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Sample records for human t-cell line

  1. T cell binding to B lymphoid cell lines in humans: a marker for T-B cell interaction?

    PubMed

    Goust, J M; Fudenberg, H H

    1983-04-15

    Binding of human circulating T cells to established normal and malignant B cell lines results in rosette formation. The percentage of B cells, circulating T cells, and thymocytes able to bind to the B-LCL Raji were 0%, 59 +/- 4% and 61 +/- 6%, respectively. The percentage of rosettes formed between Raji cells and circulating mononuclear cells from 92 normal individuals was 27.8 +/- 5.3%, and remained stable over several months. This phenomenon seems to involve relatively mature B cells, and a T cell marker which appears early in T cell ontogeny. In the peripheral blood, most of the B-LCL binding T cells exhibit a 'helper-inducer' phenotype, as determined with the monoclonal antibodies Leu 3a and OKT4. However, a significant percentage of T cells with so-called 'cytotoxic-suppressor' markers (Leu 2a and OKT8) also bind to B-LCL. The T cells involved in this morphological interactive reaction with B cells might conceivably be specifically involved in regulating B cell functions. Enumeration of this particular subset may be useful in conditions where abnormal T-B cell interactions are suspected. PMID:6601166

  2. Multiplexed gene transfer to a human T-cell line by combining Sleeping Beauty transposon system with methotrexate selection.

    PubMed

    Kacherovsky, Nataly; Liu, Gary W; Jensen, Michael C; Pun, Suzie H

    2015-07-01

    Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance T-cell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, non-viral gene transfer to a human T-cell line with efficient selection (∼ 50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade T-cells housing multiplexed genetic modifications. PMID:25808830

  3. Inhibition of proliferation by agricultural plant extracts in seven human adult T-cell leukaemia (ATL)-related cell lines.

    PubMed

    Kai, Hisahiro; Akamatsu, Ena; Torii, Eri; Kodama, Hiroko; Yukizaki, Chizuko; Sakakibara, Yoichi; Suiko, Masahito; Morishita, Kazuhiro; Kataoka, Hiroaki; Matsuno, Koji

    2011-07-01

    Adult T-cell leukaemia (ATL) is caused by human T-cell leukaemia virus type I (HTLV-I) infection and is resistant to conventional chemotherapy. We evaluated the inhibitory effects of agricultural plants on the proliferation of seven ATL-related human leukaemia cells, using three ATL cell lines (ED, Su9T01 and S1T), two human T-cell lines transformed by HTLV-I infection (HUT-102 and MT-2) and two HTLV-I-negative human T-cell acute lymphoblastic leukaemia cell lines (Jurkat and MOLT-4). A total of 52 samples of 80% ethanol extracts obtained from 30 types of agricultural plants were examined. On the basis of IC(50) values, we selected samples with greater activity than genistein, which was used as a positive control. The highest inhibitory effect was observed with extracts from leaves of Vaccinium virgatum Aiton (blueberry) on four cell lines (ED, Su9T01, HUT-102 and Jurkat); seeds of Momordica charantia L. (bitter gourd) exhibited the second highest activity. The bitter gourd seeds suppressed the proliferation of three cell lines (Su9T01, HUT-102 and Jurkat). The extracts from edible parts of Ipomea batatas LAM. (sweet potato), edible parts of Colocasia esculenta (L.) Schott (taro), skin of taro and seeds of Prunus mume Sieb. et Zucc. (mume) showed markedly greater inhibitory effects on Su9T01 than genistein. These findings suggest that ATL-preventative bioactive compounds may exist in these agricultural plants, which are considered to be functional foods. PMID:21293936

  4. CHARACTERIZATION OF NORMAL HUMAN LUNG LYMPHOCYTES AND INTERLEUKIN-2-INDUCED LUNG T CELL LINES

    EPA Science Inventory

    Lymphocytes from the lower respiratory tract were obtained by bronchoalveolar lavage of healthy, non-smoking individuals. arious monoclonal antibodies characterizing activated T cells, helper-inducer and suppressor-inducer T cell subsets, and naive versus memory cells were used t...

  5. Increasing cellular uptake of mesoporous silica nanoparticles in human embryonic kidney cell line 293T cells by using Lipofectamine 2000.

    PubMed

    Wang, Jiandong; Teng, Zhaogang; Tian, Ying; Fang, Tian; Ma, Jie; Sun, Jin; Zhu, Feipeng; Wu, Jinrong; Wang, Xin; Yang, Nannan; Zhou, Xiaojun; Yun, Shifeng; Lu, Guangming

    2013-11-01

    Mesoporous silica nanoparticles (MSNs) are ideal nanocarriers that have recently gained attention in important bioapplications such as drug, gene, and protein delivery. The efficacy of endocytosis greatly affects the biological functions of MSNs. In the present study, we investigated the effect of cationic liposomes of Lipofectamine 2000 on cellular uptake of MSNs and the cytotoxicity of cationic liposomes combining with MSNs both in vitro and in vivo. Therefore, mesoporous silica nanoparticles with an average diameter of 130 nm and negative surface charge were synthesized and characterized. The possible role of Lipofectamine 2000 in cellular uptake of MSNs was evaluated in human embryonic kidney cell line 293T cells by transmission electron microscopy (TEM) and with inductively coupled plasma (ICP) analysis. The toxicities of liposomes combining with MSNs were tested in vitro via cell apoptosis assay and MTT cell viability assay, and in vivo by histological examination of six organs of mice after intravenous injection. The endocytosis efficiency of MSNs in human embryonic kidney 293T cells was greatly increased using Lipofectamine 2000 compared with controls (P < 0.001). No apparent in vitro or in vivo cytotoxicity was found for Lipofectamine 2000 combining with MSNs. Our data indicate that cationic liposomes of Lipofectamine 2000 has the potential to greatly increase cellular uptake of MSNs with negative surface charge in human renal 293T cells without apparent toxicity. PMID:24059087

  6. A Human Embryonic Kidney 293T Cell Line Mutated at the Golgi α-Mannosidase II Locus*

    PubMed Central

    Crispin, Max; Chang, Veronica T.; Harvey, David J.; Dwek, Raymond A.; Evans, Edward J.; Stuart, David I.; Jones, E. Yvonne; Lord, J. Michael; Spooner, Robert A.; Davis, Simon J.

    2009-01-01

    Disruption of Golgi α-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi α-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi α-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia. PMID:19465480

  7. Phenotypic and Genotypic Comparisons of Human T-Cell Leukemia Virus Type 1 Reverse Transcriptases from Infected T-Cell Lines and Patient Samples▿

    PubMed Central

    Mitchell, Michael S.; Bodine, Ellen T.; Hill, Shawn; Princler, Gerald; Lloyd, Patricia; Mitsuya, Hiroaki; Matsuoka, Masao; Derse, David

    2007-01-01

    It is well established that cell-free infection with human T-cell leukemia virus type 1 (HTLV-1) is less efficient than that with other retroviruses, though the specific infectivities of only a limited number of HTLV-1 isolates have been quantified. Earlier work indicated that a postentry step in the infectious cycle accounted for the poor cell-free infectivity of HTLV-1. To determine whether variations in the pol gene sequence correlated with virus infectivity, we sequenced and phenotypically tested pol genes from a variety of HTLV-1 isolates derived from primary sources, transformed cell lines, and molecular clones. The pol genes and deduced amino acid sequences from 23 proviruses were sequenced and compared with 14 previously published sequences, revealing a limited number of amino acid variations among isolates. The variations appeared to be randomly dispersed among primary isolates and proviruses from cell lines and molecular clones. In addition, there was no correlation between reverse transcriptase sequence and the disease phenotype of the original source of the virus isolate. HTLV-1 pol gene fragments encoding reverse transcriptase were amplified from a variety of isolates and were subcloned into HTLV-1 vectors for both single-cycle infection and spreading-infection assays. Vectors carrying pol genes that matched the consensus sequence had the highest titers, and those with the largest number of variations from the consensus had the lowest titers. The molecular clone from CS-1 cells had four amino acid differences from the consensus sequence and yielded infectious titers that were approximately eight times lower than those of vectors encoding a consensus reverse transcriptase. PMID:17287279

  8. FoxO1 regulates apoptosis induced by asbestos in the MT-2 human T-cell line.

    PubMed

    Matsuzaki, Hidenori; Lee, Suni; Maeda, Megumi; Kumagai-Takei, Naoko; Nishimura, Yasumitsu; Otsuki, Takemi

    2016-09-01

    Asbestos is known to cause malignant mesothelioma and lung cancer. Recent studies implicate tumor immunity in the development of various tumors, including malignant mesothelioma. In order to establish an in vitro T-cell model to clarify the effects of long-term exposure of asbestos on tumor immunity, in this study, human T-cell line MT-2 cells were cultured with asbestos for longer than 8 months and the resultant cells (MT-2Rst) were assessed for the expression of forkhead transcription factor FoxO1. Gene expression analysis revealed that the amount of FoxO1 mRNA decreased after long-term exposure of the MT-2 cells to asbestos. In accordance with this reduction in FoxO1, pro-apoptotic Foxo1 target genes Puma, Fas ligand and Bim were also seen to be down-regulated in MT-2Rst cells. Furthermore, shRNA-mediated knock-down of FoxO1 reduced the number of apoptotic parental MT-2 cells after treatment with asbestos. On the other hand, over-expression of FoxO1 did not affect asbestos-induced apoptosis in MT-2Rst cells. These results suggested that FoxO1 played an important role in regulating asbestos-induced apoptosis and confirmed the presence of multiple pathways regulating resistance to asbestos in MT-2Rst cells. PMID:27042963

  9. Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line.

    PubMed

    Zanette, Caterina; Pelin, Marco; Crosera, Matteo; Adami, Gianpiero; Bovenzi, Massimo; Larese, Francesca Filon; Florio, Chiara

    2011-08-01

    For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products designed to come in direct contact with the skin. In this study we investigated the effects of Ag NPs on skin using the human-derived keratinocyte HaCaT cell line model. Ag NPs caused a concentration- and time-dependent decrease of cell viability, with IC(50) values of 6.8 ± 1.3 μM (MTT assay) and 12 ± 1.2 μM (SRB assay) after 7 days of contact. A 24h treatment, followed by a 6 day recovery period in Ag NPs-free medium, reduced cell viability with almost the same potency (IC(50)s of 15.3 ± 4.6 and 35 ± 20 μM, MTT and SRB assays, respectively). Under these conditions, no evidence of induction of necrotic events (propidium iodide assay) was found. Apocynin, NADPH-oxidase inhibitor, or N(G)-monomethyl-L-argynine, nitric oxide synthase inhibitor, did not prevent NPs-induced reduction of cell viability. TEM analysis of cells exposed to NPs for 24h revealed alteration of nuclear morphology but only a marginal presence of individual NPs inside the cells. These results demonstrate that on HaCaT keratinocytes a relatively short time of contact with Ag NPs causes a long-lasting inhibition of cell growth, not associated with consistent Ag NPs internalization. PMID:21501681

  10. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    NASA Technical Reports Server (NTRS)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  11. Approaches to Study Human T Cell Development.

    PubMed

    Dolens, Anne-Catherine; Van de Walle, Inge; Taghon, Tom

    2016-01-01

    Not only is human T cell development characterized by unique changes in surface marker expression, but it also requires specific growth factors and conditions to mimic and study T cell development in vitro. In this chapter, we provide an overview of the specific aspects that need attention when performing T cell differentiation cultures with human progenitors. PMID:26294413

  12. NKG2D- and T-cell receptor-dependent lysis of malignant glioma cell lines by human γδ T cells: Modulation by temozolomide and A disintegrin and metalloproteases 10 and 17 inhibitors

    PubMed Central

    Chitadze, Guranda; Lettau, Marcus; Luecke, Stefanie; Wang, Ting; Janssen, Ottmar; Fürst, Daniel; Mytilineos, Joannis; Wesch, Daniela; Oberg, Hans-Heinrich; Held-Feindt, Janka; Kabelitz, Dieter

    2016-01-01

    ABSTRACT The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) and UL-16 binding protein (ULBP) family members expressed on tumor cells with the corresponding NKG2D receptor triggers cytotoxic effector functions in NK cells and γδ T cells. However, as a mechanism of tumor immune escape, NKG2D ligands (NKG2DLs) can be released from the cell surface. In this study, we investigated the NKG2DL system in different human glioblastoma (GBM) cell lines, the most lethal brain tumor in adults. Flow cytometric analysis and ELISA revealed that despite the expression of various NKG2DLs only ULBP2 is released as a soluble protein via the proteolytic activity of “a disintegrin and metalloproteases” (ADAM) 10 and 17. Moreover, we report that temozolomide (TMZ), a chemotherapeutic agent in clinical use for the treatment of GBM, increases the cell surface expression of NKG2DLs and sensitizes GBM cells to γδ T cell-mediated lysis. Both NKG2D and the T-cell receptor (TCR) are involved. The cytotoxic activity of γδ T cells toward GBM cells is strongly enhanced in a TCR-dependent manner by stimulation with pyrophosphate antigens. These data clearly demonstrate the complexity of mechanisms regulating NKG2DL expression in GBM cells and further show that treatment with TMZ can increase the immunogenicity of GBM. Thus, TMZ might enhance the potential of the adoptive transfer of ex vivo expanded γδ T cells for the treatment of malignant glioblastoma. PMID:27141377

  13. Human gamma delta T-cell recognition of Yersinia enterocolitica.

    PubMed Central

    Young, J L; Goodall, J C; Beacock-Sharp, H; Gaston, J S

    1997-01-01

    We have studied the human gamma delta T-cell response to Yersinia enterocolitica, a facultative intracellular bacterium which causes gastroenteritis and, particularly in human leucocyte antigen (HLA)-B27+ individuals, reactive arthritis (ReA). A marked proliferation of that cytotoxic gamma delta T cells is seen when Yersinia-infected lymphoblastoid cell lines or fixed intact Yersinia are added to cultures of mononuclear cells derived from the synovial fluid of ReA patients or from the peripheral blood of healthy donors. In contrast, heat-inactivated Yersinia fail to stimulate the gamma delta T-cell response. The gamma delta T-cell lines generated killed both autologous and allogeneic infected cell lines. Interestingly, a T-cell line generated from synovial fluid mononuclear cells (SFMC) killed infected autologous cell lines and a cell line matched for HLA-B27 less well than infected allogeneic target cells. gamma delta T-cell clones isolated from this line were found to express V gamma 9V delta 2 T-cell receptor (TCR) and also killed infected mismatched cells more efficiently than autologous targets. Moreover, from experiments using major histocompatability complex (MHC)-deficient cell lines, it was apparent that target cell recognition was MHC independent. Our results suggest that gamma delta T cells can be involved in immunity to Yersinia enterocolitica and should be taken into account when considering immunopathological mechanisms leading to reactive arthritis. PMID:9378487

  14. T-cell receptor alpha-chain gene is split in a human T-cell leukemia cell line with a t(11;14)(p15;q11).

    PubMed Central

    Le Beau, M M; McKeithan, T W; Shima, E A; Goldman-Leikin, R E; Chan, S J; Bell, G I; Rowley, J D; Diaz, M O

    1986-01-01

    Chromosomal rearrangements in malignant T-cell disease frequently involve the chromosome bands containing the T-cell receptor genes. The RPMI 8402 cell line, which was established from the leukemia cells of a patient with T-cell acute lymphoblastic leukemia, is characterized by a translocation involving chromosome 14 (band q11) and chromosome 11 (band p15) [t(11;14)(p15;q11)]. By using in situ chromosomal hybridization and Southern blot analysis to examine RPMI 8402 cells, we determined that the break at 14q11 occurs within the variable region sequences of the T-cell receptor alpha-chain gene (TCRA); the break at 11p15 occurs between the HRAS1 gene and the genes for insulin and the insulin-like growth factor 2. These results suggest that the TCRA sequences activate a cellular gene located at 11p15 in malignant T-cell disorders. Images PMID:3540949

  15. Human regulatory T cells control TCR signaling and susceptibility to suppression in CD4+ T cells.

    PubMed

    Chellappa, Stalin; Lieske, Nora V; Hagness, Morten; Line, Pål D; Taskén, Kjetil; Aandahl, Einar M

    2016-07-01

    Human CD4(+)CD25(hi)FOXP3(+) regulatory T cells maintain immunologic tolerance and prevent autoimmune and inflammatory immune responses. Regulatory T cells undergo a similar activation cycle as conventional CD4(+) T cells upon antigen stimulation. Here, we demonstrate that T cell receptors and costimulation are required to activate the regulatory T cell suppressive function. Regulatory T cells suppressed the T cell receptor signaling in effector T cells in a time-dependent manner that corresponded with inhibition of cytokine production and proliferation. Modulation of the activation level and thereby the suppressive capacity of regulatory T cells imposed distinct T cell receptor signaling signatures and hyporesponsiveness in suppressed and proliferating effector T cells and established a threshold for effector T cell proliferation. The immune suppression of effector T cells was completely reversible upon removal of regulatory T cells. However, the strength of prior immune suppression by regulatory T cells and corresponding T cell receptor signaling in effector T cells determined the susceptibility to suppression upon later reexposure to regulatory T cells. These findings demonstrate how the strength of the regulatory T cell suppressive function determines intracellular signaling, immune responsiveness, and the later susceptibility of effector T cells to immune suppression and contribute to unveiling the complex interactions between regulatory T cells and effector T cells. PMID:26715685

  16. Radiation-induced interphase death observed in human T-cell lymphoma cells established as a nude mouse tumor line

    SciTech Connect

    Igarashi, T.; Yoshida, S.; Miyamoto, T. )

    1990-08-01

    Interphase death of cells occurs physiologically in healthy animal tissues as well as in tissues pathologically injured by radiation or drugs. An active self-destruction process has been found to play a major role in the interphase death of highly radiosensitive cells. However, the mechanism of this radiation-induced interphase death in human lymphoma has not yet been studied in detail. In the present study, we examined a lymphoma derived from a child lymphoblastic lymphoma bearing CD1, CD4, and CD8 antigens and established in nude mice. Low-dose x-irradiation of this lymphoma induced interphase cell death with characteristic morphological and biological changes of an active self-destruction process, i.e., changes in cell surface appearance seen using scanning electron microscopy and nuclear fragmentation accompanied with an increase in free DNA. The process was proved to require protein synthesis. It was concluded that the radiosensitivity of this T-cell lymphoma of common thymic type is mainly due to the occurrence of the active self-destruction process.

  17. Human immunodeficiency virus type 1 NL4-3 replication in four T-cell lines: rate and efficiency of entry, a major determinant of permissiveness.

    PubMed Central

    Srivastava, K K; Fernandez-Larsson, R; Zinkus, D M; Robinson, H L

    1991-01-01

    Single-cycle infections have been used to study the human immunodeficiency virus type 1 (HIV-1) life cycle in CD4+ T-cell lines that differ in their permissiveness for infection. In single-cycle infections of highly permissive C8166 cells, 50% of the infectious units escaped being blocked by a monoclonal antibody against the virus binding site on CD4 (leu3a) within 30 min. In contrast, 50% of the infectious units for three less permissive cell lines (H9, A3.01, and Jurkat) required 4 h to escape the leu3a block. Entry was also more efficient in the highly permissive cells, with NL4-3 stocks having three times more infectious units for C8166 cells than for H9, A3.01, or Jurkat cells. Postentry steps up through reverse transcription required approximately 3.5 h in each of the cell lines. The times lapsing between reverse transcription and the expression of reverse transcripts ranged from 17 to 25 h in the different cell lines. Virus production per cell was also similar in the different cell lines (within 1.5-fold of each other). These results indicate that a major determinant of the permissiveness of growing T cells for HIV-1 is the rate and efficiency of virus entry. PMID:1674969

  18. A novel 2,3-benzodiazepine-4-one derivative AMPA antagonist inhibits G2/M transition and induces apoptosis in human leukemia Jurkat T cell line.

    PubMed

    Parenti, S; Casagrande, G; Montanari, M; Espahbodinia, M; Ettari, R; Grande, A; Corsi, L

    2016-05-01

    It has been shown that the antagonism of glutamate receptors activity was able inhibit proliferation and induce apoptosis in several neuronal and non-neuronal cancer cell lines. In addition, it has been shown that glutamate might facilitate the spread and growth of leukemia T cells through interactions with AMPA receptors. The aim of the present study was to investigate the modulation of cell cycle elicited by a novel 2,3-benzodiazepine-4-one non-competitive AMPA antagonist derivative in the human leukemia Jurkat T cells. Our results indicated that the 1-(4-amino-3,5-dimethylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4h-2,3-benzodiazepin-4-one, named 1g, exerted a significant growth inhibition of leukemia Jurkat T cells in a time and dose dependent manner, arresting the transition of G2/M phase through activation of Myt-1. The molecule also induced apoptosis through the enhanced expression of the pro-apoptotic p53, and the inhibition of Bcl-2, and Bcl-xl, followed by the activation of caspase-3. The results suggested that compound 1g might act mostly as a cytostatic rather than cytotoxic compound. Although further studies are necessary, in order to identify others specific pathways involved in the activity of the present molecule, the presented results identified a novel molecule acting on specific G2/M checkpoint regulation pathway. Finally, our data suggest that compound 1g might be a good molecule for future development in the cancer research. PMID:27178220

  19. Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

    PubMed

    Pinz, K; Liu, H; Golightly, M; Jares, A; Lan, F; Zieve, G W; Hagag, N; Schuster, M; Firor, A E; Jiang, X; Ma, Y

    2016-03-01

    Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs. PMID:26526988

  20. Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines

    SciTech Connect

    Mizukoshi, Terumi; Komori, Hideyuki; Mizuguchi, Mariko; Abdelaziz, Hussein; Hara, Toshifumi; Higuchi, Masaya; Tanaka, Yuetsu; Ohara, Yoshiro; Funato, Noriko; Fujii, Masahiro; Nakamura, Masataka

    2013-09-01

    Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA binding was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection. - Highlights: • HTLV-1 Tax1 does not activate RelA of NF-κB in non-hematopoietic cell lines. • Tax1 activates the NF-κB non-canonical pathway in non-hematopoietic cell lines. • Tax1 does not induce RelA nuclear translocation in those cell lines, unlike TNFα. • The OX40L promoter κB site is activated by ectopic, but not endogenous, RelA.

  1. Identification of TL-Om1, an Adult T-Cell Leukemia (ATL) Cell Line, as Reference Material for Quantitative PCR for Human T-Lymphotropic Virus 1

    PubMed Central

    Okuma, Kazu; Yamagishi, Makoto; Yamochi, Tadanori; Firouzi, Sanaz; Momose, Haruka; Mizukami, Takuo; Takizawa, Kazuya; Araki, Kumiko; Sugamura, Kazuo; Yamaguchi, Kazunari; Watanabe, Toshiki

    2014-01-01

    Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR. PMID:25502533

  2. Protective effect of Vaccinium myrtillus extract against UVA- and UVB-induced damage in a human keratinocyte cell line (HaCaT cells).

    PubMed

    Calò, Rossella; Marabini, Laura

    2014-03-01

    Recently, the field of skin protection have shown a considerable interest in the use of botanicals. Vaccinium myrtillus contains several polyphenols and anthocyanins with multiple pharmacological properties. The purpose of our study was to examine whether a water-soluble V. myrtillus extract (dry matter 12.4%; total polyphenols 339.3mg/100 g fw; total anthocyanins 297.4 mg/100 g fw) was able to reduce UVA- and UVB-induced damage using a human keratinocyte cell line (HaCaT). HaCaT cells were pretreated for 1h with extract in a serum-free medium and then irradiated with UVA (8-40 J/cm(2)) and UVB (0.008-0.72 J/cm(2)) rays. All experiments were performed 24h after the end of irradiation, except for oxidative stress tests. The extract was able to reduce the UVB-induced cytotoxicity and genotoxicity (studied by comet and micronucleous assays) at lower doses. V. myrtillus extract reduced lipid peroxidation UVB-induced, but had no effect against the ROS UVB-produced. With UVA-induced damage V. myrtillus reduced genotoxicity as well as the unbalance of redox intracellular status. Moreover our extract reduced the UVA-induced apoptosis, but had no effect against the UVB one. V. myrtillus extract showed its free radical scavenging properties reducing oxidative stress and apoptotic markers, especially in UVA-irradiated cells. PMID:24577051

  3. Characterization of the EBV/C3d receptor on the human Jurkat T cell line: evidence for a novel transcript.

    PubMed

    Sinha, S K; Todd, S C; Hedrick, J A; Speiser, C L; Lambris, J D; Tsoukas, C D

    1993-06-15

    EBV is a human herpes virus that causes mononucleosis and is associated with various tumors. EBV infects cells via the CR2 that was previously thought to be expressed only on the surface of B cells and certain epithelial cells. Recent findings in our laboratory and those of others, however, have shown that the EBV receptor is also present on T cells. Our study shows that Jurkat human T cells have a molecule that reacts with both anti-CR2 antibodies and the third component of complement, C3. Furthermore, the data indicate that this molecule binds EBV detected by incubation with biotin-conjugated virus and streptavidin phycoerythrin. Viral binding is specific, as it is inhibited by nonconjugated virus, with anti-CR2 antibodies, and with an antibody reactive with the glycoprotein (gp350) that EBV uses to bind CR2. In addition, EBV variably infects Jurkat cells as demonstrated by the presence of transcripts of Epstein Barr nuclear Ag (EBNA-1) using the polymerase chain reaction. Immunoprecipitation experiments with anti-CR2 antibodies and SDS-PAGE analysis reveal a protein with an apparent molecular mass of 155 kDa which is higher than the one seen in B cells. The size of this molecule is reduced to 119 kDa upon endoglycosidase F treatment. Northern blot analysis of Jurkat poly(A)+ RNA shows a transcript of 4.7 kb upon probing with the B cell CR2 cDNA. This size is consistent with that of B cell CR2 mRNA. Two cDNA clones were identified upon screening of a Jurkat cell cDNA library with the B cell CR2 cDNA. One of the clones possesses an identical nucleotide sequence to the one corresponding to B cell CR2, whereas the other represents a differentially spliced transcript which lacks the exon 8b of B cell CR2. Analysis of Jurkat and Raji mRNA by PCR demonstrated the presence of this novel splice variant in both cell lines. PMID:8390533

  4. Cell Cycle- and Vpr-Mediated Regulation of Human Immunodeficiency Virus Type 1 Expression in Primary and Transformed T-Cell Lines

    PubMed Central

    Gummuluru, Suryaram; Emerman, Michael

    1999-01-01

    Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) transiently arrests cells in the G2 phase of the cell cycle and is a weak transcriptional transactivator. We found that Vpr increased HIV-1 long terminal repeat (LTR) activity in all cells examined but, when expressed at high levels, decreased HIV-1 LTR expression due to cytotoxic effects. Moreover, Vpr-mediated enhancement of HIV-1 LTR-driven transcription was observed in cycling primary human CD4+ T cells but not in terminally differentiated, noncycling primary human macrophages. In single-round infection experiments using primary human CD4+ T cells, proviral clones expressing either wild-type Vpr or Vpr mutants that retained the ability to cause a G2 arrest replicated to higher levels than proviruses lacking Vpr or expressing mutants of Vpr that did not cause an arrest. In support of the hypothesis that enhancement of HIV-1 LTR transcription by Vpr is an indirect effect of the ability of Vpr to delay cells in G2, counterflow centrifugal elutriation of cells into different phases of the cell cycle demonstrated that HIV-1 LTR expression was highest in G2. Finally, the ability of Vpr to upregulate viral transcription was dependent on a minimal promoter containing a functional TATA box and an enhancer. PMID:10364289

  5. Neoantigen landscape dynamics during human melanoma-T cell interactions.

    PubMed

    Verdegaal, Els M E; de Miranda, Noel F C C; Visser, Marten; Harryvan, Tom; van Buuren, Marit M; Andersen, Rikke S; Hadrup, Sine R; van der Minne, Caroline E; Schotte, Remko; Spits, Hergen; Haanen, John B A G; Kapiteijn, Ellen H W; Schumacher, Ton N; van der Burg, Sjoerd H

    2016-08-01

    Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy. Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens. However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance. PMID:27350335

  6. T CELL REPLICATIVE SENESCENCE IN HUMAN AGING

    PubMed Central

    Chou, Jennifer P.; Effros, Rita B.

