demonstrate that the maintenance of CD4 T cell memory subsets in healthy mice is unexpectedly and strikingly reliant on this replenishment.
Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1-specific T-cell effector function in an activation-dependent manner.

PubMed

Schmetterer, Klaus G; Haiderer, Daniela; Leb-Reichl, Victoria M; Neunkirchner, Alina; Jahn-Schmid, Beatrice; Küng, Hans J; Schuch, Karina; Steinberger, Peter; Bohle, Barbara; Pickl, Winfried F

2011-01-01

Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ-chains. cDNAs encoding the α and β-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become
Distinct HIV-1 escape patterns selected by cytotoxic T cells with identical epitope specificity.

PubMed

Yagita, Yuichi; Kuse, Nozomi; Kuroki, Kimiko; Gatanaga, Hiroyuki; Carlson, Jonathan M; Chikata, Takayuki; Brumme, Zabrina L; Murakoshi, Hayato; Akahoshi, Tomohiro; Pfeifer, Nico; Mallal, Simon; John, Mina; Ose, Toyoyuki; Matsubara, Haruki; Kanda, Ryo; Fukunaga, Yuko; Honda, Kazutaka; Kawashima, Yuka; Ariumi, Yasuo; Oka, Shinichi; Maenaka, Katsumi; Takiguchi, Masafumi

2013-02-01

Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.
Distinct HIV-1 Escape Patterns Selected by Cytotoxic T Cells with Identical Epitope Specificity

PubMed Central

Yagita, Yuichi; Kuse, Nozomi; Kuroki, Kimiko; Gatanaga, Hiroyuki; Carlson, Jonathan M.; Chikata, Takayuki; Brumme, Zabrina L.; Murakoshi, Hayato; Akahoshi, Tomohiro; Pfeifer, Nico; Mallal, Simon; John, Mina; Ose, Toyoyuki; Matsubara, Haruki; Kanda, Ryo; Fukunaga, Yuko; Honda, Kazutaka; Kawashima, Yuka; Ariumi, Yasuo; Oka, Shinichi; Maenaka, Katsumi

2013-01-01

Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication. PMID:23236061
T-Cell Warriors—Equipped to Kill Cancer Cells | Center for Cancer Research

Cancer.gov

When the body recognizes tumor cells as foreign, a natural immune response arises to attack them. Unfortunately, tumors have ways to evade immune surveillance systems and antitumor responses are often too weak to defeat the disease. Rather than relying on the body’s natural response, scientists can now manipulate a patient’s own immune cells so that they latch on to tumor cells by recognizing specific proteins on their surface. A type of immune cell that has been explored for this purpose is the killer (cytotoxic) T cell, which eliminates cells infected by viruses, damaged cells, and tumor cells.
Multichannel series piezoelectric quartz crystal cell sensor for real time and quantitative monitoring of the living cell and assessment of cytotoxicity.

PubMed

Tong, Feifei; Lian, Yan; Zhou, Huang; Shi, Xiaohong; He, Fengjiao

2014-10-21

A new multichannel series piezoelectric quartz crystal (MSPQC) cell sensor for real time monitoring of living cells in vitro was reported in this paper. The constructed sensor was used successfully to monitor adhesion, spreading, proliferation, and apoptosis of MG63 osteosarcoma cells and investigate the effects of different concentrations of cobalt chloride on MG63 cells. Quantitative real time and dynamic cell analyses data were conducted using the MSPQC cell sensor. Compared with methods such as fluorescence staining and morphology observation by microscopy, the MSPQC cell sensor is noninvasive, label free, simple, cheap, and capable of online monitoring. It can automatically record the growth status of cells and quantitatively evaluate cell proliferation and the apoptotic response to drugs. It will be a valuable detection and analysis tool for the acquisition of cellular level information and is anticipated to have application in the field of cell biology research or cytotoxicity testing in the future.
The neuro-steroid, 3β androstene 17α diol exhibits potent cytotoxic effects on human malignant glioma and lymphoma cells through different programmed cell death pathways