    2013-01-01

    The decline of the immune system appears to be an intractable consequence of aging, leading to increased susceptibility to infections, reduced effectiveness of vaccination and higher incidences of many diseases including osteoporosis and cancer in the elderly. These outcomes can be attributed, at least in part, to a phenomenon known as T cell replicative senescence, a terminal state characterized by dysregulated immune function, loss of the CD28 costimulatory molecule, shortened telomeres and elevated production of pro-inflammatory cytokines. Senescent CD8 T cells, which accumulate in the elderly, have been shown to frequently bear antigen specificity against cytomegalovirus (CMV), suggesting that this common and persistent infection may drive immune senescence and result in functional and phenotypic changes to the T cell repertoire. Senescent T cells have also been identified in patients with certain cancers, autoimmune diseases and chronic infections, such as HIV. This review discusses the in vivo and in vitro evidence for the contribution of CD8 T cell replicative senescence to a plethora of age-related pathologies and a few possible therapeutic avenues to delay or prevent this differentiative end-state in T cells. The age-associated remodeling of the immune system, through accumulation of senescent T cells has far-reaching consequences on the individual and society alike, for the current healthcare system needs to meet the urgent demands of the increasing proportions of the elderly in the US and abroad. PMID:23061726

  7. Resident memory T cells in human health and disease

    PubMed Central

    Clark, Rachael A.

    2015-01-01

    Resident memory T cells are non-recirculating memory T cells that persist long term in epithelial barrier tissues, including the gastrointestinal tract, lung, skin and reproductive tract. Resident memory T cells persist in the absence of antigens, have impressive effector functions and provide rapid on-site immune protection against known pathogens in peripheral tissues. A fundamentally distinct gene expression program differentiates resident memory T cells from circulating T cells. Although these cells likely evolved to provide rapid immune protection against pathogens, autoreactive, aberrantly activated and malignant resident memory cells contribute to numerous human inflammatory diseases including mycosis fungoides and psoriasis. This review will discuss both the science and medicine of resident memory T cells, exploring how these cells contribute to healthy immune function and discussing what is known about how these cells contribute to human inflammatory and autoimmune diseases. PMID:25568072

  8. Interaction of Staphylococcus aureus toxin "superantigens" with human T cells.

    PubMed Central

    Choi, Y W; Kotzin, B; Herron, L; Callahan, J; Marrack, P; Kappler, J

    1989-01-01

    A modification of the polymerase chain reaction has been used to establish the fact that a collection of Staphylococcus aureus toxins are "superantigens," each of which interacts with the T-cell alpha beta receptor of human T cells by means of a specific set of V beta elements. Images PMID:2479030

  9. Induction of T-cell apoptosis by human herpesvirus 6.

    PubMed Central

    Inoue, Y; Yasukawa, M; Fujita, S

    1997-01-01

    The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection. PMID:9094650

  10. Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients

    PubMed Central

    Tree, T I M; O'Byrne, D; Tremble, J M; Macfarlane, W M; Haskins, K; James, R F L; Docherty, K; Hutton, J C; Banga, J P

    2000-01-01

    Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet β-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet β-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet β-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in β-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet β-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis. PMID:10886245

  11. Plasticity of human CD4 T cell subsets.

    PubMed

    Geginat, Jens; Paroni, Moira; Maglie, Stefano; Alfen, Johanna Sophie; Kastirr, Ilko; Gruarin, Paola; De Simone, Marco; Pagani, Massimiliano; Abrignani, Sergio

    2014-01-01

    Human beings are exposed to a variety of different pathogens, which induce tailored immune responses and consequently generate highly diverse populations of pathogen-specific T cells. CD4(+) T cells have a central role in adaptive immunity, since they provide essential help for both cytotoxic T cell- and antibody-mediated responses. In addition, CD4(+) regulatory T cells are required to maintain self-tolerance and to inhibit immune responses that could damage the host. Initially, two subsets of CD4(+) helper T cells were identified that secrete characteristic effector cytokines and mediate responses against different types of pathogens, i.e., IFN-γ secreting Th1 cells that fight intracellular pathogens, and IL-4 producing Th2 cells that target extracellular parasites. It is now well established that this dichotomy is insufficient to describe the complexity of CD4(+) T cell differentiation, and in particular the human CD4 compartment contains a myriad of T cell subsets with characteristic capacities to produce cytokines and to home to involved tissues. Moreover, it has become increasingly clear that these T cell subsets are not all terminally differentiated cells, but that the majority is plastic and that in particular central memory T cells can acquire different properties and functions in secondary immune responses. In addition, there is compelling evidence that helper T cells can acquire regulatory functions upon chronic stimulation in inflamed tissues. The plasticity of antigen-experienced human T cell subsets is highly relevant for translational medicine, since it opens new perspectives for immune-modulatory therapies for chronic infections, autoimmune diseases, and cancer. PMID:25566245

  12. Plasticity of Human CD4 T Cell Subsets

    PubMed Central

    Geginat, Jens; Paroni, Moira; Maglie, Stefano; Alfen, Johanna Sophie; Kastirr, Ilko; Gruarin, Paola; De Simone, Marco; Pagani, Massimiliano; Abrignani, Sergio

    2014-01-01

    Human beings are exposed to a variety of different pathogens, which induce tailored immune responses and consequently generate highly diverse populations of pathogen-specific T cells. CD4+ T cells have a central role in adaptive immunity, since they provide essential help for both cytotoxic T cell- and antibody-mediated responses. In addition, CD4+ regulatory T cells are required to maintain self-tolerance and to inhibit immune responses that could damage the host. Initially, two subsets of CD4+ helper T cells were identified that secrete characteristic effector cytokines and mediate responses against different types of pathogens, i.e., IFN-γ secreting Th1 cells that fight intracellular pathogens, and IL-4 producing Th2 cells that target extracellular parasites. It is now well established that this dichotomy is insufficient to describe the complexity of CD4+ T cell differentiation, and in particular the human CD4 compartment contains a myriad of T cell subsets with characteristic capacities to produce cytokines and to home to involved tissues. Moreover, it has become increasingly clear that these T cell subsets are not all terminally differentiated cells, but that the majority is plastic and that in particular central memory T cells can acquire different properties and functions in secondary immune responses. In addition, there is compelling evidence that helper T cells can acquire regulatory functions upon chronic stimulation in inflamed tissues. The plasticity of antigen-experienced human T cell subsets is highly relevant for translational medicine, since it opens new perspectives for immune-modulatory therapies for chronic infections, autoimmune diseases, and cancer. PMID:25566245

  13. Allergen-Specific CD4(+) T Cells in Human Asthma.

    PubMed

    Ling, Morris F; Luster, Andrew D

    2016-03-01

    In allergic asthma, aeroallergen exposure of sensitized individuals mobilizes robust innate and adaptive airway immune responses, stimulating eosinophilic airway inflammation and the activation and infiltration of allergen-specific CD4(+) T cells into the airways. Allergen-specific CD4(+) T cells are thought to be central players in the asthmatic response as they specifically recognize the allergen and initiate and orchestrate the asthmatic inflammatory response. In this article, we briefly review the role of allergen-specific CD4(+) T cells in the pathogenesis of human allergic airway inflammation in allergic individuals, discuss the use of allergen-major histocompatibility complex class II tetramers to characterize allergen-specific CD4(+) T cells, and highlight current gaps in knowledge and directions for future research pertaining to the role of allergen-specific CD4(+) T cells in human asthma. PMID:27027948

  14. Targeting human melanoma neoantigens by T cell receptor gene therapy.

    PubMed

    Leisegang, Matthias; Kammertoens, Thomas; Uckert, Wolfgang; Blankenstein, Thomas

    2016-03-01

    In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy. PMID:26808500

  15. Human autoreactive T cells recognize CD1b and phospholipids

    PubMed Central

    Van Rhijn, Ildiko; van Berlo, Twan; Hilmenyuk, Tamara; Cheng, Tan-Yun; Wolf, Benjamin J.; Tatituri, Raju V. V.; Uldrich, Adam P.; Napolitani, Giorgio; Cerundolo, Vincenzo; Altman, John D.; Willemsen, Peter; Huang, Shouxiong; Rossjohn, Jamie; Besra, Gurdyal S.; Brenner, Michael B.; Godfrey, Dale I.; Moody, D. Branch

    2016-01-01

    In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids. PMID:26621732

  16. Acetylcholine receptor-specific suppressive T-cell factor from a retrovirally transformed T-cell line.

    PubMed Central

    Sinigaglia, F; Gotti, C; Castagnoli, R; Clementi, F

    1984-01-01

    In both experimental and human myasthenia gravis an impairment in the immune regulation leads to an increased synthesis of antibodies against the nicotinic acetylcholine receptor (AcChoR). The present work reports the establishment of an AcChoR-specific suppressive T-cell line obtained by viral transformation of AcChoR-enriched murine T lymphocytes. Enriched T cells from Torpedo AcChoR-primed mice, prestimulated in vitro with antigen, were infected with radiation leukemia viruses and injected intravenously in congeneic recipient mice. Six months later lymphomas were observed in 20% of the injected mice and two of them, of donor origin, were established as permanent continuous cell lines in vitro. One of these lines, named LA41, expresses Thy-1.2, Lyt-2, and I-Jb surface markers. Culture supernatants of LA41 cells suppress the antigen-specific in vitro proliferation of Torpedo AcChoR-primed lymphocytes. This suppression is antigen-specific since the response induced by fetal calf AcChoR and by other antigens is not affected by addition of LA41 culture supernatant in the proliferative assay. LA41 culture supernatant injected in vivo at the time of antigen-priming suppresses also significantly the production of anti-AcChoR antibodies but not the synthesis of antibodies against other antigens--i.e., fetal calf AcChoR or alpha-bungarotoxin. These data show that LA41 cells constitutively produce Torpedo AcChoR-specific suppressor factor. PMID:6095305

  17. Optimization of methods for the genetic modification of human T cells.

    PubMed

    Bilal, Mahmood Y; Vacaflores, Aldo; Houtman, Jon Cd

    2015-11-01

    CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells. PMID:26027856

  18. Human T cell lymphotropic virus-associated leukemia/lymphoma

    PubMed Central

    Ratner, Lee

    2009-01-01

    Purpose of review This article summarizes the current pathophysiologic basis for human T cell lymphotropic virus-associated leukemia/lymphoma as well as past, present, and future therapeutic options. Recent findings New studies have been published on allogeneic stem cell transplantation, arsenic trioxide, and bortezomib for this condition. Summary Studies of the molecular biology of human T cell lymphotropic virus-1-induced T cell leukemia/lymphoma have defined a critical role for oncoprotein, Tax, and activation of nuclear factor κB transcription pathways, which have provided rational approaches to improved therapy for T cell leukemia/lymphoma as well as a model for other hematopoietic malignancies characterized by nuclear factor κB activation. PMID:16093798

  19. Dual antigenic recognition by cloned human gamma delta T cells.

    PubMed Central

    Holoshitz, J; Vila, L M; Keroack, B J; McKinley, D R; Bayne, N K

    1992-01-01

    The function of gamma delta T cells is still elusive. The nature of the antigens that they recognize and the mode of presentation of these antigens are largely unknown. The majority of human peripheral gamma delta T cells bear a V gamma 9/V delta 2 T cell receptor, and display nonclonal reactivity to mycobacteria, without restriction by MHC. It is unknown whether these cells have clonal antigenic specificity as well. Here we describe rheumatoid arthritis-derived V gamma 9/V delta 2 T cell clones, displaying dual antigenic recognition: a nonclonal, MHC-unrestricted recognition of mycobacteria, and a clonal recognition of a short tetanus toxin peptide presented by HLA-DRw53, a nonpolymorphic class II MHC molecule associated with susceptibility to rheumatoid arthritis. This is the first evidence that V gamma 9/V delta 2 T cells can recognize nominal antigenic peptides presented by class II MHC molecules. These results suggest that much like alpha beta T cells, V gamma 9/V delta 2 cells may contribute to the immune response against foreign antigens in an antigen-specific and MHC-restricted manner. The reactivity of these gamma delta T cells to mycobacteria may represent a superantigen-like phenomenon. PMID:1345917

  20. Direct recognition of SLA- and HLA-like class II antigens on porcine endothelium by human T cells results in T cell activation and release of interleukin-2.

    PubMed

    Bravery, C A; Batten, P; Yacoub, M H; Rose, M L

    1995-11-15

    To investigate whether human T cells can directly recognize pig xenoantigens, highly purified human CD4+ and CD8+ T cells were incubated with pig aortic endothelial cells (PAEC). The response was measured by [3H]thymidine uptake and release of bioactive interleukin-2. A detailed examination of MHC expression by cultured PAEC and tissue sections of porcine aorta and heart showed porcine endothelial cells (EC) to be constitutively positive for SLA class II and antigens that crossreact with HLA class II molecules. Low level expression of B7 receptors was detected by binding of both human and mouse CTLA-4-Ig to untreated PAEC, which was enhanced significantly by treatment with recombinant porcine interferon-gamma. Human T cells, purified by positive selection and residual DR+ cells removed by lymphocytolysis, were shown to be functionally free of monocytes. Untreated PAEC elicited strong proliferation by human CD4+ T cells: CD8+ T cells also proliferated, but more weakly. This response was inhibited by CTLA-4-Ig. Blocking studies were performed with mAbs that bind to PAEC and not human EC (MSA3, TH16B), an mAb that binds to human and porcine EC (DA6.231), and L243, which binds to human and not porcine EC. The proliferative response of CD4+ T cells to PAEC was inhibited significantly by mAbs against swine and human determinants. In contrast, the response of CD4+ T cells to human EC was inhibited only by mAbs against human determinants. Experiments that directly compared the CD4+ and CD8+ T cell responses to PAEC and the human EC line EAhy.926, both with and without prior treatment with species-specific interferon gamma, demonstrated greater proliferation and 5-10 times more interleukin-2 in response to pig EC than to human EC. PMID:7491676

  1. Human T-cell leukemia virus type 1 tax dysregulates beta-catenin signaling.

    PubMed

    Tomita, Mariko; Kikuchi, Akira; Akiyama, Tetsu; Tanaka, Yuetsu; Mori, Naoki

    2006-11-01

    Dysregulation of beta-catenin signaling has been implicated in the malignant transformation of cells. However, the role of beta-catenin in the human T-cell leukemia virus type 1 (HTLV-1)-induced transformation of T cells is unknown. Here we found that beta-catenin protein was overexpressed in the nucleus and that beta-catenin-dependent transcription was significantly enhanced in Tax-positive HTLV-1-infected T-cell lines compared to that in Tax-negative HTLV-1-infected T-cell lines. Transfection with beta-catenin-specific small interfering RNA inhibited the growth of the Tax-positive HTLV-1-infected T-cell line HUT-102. Transient transfection of Tax appeared to enhance beta-catenin-dependent transcription by stabilizing the beta-catenin protein via activation of the cyclic AMP (cAMP) response element-binding protein. HTLV-1-infected T-cell lines overexpressing beta-catenin also showed increased Akt activity via Tax activation of the cAMP response element-binding protein, resulting in the phosphorylation and inactivation of glycogen synthase kinase 3beta, which phosphorylates beta-catenin for ubiquitination. The phosphatidylinositol 3-kinase inhibitor LY294002 reduced beta-catenin expression in Tax-positive T-cell lines, and inactivation of glycogen synthase kinase 3beta by lithium chloride restored beta-catenin expression in Tax-negative T-cell lines. Finally, we showed that dominant-negative Akt inhibited Tax-induced beta-catenin-dependent transcription. These results indicate that Tax activates beta-catenin through the Akt signaling pathway. Our findings suggest that activation of beta-catenin by Tax may be important in the transformation of T cells by HTLV-1 infection. PMID:16920823

  2. HTLV-I associated arthritis: characteristics of an HTLV-I virus infected T cell line from synovial fluid.

    PubMed Central

    Eguchi, K; Nakamura, T; Mine, M; Ida, H; Kawakami, A; Migita, K; Nagasato, K; Kurata, A; Fukuda, T; Nagataki, S

    1992-01-01

    A T cell line from mononuclear cells in the synovial fluid of a patient with polyarthritis was established. The T cell line reacted with serum samples positive for antibodies to human T cell lymphotropic virus type I (HTLV-I) and with monoclonal antibody to HTLV-I p19. In Southern blotting with an env-pX-LTR HTLV-I probe and digestion of T cell line DNA with the restriction enzymes ClaI, DraI, and PstI generated fragments that were identical to those found in two HTLV-I infected T cell lines established from adult T cell leukaemia or HTLV-I associated myelopathy. The T cell line expressed CD2, CD3, CD4, CD45RA, CD29, HLA-DR, CD25, and CD26 antigens, but not CD8 and CD20 antigens. Large amounts of interleukin 6, interferon gamma, and tumour necrosis factor alpha were secreted in the culture supernatants of this cell line. This line helped immunoglobulin production by B cells, but not K562, Raji, and synovial cell lysis. Images PMID:1616338

  3. Human Immunodeficiencies Related to Defective APC/T Cell Interaction

    PubMed Central

    Kallikourdis, Marinos; Viola, Antonella; Benvenuti, Federica

    2015-01-01

    The primary event for initiating adaptive immune responses is the encounter between T lymphocytes and antigen presenting cells (APCs) in the T cell area of secondary lymphoid organs and the formation of highly organized intercellular junctions referred to as immune synapses (IS). In vivo live-cell imaging of APC–T cell interactions combined to functional studies unveiled that T cell fate is dictated, in large part, by the stability of the initial contact. Immune cell interaction is equally important during delivery of T cell help to B cells and for the killing of target cells by cytotoxic T cells and NK cells. The critical role of contact dynamics and synapse stability on the immune response is well illustrated by human immune deficiencies in which disease pathogenesis is linked to altered adhesion or defective cross-talk between the synaptic partners. The Wiskott–Aldrich syndrome (WAS) is a severe primary immunodeficiency caused by mutations in the Wiskott–Aldrich syndrome protein (WASp), a scaffold that promotes actin polymerization and links TCR stimulation to T cell activation. Absence or mutations in WASp affects intercellular APC–T cell communications by interfering with multiple mechanisms on both sides of the IS. The warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is caused by mutations in CXCR4, a chemokine receptor that in mutant form leads to impairment of APC–T cell interactions. Present evidences suggest that other recently characterized primary immune deficiencies caused by mutation in genes linked to actin cytoskeletal reorganization, such as WIP and DOCK8, may also depend on altered synapse stability. Here, we will discuss in details the mechanisms of disturbed APC–T cell interactions in WAS and WHIM. Moreover, we will summarize the evidence pointing to a compromised conjugate formation in WIP, DOCK8, and X-linked lymphoproliferative syndrome. PMID:26379669

  4. Variant Human T-cell Lymphotropic Virus Type 1c and Adult T-cell Leukemia, Australia

    PubMed Central

    Cassar, Olivier; Bardy, Peter; Kearney, Daniel; Gessain, Antoine

    2013-01-01

    Human T-cell lymphotropic virus type 1 is endemic to central Australia among Indigenous Australians. However, virologic and clinical aspects of infection remain poorly understood. No attempt has been made to control transmission to indigenous children. We report 3 fatal cases of adult T-cell leukemia/lymphoma caused by human T-cell lymphotropic virus type 1 Australo-Melanesian subtype c. PMID:24047544

  5. Suppressive effects of tumor cell-derived 5'-deoxy-5'-methylthioadenosine on human T cells.

    PubMed

    Henrich, Frederik C; Singer, Katrin; Poller, Kerstin; Bernhardt, Luise; Strobl, Carolin D; Limm, Katharina; Ritter, Axel P; Gottfried, Eva; Völkl, Simon; Jacobs, Benedikt; Peter, Katrin; Mougiakakos, Dimitrios; Dettmer, Katja; Oefner, Peter J; Bosserhoff, Anja-Katrin; Kreutz, Marina P; Aigner, Michael; Mackensen, Andreas

    2016-08-01

    The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5'-deoxy-5'-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting. PMID:27622058

  6. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. PMID:27217403

  7. Antiviral activity of human Vδ2 T-cells against WNV includes both cytolytic and non-cytolytic mechanisms.

    PubMed

    Agrati, Chiara; Castilletti, Concetta; Cimini, Eleonora; Romanelli, Antonella; Lapa, Daniele; Quartu, Serena; Martini, Federico; Capobianchi, Maria Rosaria

    2016-04-01

    West Nile virus (WNV) causes a severe central nervous system infection in humans, primarily in the elderly and immunocompromised subjects. Human γδ T-cells play a critical role in the immune response against viruses, and studies of WNV meningoencephalitis in laboratory mice described a role of γδ T-cells in the protective immune response. Aim of this study was to analyze the cytolytic and non-cytolytic antiviral activity of human Vδ2 T-cells against WNV replication. The anti-WNV activity of soluble factor released by zoledronic acid (ZA)-activated Vδ2 T-cell lines and the cytotoxic capability of Vδ2 T-cell lines against WNV-infected cells were tested in vitro. The activation of Vδ2 T-cell lines was able to inhibit WNV replication through the release of soluble factors. IFN-γ is massively released by activated Vδ2 T-cell lines and is involved in the anti-WNV activity. Moreover, the Vδ2 T-cell lines can efficiently kill WNV-infected cells possibly through perforin-mediated mechanism. Altogether, our results provide insight into the effector functions of human Vδ2 T-cells against WNV. The possibility to target these cells by ZA, a commercially available drug used in humans, could potentially offer a new immunotherapeutic strategy for WNV infection. PMID:27196553

  8. Features of Human CD3+CD20+ T Cells.

    PubMed

    Schuh, Elisabeth; Berer, Kerstin; Mulazzani, Matthias; Feil, Katharina; Meinl, Ingrid; Lahm, Harald; Krane, Markus; Lange, Rüdiger; Pfannes, Kristina; Subklewe, Marion; Gürkov, Robert; Bradl, Monika; Hohlfeld, Reinhard; Kümpfel, Tania; Meinl, Edgar; Krumbholz, Markus

    2016-08-15

    Monoclonal Abs against CD20 reduce the number of relapses in multiple sclerosis (MS); commonly this effect is solely attributed to depletion of B cells. Recently, however, a subset of CD3(+)CD20(+) T cells has been described that is also targeted by the anti-CD20 mAb rituximab. Because the existence of cells coexpressing CD3 and CD20 is controversial and features of this subpopulation are poorly understood, we studied this issue in detail. In this study, we confirm that 3-5% of circulating human T cells display CD20 on their surface and transcribe both CD3 and CD20. We report that these CD3(+)CD20(+) T cells pervade thymus, bone marrow, and secondary lymphatic organs. They are found in the cerebrospinal fluid even in the absence of inflammation; in the cerebrospinal fluid of MS patients they occur at a frequency similar to B cells. Phenotypically, these T cells are enriched in CD8(+) and CD45RO(+) memory cells and in CCR7(-) cells. Functionally, they show a higher frequency of IL-4-, IL-17-, IFN-γ-, and TNF-α-producing cells compared with T cells lacking CD20. CD20-expressing T cells respond variably to immunomodulatory treatments given to MS patients: they are reduced by fingolimod, alemtuzumab, and dimethyl fumarate, whereas natalizumab disproportionally increases them in the blood. After depletion by rituximab, they show earlier and higher repopulation than CD20(+) B cells. Taken together, human CD3(+)CD20(+) T cells pervade lymphatic organs and the cerebrospinal fluid, have a strong ability to produce different cytokines, and respond to MS disease modifying drugs. PMID:27412413

  9. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

    PubMed

    Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

    2016-03-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. PMID:26826277

  10. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

    PubMed Central

    Theaker, Sarah M.; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J.; Cole, David K.; Peakman, Mark; Sewell, Andrew K.; Dolton, Garry

    2016-01-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. PMID:26826277

  11. DMBA induces tyrosine phosphorylation of PLC-[gamma]1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W. . Coll. of Pharmacy)

    1993-01-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP[sub 3] and the release of intracellular Ca[sup 2+]. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP[sub 3] formation and Ca[sup 2+] release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10[mu]M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-[gamma]1 that correlated with our earlier findings of IP[sub 3] formation and Ca[sup 2+] release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-[gamma]1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-[gamma]1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-[gamma]1, release of IP[sub 3], and mobilization of intracellular Ca[sup 2+].

  12. DMBA induces tyrosine phosphorylation of PLC-{gamma}1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W.

    1993-02-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP{sub 3} and the release of intracellular Ca{sup 2+}. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP{sub 3} formation and Ca{sup 2+} release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10{mu}M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-{gamma}1 that correlated with our earlier findings of IP{sub 3} formation and Ca{sup 2+} release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-{gamma}1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-{gamma}1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-{gamma}1, release of IP{sub 3}, and mobilization of intracellular Ca{sup 2+}.

  13. Human Peripheral CD4(+) Vδ1(+) γδT Cells Can Develop into αβT Cells.

    PubMed

    Ziegler, Hendrik; Welker, Christian; Sterk, Marco; Haarer, Jan; Rammensee, Hans-Georg; Handgretinger, Rupert; Schilbach, Karin

    2014-01-01

    The lifelong generation of αβT cells enables us to continuously build immunity against pathogens and malignancies despite the loss of thymic function with age. Homeostatic proliferation of post-thymic naïve and memory T cells and their transition into effector and long-lived memory cells balance the decreasing output of naïve T cells, and recent research suggests that also αβT-cell development independent from the thymus may occur. However, the sites and mechanisms of extrathymic T-cell development are not yet understood in detail. γδT cells represent a small fraction of the overall T-cell pool, and are endowed with tremendous phenotypic and functional plasticity. γδT cells that express the Vδ1 gene segment are a minor population in human peripheral blood but predominate in epithelial (and inflamed) tissues. Here, we characterize a CD4(+) peripheral Vδ1(+) γδT-cell subpopulation that expresses stem-cell and progenitor markers and is able to develop into functional αβT cells ex vivo in a simple culture system and in vivo. The route taken by this process resembles thymic T-cell development. However, it involves the re-organization of the Vδ1(+) γδTCR into the αβTCR as a consequence of TCR-γ chain downregulation and the expression of surface Vδ1(+)Vβ(+) TCR components, which we believe function as surrogate pre-TCR. This transdifferentiation process is readily detectable in vivo in inflamed tissue. Our study provides a conceptual framework for extrathymic T-cell development and opens up a new vista in immunology that requires adaptive immune responses in infection, autoimmunity, and cancer to be reconsidered. PMID:25709606

  14. The DR1 and DR6 First Exons of Human Herpesvirus 6A Are Not Required for Virus Replication in Culture and Are Deleted in Virus Stocks That Replicate Well in T-Cell Lines ▿ †

    PubMed Central

    Borenstein, Ronen; Zeigerman, Haim; Frenkel, Niza

    2010-01-01

    Human herpesvirus 6A (HHV-6A) and HHV-6B are lymphotropic viruses which replicate in cultured activated cord blood mononuclear cells (CBMCs) and in T-cell lines. Viral genomes are composed of 143-kb unique (U) sequences flanked by ∼8- to 10-kb left and right direct repeats, DRL and DRR. We have recently cloned HHV-6A (U1102) into bacterial artificial chromosome (BAC) vectors, employing DNA replicative intermediates. Surprisingly, HHV-6A BACs and their parental DNAs were found to contain short ∼2.7-kb DRs. To test whether DR shortening occurred during passaging in CBMCs or in the SupT1 T-cell line, we compared packaged DNAs from various passages. Restriction enzymes, PCR, and sequencing analyses have shown the following. (i) Early (1992) viral preparations from CBMCs contained ∼8-kb DRs. (ii) Viruses currently propagated in SupT1 cells contained ∼2.7-kb DRs. (iii) The deletion spans positions 60 to 5545 in DRL, including genes encoded by DR1 through the first exon of DR6. The pac-2-pac-1 packaging signals, the DR7 open reading frame (ORF), and the DR6 second exon were not deleted. (iv) The DRR sequence was similarly shortened by 5.4 kb. (v) The DR1 through DR6 first exon sequences were deleted from the entire HHV-6A BACs, revealing that they were not translocated into other genome locations. (vi) When virus initially cultured in CBMCs was passaged in SupT1 cells no DR shortening occurred. (vii) Viral stocks possessing short DRs replicated efficiently, revealing the plasticity of herpesvirus genomes. We conclude that the DR deletion occurred once, producing virus with advantageous growth “conquering” the population. The DR1 gene and the first DR6 exon are not required for propagation in culture. PMID:20053742

  15. Human endogenous retrovirus envelope proteins target dendritic cells to suppress T-cell activation.

    PubMed

    Hummel, Jonas; Kämmerer, Ulrike; Müller, Nora; Avota, Elita; Schneider-Schaulies, Sibylle

    2015-06-01

    Though mostly defective, human endogenous retroviruses (HERV) can retain open reading frames, which are especially expressed in the placenta. There, the envelope (env) proteins of HERV-W (Syncytin-1), HERV-FRD (Syncytin-2), and HERV-K (HML-2) were implicated in tolerance against the semi-allogenic fetus. Here, we show that the known HERV env-binding receptors ASCT-1 and -2 and MFSD2 are expressed by DCs and T-cells. When used as effectors in coculture systems, CHO cells transfected to express Syncytin-1, -2, or HML-2 did not affect T-cell expansion or overall LPS-driven phenotypic DC maturation, however, promoted release of IL-12 and TNF-α rather than IL-10. In contrast, HERV env expressing choriocarcinoma cell lines suppressed T-cell proliferation and LPS-induced TNF-α and IL-12 release, however, promoted IL-10 accumulation, indicating that these effects might not rely on HERV env interactions. However, DCs conditioned by choriocarcinoma, but also transgenic CHO cells failed to promote allogenic T-cell expansion. This was associated with a loss of DC/T-cell conjugate frequencies, impaired Ca(2+) mobilization, and aberrant patterning of f-actin and tyrosine phosphorylated proteins in T-cells. Altogether, these findings suggest that HERV env proteins target T-cell activation indirectly by modulating the stimulatory activity of DCs. PMID:25752285

  16. Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire

    PubMed Central

    Dimova, Tanya; Brouwer, Margreet; Gosselin, Françoise; Tassignon, Joël; Leo, Oberdan; Donner, Catherine; Marchant, Arnaud; Vermijlen, David

    2015-01-01

    γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αβ T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1+ and Vδ3+ γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated. PMID:25617367

  17. 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. PMID:27168241

  18. Polyfunctional T cells accumulate in large human cytomegalovirus-specific T cell responses.

    PubMed

    Lachmann, Raskit; Bajwa, Martha; Vita, Serena; Smith, Helen; Cheek, Elizabeth; Akbar, Arne; Kern, Florian

    2012-01-01

    Large cytomegalovirus (CMV)-specific CD8 T-cell responses are observed in both young and, somewhat more often, old people. Frequent CMV reactivation is thought to exhaust these cells and render them dysfunctional so that larger numbers of them are needed to control CMV. Expansions of CMV-specific CD4 T cells are also seen but are less well studied. In this study, we examined the T-cell response to the dominant CMV pp65 and IE-1 antigens in healthy CMV-infected people across a wide age range (20 to 84 years) by using multicolor flow cytometry. CMV-specific T cells were characterized by the activation markers CD40 ligand (CD40L), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) and the memory markers CD27 and CD45RA. The proportions of effector memory T cells increased in large responses, as did the proportions of polyfunctional CD8 (IFN-γ(+) IL-2(+/-) TNF-α(+)) and CD4 (CD40L(+/-) IFN-γ(+) IL-2(+) TNF-α(+)) T-cell subsets, while the proportion of naïve T cells decreased. The bigger the CD4 or CD8 T-cell response to pp65, the larger was the proportion of T cells with an advanced memory phenotype in the entire (including non-CMV-specific) T-cell compartment. In addition, the number of activation markers per cell correlated with the degree of T-cell receptor downregulation, suggesting increased antigen sensitivity in polyfunctional cells. In summary, our findings show that polyfunctional CMV-specific T cells were not superseded by dysfunctional cells, even in very large responses. At the same time, however, the memory subset composition of the entire T-cell compartment correlated with the size of the T-cell response to CMV pp65, confirming a strong effect of CMV infection on the immune systems of some, but not all, infected people. PMID:22072753

  19. A New Type of Metal Recognition by Human T Cells

    PubMed Central

    Gamerdinger, Katharina; Moulon, Corinne; Karp, David R.; van Bergen, Jeroen; Koning, Frits; Wild, Doris; Pflugfelder, Ulrike; Weltzien, Hans Ulrich

    2003-01-01

    In spite of high frequencies of metal allergies, the structural basis for major histocompatibility complex (MHC)-restricted metal recognition is among the unanswered questions in the field of T cell activation. For the human T cell clone SE9, we have identified potential Ni contact sites in the T cell receptor (TCR) and the restricting human histocompatibility leukocyte antigen (HLA)-DR structure. The specificity of this HLA-DR–promiscuous VA22/VB17+ TCR is primarily harbored in its α chain. Ni reactivity is neither dependent on protein processing in antigen-presenting cells nor affected by the nature of HLA-DR–associated peptides. However, SE9 activation by Ni crucially depends on Tyr29 in CDR1α, an N-nucleotide–encoded Tyr94 in CDR3α, and a conserved His81 in the HLA-DR β chain. These data indicate that labile, nonactivating complexes between the SE9 TCR and most HLA-DR/peptide conjugates might supply sterically optimized coordination sites for Ni ions, three of which were identified in this study. In such complexes Ni may effectively bridge the TCR α chain to His81 of most DR molecules. Thus, in analogy to superantigens, Ni may directly link TCR and MHC in a peptide-independent manner. However, unlike superantigens, Ni requires idiotypic, i.e., CDR3α-determined TCR amino acids. This new type of TCR–MHC linkage might explain the high frequency of Ni-reactive T cells in the human population. PMID:12756270

  20. Deep Profiling Human T Cell Heterogeneity by Mass Cytometry.

    PubMed

    Cheng, Y; Newell, E W

    2016-01-01

    Advances of mass cytometry and high-dimensional single-cell data analysis have brought cellular immunological research into a new generation. By coupling these two powerful technology platforms, immunologists now have more tools to resolve the tremendous diversity of immune cell subsets, and their heterogeneous functionality. Since the first introduction of mass cytometry, many reports have been published using this novel technology to study a range of cell types. At the outset, studies of human hematopoietic stem cell and peripheral CD8(+) T cells using mass cytometry have shad the light of future experimental approach in interrogating immune cell phenotypic and functional diversity. Here, we briefly revisit the past and present understanding of T cell heterogeneity, and the technologies that facilitate this knowledge. In addition, we review the current progress of mass cytometry and high-dimensional cytometric analysis, including the methodology, panel design, experimental procedure, and choice of computational algorithms with a special focus on their utility in exploration of human T cell immunology. PMID:27235682

  1. First Insight into the Kinome of Human Regulatory T Cells

    PubMed Central

    König, Sebastian; Probst-Kepper, Michael; Reinl, Tobias; Jeron, Andreas; Huehn, Jochen; Schraven, Burkhart; Jänsch, Lothar

    2012-01-01

    Regulatory T cells (Tregs) are essential for controlling peripheral tolerance by the active suppression of various immune cells including conventional T effector cells (Teffs). Downstream of the T cell receptor (TCR), more than 500 protein kinases encoded by the human genome have to be considered in signaling cascades regulating the activation of Tregs and Teffs, respectively. Following TCR engagement, Tregs posses a number of unique attributes, such as constitutive expression of Foxp3, hyporesponsiveness and poor cytokine production. Furthermore, recent studies showed that altered regulation of protein kinases is important for Treg function. These data indicate that signaling pathways in Tregs are distinctly organized and alterations at the level of protein kinases contribute to the unique Treg phenotype. However, kinase-based signaling networks in Tregs are poorly understood and necessitate further systematic characterization. In this study, we analyzed the differential expression of kinases in Tregs and Teffs by using a kinase-selective proteome strategy. In total, we revealed quantitative information on 185 kinases expressed in the human CD4+ T cell subsets. The majority of kinases was equally abundant in both T cell subsets, but 11 kinases were differentially expressed in Tregs. Most strikingly, Tregs showed an altered expression of cell cycle kinases including CDK6. Quantitative proteomics generates first comparative insight into the kinase complements of the CD4+ Teff and Treg subset. Treg-specific expression pattern of 11 protein kinases substantiate the current opinion that TCR-mediated signaling cascades are altered in Tregs and further suggests that Tregs exhibit significant specificities in cell-cycle control and progression. PMID:22815858

  2. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

    NASA Technical Reports Server (NTRS)

    Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

    1986-01-01

    Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

  3. Induction of Human Regulatory T Cells with Bacterial Superantigens.

    PubMed

    Caserta, Stefano; Taylor, Amanda L; Terrazzini, Nadia; Llewelyn, Martin J

    2016-01-01

    Regulatory T cells (Tregs) that suppress the activation of immune effector cells limit immunopathology and are fast emerging as therapeutic targets for autoimmune and cancer disease. Tools enabling Treg in vitro-induction, expansion, and characterization and manipulation will help future clinical developments. In this chapter, we describe in detail how to use bacterial superantigens to induce human Tregs efficiently from peripheral blood mononuclear cells. How to assess human Treg phenotype and suppressive capacity are also described. Technical details, variations, and alternative experimental conditions are provided. PMID:26676048

  4. Phenotypical characterization of regulatory T cells in humans and rodents.

    PubMed

    Rodríguez-Perea, A L; Arcia, E D; Rueda, C M; Velilla, P A

    2016-09-01

    Regulatory T cells (Tregs ) constitute a fascinating subpopulation of CD4(+) T cells due to their ability to limit the immune response against self and non-self antigens. Murine models and antibodies directed against surface and intracellular molecules have allowed elucidation of the mechanisms that govern their development and function. However, these markers used to their classification lack of specificity, as they can be expressed by activated T cells. Similarly, there are slight differences between animal models, in steady state and pathological conditions, anatomical localization and strategy of analysis by flow cytometry. Here, we revised the most common markers utilized for Treg typification by flow cytometry such as CD25, forkhead box protein 3 (FoxP3) and CD127, along with our data obtained in different body compartments of humans, mice and rats. Furthermore, we revised and determined the expression of other molecules important for the phenotypical characterization of Treg cells. We draw attention to the drawbacks of those markers used in chronic states of inflammation. However, until a specific marker for the identification of Tregs is discovered, the best combination of markers will depend upon the tissue or the degree of inflammation from which Tregs derive. PMID:27124481

  5. Thymic output, T-cell diversity, and T-cell function in long-term human SCID chimeras

    PubMed Central

    Win, Chan M.; Parrott, Roberta E.; Cooney, Myriah; Moser, Barry K.; Roberts, Joseph L.; Sempowski, Gregory D.; Buckley, Rebecca H.

    2009-01-01

    Severe combined immunodeficiency (SCID) is a syndrome of diverse genetic cause characterized by profound deficiencies of T, B, and sometimes NK-cell function. Nonablative human leukocyte antigen–identical or rigorously T cell–depleted haploidentical parental bone marrow transplantation (BMT) results in thymus-dependent genetically donor T-cell development in the recipients, leading to long-term survival. We reported previously that normal T-cell numbers, function, and repertoire developed by 3 to 4 months after transplantation in SCID patients, and the repertoire remained highly diverse for the first 10 years after BMT. The T-cell receptor diversity positively correlated with T-cell receptor excision circle levels, a reflection of thymic output. However, the fate of thymic function in SCID patients beyond 10 to 12 years after BMT remained to be determined. In this greater than 25-year follow-up study of 128 patients with 11 different molecular types of SCID after nonconditioned BMT, we provide evidence that T-cell function, thymic output, and T-cell clonal diversity are maintained long-term. PMID:19433858

  6. Heterosubtypic T-Cell Immunity to Influenza in Humans: Challenges for Universal T-Cell Influenza Vaccines

    PubMed Central

    Sridhar, Saranya

    2016-01-01

    Influenza A virus (IAV) remains a significant global health issue causing annual epidemics, pandemics, and sporadic human infections with highly pathogenic avian or swine influenza viruses. Current inactivated and live vaccines are the mainstay of the public health response to influenza, although vaccine efficacy is lower against antigenically distinct viral strains. The first pandemic of the twenty-first century underlined the urgent need to develop new vaccines capable of protecting against a broad range of influenza strains. Such “universal” influenza vaccines are based on the idea of heterosubtypic immunity, wherein immune responses to epitopes conserved across IAV strains can confer protection against subsequent infection and disease. T-cells recognizing conserved antigens are a key contributor in reducing viral load and limiting disease severity during heterosubtypic infection in animal models. Recent studies undertaken during the 2009 H1N1 pandemic provided key insights into the role of cross-reactive T-cells in mediating heterosubtypic protection in humans. This review focuses on human influenza to discuss the epidemiological observations that underpin cross-protective immunity, the role of T-cells as key players in mediating heterosubtypic immunity including recent data from natural history cohort studies and the ongoing clinical development of T-cell-inducing universal influenza vaccines. The challenges and knowledge gaps for developing vaccines to generate long-lived protective T-cell responses is discussed. PMID:27242800

  7. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

    PubMed Central

    Dik, Willem A.; Pike-Overzet, Karin; Weerkamp, Floor; de Ridder, Dick; de Haas, Edwin F.E.; Baert, Miranda R.M.; van der Spek, Peter; Koster, Esther E.L.; Reinders, Marcel J.T.; van Dongen, Jacques J.M.; Langerak, Anton W.; Staal, Frank J.T.

    2005-01-01

    To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before. PMID:15928199

  8. Heterosubtypic T-Cell Immunity to Influenza in Humans: Challenges for Universal T-Cell Influenza Vaccines.

    PubMed

    Sridhar, Saranya

    2016-01-01

    Influenza A virus (IAV) remains a significant global health issue causing annual epidemics, pandemics, and sporadic human infections with highly pathogenic avian or swine influenza viruses. Current inactivated and live vaccines are the mainstay of the public health response to influenza, although vaccine efficacy is lower against antigenically distinct viral strains. The first pandemic of the twenty-first century underlined the urgent need to develop new vaccines capable of protecting against a broad range of influenza strains. Such "universal" influenza vaccines are based on the idea of heterosubtypic immunity, wherein immune responses to epitopes conserved across IAV strains can confer protection against subsequent infection and disease. T-cells recognizing conserved antigens are a key contributor in reducing viral load and limiting disease severity during heterosubtypic infection in animal models. Recent studies undertaken during the 2009 H1N1 pandemic provided key insights into the role of cross-reactive T-cells in mediating heterosubtypic protection in humans. This review focuses on human influenza to discuss the epidemiological observations that underpin cross-protective immunity, the role of T-cells as key players in mediating heterosubtypic immunity including recent data from natural history cohort studies and the ongoing clinical development of T-cell-inducing universal influenza vaccines. The challenges and knowledge gaps for developing vaccines to generate long-lived protective T-cell responses is discussed. PMID:27242800

  9. Targeting ALDHbright human carcinoma initiating cells with ALDH1A1- specific CD8+ T cells

    PubMed Central

    Visus, Carmen; Wang, Yangyang; Lozano-Leon, Antonio; Ferris, Robert L.; Silver, Susan; Szczepanski, Miroslaw J.; Brand, Randall E.; Ferrone, Cristina R.; Whiteside, Theresa L.; Ferrone, Soldano; DeLeo, Albert B.; Wang, Xinhui

    2011-01-01

    Purpose Tumor cells expressing elevated aldehyde dehydrogenase (ALDH) activity attributed to ALDH1/3 isoforms have been identified as ALDHbright cells and have the properties attributed to cancer initiating cells (CIC). CIC represent the subpopulation of tumor cells that are resistant to conventional cancer treatments and highly tumorigenic in immunodeficient mice. They are considered to be responsible for tumor recurrence and metastasis. The ALDH1A1 isoform was previously identified as a tumor antigen recognized by CD8+ T cells. This study examines the ability of ALDH1A1-specific CD8+ T cells to eliminate ALDHbright cells and control tumor growth and metastases. Experimental Design ALDHbright cells were isolated by flow cytometry from HLA-A2+ human head and neck, breast and pancreas carcinoma cell lines using ALDEFLUOR® and tested for their tumorigenicity in immunodeficient mice. ALDH1A1-specific CD8+ T cells were generated in vitro and tested for their ability to eliminate CIC in vitro and in vivo by adoptive transfer to immunodeficient mice bearing human tumor xenografts. Results ALDHbright cells isolated by flow cytometry from HLA-A2+ breast, head and neck and pancreas carcinoma cell lines at low numbers (500 cells) were tumorigenic in immunodeficient mice. ALDHbright cells present in these cell lines, xenografts or surgically removed lesions were recognized by ALDH1A1-specific CD8+ T cells in vitro. Adoptive therapy with ALDH1A1-specific CD8+ T cells eliminated ALDHbright cells, inhibited tumor growth, metastases or prolonged survival of xenograft-bearing immunodeficient mice. Conclusions The results of this translational study strongly support the potential of ALDH1A1-based immunotherapy to selectively target CIC in human cancer. PMID:21856769

  10. Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

    PubMed Central

    Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

    2016-01-01

    A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

  11. Characterization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell lymphoma with large granular cell morphology.

    PubMed

    Bonnefont-Rebeix, Catherine; Fournel-Fleury, Corinne; Ponce, Frédérique; Belluco, Sara; Watrelot, Dorothée; Bouteille, Sylvie E; Rapiteau, Sylvie; Razanajaona-Doll, Diane; Pin, Jean-Jacques; Leroux, Caroline; Marchal, Thierry

    2016-01-01

    Dogs with lymphoma are established as good model for human non-Hodgkin lymphoma studies. Canine cell lines derived from lymphomas may be valuable tools for testing new therapeutic drugs. In this context, we established a canine T-cell line, PER-VAS, from a primary aggressive T-cell lymphoma with large granular morphology. Flow cytometric analysis revealed a stable immunophenotype: PER-VAS cells were positively labelled for CD5, CD45, MHC II and TLR3, and were negative for CD3, CD4 and CD8 expression. Although unstable along the culture process, IL-17 and MMP12 proteins were detectable as late as at passages 280 and 325i.e. respectively 24 and 29 months post isolation. At passage 325, PER-VAS cells maintained the expression of IL-17, CD3, CD56, IFNγ and TNFα mRNAs as shown by RT-PCR analysis. Stable rearrangement of the TCRγ gene has been evidenced by PCR. PER-VAS cells have a high proliferation index with a doubling time of 16.5h and were tumorigenic in Nude mice. Compared to the canine cell lines already reported, PER-VAS cells display an original expression pattern, close to NKT cells, which makes them valuable tools for in vitro comparative research on lymphomas. PMID:26345430

  12. Sequence variation in the human T-cell receptor loci.

    PubMed

    Mackelprang, Rachel; Carlson, Christopher S; Subrahmanyan, Lakshman; Livingston, Robert J; Eberle, Michael A; Nickerson, Deborah A

    2002-12-01

    Identifying common sequence variations known as single nucleotide polymorphisms (SNPs) in human populations is one of the current objectives of the human genome project. Nearly 3 million SNPs have been identified. Analysis of the relative allele frequency of these markers in human populations and the genetic associations between these markers, known as linkage disequilibrium, is now underway to generate a high-density genetic map. Because of the central role T cells play in immune reactivity, the T-cell receptor (TCR) loci have long been considered important candidates for common disease susceptibility within the immune system (e.g., asthma, atopy and autoimmunity). Over the past two decades, hundreds of SNPs in the TCR loci have been identified. Most studies have focused on defining SNPs in the variable gene segments which are involved in antigenic recognition. On average, the coding sequence of each TCR variable gene segment contains two SNPs, with many more found in the 5', 3' and intronic sequences of these segments. Therefore, a potentially large repertoire of functional variants exists in these loci. Association between SNPs (linkage disequilibrium) extends approximately 30 kb in the TCR loci, although a few larger regions of disequilibrium have been identified. Therefore, the SNPs found in one variable gene segment may or may not be associated with SNPs in other surrounding variable gene segments. This suggests that meaningful association studies in the TCR loci will require the analysis and typing of large marker sets to fully evaluate the role of TCR loci in common disease susceptibility in human populations. PMID:12493004

  13. Differentiation of human alloreactive CD8+ T cells in vitro

    PubMed Central

    Rentenaar, Rob J; Vosters, Jelle L G; Van Diepen, Frank N J; Remmerswaal, Ester B M; Van Lier, René A W; Ten Berge, Ineke J M

    2002-01-01

    Expansion and differentiation of alloantigen-reactive CD8+ T cells in mixed lymphocyte cultures was followed by measurement of the loss of carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence of responder cells. Proliferation of CD8+ T cells became detectable on day 4 of culture and, 2 days later, > 60% of the CD8+ T cells in culture were dividing alloreactive lymphocytes. In parallel with expansion, CD8+ T-cell differentiation was initiated, as evidenced by an increase in the number of CD45RA− and CD27− T cells and acquisition of the ability to produce interferon-γ after restimulation with the specific alloantigen. Finally, although short-term stimulation and measurement of intracellular cytokine production allowed visualization of alloreactive CD8+ T cells expanded in vitro, this procedure did not detect circulating alloreactive CD8+ T cells activated in vivo in recipients of allogeneic kidney grafts. PMID:11918689

  14. Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma.