PubMed Central

Graf, M R; Jia, W; Loria, R M

2007-01-01

The neuro-steroids 3β-androstene-17α-diol (17α-AED), 3β-androstene-17β-diol (17β-AED), 3β-androstene-7α,-17β-triol (7α-AET) and 3β-androstene-7β,-17β-triol (7β-AET) are metabolites of dehydroepiandrosterone and are produced in neuro-ectodermal tissue. Both epimers of androstenediols (17α-AED and 17β-AED) and androstenetriols (7α-AET and 7β-AET) have markedly different biological functions of their chemical analogue. We investigated the cytotoxic activity of these neuro-steroids on human T98G and U251MG glioblastoma and U937 lymphoma cells. Proliferation studies showed that 17α-AED is the most potent inhibitor, with an IC50 ∼15 μM. For T98G glioma, 90% inhibition was achieved with 25 μM of 17α-AED. Other neuro-steroids tested only marginally suppressed cell proliferation. Reduced cell adherence and viability could be detected after 18 h of 17α-AED exposure. Treatment with 17α-AED induced a significant level of apoptosis in U937 lymphoma cells, but not in the glioma cells. Cytopathology of 17α-AED-treated T98G cells revealed the presence of multiple cytoplasmic vacuoles. Acridine orange staining demonstrated the formation of acidic vesicular organelles in 17α-AED-treated T98G and U251MG, which was inhibited by bafilomycin A1. These findings indicate that 17α-AED bears the most potent cytotoxic activity of the neuro-steroids tested, and the effectiveness may depend on the number of hydroxyls and their position on the androstene molecule. These cytotoxic effects may utilize a non-apoptotic pathway in malignant glioma cells. PMID:17637679
Chimeric antigen receptor T cells: a novel therapy for solid tumors.

PubMed

Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

2017-03-29

The chimeric antigen receptor T (CAR-T) cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs) expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII) was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2), and mesothelin (MSLN), as well as the challenges for CAR-T cell therapy.

γδ T cells as a potential tool in colon cancer immunotherapy.

PubMed

Ramutton, Thiranut; Buccheri, Simona; Dieli, Francesco; Todaro, Matilde; Stassi, Giorgio; Meraviglia, Serena

2014-01-01

γδ T cells are capable of recognizing tumor cells and exert potent cellular cytotoxicity against a large range of tumors, including colon cancer. However, tumors utilize numerous strategies to escape recognition or killing by patrolling γδ T cells, such a downregulation of NKG2D ligands, MICA/B and ULBPs. Therefore, the combined upregulation of T-cell receptorand NKG2D ligands on tumor cells and induction of NKG2D expression on γδ T cells may greatly enhance tumor killing and unlock the functions of γδ T cells. Here, we briefly review current data on the mechanisms of γδ T-cell recognition and killing of colon cancer cells and propose that γδ T cells may represent a promising target for the design of novel and highly innovative immunotherapy in patients with colon cancer.
Natural Killer Dendritic Cells Enhance Immune Responses Elicited by α -Galactosylceramide-Stimulated Natural Killer T Cells.

PubMed

Lee, Sung Won; Park, Hyun Jung; Kim, Nayoung; Hong, Seokmann

2013-01-01

Natural killer dendritic cells (NKDCs) possess potent anti-tumor activity, but the cellular effect of NKDC interactions with other innate immune cells is unclear. In this study, we demonstrate that the interaction of NKDCs and natural killer T (NKT) cells is required for the anti-tumor immune responses that are elicited by α -galactosylceramide ( α -GC) in mice. The rapid and strong expression of interferon- γ by NKDCs after α -GC stimulation was dependent on NKT cells. Various NK and DC molecular markers and cytotoxic molecules were up-regulated following α -GC administration. This up-regulation could improve NKDC presentation of tumor antigens and increase cytotoxicity against tumor cells. NKDCs were required for the stimulation of DCs, NK cells, and NKT cells. The strong anti-tumor immune responses elicited by α -GC may be due to the down-regulation of regulatory T cells. Furthermore, the depletion of NKDCs dampened the tumor clearance mediated by α -GC-stimulated NKT cells in vivo. Taken together, these results indicate that complex interactions of innate immune cells might be required to achieve optimal anti-tumor immune responses during the early stages of tumorigenesis.
Impact of diffusion barriers to small cytotoxic molecules on the efficacy of immunotherapy in breast cancer.