    PubMed

    Mizukoshi, Eishiro; Nakamoto, Yasunari; Marukawa, Yohei; Arai, Kuniaki; Yamashita, Tatsuya; Tsuji, Hirokazu; Kuzushima, Kiyotaka; Takiguchi, Masafumi; Kaneko, Shuichi

    2006-06-01

    Human telomerase reverse transcriptase, hTERT, has been identified as the catalytic enzyme required for telomere elongation. hTERT is expressed in most tumor cells but seldom expressed in most human adult cells. It has been reported that 80% to 90% of hepatocellular carcinomas (HCCs) express hTERT, making the enzyme a potential target in immunotherapy for HCC. In the current study, we identified hTERT-derived, HLA-A*2402-restricted cytotoxic T cell (CTL) epitopes and analyzed hTERT-specific CTL responses in patients with HCC. Peptides containing the epitopes showed high affinity to bind HLA-A*2402 in a major histocompatibility complex binding assay and were able to induce hTERT-specific CTLs in both hTERT cDNA-immunized HLA-A*2402/Kb transgenic mice and patients with HCC. The CTLs were able to kill hepatoma cell lines depending on hTERT expression levels in an HLA-A*2402-restricted manner and induced irrespective of hepatitis viral infection. The number of single hTERT epitope-specific T cells detected by ELISPOT assay was 10 to 100 specific cells per 3 x 10(5) PBMCs, and positive T cell responses were observed in 6.9% to 12.5% of HCC patients. hTERT-specific T cell responses were observed even in the patients with early stages of HCC. The frequency of hTERT/tetramer+ CD8+ T cells in the tumor tissue of patients with HCC was quite high, and they were functional. In conclusion, these results suggest that hTERT is an attractive target for T-cell-based immunotherapy for HCC, and the identified hTERT epitopes may be valuable both for immunotherapy and for analyzing host immune responses to HCC. PMID:16729333

  15. Targeting ß2 adrenergic receptors regulate human T cell function directly and indirectly.

    PubMed

    Zalli, A; Bosch, J A; Goodyear, O; Riddell, N; McGettrick, H M; Moss, P; Wallace, G R

    2015-03-01

    It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay. The results show that Sal reduced the percentage of IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA(*009) (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells. Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells. PMID:25526818

  16. T-cell function, T-cell phenotype and its role in responsiveness to recombinant human erythropoietin in hemodialysis patients.

    PubMed

    Abdalla, M; Yassein, S; Darwish, K H

    2010-01-01

    Resistance to recombinant human erythropoietin (Epo) occurs in a small proportion of hemodialysis (HD) patients. In this study we investigated the relationship between T-cell phenotype (using flow cytometry), T-cell function (by measuring in vitro cytokine production) and responsiveness to Epo in HD patients and to compare the results with those from healthy controls. T-cell phenotypes were assessed and T-cell function was studied. The study included 24 chronic renal failure (CRF) patients on HD treated with rHuEPO as well as 14 normal control subjects. Dual-colour immunofluorescence and flow cytometry were used to compare the surface antigen expression on freshly isolated CD4+ and CD8+ T-cells from PBMC of the studied groups. Levels of a panel of selected cytokines (IL-4, IFN-gamma, slL-2R and IL-10) were determined in PBMC culture supernatants and in plasma samples (TNF-alpha, IFN-gamma, IL-6, slL-2R) using (ELISA) kits. Patients were followed-up for 24 months and a survival study was carried out. T-cells from poor responders showed increased proportions of CD4+/CD28- cells and CD8+/CD28- cells compared with both good responders and controls. Compared with their CD28+ counterparts, CD4+/CD28- T-cells produced significantly more IFN-gamma, enabling them to function as pro-inflammatory cells. There was no difference in secretion of IFN-gamma, slL-2R or IL-4 in PBMC cultures obtained from HD patients and controls. However, Unstimulated PBMC from poor responders generated increased levels of IL-10 poor compared with both good responders and controls. Plasma slL-2R and IL-6 were significantly elevated in both good and poor responders compared with controls. Plasma levels of IFN-gamma and TNF-alpha were undetectable in both HD patients and controls. In the follow up period, more deaths were occurring among the poor responders than the good responders. Based on the finding of the this study we may suggest that, in the absence of any obvious cause, poor response to Epo

  17. Assessment of Newcastle disease-specific T cell proliferation in different inbred MHC chicken lines.

    PubMed

    Norup, L R; Dalgaard, T S; Pedersen, A R; Juul-Madsen, H R

    2011-07-01

    In this study, we have described the establishment of an antigen-specific T cell proliferation assay based on recall stimulation with Newcastle disease (ND) antigen; further, we have described the results obtained after recall stimulation of animals containing different major histocompatibility complex (MHC) haplotypes, vaccinated against ND. First optimization of the assay was performed to lower unspecific proliferation and to enhance antigen-specific T cell proliferation. These two issues were achieved using ethylene diamine tetra acetic acid as stabilizing agent in blood samples and autologous immune serum in culture medium. The optimized assay was used to screen chickens with different MHC haplotypes for their ability to perform T cell proliferation. Results showed that the antigen-specific response of CD4(+) and CD8(+) T cells from B12 chickens was generally low, whereas B13, B130 and B201 chickens were medium in CD4(+) or CD8(+) T cell responses. High responses were seen only in few animals of each haplotype and not in general. A polymorphism in the chicken CD8α gene was found in our experimental chicken lines, resulting in incapability to detect CD8α(+) T cells using antibodies from the CT8 clone. Screening chickens with alternative antibodies showed that antibodies from the 2-398 clone were able to discriminate all CD8α(+) cells from CD8α(-) cells, and consequently this antibody was used in a second vaccination experiment performed with chickens of the haplotypes B13 and B130. This experiment showed a significant difference in antigen-specific proliferation of CD4(+) T cells between the two lines, but not in CD8α(+) T cell proliferation. PMID:21332569

  18. Human T-cell lymphotropic virus type 1 Tax mediates enhanced transcription in CD4+ T lymphocytes.

    PubMed Central

    Newbound, G C; Andrews, J M; O'Rourke, J P; Brady, J N; Lairmore, M D

    1996-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma and is associated with a variety of immunoregulatory disorders. HTLV-1 has been shown to bind to and infect a variety of hematopoietic and nonhematopoietic cells. However, both in vivo and in vitro, the provirus is mostly detected in and preferentially transforms CD4+ T cells. The molecular mechanism that determines the CD4+ T-cell tropism of HTLV-1 has not been determined. Using cocultures of purified CD4+ and CD8+ T cells with an HTLV-1 producing cell line, we measured viral transcription by using Northern (RNA) blot analysis, protein production by using a p24 antigen capture assay and flow cytometric analysis for viral envelope, and proviral integration by using DNA slot blot analysis. We further measured HTLV-1 long terminal repeat-directed transcription in purified CD4+ and CD8+ T cells by using transient transfection assays and in vitro transcription. We demonstrate a higher rate of viral transcription in primary CD4+ T cells than in CD8+ T cells. HTLV-1 protein production was 5- to 25-fold greater in CD4+ cocultures and mRNA levels were 5-fold greater in these cultures than in the CD8+ cocultures. Transient transfection and in vitro transcription indicated a modest increase in basal transcription in CD4+ T cells, whereas there was a 20-fold increase in reporter gene activity in CD4+ T cells cotransfected with tax. These data suggest that unique or activated transcription factors, particularly Tax-responsive factors in CD4+ T cells, recognize regulatory sequences within the HTLV-1 long terminal repeat, and this mediates the observed enhanced viral transcription and ultimately the cell tropism and leukemogenic potential of the virus. PMID:8642630

  19. Photoprotection by Punica granatum seed oil nanoemulsion entrapping polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in human keratinocyte (HaCaT) cell line.

    PubMed

    Baccarin, Thaisa; Mitjans, Montserrat; Ramos, David; Lemos-Senna, Elenara; Vinardell, Maria Pilar

    2015-12-01

    There has been an increase in the use of botanicals as skin photoprotective agents. Pomegranate (Punica granatum L.) is well known for its high concentration of polyphenolic compounds and for its antioxidant and anti-inflammatory properties. The aim of this study was to analyze the photoprotection provided by P. granatum seed oil nanoemulsion entrapping the polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in the keratinocyte HaCaT cell line. For this purpose, HaCaT cells were pretreated for 1h with nanoemulsions in a serum-free medium and then irradiated with UVB (90-200 mJ/cm(2)) rays. Fluorescence microscopy analysis provided information about the cellular internalization of the nanodroplets. We also determined the in vitro SPF of the nanoemulsions and evaluated their phototoxicity using the 3T3 Neutral Red Uptake Phototoxicity Test. The nanoemulsions were able to protect the cells' DNA against UVB-induced damage in a concentration dependent manner. Nanodroplets were internalized by the cells but a higher proportion was detected along the cell membrane. The SPF obtained (~25) depended on the concentration of the ethyl acetate fraction and pomegranate seed oil in the nanoemulsion. The photoprotective formulations were classified as non-phototoxic. In conclusion, nanoemulsions entrapping the polyphenol-rich ethyl acetate fraction show potential for use as a sunscreen product. PMID:26406978

  20. Oxidative phenomena are implicated in human T-cell stimulation.

    PubMed Central

    Sekkat, C; Dornand, J; Gerber, M

    1988-01-01

    Phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and PHA + PMA stimulation of T-enriched peripheral blood lymphocytes (PBL) and the Jurkat malignant T-cell line leads to oxidative-product formation, as evaluated by flow cytofluorometric studies, an increase in K+ flux across the membrane, cGMP production and a depolarization of the cell membrane. Irradiation (20 Gy), which enhances IL-2 synthesis by activated T-enriched PBL and Jurkat cells, also increases oxidative product formation, K+ flux, cGMP production, and induces cell membrane depolarization. Conversely, irradiation does not produce a rise in intracellular free Ca2+, as measured in PHA-stimulated Jurkat cells. PMA is also without effect on intracellular free Ca2+, added before or after PHA stimulation. Thus, except for the rise in intracellular free Ca2+, irradiation and stimulation exert similar effects on some of the events observed in IL-2-producing Jurkat cells, but these effects are not additive. Stimulation and irradiation effects are shown to be additive or synergistic only for cGMP production. It is proposed that irradiation may increase IL-2 synthesis by participating in an additional signal related to the oxidative metabolism of arachidonic acid (AA). PMID:3258279

  1. Oxidative phenomena are implicated in human T-cell stimulation.

    PubMed

    Sekkat, C; Dornand, J; Gerber, M

    1988-03-01

    Phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and PHA + PMA stimulation of T-enriched peripheral blood lymphocytes (PBL) and the Jurkat malignant T-cell line leads to oxidative-product formation, as evaluated by flow cytofluorometric studies, an increase in K+ flux across the membrane, cGMP production and a depolarization of the cell membrane. Irradiation (20 Gy), which enhances IL-2 synthesis by activated T-enriched PBL and Jurkat cells, also increases oxidative product formation, K+ flux, cGMP production, and induces cell membrane depolarization. Conversely, irradiation does not produce a rise in intracellular free Ca2+, as measured in PHA-stimulated Jurkat cells. PMA is also without effect on intracellular free Ca2+, added before or after PHA stimulation. Thus, except for the rise in intracellular free Ca2+, irradiation and stimulation exert similar effects on some of the events observed in IL-2-producing Jurkat cells, but these effects are not additive. Stimulation and irradiation effects are shown to be additive or synergistic only for cGMP production. It is proposed that irradiation may increase IL-2 synthesis by participating in an additional signal related to the oxidative metabolism of arachidonic acid (AA). PMID:3258279

  2. Human regulatory T cells suppress proliferation of B lymphoma cells.

    PubMed

    Grygorowicz, Monika Anna; Biernacka, Marzena; Bujko, Mateusz; Nowak, Eliza; Rymkiewicz, Grzegorz; Paszkiewicz-Kozik, Ewa; Borycka, Ilona Sara; Bystydzienski, Zbigniew; Walewski, Jan; Markowicz, Sergiusz

    2016-08-01

    Activated regulatory T cells (Tregs) suppress proliferation and differentiation of normal B cells. In our study, allogeneic polyclonal CD4 (+) CD25 (+) Tregs and CD4 (+) CD25 (+) CD127(lo)Tregs expanded in vitro in the presence of rapamycin and low dose IL-2 suppressed proliferation of 11 out of 12 established lymphoma B-cell lines. The effect of expanded CD4 (+) CD25 (+) Tregs on survival of freshly isolated lymphoma B cells maintained in culture with soluble multimeric CD40L and IL-4 was variable across lymphoma entities. The survival of freshly isolated follicular lymphoma cells usually decreased in cocultures with CD4 (+) CD25 (+) Tregs. Treg effect on chronic lymphocytic leukemia/small lymphocytic lymphoma cells ranged from suppression to help in individual patients. CD4 (+) CD25 (+) Tregs or CD4 (+) CD25 (+) CD127(lo)Tregs expanded ex vivo with rapamycin could be used to suppress regrowth of residual lymphoma after autologous hematopoietic cell transplantation (HCT), and to counteract both graft-versus-host disease and lymphoma re-growth after allogeneic HCT in select patients with lymphoma susceptible to the regulation by Tregs. PMID:26758248

  3. Unconventional Human T Cells Accumulate at the Site of Infection in Response to Microbial Ligands and Induce Local Tissue Remodeling

    PubMed Central

    Liuzzi, Anna Rita; Kift-Morgan, Ann; Lopez-Anton, Melisa; Friberg, Ida M.; Zhang, Jingjing; Brook, Amy C.; Roberts, Gareth W.; Donovan, Kieron L.; Colmont, Chantal S.; Toleman, Mark A.; Bowen, Timothy; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Fraser, Donald J.

    2016-01-01

    The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vγ9/Vδ2+ γδ T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-γ and TNF-α in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vγ9/Vδ2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response. PMID:27527598

  4. Regulation of human tonsillar T-cell proliferation by the active metabolite of vitamin D3.

    PubMed Central

    Nunn, J D; Katz, D R; Barker, S; Fraher, L J; Hewison, M; Hendy, G N; O'Riordan, J L

    1986-01-01

    We have examined the effects of 1,25(OH)2D3 on T-cell populations isolated by buoyant density and E rosetting from human tonsils. Cell proliferation was assessed by measuring the incorporation of 125iododeoxyuridine; interleukin-2 (IL-2) production was measured using an IL-2-dependent cell line, and the number of 1,25(OH)2D3 receptors was measured by whole-cell nuclear association assay. At a concentration of 10(-7) M, 1,25(OH)2D3 inhibited mitogen-induced T-cell proliferation in all E+ T-cell populations. This effect was more pronounced in the cells from the intermediate and high density layers and was reflected both in cell proliferative responses and in relative IL-2 synthesis. By adding the 1,25(OH)2D3 during the course of the mitogen assay, we demonstrated that activation of the T cell precedes the 1,25(OH)2D3-mediated inhibition. Cells that had been preincubated with mitogen in the presence of the 1,25(OH)2D3 were refractory to further stimulation by mitogens. Receptors for 1,25(OH)2D3 could not be detected in unstimulated T cells. However, activation led to the expression of high-affinity receptors for 1,25(OH)2D3. Co-incubation of the cells with mitogen and 1,25(OH)2D3 increased the number of receptors compared with mitogen alone. The effects provide further evidence for the hypothesis that 1,25(OH)2D3 is an important potential modulator of the immune system through its action on T cells. Taking our observations in conjunction with the known capacity of monocytes to hydroxylate the precursor metabolite (and thus synthesize the active form of cholecalciferol), the results support the suggestion that 1,25(OH)2D3 plays a role as a local mediator of mononuclear phagocyte-T cell interaction in human lymphomedullary tissues. PMID:3026959

  5. T cell receptor diversity in the human thymus.

    PubMed

    Vanhanen, Reetta; Heikkilä, Nelli; Aggarwal, Kunal; Hamm, David; Tarkkila, Heikki; Pätilä, Tommi; Jokiranta, T Sakari; Saramäki, Jari; Arstila, T Petteri

    2016-08-01

    A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCRα and TCRβ gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1 to 3×10(6) unique TCRβ and 0.5×10(6) TCRα sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3×10(6) unique TCRβ sequences and 3.7×10(6) TCRα sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results provide an estimate of a thymic repertoire consisting of 40 to 70×10(6) unique TCRβ sequences and 60 to 100×10(6) TCRα sequences. The thymic repertoire is thus extremely diverse. Moreover, extrapolation of the data and comparison with earlier estimates of peripheral diversity also suggest that the thymic repertoire is transient, with different clones produced at different times. PMID:27442982

  6. Human T-cell lymphotropic virus in volunteer blood donors.

    PubMed

    Taylor, P E; Stevens, C E; Pindyck, J; Schrode, J; Steaffens, J W; Lee, H

    1990-01-01

    Serum samples collected in 1985 and 1986 from 18,257 donors to the Greater New York Blood Program were screened by enzyme-linked immunoassay for antibody to human T-cell lymphotropic virus (anti-HTLV). Fifteen samples (0.08%) were confirmed positive: 7 by radioimmunoprecipitation assay (RIPA) alone, 6 by Western blot alone, and 2 by combined results from both tests. One donor, whose original test result was uninterpretable because multiple nonspecific bands were present on RIPA, clearly tested positive on subsequent specimens. Follow-up testing of individuals with this type of result may be needed to resolve their HTLV status. Anti-HTLV prevalence increased with age and was significantly more common in black or Hispanic donors and in those born in the Caribbean than in other donors. All anti-HTLV-positive donors were negative for antibody to HIV-1, and only one donor (7% of those positive) would have been excluded by any of the routine donor screening tests used at that time. PMID:2173176

  7. Human T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    Multiple DNA and protein sequence alignments have been constructed for the human T-cell receptor {alpha}/{delta}, {beta}, and {gamma} (TCRA/D, B, and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence, a name was derived which complies with the standard nomenclature. The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments. By discriminating between alleles at the same locus versus genes from different loci, we were able to reduce the number of more than 150 different TCRBV sequences in the database to a repertoire of only 47 functional TCRBV gene segments. An extension of this analysis to the over 100 TCRAV sequences results in a predicted repertoire of 42 functional TCRAV gene segments. Our alignment revealed two residues that distinguish between the highly homologous V{delta} and V{alpha}, one at a site that in V{sub H} contacts the constant region, the other at the interface between immunoglobulin V{sub H} and V{sub L}. This site may be responsible for restricted pairing between certain V{delta} and V{gamma} chains. On the other hand, V{beta} and V{gamma} appear to be related by the fact that their CDR2 length is increased by four residues as compared with that of V{alpha}/{delta} peptides. 150 refs., 12 figs., 5 tabs.

  8. Epigenetic biomarkers of T-cells in human glioma.

    PubMed

    Wiencke, John K; Accomando, William P; Zheng, Shichun; Patoka, Joe; Dou, Xiaoqin; Phillips, Joanna J; Hsuang, George; Christensen, Brock C; Houseman, E Andres; Koestler, Devin C; Bracci, Paige; Wiemels, Joseph L; Wrensch, Margaret; Nelson, Heather H; Kelsey, Karl T

    2012-12-01

    Immune factors are thought to influence glioma risk and outcomes, but immune profiling studies to further our understanding of the immune response are limited by current immunodiagnostic methods. We developed a new assay to capture glioma immune biology based on quantitative methylation specific PCR (qMSP) of two T-cell genes (CD3Z: T-cells, and FOXP3: Tregs). Flow cytometry of T-cells correlated well with the CD3Z demethylation assay (r = 0.93; p < 2.2 × 10 (-16) ), demonstrating the validity of the assay. Furthermore, there was a high correlation between qMSP and immunohistochemistry (IHC) in quantifying tumor infiltrating T-cells (r = 0.85; p = 3.4 × 10 (-11) ). Applying our qMSP methods to archival whole blood from 65 glioblastoma multiforme (GBM) cases and 94 non-diseased controls, GBM cases had highly statistically significantly lower T-cells (p = 1.7 × 10 (-9) ) as well as Tregs (p = 5.2 × 10 (-11) ) and a modestly lower ratio of Tregs/T-cells (p = 0.024). Applying the methods to 120 excised glioma tumors, we observed that tumor infiltrating CD3+ T-cells were positively correlated with glioma tumor grade (p = 5.7 × 10 (-7) ), and that Tregs were enriched in tumors compared with peripheral blood indicating active chemoattraction of suppressive Tregs into the tumor compartment. Poorer patient survival was correlated with higher levels of tumor infiltrating T-cells (p = 0.01) and Tregs (p = 0.04). DNA methylation based immunodiagnostics represent a new generation of powerful laboratory tools offering many advantages over conventional methods that will facilitate large clinical epidemiologic studies and capitalize on stored archival blood and tissue banks. PMID:23108258

  9. Epigenetic biomarkers of T-cells in human glioma

    PubMed Central

    Wiencke, John K.; Accomando, William P.; Zheng, Shichun; Patoka, Joe; Dou, Xiaoqin; Phillips, Joanna J.; Hsuang, George; Christensen, Brock C.; Houseman, E. Andres; Koestler, Devin C.; Bracci, Paige; Wiemels, Joseph L.; Wrensch, Margaret; Nelson, Heather H.; Kelsey, Karl T.

    2012-01-01

    Immune factors are thought to influence glioma risk and outcomes, but immune profiling studies to further our understanding of the immune response are limited by current immunodiagnostic methods. We developed a new assay to capture glioma immune biology based on quantitative methylation specific PCR (qMSP) of two T-cell genes (CD3Z: T-cells, and FOXP3: Tregs). Flow cytometry of T-cells correlated well with the CD3Z demethylation assay (r = 0.93; p < 2.2 × 10−16), demonstrating the validity of the assay. Furthermore, there was a high correlation between qMSP and immunohistochemistry (IHC) in quantifying tumor infiltrating T-cells (r = 0.85; p = 3.4 × 10−11). Applying our qMSP methods to archival whole blood from 65 glioblastoma multiforme (GBM) cases and 94 non-diseased controls, GBM cases had highly statistically significantly lower T-cells (p = 1.7 × 10−9) as well as Tregs (p = 5.2 × 10−11) and a modestly lower ratio of Tregs/T-cells (p = 0.024). Applying the methods to 120 excised glioma tumors, we observed that tumor infiltrating CD3+ T-cells were positively correlated with glioma tumor grade (p = 5.7 × 10−7), and that Tregs were enriched in tumors compared with peripheral blood indicating active chemoattraction of suppressive Tregs into the tumor compartment. Poorer patient survival was correlated with higher levels of tumor infiltrating T-cells (p = 0.01) and Tregs (p = 0.04). DNA methylation based immunodiagnostics represent a new generation of powerful laboratory tools offering many advantages over conventional methods that will facilitate large clinical epidemiologic studies and capitalize on stored archival blood and tissue banks. PMID:23108258

  10. Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

    PubMed Central

    Kishino, Yoshikazu; Seki, Tomohisa; Yuasa, Shinsuke; Fujita, Jun; Fukuda, Keiichi

    2015-01-01

    Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus. PMID:26650709

  11. T cell stimulator cells, an efficient and versatile cellular system to assess the role of costimulatory ligands in the activation of human T cells

    PubMed Central

    Leitner, Judith; Kuschei, Werner; Grabmeier-Pfistershammer, Katharina; Woitek, Ramona; Kriehuber, Ernst; Majdic, Otto; Zlabinger, Gerhard; Pickl, Winfried F.; Steinberger, Peter

    2010-01-01

    It is well established that full activation of T cells requires the interaction of the TCR complex with the peptide–MHC complex (Signal 1) and additional signals (Signal 2). These second signals are generated by the interaction of costimulatory ligands expressed on antigen presenting cells with activating receptors on T cells. In addition, T cell responses are negatively regulated by inhibitory costimulatory pathways. Since professional antigen presenting cells (APC) harbour a plethora of stimulating and inhibitory surface molecules, the contribution of individual costimulatory molecules is difficult to assess on these cells. We have developed a system of stimulator cells that can give signal 1 to human T cells via a membrane bound anti-CD3 antibody fragment. By expressing human costimulatory ligands on these cells, their role in T cell activation processes can readily be analyzed. We demonstrate that T cell stimulator cells are excellent tools to study various aspects of human T cell costimulation, including the effects of immunomodulatory drugs or how costimulatory signals contribute to the in vitro expansion of T cells. T cell stimulator cells are especially suited for the functional evaluation of ligands that are implicated in costimulatory processes. In this study we have evaluated the role of the CD2 family member CD150 (SLAM) and the TNF family member TL1A (TNFSF15) in the activation of human T cells. Whereas our results do not point to a significant role of CD150 in T cell activation we found TL1A to potently costimulate human T cells. Taken together our results demonstrate that T cell stimulator cells are excellent tools to study various aspects of costimulatory processes. PMID:20858499

  12. Potential role of natural killer cells in controlling tumorigenesis by human T-cell leukemia viruses.

    PubMed Central

    Feuer, G; Stewart, S A; Baird, S M; Lee, F; Feuer, R; Chen, I S

    1995-01-01

    Human T-cell leukemia virus (HTLV) is the etiologic agent of adult T-cell leukemia (ATL), a malignancy of T lymphocytes that is characterized by a long latency period after virus exposure. Intraperitoneal inoculation of severe combined immunodeficient (SCID) mice with HTLV-transformed cell lines and ATL tumor cells was employed to investigate the tumorigenic potential of HTLV type I (HTLV-I)-infected cells. In contrast to inoculation of ATL (RV-ATL) cells into SCID mice, which resulted in the formation of lymphomas, inoculation of HTLV-I- and HTLV-II-transformed cell lines (SLB-I and JLB-II cells, respectively) did not result in tumor formation. Immunosuppression of SCID mice, either by whole-body irradiation or by treatment with an antiserum, anti-asialo GM1 (alpha-AGM1), which transiently abrogates natural killer cell activity in vivo, was necessary to establish the growth of tumors derived from HTLV-transformed cell lines. PCR and flow cytometric studies reveal that HTLV-I-transformed cells are eliminated from the peritoneal cavities of inoculated mice by 3 days postinoculation; in contrast, RV-ATL cells persist and are detected until the mice succumb to lymphoma development. The differing behaviors of HTLV-infected cell lines and ATL tumor cells in SCID mice suggest that ATL cells have a higher tumorigenic potential in vivo than do HTLV-infected cell lines because of their ability to evade natural killer cell-mediated cytolysis. PMID:7815516

  13. Human CD141+ DCs induce CD4+ T cells to produce type 2 cytokines1

    PubMed Central

    Yu, Chun I; Becker, Christian; Metang, Patrick; Marches, Florentina; Wang, Yuanyuan; Toshiyuki, Hori; Banchereau, Jacques; Merad, Miriam; Palucka, Karolina

    2014-01-01

    Dendritic cells (DCs) play the central role in the priming of naïve T cells and the differentiation of unique effector T cells. Here, using lung tissues and blood from both humans and humanized mice, we analyzed the response of human CD1c+ and CD141+ DC subsets to live-attenuated influenza virus (LAIV). Specifically, we analyzed the type of CD4+ T cell immunity elicited by LAIV-exposed DCs. Both DC subsets induce proliferation of allogeneic naïve CD4+ T cells with capacity to secrete IFN-γ. However, CD141+ DCs are uniquely able to induce the differentiation of IL-4 and IL-13 producing CD4+ T cells. CD141+ DCs induce IL-4 and IL-13 secreting CD4+ T cells through OX40L. Thus, CD141+ DCs demonstrate remarkable plasticity in guiding adaptive immune responses. PMID:25246496

  14. Characterization of an infectious molecular clone of human T-cell leukemia virus type I.

    PubMed Central

    Zhao, T M; Robinson, M A; Bowers, F S; Kindt, T J

    1995-01-01

    An infectious molecular clone of human T-cell leukemia virus type I (HTLV-I) was derived from an HTLV-I-transformed rabbit T-cell line, RH/K30, obtained by coculture of rabbit peripheral blood mononuclear cells (PBMC) with the human HTLV-I-transformed cell line MT-2. The RH/K30 cell line contained two integrated proviruses, an intact HTLV-I genome and an apparently defective provirus with an in-frame stop codon in the env gene. A genomic DNA fragment containing the intact HTLV-I provirus was cloned into bacteriophage lambda (K30 phi) and subcloned into a plasmid vector (K30p). HTLV-I p24gag protein was detected in culture supernatants of human and rabbit T-cell and fibroblast lines transfected with these clones, at levels comparable to those of the parental cell line RH/K30. Persistent expression of virus was observed in one of these lines, RL-5/K30p, for more than 24 months. Biologic characterization of this cell line revealed the presence of integrated HTLV-I provirus, spliced and unspliced mRNA transcripts, and typical extracellular type C retrovirus particles. As expected, these virus particles contained HTLV-I RNA and reverse transcriptase activity. The transfected cells also expressed surface major histocompatibility complex class II, whereas no expression of this molecule was detected in the parental RL-5 cell line. Virus was passaged by cocultivation of irradiated RL-5/K30p cells with either rabbit PBMC or human cord blood mononuclear cells, demonstrating in vitro infectivity. The virus produced in these cells was also infectious in vivo, since rabbits injected with RL-5/K30p cells became productively infected, as evidenced by seroconversion, amplification of HTLV-I-specific sequences by PCR from PBMC DNA, and virus isolation from PBMC. Availability of infectious molecular clones will facilitate functional studies of HTLV-I genes and gene products. PMID:7884847

  15. T Cells and Stromal Fibroblasts in Human Tumor Microenvironments Represent Potential Therapeutic Targets

    PubMed Central

    Barnas, Jennifer L.; Simpson-Abelson, Michelle R.; Yokota, Sandra J.; Kelleher, Raymond J.

    2010-01-01

    The immune system of cancer patients recognizes tumor-associated antigens expressed on solid tumors and these antigens are able to induce tumor-specific humoral and cellular immune responses. Diverse immunotherapeutic strategies have been used in an attempt to enhance both antibody and T cell responses to tumors. While several tumor vaccination strategies significantly increase the number of tumor-specific lymphocytes in the blood of cancer patients, most vaccinated patients ultimately experience tumor progression. CD4+ and CD8+ T cells with an effector memory phenotype infiltrate human tumor microenvironments, but most are hyporesponsive to stimulation via the T cell receptor (TCR) and CD28 under conditions that activate memory T cells derived from the peripheral blood of the cancer patients or normal donors. Attempts to identify cells and molecules responsible for the TCR signaling arrest of tumor-infiltrating T cells have focused largely upon the immunosuppressive effects of tumor cells, tolerogenic dendritic cells and regulatory T cells. Here we review potential mechanisms by which human T cell function is arrested in the tumor microenvironment with a focus on the immunomodulatory effects of stromal fibroblasts. Determining in vivo which cells and molecules are responsible for the TCR arrest in human tumor-infiltrating T cells will be necessary to formulate and test strategies to prevent or reverse the signaling arrest of the human T cells in situ for a more effective design of tumor vaccines. These questions are now addressable using novel human xenograft models of tumor microenvironments. PMID:21209773

  16. Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones

    NASA Astrophysics Data System (ADS)

    Riddell, Stanley R.; Watanabe, Kathe S.; Goodrich, James M.; Li, Cheng R.; Agha, Mounzer E.; Greenberg, Philip D.

    1992-07-01

    The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8^+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8^+ cytomegalovirus-specific CTL responses.

  17. The tetraspanin CD9 is preferentially expressed on the human CD4+CD45RA+ naive T cell population and is involved in T cell activation

    PubMed Central

    KOBAYASHI, H; HOSONO, O; IWATA, S; KAWASAKI, H; KUWANA, M; TANAKA, H; DANG, N H; MORIMOTO, C

    2004-01-01

    Human CD4+ T cells can be divided into reciprocal memory and naive T cell subsets based on their expression of CD45 isoforms and CD29/integrin beta1 subunit. To identify unique cell surface molecules on human T cells, we developed a new monoclonal antibody termed anti5H9. Binding of anti5H9 triggers a co-stimulatory response in human peripheral blood T cells. Retrovirus-mediated expression cloning has revealed that the antigen recognized by anti5H9 is identical to the tetraspanin CD9. We now show that human CD9 is preferentially expressed on the CD4+CD45RA+ naive T cell subset, and that CD9+CD45RA+ T cells respond preferentially to the recombinant beta2-glycoprotein I, compared to CD9–CD45RA+ T cells. Furthermore, anti5H9 inhibits both the recombinant beta2-glycoprotein I- and the recall antigen tetanus toxoid-specific T cell proliferation. These results suggest that the tetraspanin CD9 plays an important role in T cell activation. PMID:15196249

  18. Neonatal thymectomy reveals differentiation and plasticity within human naive T cells.

    PubMed

    van den Broek, Theo; Delemarre, Eveline M; Janssen, Willemijn J M; Nievelstein, Rutger A J; Broen, Jasper C; Tesselaar, Kiki; Borghans, Jose A M; Nieuwenhuis, Edward E S; Prakken, Berent J; Mokry, Michal; Jansen, Nicolaas J G; van Wijk, Femke

    2016-03-01

    The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanding on the identification of truly naive T cells and recent thymic emigrants. PMID:26901814

  19. Tissue distribution of human gamma delta T cells: no evidence for general epithelial tropism.

    PubMed Central

    Vroom, T M; Scholte, G; Ossendorp, F; Borst, J

    1991-01-01

    In man and mice only a small proportion of T cells in the peripheral lymphoid compartment express the gamma delta T cell receptor (TCR). In mice, however, gamma delta T cells comprise the predominant population at particular epithelial sites--in epidermis and epithelia of intestine, reproductive organs, and tongue. The distribution of gamma delta T cells in normal human tissues was investigated, paying particular attention to epithelial layers. In all lymphatic organs and in epithelia of a wide variety of non-lymphatic organs, including the respiratory tract, male and female reproductive organs and tongue, gamma delta T cells constituted less than 5% of total T cells, with the remainder expressing TCR alpha beta. The only exception was the intestine, where gamma delta T cells were preferentially situated in the columnar epithelium of the crypts, rather than in the lamina propria. It is concluded, therefore, that human gamma delta T cells do not display a general epithelial tropism and are, in terms of relative numbers, no more able than alpha beta T cells to carry out continuous surveillance of the immune system against infection or transformation in epithelia. gamma delta T cells may, however, have a specialised function in the epithelium of the intestinal tract. Images PMID:1838746

  20. Neonatal thymectomy reveals differentiation and plasticity within human naive T cells

    PubMed Central

    van den Broek, Theo; Delemarre, Eveline M.; Janssen, Willemijn J.M.; Nievelstein, Rutger A.J.; Broen, Jasper C.; Tesselaar, Kiki; Borghans, Jose A.M.; Nieuwenhuis, Edward E.S.; Prakken, Berent J.; Mokry, Michal; Jansen, Nicolaas J.G.

    2016-01-01

    The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanding on the identification of truly naive T cells and recent thymic emigrants. PMID:26901814

  1. DRUG RESISTANT ALLOREACTIVE T CELLS MAY CONTRIBUTE TO HUMAN GRAFT REJECTION

    EPA Science Inventory

    The objective of our study was to determine whether resistance to immunosuppressive drugs by transplant recipient's T cells could contribute to continued graft rejection, in spite of immunosuppressive therapy. he T cell lines used in this series of experiments were originally est...

  2. Animals Models of Human T Cell Leukemia Virus Type I Leukemogenesis.

    PubMed

    Niewiesk, Stefan

    2016-03-31

    Infection with human T cell leukemia virus type I (HTLV-I) causes adult T cell leukemia (ATL) in a minority of infected individuals after long periods of viral persistence. The various stages of HTLV-I infection and leukemia development are studied by using several different animal models: (1) the rabbit (and mouse) model of persistent HTLV-I infection, (2) transgenic mice to model tumorigenesis by HTLV-I specific protein expression, (3) ATL cell transfers into immune-deficient mice, and (4) infection of humanized mice with HTLV-I. After infection, virus replicates without clinical disease in rabbits and to a lesser extent in mice. Transgenic expression of both the transactivator protein (Tax) and the HTLV-I bZIP factor (HBZ) protein have provided insight into factors important in leukemia/lymphoma development. To investigate factors relating to tumor spread and tissue invasion, a number of immune-deficient mice based on the severe combined immunodeficiency (SCID) or non-obese diabetic/SCID background have been used. Inoculation of adult T cell leukemia cell (lines) leads to lymphoma with osteolytic bone lesions and to a lesser degree to leukemia development. These mice have been used extensively for the testing of anticancer drugs and virotherapy. A recent development is the use of so-called humanized mice, which, upon transfer of CD34(+)human umbilical cord stem cells, generate human lymphocytes. Infection with HTLV-I leads to leukemia/lymphoma development, thus providing an opportunity to investigate disease development with the aid of molecularly cloned viruses. However, further improvements of this mouse model, particularly in respect to the development of adaptive immune responses, are necessary. PMID:27034390

  3. Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

    PubMed Central

    Thomas, Simone; Klobuch, Sebastian; Podlech, Jürgen; Plachter, Bodo; Hoffmann, Petra; Renzaho, Angelique; Theobald, Matthias

    2015-01-01

    Reactivation of human cytomegalovirus (HCMV) can cause severe disease in recipients of hematopoietic stem cell transplantation. Although preclinical research in murine models as well as clinical trials have provided 'proof of concept' for infection control by pre-emptive CD8 T-cell immunotherapy, there exists no predictive model to experimentally evaluate parameters that determine antiviral efficacy of human T cells in terms of virus control in functional organs, prevention of organ disease, and host survival benefit. We here introduce a novel mouse model for testing HCMV epitope-specific human T cells. The HCMV UL83/pp65-derived NLV-peptide was presented by transgenic HLA-A2.1 in the context of a lethal infection of NOD/SCID/IL-2rg-/- mice with a chimeric murine CMV, mCMV-NLV. Scenarios of HCMV-seropositive and -seronegative human T-cell donors were modeled by testing peptide-restimulated and T-cell receptor-transduced human T cells, respectively. Upon transfer, the T cells infiltrated host tissues in an epitope-specific manner, confining the infection to nodular inflammatory foci. This resulted in a significant reduction of viral load, diminished organ pathology, and prolonged survival. The model has thus proven its potential for a preclinical testing of the protective antiviral efficacy of HCMV epitope-specific human T cells in the evaluation of new approaches to an immunotherapy of CMV disease. PMID:26181057

  4. The BMP Pathway Participates in Human Naive CD4+ T Cell Activation and Homeostasis

    PubMed Central

    Martínez, Víctor G.; Sacedón, Rosa; Hidalgo, Laura; Valencia, Jaris; Fernández-Sevilla, Lidia M.; Hernández-López, Carmen

    2015-01-01

    Bone Morphogenetic Proteins (BMPs) form a group of secreted factors that belongs to the TGF-β superfamily. Among different roles in a number of immune cell types, BMPs are known to regulate T cell development within the thymus, although the role of BMP signaling in human mature T cells remains elusive. In this study, we demonstrate that canonical BMP signaling is necessary during two critical events that regulate the size and function of human naive CD4+ T cell population: activation and homeostasis. Upon stimulation via TCR, naive CD4+ T cells upregulate the expression of BMP ligands triggering canonical BMP signaling in CD25+ cells. Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling. Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis. Moreover, upregulation of two critical receptors for T cell homeostasis, CXCR4 and CCR9, triggered by IL-7 is also abrogated in the absence of BMP signaling. Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application. PMID:26110906

  5. Human lymph-node CD8+ T cells display an altered phenotype during systemic autoimmunity

    PubMed Central

    Ramwadhdoebe, Tamara H; Hähnlein, Janine; van Kuijk, Bo J; Choi, Ivy Y; van Boven, Leonard J; Gerlag, Danielle M; Tak, Paul P; van Baarsen, Lisa G

    2016-01-01

    Although many studies are focused on auto-reactive CD4+ T cells, the precise role of CD8+ T cells in autoimmunity is poorly understood. The objective of this study is to provide more insight into the phenotype and function CD8+ T cells during the development of autoimmune disease by studying CD8+ T cells in human lymph-node biopsies and peripheral blood obtained during the earliest phases of rheumatoid arthritis (RA). Here, we show that lymphoid pro-inflammatory CD8+ T cells exhibit a less-responsive phenotype already during the earliest phases of autoimmunity compared with healthy individuals. We found an increase in CD8+ memory T cells in lymphoid tissue during the earliest phases of autoimmunity, even before clinical onset of RA, accompanied by an increased frequency of non-circulating or recently activated (CD69+) CD8+ T cells in lymphoid tissue and peripheral blood. Importantly, lymphoid pro-inflammatory CD8+IL-17A+ T cells displayed a decreased capacity of cytokine production, which was related to disease activity in early RA patients. In addition, a decreased frequency of regulatory CD8+IL-10+ T cells in peripheral blood was also related to disease activity in early RA patients. Our results suggest that different CD8+ T-cell subsets are affected already during the earliest phases of systemic autoimmunity. PMID:27195110

  6. Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

    PubMed Central

    Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

    2015-01-01

    The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

  7. CD8(+) T cells in human autoimmune arthritis: the unusual suspects.

    PubMed

    Petrelli, Alessandra; van Wijk, Femke

    2016-07-01

    CD8(+) T cells are key players in the body's defence against viral infections and cancer. To date, data on the role of CD8(+) T cells in autoimmune diseases have been scarce, especially when compared with the wealth of research on CD4(+) T cells. However, growing evidence suggests that CD8(+) T-cell homeostasis is impaired in human autoimmune diseases. The contribution of CD8(+) T cells to autoimmune arthritis is indicated by the close association of MHC class I polymorphisms with disease risk, as well as the correlation between CD8(+) T-cell phenotype and disease outcome. The heterogeneous phenotype, resistance to regulation and impaired regulatory function of CD8(+) T cells - especially at the target organ - might contribute to the persistence of autoimmune inflammation. Moreover, newly identified populations of tissue-resident CD8(+) T cells and their interaction with antigen-presenting cells might have a key role in disease pathology. In this Review, we assess the link between CD8(+) T cells, autoimmune arthritis and the basis of their homeostatic changes under inflammatory conditions. Improved insight into CD8(+) T cell-specific pathogenicity will be essential for a better understanding of autoimmune arthritis and the identification of new therapeutic targets. PMID:27256711

  8. Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

    SciTech Connect

    Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

    1987-12-01

    The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2/sup +/, CD3/sup +/, CD4/sup +/ or CD2/sup +/, CD3/sup +/, CD8/sup +/) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by /sup 51/Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.

  9. Human memory T cells with a naive phenotype accumulate with aging and respond to persistent viruses.

    PubMed

    Pulko, Vesna; Davies, John S; Martinez, Carmine; Lanteri, Marion C; Busch, Michael P; Diamond, Michael S; Knox, Kenneth; Bush, Erin C; Sims, Peter A; Sinari, Shripad; Billheimer, Dean; Haddad, Elias K; Murray, Kristy O; Wertheimer, Anne M; Nikolich-Žugich, Janko

    2016-08-01

    The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens. PMID:27270402

  10. TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

    PubMed

    Lehnert, Corinna; Weiswange, Maxi; Jeremias, Irmela; Bayer, Carina; Grunert, Michaela; Debatin, Klaus-Michael; Strauss, Gudrun

    2014-10-15

    The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses. PMID:25217163

  11. CD4+CD25bright T cells in human intestinal lamina propria as regulatory cells.

    PubMed

    Makita, Shin; Kanai, Takanori; Oshima, Shigeru; Uraushihara, Koji; Totsuka, Teruji; Sawada, Taisuke; Nakamura, Tetsuya; Koganei, Kazutaka; Fukushima, Tsuneo; Watanabe, Mamoru

    2004-09-01

    It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4(+)CD25(bright) T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4(+) T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4(+)CD25(-) T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4(+)CD25(+) T cells, however, cells expressing CD25 at high levels (CD4(+)CD25(bright)) suppressed the proliferation and various cytokine productions of CD4(+)CD25(-) T cells. LP CD4(+)CD25(bright) T cells by themselves produced fewer amounts of IL-2, IFN-gamma, and IL-10. Interestingly, LP CD4(+)CD25(bright) T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4(+)CD25(bright) T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis. PMID:15322172

  12. A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells

    PubMed Central

    Schaerli, Patrick; Ebert, Lisa; Willimann, Katharina; Blaser, Andrea; Roos, Regula Stuber; Loetscher, Pius; Moser, Bernhard

    2004-01-01

    Effective immune surveillance is essential for maintaining protection and homeostasis of peripheral tissues. However, mechanisms controlling memory T cell migration to peripheral tissues such as the skin are poorly understood. Here, we show that the majority of human T cells in healthy skin express the chemokine receptor CCR8 and respond to its selective ligand I-309/CCL1. These CCR8+ T cells are absent in small intestine and colon tissue, and are extremely rare in peripheral blood, suggesting healthy skin as their physiological target site. Cutaneous CCR8+ T cells are preactivated and secrete proinflammatory cytokines such as tumor necrosis factor–α and interferon-γ, but lack markers of cytolytic T cells. Secretion of interleukin (IL)-4, IL-10, and transforming growth factor–β was low to undetectable, arguing against a strict association of CCR8 expression with either T helper cell 2 or regulatory T cell subsets. Potential precursors of skin surveillance T cells in peripheral blood may correspond to the minor subset of CCR8+CD25− T cells. Importantly, CCL1 is constitutively expressed at strategic cutaneous locations, including dermal microvessels and epidermal antigen-presenting cells. For the first time, these findings define a chemokine system for homeostatic T cell traffic in normal human skin. PMID:15123746

  13. Azathioprine therapy selectively ablates human Vδ2⁺ T cells in Crohn's disease.

    PubMed

    McCarthy, Neil E; Hedin, Charlotte R; Sanders, Theodore J; Amon, Protima; Hoti, Inva; Ayada, Ibrahim; Baji, Vidya; Giles, Edward M; Wildemann, Martha; Bashir, Zora; Whelan, Kevin; Sanderson, Ian; Lindsay, James O; Stagg, Andrew J

    2015-08-01

    Tumor-derived and bacterial phosphoantigens are recognized by unconventional lymphocytes that express a Vγ9Vδ2 T cell receptor (Vδ2 T cells) and mediate host protection against microbial infections and malignancies. Vδ2 T cells are absent in rodents but readily populate the human intestine, where their function is largely unknown. Here, we assessed Vδ2 T cell phenotype and function by flow cytometry in blood and intestinal tissue from Crohn's disease patients (CD patients) and healthy controls. Blood from CD patients included an increased percentage of gut-tropic integrin β7-expressing Vδ2 T cells, while "Th1-committed" CD27-expressing Vδ2 T cells were selectively depleted. A corresponding population of CD27+ Vδ2 T cells was present in mucosal biopsies from CD patients and produced elevated levels of TNFα compared with controls. In colonic mucosa from CD patients, Vδ2 T cell production of TNFα was reduced by pharmacological blockade of retinoic acid receptor-α (RARα) signaling, indicating that dietary vitamin metabolites can influence Vδ2 T cell function in inflamed intestine. Vδ2 T cells were ablated in blood and tissue from CD patients receiving azathioprine (AZA) therapy, and posttreatment Vδ2 T cell recovery correlated with time since drug withdrawal and inversely correlated with patient age. These results indicate that human Vδ2 T cells exert proinflammatory effects in CD that are modified by dietary vitamin metabolites and ablated by AZA therapy, which may help resolve intestinal inflammation but could increase malignancy risk by impairing systemic tumor surveillance. PMID:26168223

  14. Azathioprine therapy selectively ablates human Vδ2+ T cells in Crohn’s disease

    PubMed Central

    McCarthy, Neil E.; Hedin, Charlotte R.; Sanders, Theodore J.; Amon, Protima; Hoti, Inva; Ayada, Ibrahim; Baji, Vidya; Giles, Edward M.; Wildemann, Martha; Bashir, Zora; Whelan, Kevin; Sanderson, Ian; Lindsay, James O.; Stagg, Andrew J.

    2015-01-01

    Tumor-derived and bacterial phosphoantigens are recognized by unconventional lymphocytes that express a Vγ9Vδ2 T cell receptor (Vδ2 T cells) and mediate host protection against microbial infections and malignancies. Vδ2 T cells are absent in rodents but readily populate the human intestine, where their function is largely unknown. Here, we assessed Vδ2 T cell phenotype and function by flow cytometry in blood and intestinal tissue from Crohn’s disease patients (CD patients) and healthy controls. Blood from CD patients included an increased percentage of gut-tropic integrin β7–expressing Vδ2 T cells, while “Th1-committed” CD27-expressing Vδ2 T cells were selectively depleted. A corresponding population of CD27+ Vδ2 T cells was present in mucosal biopsies from CD patients and produced elevated levels of TNFα compared with controls. In colonic mucosa from CD patients, Vδ2 T cell production of TNFα was reduced by pharmacological blockade of retinoic acid receptor-α (RARα) signaling, indicating that dietary vitamin metabolites can influence Vδ2 T cell function in inflamed intestine. Vδ2 T cells were ablated in blood and tissue from CD patients receiving azathioprine (AZA) therapy, and posttreatment Vδ2 T cell recovery correlated with time since drug withdrawal and inversely correlated with patient age. These results indicate that human Vδ2 T cells exert proinflammatory effects in CD that are modified by dietary vitamin metabolites and ablated by AZA therapy, which may help resolve intestinal inflammation but could increase malignancy risk by impairing systemic tumor surveillance. PMID:26168223

  15. DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice.

    PubMed

    Randall, Katrina L; Chan, Stephanie S-Y; Ma, Cindy S; Fung, Ivan; Mei, Yan; Yabas, Mehmet; Tan, Andy; Arkwright, Peter D; Al Suwairi, Wafaa; Lugo Reyes, Saul Oswaldo; Yamazaki-Nakashimada, Marco A; Garcia-Cruz, Maria de la Luz; Smart, Joanne M; Picard, Capucine; Okada, Satoshi; Jouanguy, Emmanuelle; Casanova, Jean-Laurent; Lambe, Teresa; Cornall, Richard J; Russell, Sarah; Oliaro, Jane; Tangye, Stuart G; Bertram, Edward M; Goodnow, Christopher C

    2011-10-24

    In humans, DOCK8 immunodeficiency syndrome is characterized by severe cutaneous viral infections. Thus, CD8 T cell function may be compromised in the absence of DOCK8. In this study, by analyzing mutant mice and humans, we demonstrate a critical, intrinsic role for DOCK8 in peripheral CD8 T cell survival and function. DOCK8 mutation selectively diminished the abundance of circulating naive CD8 T cells in both species, and in DOCK8-deficient humans, most CD8 T cells displayed an exhausted CD45RA(+)CCR7(-) phenotype. Analyses in mice revealed the CD8 T cell abnormalities to be cell autonomous and primarily postthymic. DOCK8 mutant naive CD8 T cells had a shorter lifespan and, upon encounter with antigen on dendritic cells, exhibited poor LFA-1 synaptic polarization and a delay in the first cell division. Although DOCK8 mutant T cells underwent near-normal primary clonal expansion after primary infection with recombinant influenza virus in vivo, they showed greatly reduced memory cell persistence and recall. These findings highlight a key role for DOCK8 in the survival and function of human and mouse CD8 T cells. PMID:22006977

  16. Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes

    PubMed Central

    Moroz, Maxim A.; Zhang, Hanwen; Lee, Jason; Moroz, Ekaterina; Zurita, Juan; Shenker, Larissa; Serganova, Inna; Blasberg, Ronald; Ponomarev, Vladimir

    2015-01-01

    Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. Methods Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodiumiodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 105 to 3 × 106 reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (back-ground) was measured. Results The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-18F-fluorobenzylguanidine (18F-MFBG) reporter system could detect less than 1 × 105 T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/124I-iodide reporter system could detect approximately 1 × 106 T cells; 124I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2′-18F-fluoro-5-ethyl-1-β-D-arabinofuranosyluracil (18F-FEAU) and hsvTK/18F-FEAU reporter systems detected approximately 3 × 105 T cells

  17. A Diverse Repertoire of CD4 T Cells Targets the Immediate-Early 1 Protein of Human Cytomegalovirus

    PubMed Central

    Ameres, Stefanie; Liang, Xiaoling; Wiesner, Martina; Mautner, Josef; Moosmann, Andreas

    2015-01-01

    T-cell responses to the immediate-early 1 (IE-1) protein of human cytomegalovirus (HCMV) are associated with protection from viral disease. Thus, IE-1 is a promising target for immunotherapy. CD8 T-cell responses to IE-1 are generally strong. In contrast, CD4 T-cell responses to IE-1 were described to be comparatively infrequent or undetectable in HCMV carriers, and information on their target epitopes and their function has been limited. To analyze the repertoire of IE-1-specific CD4 T cells, we expanded them from healthy donors with autologous IE-1-expressing mini-Epstein–Barr virus-transformed B-cell lines and established IE-1-specific CD4 T-cell clones. Clones from seven out of seven HCMV-positive donors recognized endogenously processed IE-1 epitopes restricted through HLA-DR, DQ, or DP. Three to seven IE-1 epitopes were recognized per donor. Cumulatively, about 27 different HLA/peptide class II complexes were recognized by 117 IE-1-specific clones. Our results suggest that a highly diversified repertoire of IE-1-specific CD4 T cells targeting multiple epitopes is usually present in healthy HCMV carriers. Therefore, multiepitope approaches to immunomonitoring and immunotherapy will make optimal use of this potentially important class of HCMV-specific effector cells. PMID:26635812

  18. Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

    PubMed

    Estrada, Leonardo D; Ağaç, Didem; Farrar, J David

    2016-08-01

    Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection. PMID:27222010

  19. T cell immunity. Functional heterogeneity of human memory CD4⁺ T cell clones primed by pathogens or vaccines.

    PubMed

    Becattini, Simone; Latorre, Daniela; Mele, Federico; Foglierini, Mathilde; De Gregorio, Corinne; Cassotta, Antonino; Fernandez, Blanca; Kelderman, Sander; Schumacher, Ton N; Corti, Davide; Lanzavecchia, Antonio; Sallusto, Federica

    2015-01-23

    Distinct types of CD4(+) T cells protect the host against different classes of pathogens. However, it is unclear whether a given pathogen induces a single type of polarized T cell. By combining antigenic stimulation and T cell receptor deep sequencing, we found that human pathogen- and vaccine-specific T helper 1 (T(H)1), T(H)2, and T(H)17 memory cells have different frequencies but comparable diversity and comprise not only clones polarized toward a single fate, but also clones whose progeny have acquired multiple fates. Single naïve T cells primed by a pathogen in vitro could also give rise to multiple fates. Our results unravel an unexpected degree of interclonal and intraclonal functional heterogeneity of the human T cell response and suggest that polarized responses result from preferential expansion rather than priming. PMID:25477212

  20. Identification and Phylogeny of the First T Cell Epitope Identified from a Human Gut Bacteroides Species

    PubMed Central

    Perez-Muñoz, Maria Elisa; Joglekar, Payal; Shen, Yi-Ji; Chang, Kuan Y.; Peterson, Daniel A.