PubMed

Das, Hiranmoy; Wang, Zhihui; Niazi, M Khalid Khan; Aggarwal, Reeva; Lu, Jingwei; Kanji, Suman; Das, Manjusri; Joseph, Matthew; Gurcan, Metin; Cristini, Vittorio

2013-01-01

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.
In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. III. Spontaneous and natural cytotoxic effector cells.

PubMed Central

Herbert, A G; Le Gros, G S; Bidawid, S; Watson, J D

1984-01-01

Cytotoxic effector cell populations in murine spleen can be characterized by the phenotype of the cytotoxic cells or the nature of target cells. Lytic events can be antigen-specific, MHC-restricted and clonal, or target cell-specific but apparently non-MHC-restricted. Two cytotoxic effectors of this latter category are spontaneous and natural killers. Normal spleen cells from (BALB/c X DBA/2J)F1 mice (CDF1) cultured without added antigen develop a population of Thy-1+ spontaneous cytotoxic lymphocytes (SCTL) that lyse the DBA/2J mastocytoma P815, as well as the BALB/c-derived plasmacytomas MOPC-11 and SP2/0. Cold target competition experiments reveal the BALB/c-derived plasmacytomas MOPC-11, SP2/0, J558 and the A strain-derived T cell lymphoma YAC-1, but not normal lymphoblasts, block the lysis of P815 target cells. Thus, while these tumour cells appear to express common antigens which are recognized by SCTL cells, plasmacytomas such as J558 are not susceptible to lysis by SCTL. The relationship of SCTL to natural killer (NK) cells was examined. In-vivo treatment of mice with monoclonal anti-Thy-1 antibody leads to a rapid loss of SCTL and precursors from the spleen, but there is a concomitant increase in NK cell activity. PMID:6607213
NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability

PubMed Central

Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

2013-01-01

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy. PMID:22907642
Cytotoxic-T-lymphocyte antigen 4 receptor signaling for lymphocyte adhesion is mediated by C3G and Rap1.

PubMed

Kloog, Yoel; Mor, Adam

2014-03-01

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.
Cytotoxic-T-Lymphocyte Antigen 4 Receptor Signaling for Lymphocyte Adhesion Is Mediated by C3G and Rap1

PubMed Central

Kloog, Yoel

2014-01-01

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders. PMID:24396067
In vitro cytotoxicity of orthodontic primers.

PubMed

D'Antò, Vincenzo; Spagnuolo, Gianrico; Polito, Ilaria; Paduano, Sergio; Ambrosio, Luigi; Valletta, Rosa

2009-01-01

The aim of this study was to evaluate the cytotoxicity of four orthodontic primers: Transbond XT and Transbond MIP (3M, USA), Eagle Fluorsure (American Orthodontics, USA) and Ortho Solo (Ormco, USA). Balb 3T3 cells were exposed to different concentrations of primers (0-0.25 mg/ml). Mitochondrial dehydrogenase activity was evaluated by MTT assay and cell necrosis was measured by flow cytometry (propidium iodide staining). All the materials decreased cell viability in a dose related manner. Cytotoxicity of orthodontic primers based on concentrations which caused a 50% decrease of mitochondrial activity was ranked as follows: Transbond XT (45.57 mg/ml) > Eagle Fluorsure (49.27 mg/ml) > Transbond MIP (64.35 mg/ml) > Ortho solo (70.09 mg/ml). Our results suggest that the cytotoxic potencies demonstrated by orthodontic primers might be of clinical relevance since they disturbed cell metabolism and induced cell death in monolayer cultures.
Cadmium-coordinated supramolecule suppresses tumor growth of T-cell leukemia in mice

PubMed Central

Zhou, Xiaoping; Koizumi, Yukio; Zhang, Muxin; Natsui, Miyuki; Koyota, Souichi; Yamada, Manabu; Kondo, Yoshihiko; Hamada, Fumio; Sugiyama, Toshihiro

2015-01-01

Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal-coordinating ability has been postulated to contribute to the cytotoxic effects of anti-tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p-alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium-coordinated thiacalix[4]arene tetrasulfate (TC4ATS-Cd) exhibits an anti-proliferative effect against T-cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 μM against epithelia-derived cancer cell lines, while TC4ATS-Cd elicited no significant cytotoxicity (IC50 > 947 μM). However, a number of T-cell leukemia cell lines exhibited marked sensitivity to TC4ATS-Cd. In Jurkat cells, toxicity of TC4ATS-Cd occurred with an IC50 of 6.9 μM, which is comparable to that of 6.5 μM observed for cadmium alone. TC4ATS-Cd induced apoptotic cell death through activation of caspase-3 in Jurkat cells. In a xenograft model, TC4ATS-Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS-Cd-treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium-treated mice. These results suggest that cadmium-coordinated supramolecules may have therapeutic potential for treatment of T-cell leukemia. PMID:25735932
Cytotoxic T-lymphocyte-associated protein 4 gene polymorphism is related to rheumatoid arthritis in Egyptian population.