    2015-01-01

    Host T cell reactivity toward gut bacterial epitopes has been recognized as part of disease pathogenesis. However, the specificity of T cells that recognize this vast number of epitopes has not yet been well described. After colonizing a C57BL/6J germ-free mouse with the human gut symbiotic bacteria Bacteroides thetaiotaomicron, we isolated a T cell that recognized these bacteria in vitro. Using this T cell, we mapped the first known non-carbohydrate T cell epitope within the phylum Bacteroidetes. The T cell also reacted to two other additional Bacteroides species. We identified the peptide that stimulated the T cell by using a genetic approach. Genomic data from the epitope-positive and epitope-negative bacteria explain the cross-reactivity of the T cell to multiple species. This epitope degeneracy should shape our understanding of the T cell repertoire stimulated by the complex microbiome residing in the gastrointestinal tract in both healthy and disease states. PMID:26637014

  1. Human RHOH deficiency causes T cell defects and susceptibility to EV-HPV infections

    PubMed Central

    Crequer, Amandine; Troeger, Anja; Patin, Etienne; Ma, Cindy S.; Picard, Capucine; Pedergnana, Vincent; Fieschi, Claire; Lim, Annick; Abhyankar, Avinash; Gineau, Laure; Mueller-Fleckenstein, Ingrid; Schmidt, Monika; Taieb, Alain; Krueger, James; Abel, Laurent; Tangye, Stuart G.; Orth, Gérard; Williams, David A.; Casanova, Jean-Laurent; Jouanguy, Emmanuelle

    2012-01-01

    Epidermodysplasia verruciformis (EV) is a rare genetic disorder characterized by increased susceptibility to specific human papillomaviruses, the betapapillomaviruses. These EV-HPVs cause warts and increase the risk of skin carcinomas in otherwise healthy individuals. Inactivating mutations in epidermodysplasia verruciformis 1 (EVER1) or EVER2 have been identified in most, but not all, patients with autosomal recessive EV. We found that 2 young adult siblings presenting with T cell deficiency and various infectious diseases, including persistent EV-HPV infections, were homozygous for a mutation creating a stop codon in the ras homolog gene family member H (RHOH) gene. RHOH encodes an atypical Rho GTPase expressed predominantly in hematopoietic cells. Patients’ circulating T cells contained predominantly effector memory T cells, which displayed impaired TCR signaling. Additionally, very few circulating T cells expressed the β7 integrin subunit, which homes T cells to specific tissues. Similarly, Rhoh-null mice exhibited a severe overall T cell defect and abnormally small numbers of circulating β7-positive cells. Expression of the WT, but not of the mutated RHOH, allele in Rhoh–/– hematopoietic stem cells corrected the T cell lymphopenia in mice after bone marrow transplantation. We conclude that RHOH deficiency leads to T cell defects and persistent EV-HPV infections, suggesting that T cells play a role in the pathogenesis of chronic EV-HPV infections. PMID:22850876

  2. Sleeping Beauty system to redirect T-cell specificity for human applications

    PubMed Central

    Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Dawson, Margaret; Figliola, Matthew; Olivares, Simon; Rao, Pullavathi; Jue, Yi; Multani, Asha; Yang, Ge; Zhang, Ling; Kellar, Denise; Ang, Sonny; Torikai, Hiroki; Rabinovich, Brian; Lee, Dean A.; Kebriaei, Partow; Hackett, Perry; Champlin, Richard E.; Cooper, Laurence J.N.

    2013-01-01

    The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electro-transfer of CD19-specific SB DNA plasmids in PBMC and propagation on CD19+ artificial antigen presenting cells (aAPC) was used to numerically expand CD3+ T cells expressing CAR. By Day 28 of co-culture >90% of expanded CD3+ T cells expressed CAR. CAR+ T cells specifically killed CD19+ target cells and consisted of subsets expressing biomarkers consistent with central memory, ieffector memory, and effector phenotypes. CAR+ T cells contracted numerically in the absence of CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCRVα and TCRVβ repertoire. Quantitative fluorescence in situ hybridization (Q-FISH) revealed that CAR+ T cells preserved telomere length. Quantitative PCR (Q-PCR) and FISH showed CAR transposon integrated on average once per T-cell genome. CAR+ T cells in peripheral blood can be detected by Q-PCR at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the non-viral SB system for the investigational treatment of CD19+ B-cell malignancies (currently under three INDs #: 14193, 14577, and 14739). PMID:23377665

  3. Generation of functional CD8+ T Cells by human dendritic cells expressing glypican-3 epitopes

    PubMed Central

    2010-01-01

    Background Glypican 3 (GPC-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (HCC). Since it is a potential target for T cell immunotherapy, we investigated the generation of functional, GPC-3 specific T cells from peripheral blood mononuclear cells (PBMC). Methods Dendritic cells (DC) were derived from adherent PBMC cultured at 37°C for 7 days in X-Vivo, 1% autologous plasma, and 800 u/ml GM-CSF plus 500 u/ml IL-4. Immature DC were transfected with 20 μg of in vitro synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 μF and 4 ms pulse time), or pulsed with peptide, and subsequently matured with lipopolysaccharide (LPS). Six predicted GPC-3 peptide epitopes were synthesized using standard f-moc technology and tested for their binding affinity to HLA-A2.1 molecules using the cell line T2. Results DC transfected with GPC-3 mRNA but not control DC demonstrated strong intracellular staining for GPC-3 and in vitro generated interferon-gamma expressing T cells from autologous PBMC harvested from normal subjects. One peptide, GPC-3522-530 FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope: i) it showed high affinity binding to HLA-A2, in T2 cell binding assay; ii) it was generated by the MHC class I processing pathway in DC transfected with GPC-3 mRNA, and iii) HLA-A2 positive DC loaded with the peptide stimulated proliferation in autologous T cells and generated CTL that lysed HLA-A2 and GPC-3 positive target cells. Conclusions These findings demonstrate that electroporation of GPC-3 mRNA is an efficient method to load human monocyte-derived DC with antigen because in vitro they generated GPC-3-reactive T cells that were functional, as shown by interferon-gamma production. Furthermore, this study identified a novel naturally processed, HLA-A2-restricted CTL epitope, GPC-3522-530 FLAELAYDL, which can be used to monitor HLA-A2

  4. Pseudotypes of human T-cell leukemia virus types 1 and 2: neutralization by patients' sera.

    PubMed Central

    Clapham, P; Nagy, K; Weiss, R A

    1984-01-01

    Pseudotypes of vesicular stomatitis virus (VSV) bearing envelope antigens of human T-cell leukemia virus (HTLV) types 1 and 2 were prepared by propagating VSV in cells lines productively infected with HTLV. Plaque assays of VSV (HTLV) pseudotypes were employed to determine the presence of (i) HTLV receptors on cells and (ii) neutralizing antibodies in the serum of patients with adult T-cell leukemia-lymphoma (ATLL). Cell surface receptors for HTLV-1 and HTLV-2 were found on nonlymphoid cells of human and mammalian origin. Neutralizing antibodies specific to VSV(HTLV-1) were found in sera of ATLL patients in titers varying from 1:50 to 1:30,000 and did not correlate closely with antibody titers for internal viral antigens. Sera from ATLL patients in the United Kingdom (Caribbean immigrants), United States, and Japan completely neutralized VSV (HTLV-1), indicating that the HTLV isolates from these distinct geographic regions represent a single envelope serotype. Neutralization of VSV (HTLV-1) was more specific and more sensitive than assays of syncytium inhibition. No cross-neutralization was observed between bovine leukosis virus and HTLV, and only limited cross-reaction was found for envelope antigens of HTLV-1 and HTLV-2. These studies show that VSV (HTLV) pseudotypes can be readily used to screen for neutralizing antibodies in patients' sera and to distinguish HTLV envelope serotypes. PMID:6326149

  5. Synergy between interleukin-2 and a second factor in the long-term growth of human T cells.

    PubMed Central

    Mills, B J; Shively, J E; Mitsky, P S; Hawke, D H; Forman, S J; Wright, C L; Todd, C W

    1986-01-01

    It has recently been shown that factors in addition to interleukin-2 (IL-2) are required for the proliferation or differentiation of at least some murine T-cell lines. We have previously shown that conditioned medium from human mononuclear cells stimulated with phorbol ester and staphylococcal enterotoxin A is superior to commercial sources of IL-2 for the long-term growth of human T cells. We have identified in these supernatants a non-IL-2 factor (synergistic factor, SF) which synergizes with JURKAT IL-2 in the long-term growth of human T cells. [3H]TdR incorporation by IL-2-dependent human T cells after growth in IL-2 or SF alone for 14 days was slight, but significant. By contrast, growth in a combination of SF and IL-2 for 14 days stimulated [3H]TdR incorporation 10-20-fold higher, generally equal to the high incorporation measured when cells were grown in the presence of the conditioned medium from which SF was obtained. In a standard 2-day IL-2 assay, there was no correlation between activity and long-term growth-promoting ability. These results suggest that the 14-day assay better discerns the growth-promoting activity of various factors or combinations of factors. The mechanism of this interaction between SF and IL-2 remains to be elucidated. It is clear, however, that T-cell growth factor activity, when assessed by the long-term growth of human T cells, is not due to interleukin-2 alone. PMID:3489670

  6. Cross-presentation of viral antigens in dribbles leads to efficient activation of virus-specific human memory t cells

    PubMed Central

    2014-01-01

    Background Autophagy regulates innate and adaptive immune responses to pathogens and tumors. We have reported that autophagosomes derived from tumor cells after proteasome inhibition, DRibbles (Defective ribosomal products in blebs), were excellent sources of antigens for efficient cross priming of tumor-specific CD8+ T cells, which mediated regression of established tumors in mice. But the activity of DRibbles in human has not been reported. Methods DRibbles or cell lysates derived from HEK293T or UbiLT3 cell lines expressing cytomegalovirus (CMV) pp65 protein or transfected with a plasmid encoding dominant HLA-A2 restricted CMV, Epstein-Barr virus (EBV), and Influenza (Flu) epitopes (CEF) were loaded onto human monocytes or PBMCs and the response of human CMV pp65 or CEF antigen-specific CD4+ and CD8+ memory T cells was detected by intracellular staining. The effect of cytokines (GM-CSF, IL-4, IL-12, TNF-α, IFN-α and IFN-γ) TLR agonists (Lipopolysaccharide, Polyinosinic-polycytidylic acid (poly(I:C), M52-CpG, R848, TLR2 ligand) and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored. Results In this study we showed that purified monocytes, or human PBMCs, loaded with DRibbles isolated from cells expressing CMV or CEF epitopes, could activate CMV- or CEF-specific memory T cells. DRibbles were significantly more efficient at stimulating CD8+ memory T cells compared to cell lysates expressing the same antigenic epitopes. We optimized the conditions for T-cell activation and IFN-γ production following direct loading of DRibbles onto PBMCs. We found that the addition of Poly(I:C), CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8+ T cell responses. Conclusions DRibbles containing specific viral antigens are an efficient ex vivo activator of human antigen-specific memory T cells specific for those antigens. This function could be enhanced by combining with Poly

  7. The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

    PubMed Central

    Hamaia, S; Cassé, H; Gazzolo, L; Duc Dodon, M

    1997-01-01

    The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may

  8. Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis

    PubMed Central

    Levring, Trine B.; Kongsbak, Martin; Rode, Anna K. O.; Woetmann, Anders; Ødum, Niels; Bonefeld, Charlotte Menné; Geisler, Carsten

    2015-01-01

    Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication. PMID:26392411

  9. Propagation of mouse and human T cells with defined antigen specificity and function.

    PubMed

    Cohen, P A; Fowler, D H; Kim, H; White, R L; Czerniecki, B J; Carter, C; Gress, R E; Rosenberg, S A

    1994-01-01

    Difficulties maintaining fully functional CD4+ T cells in culture have historically limited the study of their role in tumour rejection as well as other clinical applications. As the therapeutic value of current antitumour CD8+ T cell adoptive therapy becomes better defined, a strong impetus exists to determine optimal conditions for culturing antitumour CD4+ T cells. Our goal is to promote broadly polyclonal, antigen-specific CD4+ T cell responses of either Th1 or Th2 character for use in antitumour therapy or allograft facilitation, respectively. Similar obstacles exist in murine and human cultures: (1) during even brief periods of culture CD4+ T cells develop high 'background' reactivity to class II-positive antigen-presenting cells; (2) maintenance of antigen specificity as evidenced by cytokine secretion and short-term proliferation assays is insufficient to ensure bulk numerical expansion; (3) Th1-type CD4+ T cells often lose their potential for antigen-specific secretion of interleukin 2 on re-stimulation (though remain inducible by 12-O-tetradecanoylphorbol 13-acetate/ionomycin); (4) during prolonged culture selection pressure favours CD4+ subpopulations that recognize artifactual antigens such as culture medium proteins; (5) even with optimal culture conditions, cultured CD4+ T cells may function differently in vivo to uncultured CD4+ T cells. We have devised various strategies to surmount these obstacles by use of selected cytokines, antigen-presenting cells and timely culture manoeuvres. PMID:7540969

  10. TNF-α blockade induces IL-10 expression in human CD4+ T cells

    NASA Astrophysics Data System (ADS)

    Evans, Hayley G.; Roostalu, Urmas; Walter, Gina J.; Gullick, Nicola J.; Frederiksen, Klaus S.; Roberts, Ceri A.; Sumner, Jonathan; Baeten, Dominique L.; Gerwien, Jens G.; Cope, Andrew P.; Geissmann, Frederic; Kirkham, Bruce W.; Taams, Leonie S.

    2014-02-01

    IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

  11. Characterization of a novel subset of T cells from human spleen that lacks L-selectin.

    PubMed Central

    Wallace, D L; Beverley, P C

    1993-01-01

    Human L-selection (LAM-1, Leu-8, TQ1, DREG 56) is a member of the 'selection' family of adhesion molecules. Antibodies to L-selectin have been shown to block the binding of T cells to peripheral lymph node high endothelial venules (HEV). Most unstimulated peripheral blood T cells express high levels of L-selectin whilst it is only weakly expressed on the majority of T cells from secondary lymphoid organs. We show here (a) that T cells from tonsil and lymph node up-regulate L-selectin when released from their microenvironment, (b) that in contrast, spleen contains a stable L-selectin negative subset, (c) that this subset remains surface L-selection negative after stimulation even though the T cells can respond by proliferation, (d) that this subset expresses minimal levels of LAM-1 mRNA and (e) that mucosal lymphocyte antigen (MLA) positive and T-cell receptor (TcR) gamma delta positive T cells found within the L-selectin negative population are similar to subsets of T cells found amongst lamina propria (LP) and intraepithelial lymphocytes (IEL) of the gut. Images Figure 3 PMID:7684357

  12. SV40 large T antigen-specific human T cell memory responses.

    PubMed

    Coleman, Sharon; Gibbs, Allen; Butchart, Eric; Mason, Malcolm D; Jasani, Bharat; Tabi, Zsuzsanna

    2008-08-01

    The continued presence of simian virus 40 (SV40), a monkey polyomavirus, in man is confirmed by the regular detection of SV40-specific antibodies in 5-10% of children who are unlikely to have received contaminated polio-vaccines. The aim of our experiments was to find cellular immunological evidence of SV40 infection in humans by testing memory T cell responses to SV40 large T antigen (Tag). As there is some indication that the virus may be present in malignant pleural mesothelioma (MPM) cells, we analyzed T cell responses in MPM patients and in healthy donors. The frequencies of responding T cells to overlapping Tag peptides were tested by cytokine flow cytometry. CD8+ T cells from 4 of 32 MPM patients responded (above twofold of control) to SV40 Tag peptides, while no positive responses were detected in 12 healthy donors. Within SV40 Tag we identified three 15 amino acid-long immunogenic sequences and one 9 amino acid-long T cell epitope (p138) (138FPSELLSFL146), the latter including a HLA-B7-restriction motif. T cell responses to p138 were SV40-specific as T cells stimulated with p138 did not cross-react with the corresponding sequences of Tag of human polyomaviruses BKV and JCV. Similarly, the relevant BKV and JCV Tag peptides did not generate T cell responses against SV40 TAg p138. Peptide-stimulated T cells also killed SV40 Tag-transfected target cells. This article demonstrates the presence, and provides a detailed analysis, of SV40-specific T cell memory in man. PMID:18551603

  13. Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells

    SciTech Connect

    Zu, Youli; Kohno, Michiaki; Namba, Yuziro ); Kohno, Michiaki ); Kubota, Ichiro ); Nishida, Eisuke )

    1990-01-30

    The authors have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of ({sup 32}P)P{sub i}-labeled and ({sup 3}H)leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fraction studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.

  14. A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation

    PubMed Central

    Chillemi, Antonella; Quarona, Valeria; Zaccarello, Gianluca; Carrega, Paolo; Ferlazzo, Guido; Mingari, Maria Cristina; Moretta, Lorenzo

    2015-01-01

    Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD+. Melanoma cells inhibited T cell proliferation through an ADO-dependent mechanism, since such inhibition was reverted using CD38/CD73 specific inhibitors. Melanoma cells abolished the function of effector memory, central memory and reduced naïve CD4+ T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4+ T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8+ T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role. PMID:26329660

  15. Temporal Gene Regulation During HIV-1 Infection of Human CD4+ T Cells

    PubMed Central

    Corbeil, Jacques; Sheeter, Dennis; Genini, Davide; Rought, Steffney; Leoni, Lorenzo; Du, Pinyi; Ferguson, Mark; Masys, Daniel R.; Welsh, John B.; Fink, J. Lynn; Sasik, Roman; Huang, David; Drenkow, Jorg; Richman, Douglas D.; Gingeras, Thomas

    2001-01-01

    CD4+ T-cell depletion is a characteristic of human immunodeficiency virus type 1 (HIV-1) infection. In this study, modulation of mRNA expression of 6800 genes was monitored simultaneously at eight time points in a CD4+ T-cell line (CEM-GFP) during HIV infection. The responses to infection included: (1) >30% decrease at 72 h after infection in overall host-cell production of monitored mRNA synthesis, with the replacement of host-cell mRNA by viral mRNA, (2) suppression of the expression of selected mitochondrial and DNA repair gene transcripts, (3) increased expression of the proapoptotic gene and its gene p53-induced product Bax, and (4) activation of caspases 2, 3, and 9. The intense HIV-1 transcription resulted in the repression of much cellular RNA expression and was associated with the induction of apoptosis of infected cells but not bystander cells. This choreographed host gene response indicated that the subversion of the cell transcriptional machinery for the purpose of HIV-1 replication is akin to genotoxic stress and represents a major factor leading to HIV-induced apoptosis. PMID:11435401

  16. CD4-Negative Cells Bind Human Immunodeficiency Virus Type 1 and Efficiently Transfer Virus to T Cells

    PubMed Central

    Olinger, Gene G.; Saifuddin, Mohammed; Spear, Gregory T.

    2000-01-01

    The ability of human immunodeficiency virus strain MN (HIVMN), a T-cell line-adapted strain of HIV, and X4 and R5 primary isolates to bind to various cell types was investigated. In general, HIVMN bound to cells at higher levels than did the primary isolates. Virus bound to both CD4-positive (CD4+) and CD4-negative (CD4−) cells, including neutrophils, Raji cells, tonsil mononuclear cells, erythrocytes, platelets, and peripheral blood mononuclear cells (PBMC), although virus bound at significantly higher levels to PBMC. However, there was no difference in the amount of HIV that bound to CD4-enriched or CD4-depleted PBMC. Virus bound to CD4− cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. Virus bound to nucleated cells was significantly more infectious than virus bound to erythrocytes or platelets. The enhanced infection of T cells by virus bound to CD4− cells was not due to stimulatory signals provided by CD4− cells or infection of CD4− cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD4− cells is efficiently passed to CD4+ T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD4− cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD4− cells is an important route for infection of T cells in vivo. PMID:10954556

  17. Identification of novel CD8+ T cell epitopes in human herpesvirus 6B U11 and U90

    PubMed Central

    Halawi, Mustafa; Khan, Naeem; Blake, Neil

    2015-01-01

    Human herpesvirus 6B (HHV6B) infects over 90% of the population, and normally establishes a latent infection, where episodes of reactivation are asymptomatic. However, in immunocompromised patients HHV6B reactivation is associated with high morbidity and mortality. Cellular immunotherapy has been utilised against other herpesvirus in immunocompromised settings. However, limited information on the immune response against HHV6B has hampered the development of immunotherapy for HHV6B-driven disease. In this study, we have analysed the cellular immune response against four HHV6B antigens in a panel of 30 healthy donors. We show that the base-line level of T cell reactivity in peripheral blood is very low to undetectable. A short-term reactivation step enabled expansion of T cell responses, and all donors responded to at least 1 antigen, but more commonly 3 or 4. A hierarchy of immunogenicity was determined with antigens U90 and U54 being co-dominant, followed by U11 and U39. Putative CD8+ T cell epitopes were mapped to U90 and U11, predicted to be presented in the context of HLA-A1, A29, B39 and C6. T cells reactive against these novel epitopes were able to recognise virus-infected cells. Our data is supportive of the application and on-going development of T cell immunotherapy against HHVB-driven disease in the immunocompromised host. PMID:26029371

  18. Noncytotoxic T cell clones obtained from a human mixed leukocyte culture.

    PubMed

    Chu, M H; Wee, S L; Bach, F H

    1990-02-01

    Peripheral blood mononuclear cells from a DQW-1 homozygous individual were cocultured with irradiated lymphoblastoid cell line from a DQW-1 homozygous unrelated donor bearing BW35-DW1 haplotype. From T cell cloning of primary and twice-stimulated mixed leukocyte cultures (MLC), 7 and 11 T cell clones were obtained respectively. None of the 18 clones showed specific cytotoxic activity against the alloantigen of the stimulator cell as well as natural killer (NK)-like activity against K562 cells. However, most T cell clones from both primary and re-stimulated MLC demonstrated moderate cytotoxic activity in lectin-dependent cell-mediated cytolysis (LDCC) assay. Screening assay for cell-mediated lympholysis (CML) performed on growing microcultures obtained from restimulated MLC cloning confirmed the non-cytotoxic status of these T cell clones by showing that 41 out of 44 growing microcultures were not cytotoxic against the stimulator cell; the other 3 clones lyzed the target cell mildly. The cells from all 5 T cell clones detected for indirect fluorescence expressed CD3 and CD4 surface markers. Taken together, the results suggested that proliferation-regulating T cell subsets or factor(s) may be generated during the course of MLCs under the present responder-stimulator combination, and may suppress the development of alloreactive cytotoxic T cells and NK-like cells. PMID:2144231

  19. Sequestration from Immune CD4^+ T Cells of Mycobacteria Growing in Human Macrophages

    NASA Astrophysics Data System (ADS)

    Pancholi, Preeti; Mirza, Asra; Bhardwaj, Nina; Steinman, Ralph M.

    1993-05-01

    CD4^+ helper T cells mediate resistance to tuberculosis, presumably by enhancing the antimicrobial activity of macrophages within which the Mycobacterium tuberculosis organism grows. A first step in resistance should be the presentation of mycobacterial antigens by macrophages to CD4^+ T cells. However, when the antigenic stimulus is limited to organisms growing in human monocytes, the organisms become sequestered from immune CD4^+ T cells. This block in presentation is selective for growing mycobacteria and not for other stimuli. Sequestration would allow replicating organisms to persist in infected individuals and may contribute to virulence.

  20. A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation*

    PubMed Central

    Graessel, Anke; Hauck, Stefanie M.; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B.; Suttner, Kathrin

    2015-01-01

    Naive CD4+ T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. PMID:25991687

  1. Expression and regulation of Schlafen (SLFN) family members in primary human monocytes, monocyte-derived dendritic cells and T cells

    PubMed Central

    Puck, Alexander; Aigner, Regina; Modak, Madhura; Cejka, Petra; Blaas, Dieter; Stöckl, Johannes

    2015-01-01

    Schlafen (SLFN/Slfn) family members have been investigated for their involvement in fundamental cellular processes including growth regulation, differentiation and control of viral replication. However, most research has been focused on the characterization of Slfns within the murine system or in human cell lines. Since little is known about SLFNs in primary human immune cells, we set out to analyze the expression and regulation of the six human SLFN genes in monocytes, monocyte-derived dendritic cells (moDCs) and T cells. Comparison of SLFN gene expression across these three cell types showed high mRNA expression of SLFN11 in monocytes and moDCs and high SLFN5 expression in T cells, indicating functional importance within these cell types. Differentiation of monocytes to moDCs leads to the gradual upregulation of SLFN12L and SLFN13 while SLFN12 levels were decreased by differentiation stimuli. Stimulation of moDCs via human rhinovirus, lipopolysaccharide, or IFN-α lead to strong upregulation of SLFN gene expression, while peptidoglycan poorly stimulated regulation of both SLFNs and the classical interferon-stimulated gene MxA. T cell activation was found to downregulate the expression of SLFN5, SLFN12 and SLFN12L, which was reversible upon addition of exogenous IFN-α. In conclusion, we demonstrate, that SLFN gene upregulation is mainly dependent on autocrine type I interferon signaling in primary human immune cells. Rapid decrease of SLFN expression levels following T cell receptor stimulation indicates a role of SLFNs in the regulation of human T cell quiescence. PMID:26623250

  2. Human T cells in silico: Modelling their electrophysiological behaviour in health and disease.

    PubMed

    Ehling, Petra; Meuth, Patrick; Eichinger, Paul; Herrmann, Alexander M; Bittner, Stefan; Pawlowski, Matthias; Pankratz, Susann; Herty, Michael; Budde, Thomas; Meuth, Sven G

    2016-09-01

    Although various types of ion channels are known to have an impact on human T cell effector functions, their exact mechanisms of influence are still poorly understood. The patch clamp technique is a well-established method for the investigation of ion channels in neurons and T cells. However, small cell sizes and limited selectivity of pharmacological blockers restrict the value of this experimental approach. Building a realistic T cell computer model therefore can help to overcome these kinds of limitations as well as reduce the overall experimental effort. The computer model introduced here was fed off ion channel parameters from literature and new experimental data. It is capable of simulating the electrophysiological behaviour of resting and activated human CD4(+) T cells under basal conditions and during extracellular acidification. The latter allows for the very first time to assess the electrophysiological consequences of tissue acidosis accompanying most forms of inflammation. PMID:27288542

  3. Role of the MHC restriction during maturation of antigen-specific human T cells in the thymus.

    PubMed

    Hesnard, Leslie; Legoux, François; Gautreau, Laetitia; Moyon, Melinda; Baron, Olivier; Devilder, Marie-Claire; Bonneville, Marc; Saulquin, Xavier

    2016-03-01

    In the thymus, a T-cell repertoire able to confer protection against infectious and noninfectious agents in a peptide-dependent, self-MHC-restricted manner is selected. Direct detection of Ag-specific thymocytes, and analysis of the impact of the expression of the MHC-restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer-based enrichment protocol to analyze the ex vivo frequency and activation-phenotype of human thymocytes specific for self, viral and tumor-antigens presented by HLA-A*0201 (A2) in individuals expressing or not this allele. Ag-specific thymocytes were quantified within both CD4CD8 double or single-positive compartments in every donor. Our data indicate that the maturation efficiency of Ag-specific thymocytes is poorly affected by HLA-A2 expression, in terms of frequencies. Nevertheless, A2-restricted T-cell lines from A2(+) donors reacted to A2(+) cell lines in a highly peptide-specific fashion, whereas their alloreactive counterparts showed off-target activity. This first ex vivo analysis of human antigen-specific thymocytes at different stages of human T-cell development should open new perspectives in the understanding of the human thymic selection process. PMID:26635029

  4. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.

    PubMed

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R; Subramaniam, Meena; Gate, Rachel E; Haliburton, Genevieve E; Ye, Chun J; Bluestone, Jeffrey A; Doudna, Jennifer A; Marson, Alexander

    2015-08-18

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells. PMID:26216948

  5. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    PubMed Central

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; Marson, Alexander

    2015-01-01

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells. PMID:26216948

  6. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    DOE PAGESBeta

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; et al

    2015-07-27

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9more » RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

  7. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    SciTech Connect

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; Marson, Alexander

    2015-07-27

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

  8. Linking the T cell receptor to the single cell transcriptome in antigen-specific human T cells.

    PubMed

    Eltahla, Auda A; Rizzetto, Simone; Pirozyan, Mehdi R; Betz-Stablein, Brigid D; Venturi, Vanessa; Kedzierska, Katherine; Lloyd, Andrew R; Bull, Rowena A; Luciani, Fabio

    2016-07-01

    Heterogeneity of T cells is a hallmark of a successful adaptive immune response, harnessing the vast diversity of antigen-specific T cells into a coordinated evolution of effector and memory outcomes. The T cell receptor (TCR) repertoire is highly diverse to account for the highly heterogeneous antigenic world. During the response to a virus multiple individual clones of antigen specific CD8+ (Ag-specific) T cells can be identified against a single epitope and multiple epitopes are recognised. Advances in single-cell technologies have provided the potential to study Ag-specific T cell heterogeneity at both surface phenotype and transcriptome levels, thereby allowing investigation of the diversity within the same apparent sub-population. We propose a new method (VDJPuzzle) to reconstruct the native TCRαβ from single cell RNA-seq data of Ag-specific T cells and then to link these with the gene expression profile of individual cells. We applied this method using rare Ag-specific T cells isolated from peripheral blood of a subject who cleared hepatitis C virus infection. We successfully reconstructed productive TCRαβ in 56 of a total of 63 cells (89%), with double α and double β in 18, and 7% respectively, and double TCRαβ in 2 cells. The method was validated via standard single cell PCR sequencing of the TCR. We demonstrate that single-cell transcriptome analysis can successfully distinguish Ag-specific T cell populations sorted directly from resting memory cells in peripheral blood and sorted after ex vivo stimulation. This approach allows a detailed analysis of the TCR diversity and its relationship with the transcriptional profile of different clones. PMID:26860370

  9. Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells

    PubMed Central

    Watanabe, Rei; Gehad, Ahmed; Yang, Chao; Campbell, Laura; Teague, Jessica E.; Schlapbach, Christoph; Elco, Christopher; Huang, Victor; Matos, Tiago R.; Kupper, Thomas S.; Clark, Rachael A.

    2015-01-01

    The skin of an adult human contains approximately 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All non-recirculating resident memory T cells (TRM) expressed CD69, but the majority were CD4+, CD103− and located in the dermis, in contrast to studies in mice. Both CD4+ and CD8+ CD103+ TRM were enriched in the epidermis, had potent effector functions and had a limited proliferative capacity compared to CD103− TRM. TRM of both types had more potent effector functions than recirculating T cells. Induction of CD103 on human T cells was enhanced by keratinocyte contact, depended on TGFβ and was independent of T cell keratinocyte adhesive interactions. We observed two distinct populations of recirculating T cells, CCR7+/L-selectin+ central memory T cells (TCM) and CCR7+/L-selectin− T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions and TMM were depleted more slowly from skin after alemtuzumab, suggesting TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities. PMID:25787765

  10. LGIT In Vitro Latency Model in Primary and T Cell Lines to Test HIV-1 Reactivation Compounds.

    PubMed

    Jung, Ulrike; Takahashi, Mayumi; Rossi, John J; Burnett, John C

    2016-01-01

    Persistent latent HIV-1 reservoirs pose a major barrier for combinatorial antiretroviral therapy (cART) to achieve eradication of the virus. A variety of mechanisms likely contribute to HIV-1 persistence, including establishment of post-integration latency in resting CD4+ T-lymphocytes, the proliferation of these latently infected cells, and the induced or spontaneous reactivation of latent virus. To elucidate the mechanisms of latency and to investigate therapeutic strategies for reactivating and purging the latent reservoir, investigators have developed in vitro models of HIV-1 latency using primary CD4+ T-lymphocytes and CD4+ T-cell lines. Several types of in vitro latency models range from replication-competent to single-round, replication-deficient viruses exhibiting different degrees of viral genomic deletion. Working under the hypothesis that HIV-1 post-integration latency is directly linked to HIV-1 promoter activity, which can be obscured by additional proteins expressed during replication, we focus here on the creation of latently infected primary human T-cells and cell lines through the single-round, replication deficient HIV-1 LGIT model. In this model the long terminal repeat (LTR) of the HIV-1 virus drives a cassette of GFP-IRES-Tat that allows testing of reactivating components and initiates a positive feedback loop through Tat expression. PMID:26714717

  11. T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cells in vitro.

    PubMed

    Grassi, Francesco; Cattini, Luca; Gambari, Laura; Manferdini, Cristina; Piacentini, Anna; Gabusi, Elena; Facchini, Andrea; Lisignoli, Gina

    2016-04-01

    T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23653421

  12. CD8low CD100− T Cells Identify a Novel CD8 T Cell Subset Associated with Viral Control during Human Hantaan Virus Infection

    PubMed Central

    Liu, Bei; Ma, Ying; Zhang, Yusi; Zhang, Chunmei; Yi, Jing; Zhuang, Ran; Yu, Haitao; Yang, Angang

    2015-01-01

    ABSTRACT Hantaan virus (HTNV) infection can cause a severe lethal hemorrhagic fever with renal syndrome (HFRS) in humans. CD8+ T cells play a critical role in combating HTNV infections. However, the contributions of different CD8+ T cell subsets to the immune response against viral infection are poorly understood. Here, we identified a novel subset of CD8+ T cells characterized by the CD8low CD100− phenotype in HFRS patients. The CD8low CD100− subset accounted for a median of 14.3% of the total CD8+ T cells in early phase of HFRS, and this percentage subsequently declined in the late phase of infection, whereas this subset was absent in healthy controls. Furthermore, the CD8low CD100− cells were associated with high activation and expressed high levels of cytolytic effector molecules and exhibited a distinct expression profile of effector CD8+ T cells (CCR7+/− CD45RA− CD127high CD27int CD28low CD62L−). When stimulated with specific HTNV nucleocapsid protein-derived peptide pools, most responding CD8+ cells (gamma interferon [IFN-γ] positive and/or tumor necrosis factor alpha [TNF-α] positive) were CD8low CD100− cells. The frequency of CD8low CD100− cells among HTNV-specific CD8+ T cells was higher in milder cases than in more severe cases. Importantly, the proportion of the CD8low CD100− subset among CD8+ T cells in early phase of HFRS was negatively correlated with the HTNV viral load, suggesting that CD8low CD100− cells may be associated with viral clearance. The contraction of the CD8low CD100− subset in late phase of infection may be related to the consistently high expression levels of PD-1. These results may provide new insights into our understanding of CD8+ T cell-mediated protective immunity as well as immune homeostasis after HTNV infection in humans. IMPORTANCE CD8+ T cells play important roles in the antiviral immune response. We found that the proportion of CD8low CD100− cells among CD8+ T cells from HFRS patients was

  13. A novel self-lipid antigen targets human T cells against CD1c+ leukemias

    PubMed Central

    Lepore, Marco; de Lalla, Claudia; Gundimeda, S. Ramanjaneyulu; Gsellinger, Heiko; Consonni, Michela; Garavaglia, Claudio; Sansano, Sebastiano; Piccolo, Francesco; Scelfo, Andrea; Häussinger, Daniel; Montagna, Daniela; Locatelli, Franco; Bonini, Chiara; Bondanza, Attilio; Forcina, Alessandra; Li, Zhiyuan; Ni, Guanghui; Ciceri, Fabio; Jenö, Paul; Xia, Chengfeng

    2014-01-01

    T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c+ acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c+ human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy. PMID:24935257

  14. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    PubMed Central

    Pérez-Mazliah, Damián; Albareda, María C.; Alvarez, María G.; Lococo, Bruno; Bertocchi, Graciela L.; Petti, Marcos; Viotti, Rodolfo J.; Laucella, Susana A.

    2012-01-01

    Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza (Flu) virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases. PMID:23049532

  15. FOXP3+ regulatory T cells in the human immune system.

    PubMed

    Sakaguchi, Shimon; Miyara, Makoto; Costantino, Cristina M; Hafler, David A

    2010-07-01

    Forkhead box P3 (FOXP3)(+) regulatory T (T(Reg)) cells are potent mediators of dominant self tolerance in the periphery. But confusion as to the identity, stability and suppressive function of human T(Reg) cells has, to date, impeded the general therapeutic use of these cells. Recent studies have suggested that human T(Reg) cells are functionally and phenotypically diverse. Here we discuss recent findings regarding human T(Reg) cells, including the ontogeny and development of T(Reg) cell subsets that have naive or memory phenotypes, the unique mechanisms of suppression mediated by T(Reg) cell subsets and factors that regulate T(Reg) cell lineage commitment. We discuss future studies that are needed for the successful therapeutic use of human T(Reg) cells. PMID:20559327

  16. Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection.

    PubMed

    Agrati, C; Castilletti, C; Casetti, R; Sacchi, A; Falasca, L; Turchi, F; Tumino, N; Bordoni, V; Cimini, E; Viola, D; Lalle, E; Bordi, L; Lanini, S; Martini, F; Nicastri, E; Petrosillo, N; Puro, V; Piacentini, M; Di Caro, A; Kobinger, G P; Zumla, A; Ippolito, G; Capobianchi, M R

    2016-01-01

    Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells. PMID:27031961

  17. Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection

    PubMed Central

    Agrati, C; Castilletti, C; Casetti, R; Sacchi, A; Falasca, L; Turchi, F; Tumino, N; Bordoni, V; Cimini, E; Viola, D; Lalle, E; Bordi, L; Lanini, S; Martini, F; Nicastri, E; Petrosillo, N; Puro, V; Piacentini, M; Di Caro, A; Kobinger, G P; Zumla, A; Ippolito, G; Capobianchi, M R

    2016-01-01

    Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells. PMID:27031961

  18. Metabolic engineering of Salmonella vaccine bacteria to boost human Vγ2Vδ2 T cell immunity.

    PubMed

    Workalemahu, Grefachew; Wang, Hong; Puan, Kia-Joo; Nada, Mohanad H; Kuzuyama, Tomohisa; Jones, Bradley D; Jin, Chenggang; Morita, Craig T

    2014-07-15

    Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-D-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long-lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA(-) Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB(-) Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as did the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB(-) Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags. PMID:24943221

  19. Metabolic Engineering of Salmonella Vaccine Bacteria to Boost Human Vγ2Vδ2 T Cell Immunity

    PubMed Central

    Workalemahu, Grefachew; Wang, Hong; Puan, Kia-Joo; Nada, Mohanad H.; Kuzuyama, Tomohisa; Jones, Bradley D.; Jin, Chenggang; Morita, Craig T.

    2014-01-01

    Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-D-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA− Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB− Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB− Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags. PMID:24943221

  20. Regulation of the herpesvirus saimiri oncogene stpC, similar to that of T-cell activation genes, in growth-transformed human T lymphocytes.

    PubMed Central

    Fickenscher, H; Biesinger, B; Knappe, A; Wittmann, S; Fleckenstein, B

    1996-01-01

    Herpesvirus saimiri strain C488, a T-cell tumor virus of New World primates, transforms human T lymphocytes to stable interleukin-2-dependent growth without need for further stimulation by antigen or mitogen. The transformed cell lines show the phenotype of activated mature T cells and retain many essential features of the primary parental cells, e.g., antigen specificity. In contrast to transformed New World monkey T cells, the human lines do not support lytic growth of the virus, even after chemical stimulation. Here we show that many viral genes remain silent during episomal persistence. However, the viral oncogene stpC is predominantly transcribed and translated to a stable cytoplasmic protein of 20 kDa that is heterogeneously expressed in individual cells. This 1.7-kb mRNA is bicistronic, encoding also Tip, a viral protein interacting with the T-cell-specific tyrosine kinase Lck. stpC/tip transcripts are heavily induced upon stimulation by mitogen or phorbol ester. Block of protein synthesis does not abolish transcription: treatment with cycloheximide greatly induces stpC/tip mRNA levels. Thus, this gene complex is regulated similarly to early T-cell activation genes. Constitutive and induced expression engage different transcription start sites. The T-cell regulation of the viral genes stpC and tip may contribute to the T-cell tropism of growth transformation by herpesvirus saimiri. PMID:8709223

  1. Engineering Human Peripheral Blood Stem Cell Grafts That Are Depleted of Naïve T Cells and Retain Functional Pathogen-Specific Memory T cells

    PubMed Central

    Bleakley, Marie; Heimfeld, Shelly; Jones, Lori A.; Turtle, Cameron; Riddell, Stanley R.; Shlomchik, Warren

    2014-01-01

    Graft-versus-host disease (GVHD) is a frequent major complication of allogeneic hematopoietic cell transplantation (HCT). The development of approaches that selectively deplete T cells that cause GVHD from allogeneic stem cell grafts and preserve T cells specific for pathogens may improve HCT outcomes. It has been hypothesized that the majority of T cells that can cause GVHD reside within the naïve T cell (TN) subset, and previous studies performed in mouse models and with human cells in vitro support this hypothesis. As a prelude to translating these findings to the clinic, we developed and evaluated a novel, two-step, clinically compliant procedure for manipulating peripheral blood stem cells (PBSC) to remove TN, preserve CD34+ hematopoietic stem cells, and provide for a fixed dose of memory T cells (TM) that includes T cells with specificity for common opportunistic pathogens encountered after HCT. Our studies demonstrate effective and reproducible performance of the immunomagnetic cell selection procedure for depleting TN. Moreover, after cell processing the CD45RA-depleted PBSC products are enriched for CD4+ and CD8+ TM with a central memory phenotype and contain TM cells that are capable of proliferating and producing effector cytokines in response to opportunistic pathogens. PMID:24525279

  2. Efficient induction of human T-cell leukemia virus-1-specific CTL by chimeric particle without adjuvant as a prophylactic for adult T-cell leukemia.

    PubMed

    Kozako, Tomohiro; Fukada, Katsuhiko; Hirata, Shinya; White, Yohann; Harao, Michiko; Nishimura, Yasuharu; Kino, Youichiro; Soeda, Shinji; Shimeno, Hiroshi; Lemonnier, François; Sonoda, Shunro; Arima, Naomichi

    2009-12-01

    Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with the human T-cell leukemia virus-1 (HTLV-1). HTLV-1-specific cytotoxic T lymphocytes (CTLs) play an important role in suppressing proliferation of HTLV-1-infected or transformed T-cells in vitro. Efficient induction of antigen-specific CTLs is important for immunologic suppression of oncogenesis, but has evaded strategies utilizing poorly immunogenic free synthetic peptides. In the present study, we examined the efficient induction of HTLV-1-specific CD8+ T-cell response by an HTLV-1/hepatitis B virus core (HBc) chimeric particle incorporating the HLA-A*0201-restricted HTLV-1 Tax-epitope. The immunization of HLA-A*0201-transgenic mice with the chimeric particle induced antigen-specific gamma-interferon reaction, whereas immunization with epitope peptide only induced no reaction as assessed by enzyme-linked immunospot assay. Immunization with the chimeric particle also induced HTLV-1-specific CD8+ T-cells in spleen and inguinal lymph nodes. Furthermore, upon exposure of dendritic cells from HLA-A*0201-transgenic mice to the chimeric particle, the expression of CD86, HLA-A02, TLR4 and MHC class II was increased. Additionally, our results show that HTLV-1-specific CD8+ T-cells can be induced by peptide with HTLV-1/HBc particle from ATL patient, but not by peptide only and these HTLV-1-specific CD8+ T-cells were able to lyse cells presenting the peptide. These results suggest that HTLV-1/HBc chimeric particle is capable of inducing strong cellular immune responses without adjuvants via effective maturation of dendritic cells and is potentially useful as an effective carrier for therapeutic vaccines in tumors, or in infectious diseases by substituting the epitope peptide. PMID:19889459

  3. T-cell activation is required for efficient replication of human herpesvirus 6.

    PubMed Central

    Frenkel, N; Schirmer, E C; Katsafanas, G; June, C H

    1990-01-01

    We have investigated whether T-cell activation is required for the replication of the T-lymphotropic human herpesvirus 6. The virus did not replicate in quiescent peripheral blood lymphocytes but replicated efficiently following exposure of the cells to the polyclonal mitogen phytohemagglutinin (PHA). When purified T cells were treated with PHA in the absence of accessory cells, no virus replication was observed unless exogenous interleukin-2 (IL-2) was added to the medium, promoting cell division. Incubation of peripheral blood lymphocytes in the absence of PHA but in the presence of IL-2 resulted in delayed cell blastogenesis and virus replication. Cell blastogenesis and virus replication did not occur in the purified T-cell cultures incubated with IL-2 alone. Taken together, the results show that human herpesvirus 6 replication requires full progression of the cell cycle. This finding might have implications for the pathogenicity of the virus in the human host. Images PMID:2166835

  4. Suppression of experimental allergic encephalomyelitis in rats by suppressor factor from a permanent T cell line.

    PubMed

    Stĕdra, J; Holán, V; Lodin, Z

    1986-01-01

    An antigen non-specific suppressor factor (SF4) produced by a permanent mouse T cell line inhibits the mitogen- and antigen-induced proliferation of cells in vitro. The suppression of immune response is not restricted by interspecies barrier. Administration of the SF4 factor in vivo had a significant suppressive effect on the induction and manifestation of experimental allergic encephalomyelitis (EAE) in rats. Rats treated with SF4 factor, the first dose being injected on the day of EAE induction, had no clinical manifestations or developed only mild clinical signs of EAE. Administration of the SF4 starting on day 4 after EAE induction, when the immune system had been activated, depressed the course of EAE. The results obtained in this model autoimmune disease indicate that the described suppressor factor is active in vivo and that it may be used to depress the autoaggressive immune reactions. PMID:3488925

  5. Impact of nicotine on the interplay between human periodontal ligament cells and CD4+ T cells.

    PubMed

    Ge, Xin; Liu, Ying-Feng; Wong, Yong; Wu, Li-Zheng; Tan, Ling; Liu, Fen; Wang, Xiao-Jing

    2016-09-01

    Periodontitis is a common infectious disease associated with destruction of periodontal ligaments and alveolar bones. CD4(+) T cell-mediated immune response is involved in the progression of periodontitis. Tobacco consumption increases the risk of periodontal disease. However, the impact of nicotine on the interaction between human periodontal ligament (PDL) cells and CD4(+) T cells remains unrevealed. Our study aims to investigate the effect of nicotine on PDL cells and the cocultured CD4(+) T cells. The PDL cell cultures were established by explants from healthy individuals, exposed to nicotine or α-bungarotoxin (α-BTX), and incubated solely or in combination with CD4(+) T cells. Afterwards, cell viability, secreted cytokines, and matrix metalloproteinases (MMPs) were evaluated. In monoculture of PDL cells, nicotine dramatically repressed cell viability and increased apoptosis. Meanwhile, α-BTX largely reversed the nicotine-induced apoptosis and increased viability of PDL cells. Compared with the monoculture, MMP-1, MMP-3, interleukin (IL)-1β, IL-6, IL-17, and IL-21 in supernatant of cocultures were markedly elevated after treatment with nicotine. Moreover, α-BTX significantly attenuated nicotine-triggered production of these components either in mono- or co-cultures. In addition, PDL cell-derived CXCL12 following nicotine treatment recruited CD4(+) T cells. Above all, nicotine deteriorated periodontitis partially by promoting PDL cell-CD4(+) T cell-mediated inflammatory response and matrix degradation. PMID:26553320

  6. Human cytomegalovirus elicits fetal γδ T cell responses in utero

    PubMed Central

    Brouwer, Margreet; Donner, Catherine; Liesnard, Corinne; Tackoen, Marie; Van Rysselberge, Michel; Twité, Nicolas; Goldman, Michel; Marchant, Arnaud; Willems, Fabienne

    2010-01-01

    The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. γδ T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex–unrestricted activity. We show that upon CMV infection in utero, fetal γδ T cells expand and become differentiated. The expansion was restricted to Vγ9-negative γδ T cells, irrespective of their Vδ chain expression. Differentiated γδ T cells expressed high levels of IFN-γ, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vγ8Vδ1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) δ1–CALGELGDDKLIF/CDR3γ8–CATWDTTGWFKIF. Public Vγ8Vδ1-TCR–expressing cell clones produced IFN-γ upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated γδ T cells and public Vγ8Vδ1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal γδ T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life. PMID:20368575

  7. Stimulus-dependent modulation of human B cell function by T cell clones.

    PubMed

    Patel, S S; Thiele, D L; Lipsky, P E

    1993-02-01

    T cells exert both positive and negative regulatory effects on B cell function. To determine whether the nature of the stimulus could alter the immunoregulatory effects of T cells, the capacity of a battery of human T cell clones to modulate B cell function after stimulation with either pokeweed mitogen or a mAb to the CD3 molecular complex was examined. It was observed that most clones induced B cell differentiation when stimulated with immobilized mAb to CD3 (64.1) regardless of their phenotype. Moreover, the majority of clones (42 of 51) augmented the generation of immunoglobulin-secreting cells (ISC) supported by anti-CD3-stimulated blood CD4+ T cells. By contrast, none of the clones induced B cell differentiation when stimulated with PWM and 48 of 51 clones suppressed the generation of ISC induced by blood CD4+ T cells. This suppression could not be accounted for by the depletion of essential molecules or factors or by secretion of suppressive factors. Suppressive activity of clones did not correlate with the CD4 or CD8 phenotype and was not overcome by the addition of supernatants generated from mitogen-stimulated T cells or recombinant IL-2. Suppression by most clones, however, was abrogated when the clones were treated with mitomycin C or irradiated. A number of suppressive mechanisms by individual PWM-stimulated clones was identified, including direct inhibition of B cell function by cytotoxic and non-lytic means and suppression of helper T cell function. The failure of anti-CD3-stimulated clones to suppress the differentiation of B cells appeared to reflect the capacity of this stimulus to induce apoptosis by the clones after initial activation. These results indicate that T cell clones can provide help for B cell differentiation or can suppress B cell function by a variety of mechanisms depending upon the mode of stimulation. PMID:8174175

  8. Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes.