PubMed

Fattah, Shaimaa A; Ghattas, Maivel H; Saleh, Samy M; Abo-Elmatty, Dina M

2017-02-01

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a CD28-family receptor expressed on T-cells which suppresses T cell proliferation. CTLA-4 -318C/T polymorphism is involved in regulation of CTLA-4 expression. The study aimed to investigate the genetic association of CTLA-4 -318C/T polymorphism with rheumatoid arthritis (RA) and the activity and severity of the disease in the Egyptian population. A single nucleotide polymorphism (rs5742909) in CTLA-4 was genotyped in 100 RA patients and 100 healthy controls using polymerase chain reaction-restriction fragment length polymorphism. Diagnostic tests were measured for RA patients. The frequency of T allele in RA patients was significantly higher than in the control subjects (p = 0.002). CT and TT genotypes had high C-reactive protein, erythrocyte sedimentation rate and disease activity score 28 while CC genotype had a high rheumatoid factor. A minor allele of CTLA-4 rs5742909 polymorphism was associated with RA and the activity but not the severity of the disease.
Binding effect of fluorescence labeled glycyrrhetinic acid with GA receptors in hepatocellular carcinoma cells.

PubMed

Sun, Yu-Qi; Dai, Chun-Mei; Zheng, Yan; Shi, Shu-Dan; Hu, Hai-Yang; Chen, Da-Wei

2017-11-01

Glycyrrhetinic acid (GA) is a natural active component from licorice, which is broadly used in traditional Chinese medicine. Lots of glycyrrhetinic acid receptors (GA-R) are proved to locate on the surface of liver cells. Many reports about the hepatocellular carcinoma (HCC) treatment were dependent on GA modified carriers. However, the reality of GA-R in HCC cells was not clear. In this paper, 18β-glycyrrhetinic acid (18β-GA) was labeled with fluorescence (FITC) by chemical synthesis. Together with the binding effect of fluorescence labeled glycyrrhetinic acid (FITC-GA), the competitive action of 18β-GA with GA-R was investigated in HCC cells. The results showed that in HepG2 cells, 18β-GA and FITC-GA presented similar cytotoxicity. The specific binding saturation of GA showed the dissociation constant (K d ) was 7.457±2.122pmol/L and the maximum binding counts (B max ) was 2.385±0.175pmol/2.5×10 6 cells, respectively. FITC-GA bound to cytomembrane specifically and 18β-GA competed to bind the sites significantly in HepG2 cells. Therefore, there is binding effect between fluorescence labeled GA and GA-R. The GA-R on HCC cells is confirmed as expected, which provides a useful reference of active target modified by GA and a novel approach for receptors and ligands study. Copyright © 2017 Elsevier Inc. All rights reserved.
Phytochemical properties and cytotoxicity evaluation of the aqueous extracts from Rafflesia cantleyi

NASA Astrophysics Data System (ADS)

Bakoush, Sumaia Mohamed Mohamed; Yaacob, Wan Ahmad; Adam, Jumaat; Ibrahim, Nazlina

2015-09-01

In the present study, phytochemical properties and cytotoxic evaluation of aqueous extract of Rafflesia cantleyi bud parts were done. Three bud parts including disk, bract and perigone tube were extracted in water to produce crude aqueous extract. Cytotoxic activity of R. cantleyi bud parts was assessed by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against normal cells Vero, 3T3 cell lines and mice peripheral blood mononuclear cells PBMC. Phytochemical analyses revealed the presence of tannins, flavonoids, steroids and alkaloids. The CC50 value against Vero, 3T3 and PBMC cells were equal or more than 125 µg/ml indicating the non-cytotoxic effect of the bud parts extracts. The finding revealed that crude extracts of all the tested bud parts contained potential bioactive compounds which can be used for various biological activities and have no cytotoxicity to selected normal cells.
The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

PubMed Central

Awerkiew, Sabine; Schmidt, Annette; Hombach, Andreas A.; Pfister, Herbert; Abken, Hinrich

2012-01-01

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter. PMID:22292024
[Systemic EBV+ T-cell lymphoproliferative disease of childhood].

PubMed

Lemaire, Anne-Sophie; Daussay, Dorothée; Bouchindhomme, Brigitte; Grardel, Nathalie; Botte, Astrid; Copin, Marie-Christine

2014-08-01

Systemic EBV+ T-cell lymphoproliferative disease of childhood is a recent entity described in the 2008 World Health Organisation tumours of haematopoietic system and lymphoid tissues as a clonal T-cell EBV+ systemic proliferation. It occurs after acute or chronic active EBV infection. We report the case of a caucasian, immunocompetent 12-year-old girl, with no particular history, who presented with hemophagocytic lymphohistiocytosis in the aftermath of an infectious mononucleosis. Main symptoms were multiple organ failure, hepatosplenomegaly and pancytopenia. Histopathology of peripheral lymph node and bone marrow revealed a T-cell, CD8+, EBV+ lymphoproliferation. An elevated viral load was detected in blood by PCR. The patient died within 3 weeks. Since most of the cases have been reported in Asia and South America, few cases still have been described in Europe. Unlike B-cell lymphoproliferation in immunocompromised individuals, T-cell EBV+ lymphoproliferation occurs in immunocompetent patients and seems to be the consequence of a proliferative disorder of EBV-infected T-cells, attributed to a cytotoxic T-cell response deficiency. These T-cell proliferations are more frequently immunoreactive for CD8 than CD4. A key feature of the diagnosis might be EBV viral load. Copyright © 2014 Elsevier Masson SAS. All rights reserved.
Loss of the ex vivo but not the reinducible CD8+ T-cell response to Tax in human T-cell leukemia virus type 1-infected patients with adult T-cell leukemia/lymphoma.

PubMed

Arnulf, B; Thorel, M; Poirot, Y; Tamouza, R; Boulanger, E; Jaccard, A; Oksenhendler, E; Hermine, O; Pique, C

2004-01-01

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM). In asymptomatic carriers and HAM patients, HTLV-1 infection leads to a vigorous cytotoxic T-cell (CTL) response mainly directed to the regulatory Tax protein. In contrast, initial studies showed that anti-HTLV-1 CTL activities were not reproductively detected in ATLL patients, neither ex vivo, nor after in vitro restimulation. To better understand this discrepancy, we explored the anti-HTLV-1 CD8+ T-cell response of eight ATLL patients by using in vitro restimulated or freshly isolated CD8+ T cells. In all the ATLL patients, we found that mitogenic activation allowed the induction of CD8+ T cells able to lyse autologous HTLV-1-infected cells and/or to produce IFNgamma in response to Tax peptides. In contrast, only a minority of the patients possessed CD8+ cells able to respond ex vivo to the same epitopes. These findings indicate that although a restimulatable anti-HTLV-1 CTL activity persists during ATLL, the specific ex vivo response is not constantly maintained. This provides definitive evidence that the CD8+ T-cell response to HTLV-1 is affected by ATLL development and reveals that a major defect concerns the generation and/or the functionality of CD8+ effectors.
[Natural killer cell cytotoxic activity in critical pediatric patients with suspected hemophagocytic syndrome].

PubMed

Martínez, I; Fernández, L; Valentín, J; Castillo, C; Chamorro, C; Pérez-Martínez, A

2015-05-01

To determine the role of natural killer (NK) cytotoxic activity in patients with suspected hemophagocytic lymphohistiocytosis syndrome (HLH). A prospective study was conducted from September 2008 to February 2014. The study was carried out in the Hematological Oncology Laboratory of Hospital Infantil Universitario Niño Jesús, Madrid (Spain). We analyzed 30 peripheral blood samples from intensive care patients with suspected HLH. There were 18 males and 12 females, with a mean age of 4.7 years (range 0.2-22). NK cell cytotoxicity was compared with healthy controls according to age and sex. In vitro NK cell cytotoxicity against the K562 cell line was determined by time-resolved fluorescence (Europium-TDA) under resting conditions, after interleukin 15 stimulation, and following block with Fas ligand antibody. NK cell cytotoxicity. A total of 20 patients showed a significant decrease of NK cell activity compared with controls (P=.001). Nine of these patients were diagnosed with primary HLH. A total of 10 patients were diagnosed with secondary HLH. Cytotoxic activity was normal in 10 subjects. None of them were diagnosed with HLH. Interleukin 15 stimulation increased NK cell cytotoxicity in secondary HLH, and blocking Fas ligand on NK cells decreased cytotoxic activity in primary HLH patients (P=.001). In our experience, NK cell cytotoxic activity measured by time-resolved fluorescence is a simple and useful clinical diagnostic test for HLH. Interleukin 15 stimulation and Fas ligand blocking on NK cells could help differentiate between primary and secondary HLH. Copyright © 2014 Elsevier España, S.L.U. and SEMICYUC. All rights reserved.
Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy.

PubMed

Antunes, Dinler A; Rigo, Maurício M; Freitas, Martiela V; Mendes, Marcus F A; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E; Selin, Liisa K; Cornberg, Markus; Vieira, Gustavo F

2017-01-01

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient's own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide-ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide-MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC "hot-spots" for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus-derived peptides. Our
Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy

PubMed Central

Antunes, Dinler A.; Rigo, Maurício M.; Freitas, Martiela V.; Mendes, Marcus F. A.; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E.; Selin, Liisa K.; Cornberg, Markus; Vieira, Gustavo F.

2017-01-01

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient’s own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide–ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide–MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC “hot-spots” for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus
Immune mechanisms in organ allograft rejection. V. Pivotal role of the cytotoxic-suppressor T cell subset in the rejection of heart grafts bearing isolated class I disparities in the inbred rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lowry, R.P.; Forbes, R.D.; Blackburn, J.H.

1985-11-01

The cellular requirements for rejection of heart grafts bearing isolated major histocompatibility complex (MHC) subregion RT1A-encoded class I disparities was assessed by adoptive transfer. Sublethally irradiated (780 rads) (PVG X WF)F1 recipients of irradiated PVG-RT1r1 heart grafts were selectively reconstituted with spleen cells from syngeneic donors previously sensitized with two sequential PVG-RT1r1 skin grafts. PVG-RT1r1 heart grafts were rejected acutely in recipients reconstituted with 10 X 10(6) unfractionated immune spleen cells or inocula (5 X 10(6) cells) depleted of SIg+ cells, but additional depletion of cytotoxic T cells and their precursors resulted in marked prolongation of graft survival. Reducing themore » reconstituting inocula from 4 X 10(6) to 2.5 X 10(6) spleen cells prolonged graft survival to that observed in unreconstituted recipients. Additional studies were performed to define the immunologic basis for prolonged survival of PVG-RT1r1 heart grafts in homozygous PVG recipients. Although lymphoid cells of naive PVG failed to proliferate on coculture with irradiated PVG-RT1r1, bulk cultures yielding but weak and variable CTL generation, lymphoid cells from specifically sensitized PVG proliferated and generated greater cytotoxic T lymphocyte (CTL) activity under identical conditions, strongly suggesting, therefore, that prolonged heart graft survival in this strain combination is related to low CTL precursor frequency.« less
Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection

PubMed Central

Zhukovsky, Eugene A.; Morse, Richard J.; Maus, Marcela V.

2016-01-01

To realize the full potential of cancer immunotherapy, the latest generation immunotherapeutics are designed to harness the potent tumor-killing capacity of T cells. Thus, to mobilize T cells, new optimized bispecific antibody (BsAb) designs, enabling efficient polyclonal redirection of cytotoxic activity through binding to CD3 and a Tumor Associated Antigen (TAA) and refined genetically-modified T cells have recently expanded the arsenal of available options for cancer treatment. This review presents the current understanding of the parameters crucial to the design of optimal T cell redirecting BsAb and chimeric antigen receptor (CAR)-modified T cells. However, there are additional questions that require thorough elucidation. Both modalities will benefit from design changes that may increase the therapeutic window. One such approach could employ the discrimination afforded by multiple TAA to significantly increase selectivity. PMID:26963133

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