    PubMed

    Simon, Petra; Omokoko, Tana A; Breitkreuz, Andrea; Hebich, Lisa; Kreiter, Sebastian; Attig, Sebastian; Konur, Abdo; Britten, Cedrik M; Paret, Claudia; Dhaene, Karl; Türeci, Özlem; Sahin, Ugur

    2014-12-01

    The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes. PMID:25245536

  9. Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

    PubMed Central

    Andolfi, Grazia; Fousteri, Georgia; Rossetti, Maura; Magnani, Chiara F; Jofra, Tatiana; Locafaro, Grazia; Bondanza, Attilio; Gregori, Silvia; Roncarolo, Maria-Grazia

    2012-01-01

    Type 1 regulatory T (Tr1) cells are an inducible subset of CD4+ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4+ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP+ LV-IL-10–transduced human CD4+ T (CD4LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4+ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells. PMID:22692497

  10. Stereotaxic administrations of allogeneic human Vγ9Vδ2 T cells efficiently control the development of human glioblastoma brain tumors.

    PubMed

    Jarry, Ulrich; Chauvin, Cynthia; Joalland, Noémie; Léger, Alexandra; Minault, Sandrine; Robard, Myriam; Bonneville, Marc; Oliver, Lisa; Vallette, François M; Vié, Henri; Pecqueur, Claire; Scotet, Emmanuel

    2016-06-01

    Glioblastoma multiforme (GBM) represents the most frequent and deadliest primary brain tumor. Aggressive treatment still fails to eliminate deep brain infiltrative and highly resistant tumor cells. Human Vγ9Vδ2 T cells, the major peripheral blood γδ T cell subset, react against a wide array of tumor cells and represent attractive immune effector T cells for the design of antitumor therapies. This study aims at providing a preclinical rationale for immunotherapies in GBM based on stereotaxic administration of allogeneic human Vγ9Vδ2 T cells. The feasibility and the antitumor efficacy of stereotaxic Vγ9Vδ2 T cell injections have been investigated in orthotopic GBM mice model using selected heterogeneous and invasive primary human GBM cells. Allogeneic human Vγ9Vδ2 T cells survive and patrol for several days within the brain parenchyma following adoptive transfer and can successfully eliminate infiltrative GBM primary cells. These striking observations pave the way for optimized stereotaxic antitumor immunotherapies targeting human allogeneic Vγ9Vδ2 T cells in GBM patients. PMID:27471644

  11. MHC II tetramers visualize human CD4+ T cell responses to Epstein-Barr virus infection and demonstrate atypical kinetics of the nuclear antigen EBNA1 response.

    PubMed

    Long, Heather M; Chagoury, Odette L; Leese, Alison M; Ryan, Gordon B; James, Eddie; Morton, Laura T; Abbott, Rachel J M; Sabbah, Shereen; Kwok, William; Rickinson, Alan B

    2013-05-01

    Virus-specific CD4(+) T cells are key orchestrators of host responses to viral infection yet, compared with their CD8(+) T cell counterparts, remain poorly characterized at the single cell level. Here we use nine MHC II-epitope peptide tetramers to visualize human CD4(+) T cell responses to Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis (IM), a disease associated with large virus-specific CD8(+) T cell responses. We find that, while not approaching virus-specific CD8(+) T cell expansions in magnitude, activated CD4(+) T cells specific for epitopes in the latent antigen EBNA2 and four lytic cycle antigens are detected at high frequencies in acute IM blood. They then fall rapidly to values typical of life-long virus carriage where most tetramer-positive cells display conventional memory markers but some, unexpectedly, revert to a naive-like phenotype. In contrast CD4(+) T cell responses to EBNA1 epitopes are greatly delayed in IM patients, in line with the well-known but hitherto unexplained delay in EBNA1 IgG antibody responses. We present evidence from an in vitro system that may explain these unusual kinetics. Unlike other EBNAs and lytic cycle proteins, EBNA1 is not naturally released from EBV-infected cells as a source of antigen for CD4(+) T cell priming. PMID:23569328

  12. Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein.

    PubMed

    Bosque, Alberto; Dietz, Lisa; Gallego-Lleyda, Ana; Sanclemente, Manuel; Iturralde, María; Naval, Javier; Alava, María Angeles; Martínez-Lostao, Luis; Thierse, Hermann-Josef; Anel, Alberto

    2016-05-17

    We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. PMID:27086912

  13. AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation.

    PubMed

    Grant, Christian; Jain, Pooja; Nonnemacher, Michael; Flaig, Katherine E; Irish, Bryan; Ahuja, Jaya; Alexaki, Aikaterini; Alefantis, Timothy; Wigdahl, Brian

    2006-09-01

    Human T cell leukemia virus type 1 (HTLV-1) has previously been shown to infect antigen-presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV-1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV-1 viral gene expression in established monocytic cell lines was examined. U-937 promonocytic cells transiently transfected with a HTLV-1 long-terminal repeat (LTR) luciferase construct were treated with phorbol 12-myristate 13-acetate (PMA) to induce cellular differentiation. PMA-induced cellular differentiation resulted in activation of basal and Tax-mediated transactivation of the HTLV-1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA-induced cellular differentiation induced DNA-binding activity of cellular transcription factors to Tax-responsive element 1 (TRE-1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP-1) family of basic region/leucine zipper proteins (Fra-1, Fra-2, JunB, and JunD) were induced to bind to TRE-1 repeat II during cellular differentiation. Inhibition of AP-1 DNA-binding activity by overexpression of a dominant-negative c-Fos mutant (A-Fos) in transient expression analyses resulted in severely decreased levels of HTLV-1 LTR activation in PMA-induced U-937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV-1 viral gene expression may become up-regulated by AP-1 during differentiation into macrophages or dendritic cells. PMID:16829632

  14. Lipoproteins are Major Targets of the Polyclonal Human T-cell Response to M. tuberculosis1

    PubMed Central

    Seshadri, Chetan; Turner, Marie T.; Lewinsohn, David M.; Moody, D. Branch; Van Rhijn, Ildiko

    2012-01-01

    Most vaccines and basic studies of T cell epitopes in M. tuberculosis emphasize water soluble proteins that are secreted into the extracellular space and presented in the context of MHC Class II. Much less is known about the role of antigens retained within the cell wall. We used polyclonal T cells from infected humans to probe for responses to immunodominant antigens in the M. tuberculosis cell wall. We found that the magnitude of response to secreted or cell wall intrinsic compounds was similar among healthy controls, patients with latent tuberculosis, and patients with active tuberculosis. Individual responses to secreted antigens and cell wall extract were strongly correlated (r2=0.495, p=0.001), suggesting that T cells responding to cell wall and secreted antigens are present at similar frequency. Surprisingly, T cell stimulatory factors intrinsic to the cell wall partition into organic solvents; however, these responses are not explained by CD1-mediated presentation of lipids. Instead, we find that molecules soluble in organic solvents are dependent upon MHC Class II and recognized by IFN-γ secreting CD4+ T cells. We reasoned that MHC Class II dependent antigens extracting into lipid mixtures might be found among triacylated lipoproteins present in mycobacteria. We used M. tuberculosis lacking prolipoprotein signal peptidase A (lspA), an enzyme required for lipoprotein synthesis, to demonstrate loss of polyclonal T cell responses. Our results demonstrate the use of bacterial genetics to identify lipoproteins as an unexpected and immunodominant class of cell wall-associated antigens targeted by the polyclonal human T cell response to M. tuberculosis. PMID:23197260

  15. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

    PubMed

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A; Tanaka, Yuetsu

    2016-01-01

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. PMID:27409630

  16. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

    PubMed Central

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A.; Tanaka, Yuetsu

    2016-01-01

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4+ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. PMID:27409630

  17. Production and Concentration of Lentivirus for Transduction of Primary Human T Cells.

    PubMed

    Kennedy, Alan; Cribbs, Adam P

    2016-01-01

    Lentiviral vectors have emerged as efficient tools for investigating T cell biology through their ability to efficiently deliver transgene expression into both dividing and nondividing cells. Such lentiviral vectors have the potential to infect a wide variety of cell types. However, despite this advantage, the ability to transduce primary human T cells remains challenging and methods to achieve efficient gene transfer are often time consuming and expensive. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of non-standard equipment to transduce primary human T cells. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility. PMID:27317175

  18. Real-time tracking, retrieval and gene expression analysis of migrating human T cells.

    PubMed

    Mehling, Matthias; Frank, Tino; Albayrak, Cem; Tay, Savaş

    2015-03-01

    Dynamical analysis of single-cells allows assessment of the extent and role of cell-to-cell variability, however traditional dish-and-pipette techniques have hindered single-cell analysis in quantitative biology. We developed an automated microfluidic cell culture system that generates stable diffusion-based chemokine gradients, where cells can be placed in predetermined positions, monitored via single-cell time-lapse microscopy, and subsequently be retrieved based on their migration speed and directionality for further off-chip gene expression analysis, constituting a powerful platform for multiparameter quantitative studies of single-cell chemotaxis. Using this system we studied CXCL12-directed migration of individual human primary T cells. Spatiotemporally deterministic retrieval of T cell subsets in relation to their migration speed, and subsequent analysis with microfluidic droplet digital-PCR showed that the expression level of CXCR4 – the receptor of CXCL12 – underlies enhanced human T cell chemotaxis. PMID:25512266

  19. Complexity of the primary genetic response to mitogenic activation of human T cells.

    PubMed Central

    Zipfel, P F; Irving, S G; Kelly, K; Siebenlist, U

    1989-01-01

    We describe the isolation and characterization of more than 60 novel cDNA clones that constitute part of the immediate genetic response to resting human peripheral blood T cells after mitogen activation. This primary response was highly complex, both in the absolute number of inducible genes and in the diversity of regulation. Although most of the genes expressed in activated T cells were shared with the activation response of normal human fibroblasts, a significant number were more restricted in tissue specificity and thus likely encode or effect the differentiated functions of activated T cells. The activatable genes could be further differentiated on the basis of kinetics of induction, response to cycloheximide, and sensitivity to the immunosuppressive drug cyclosporin A. It is of note that cyclosporin A inhibited the expression of more than 10 inducible genes, which suggests that this drug has a broad genetic mechanism of action. Images PMID:2498643

  20. Hormone Conjugated with Antibody to CD3 Mediates Cytotoxic T Cell Lysis of Human Melanoma Cells

    NASA Astrophysics Data System (ADS)

    Liu, Margaret Ann; Nussbaum, Samuel R.; Eisen, Herman N.

    1988-01-01

    Cytotoxic T lymphocytes can be activated by antibodies to their antigen-specific receptor complex (TCR-CD3) to destroy target cells, regardless of the specificity of the cytotoxic T cells. A novel hormone-antibody conjugate, consisting of an analog of melanocyte-stimulating hormone chemically coupled to a monoclonal antibody to CD3, the invariant component of the T cell receptor complex, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes that bear no specificity for the tumor cells. As targeting components of such anti-CD3 conjugates, hormones or growth factors are expected to prove more effective than antibodies to tumor-associated antigens in focusing the destructive activity of cytotoxic T cells on tumor target cells.

  1. Complexity of the primary genetic response to mitogenic activation of human T cells

    SciTech Connect

    Zipfel, P.F.; Siebenlist, U. ); Irving, S.G.; Kelly, K. )

    1989-03-01

    The authors describe the isolation and characterization of more than 60 novel cDNA clones that constitute part of the immediate genetic response to resting human peripheral blood T cells after mitogen activation. This primary response was highly complex, both in the absolute number of inducible genes and in the diversity of regulation. Although most of the genes expressed in activated T cells were shared with the activation response of normal human fibroblasts, a significant number were more restricted in tissue specificity and thus likely encode or effect the differentiated functions of activated T cells. The activatable genes could be further differentiated on the basis of kinetics of induction, response to cycloheximide, and sensitivity to the immunosuppressive drug cylcosporin A. It is of note that cyclosporin A inhibited the expression of more than 10 inducible genes, which suggests that this drug has a broad genetic mechanism of action.

  2. A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation.

    PubMed

    Morandi, Fabio; Morandi, Barbara; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Zaccarello, Gianluca; Carrega, Paolo; Ferlazzo, Guido; Mingari, Maria Cristina; Moretta, Lorenzo; Pistoia, Vito; Malavasi, Fabio

    2015-09-22

    Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD(+ )T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4(+) T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8(+) T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role. PMID:26329660

  3. CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages

    PubMed Central

    Fergusson, Joannah R.; Smith, Kira E.; Fleming, Vicki M.; Rajoriya, Neil; Newell, Evan W.; Simmons, Ruth; Marchi, Emanuele; Björkander, Sophia; Kang, Yu-Hoi; Swadling, Leo; Kurioka, Ayako; Sahgal, Natasha; Lockstone, Helen; Baban, Dilair; Freeman, Gordon J.; Sverremark-Ekström, Eva; Davis, Mark M.; Davenport, Miles P.; Venturi, Vanessa; Ussher, James E.; Willberg, Christian B.; Klenerman, Paul

    2014-01-01

    The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a novel population known as Mucosal Associated Invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, that can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Further, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161 expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both TCR expression and cell lineage. PMID:25437561

  4. Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

    PubMed

    Blewett, Megan M; Xie, Jiji; Zaro, Balyn W; Backus, Keriann M; Altman, Amnon; Teijaro, John R; Cravatt, Benjamin F

    2016-01-01

    Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology. PMID:27625306

  5. Granzyme B produced by human plasmacytoid dendritic cells suppresses T-cell expansion

    PubMed Central

    Vollmer, Angelika; Blackwell, Sue E.; Maier, Julia; Sontheimer, Kai; Beyer, Thamara; Mandel, Birgit; Lunov, Oleg; Tron, Kyrylo; Nienhaus, G. Ulrich; Simmet, Thomas; Debatin, Klaus-Michael; Weiner, George J.

    2010-01-01

    Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB+ pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC–T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations. PMID:19965634

  6. Genetically Modified T Cells Targeting Interleukin-11 Receptor α-Chain Kill Human Osteosarcoma Cells and Induce the Regression of Established Osteosarcoma Lung Metastases

    PubMed Central

    Cooper, Laurence JN; Hollomon, Mario; Huls, Helen; Kleinerman, Eugenie S

    2013-01-01

    The treatment of osteosarcoma (OS) pulmonary metastases remains a challenge. T cells genetically modified to express a chimeric antigen receptor (CAR), which recognizes a tumor-associated antigen, have shown activity against hematopoetic malignancies in clinical trials, but this requires the identification of a specific receptor on the tumor cell. In the current study, we found that interleukin (IL)-11Rα was selectively expressed on 14 of 16 OS patients’ lung metastases and 4 different human OS cell lines, indicating that IL-11Rα may be a novel target for CAR-specific T-cell therapy. IL-11Rα expression was absent or low in normal organ tissues, with the exception of the GI track. IL-11Rα-CAR-specific T cells were obtained by non-viral gene transfer of Sleeping Beauty DNA plasmids and selectively expanded ex vivo using artificial antigen presenting cells derived from IL-11Rα + K562 cells genetically modified to co-express T-cell co-stimulatory molecules. IL-11Rα-CAR+ T cells killed all 4 OS cell lines in vitro; cytotoxicity correlated with the level of IL-11Rα expression on the tumor cells. Intravenous injection of IL-11Rα-CAR+ T cells into mice resulted in the regression of OS pulmonary metastases with no organ toxicity. Together, the data suggest that IL-11Rα-CAR T cells may represent a new therapy for OS patients with pulmonary metastases. PMID:22075555

  7. [Rhabdomyosarcoma lysis by T cells expressing a human autoantibody based chimeric receptor targeting the fetal acetylcholine receptors].

    PubMed

    Gattenlöhner, S

    2006-01-01

    Rhabdomyosarcomas (RMSs) are the most frequent malignant soft tissue tumors of childhood. Since even aggressive multimodality treatments including autologous stem cell rescue have failed to improve the < 20 % overall survival rate of children with metastatic RMS, novel treatment approaches are urgently needed. Looking for potential targets for immunotherapies, we identified the gamma subunit of the fetal acetylcholine receptor (fAChR) as a specific and overexpressed membrane antigen in RMS. Additionally we established a duplex RT-PCR with simultaneous amplification of alpha and gamma subunit message of the fAChR and the quantification of both transcripts resulting in alpha/gammaAChR ratio > 1 was 100% sensitive in alveolar and embryonal rhabdomyosarcoma. Since the fAChR was the first extracellular tumor marker that can distinguish rhabdomyosarcomas from nonrhabdomyomatous tumors and from normal muscle and therefore implies, that the fAChR may be a target for immunotherapeutic strategies, we synthesized a scFv antibody fragment directed against the fAChR and enigineered both a Pseudomonas exotoxin A based immunotoxin as well as a chimeric T cell receptor composed of the antigen-binding domain of the scFv fragment joined to the signaling domain of the T cell receptor zeta chain. The interaction of fAChzeta-transduced T cells with several RMS cell lines but not with fAChR-negative controls induced strong T cell activation, characterized by secretion of high amounts of interferon-gamma. Moreover after co-incubations with RMS cell lines fAChRzeta-transduced T cells as well fAChR specific immunotoxin induced specific receptor-concentration dependent tumor cell lysis. Therefore, fAChRzeta-transduced T cells and the fAChR specific immunotoxin respectively are promising new tools for the immunotherapy of rhabdomyosarcomas and may provide an effective complementary approach to eradicate residual or metastatic RMS cells in patients, since 1. RMS-direceted chemotherapies

  8. Tax Protein-induced Expression of Antiapoptotic Bfl-1 Protein Contributes to Survival of Human T-cell Leukemia Virus Type 1 (HTLV-1)-infected T-cells*♦

    PubMed Central

    Macaire, Héloïse; Riquet, Aurélien; Moncollin, Vincent; Biémont-Trescol, Marie-Claude; Duc Dodon, Madeleine; Hermine, Olivier; Debaud, Anne-Laure; Mahieux, Renaud; Mesnard, Jean-Michel; Pierre, Marlène; Gazzolo, Louis; Bonnefoy, Nathalie; Valentin, Hélène

    2012-01-01

    Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4+ T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-xL, and Bcl-2. Indeed, both Bfl-1 and Bcl-xL knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-xL in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-xL represent potential therapeutic targets for ATLL treatment. PMID:22553204

  9. Cloning, characterization, and mapping of human homolog of mouse T-cell death-associated gene.

    PubMed

    Kyaw, H; Zeng, Z; Su, K; Fan, P; Shell, B K; Carter, K C; Li, Y

    1998-06-01

    To establish immunologic autotolerance, self-reactive immature thymocytes are eliminated by negative selection during T-cell development in the thymus. Self-reactive clones undergo apoptosis after stimulation via the T-cell receptor (TCR). The process of cell selection is determined by the dedication of the TCR for tolerogenic antigen/major histocompatibility complex. We have cloned a novel human gene that is highly homologous in the transmembrane and G protein-coupling domains to mouse T-cell death-associated gene 8 (TDAG8). The gene, human TDAG8 (hTDAG8), which belongs to the G protein-couple receptor superfamily, encodes a protein of 337 amino acids. An expressed sequence tag (EST) corresponding to hTDAG8 was identified from a human thyroid cDNA library and subsequently used to isolate a full-length genomic clone. Northern blot analysis revealed that the hTDAG8 gene is expressed predominantly in lymphoid tissues, including peripheral blood leukocytes, spleen, lymph nodes, and thymus. Stably transfected mammalian CHO cells were generated, and heterologous expression of hTDAG8 was confirmed by Northern blot analysis. Fluorescent in situ hybridization (FISH) revealed that hTDAG8 maps to human chromosome 14q31-32.1, a region in which abnormalities associated with human T-cell lymphoma or leukemia are found. Taken together, these data implicate the hTDAG8 gene in T-cell-associated diseases in humans, but its actual physiological and pathological role in the human immune system needs further investigation. PMID:9655242

  10. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection

    PubMed Central

    Teng, Yen-Tung A.; Nguyen, Hai; Gao, Xuijuan; Kong, Young-Yun; Gorczynski, Reginald M.; Singh, Bhagirath; Ellen, Richard P.; Penninger, Josef M.

    2000-01-01

    Periodontitis, a prime cause of tooth loss in humans, is implicated in the increased risk of systemic diseases such as heart failure, stroke, and bacterial pneumonia. The mechanisms by which periodontitis and antibacterial immunity lead to alveolar bone and tooth loss are poorly understood. To study the human immune response to specific periodontal infections, we transplanted human peripheral blood lymphocytes (HuPBLs) from periodontitis patients into NOD/SCID mice. Oral challenge of HuPBL-NOD/SCID mice with Actinobacillus actinomycetemcomitans, a well-known Gram-negative anaerobic microorganism that causes human periodontitis, activates human CD4+ T cells in the periodontium and triggers local alveolar bone destruction. Human CD4+ T cells, but not CD8+ T cells or B cells, are identified as essential mediators of alveolar bone destruction. Stimulation of CD4+ T cells by A. actinomycetemcomitans induces production of osteoprotegerin ligand (OPG-L), a key modulator of osteoclastogenesis and osteoclast activation. In vivo inhibition of OPG-L function with the decoy receptor OPG diminishes alveolar bone destruction and reduces the number of periodontal osteoclasts after microbial challenge. These data imply that the molecular explanation for alveolar bone destruction observed in periodontal infections is mediated by microorganism-triggered induction of OPG-L expression on CD4+ T cells and the consequent activation of osteoclasts. Inhibition of OPG-L may thus have therapeutic value to prevent alveolar bone and/or tooth loss in human periodontitis. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R59–R67 (2000). PMID:10995794

  11. Cross-reactive human B cell and T cell epitopes between influenza A and B viruses

    PubMed Central

    2013-01-01

    Influenza A and B viruses form different genera, which were originally distinguished by antigenic differences in their nucleoproteins and matrix 1 proteins. Cross-protection between these two genera has not been observed in animal experiments, which is consistent with the low homology in viral proteins common to both viruses except for one of three polymerase proteins, polymerase basic 1 (PB1). Recently, however, antibody and CD4+ T cell epitopes conserved between the two genera were identified in humans. A protective antibody epitope was located in the stalk region of the surface glycoprotein, hemagglutinin, and a CD4+ T cell epitope was located in the fusion peptide of the hemagglutinin. The fusion peptide was also found to contain antibody epitopes in humans and animals. A short stretch of well-conserved peptide was also identified in the other surface glycoprotein, neuraminidase, and antibodies binding to this peptide were generated by peptide immunization in rabbits. Although PB1, the only protein which has relatively high overall sequence homology between influenza A and B viruses, is not considered an immunodominant protein in the T cell responses to influenza A virus infection, amino acid sequence comparisons show that a considerable number of previously identified T cell epitopes in the PB1 of influenza A viruses are conserved in the PB1 of influenza B viruses. These data indicate that B and T cell cross-reactivity exists between influenza A and B viruses, which may have modulatory effects on the disease process and recovery. Although the antibody titers and the specific T cell frequencies induced by natural infection or standard vaccination may not be high enough to provide cross protection in humans, it might be possible to develop immunization strategies to induce these cross-reactive responses more efficiently. PMID:23886073

  12. Half of the T-cell repertoire combinatorial diversity is genetically determined in humans and humanized mice.

    PubMed

    Pham, Hang-Phuong; Manuel, Manuarii; Petit, Nicolas; Klatzmann, David; Cohen-Kaminsky, Sylvia; Six, Adrien; Marodon, Gilles

    2012-03-01

    In humanized mice, the T-cell repertoire is derived from genetically identical human progenitors in distinct animals. Thus, careful comparison of the T-cell repertoires of humanized mice with those of humans may reveal the contribution of genetic determinism on T-cell repertoire formation. Here, we performed a comprehensive assessment of the distribution of V-J combinations of the human β chain of the T-cell receptor (hTRBV) in NOD.SCID.γc(-/-) (NSG) humanized mice. We observed that numerous V-J combinations were equally distributed in the thymus and in the periphery of humanized mice compared with human references. A global analysis of the data, comparing repertoire perturbation indices in humanized NSG mice and unrelated human PBMCs, reveals that 50% of the hTRBV families significantly overlapped. Using multivariate ranking and bootstrap analyses, we found that 18% of all possible V-J combinations contributed close to 50% of the expressed diversity, with significant over-representation of BV5-J1.1+1.2 and BV6-J1.1+1.2 rearrangements. Finally, comparison of CD3(-) and CD3(+) thymocyte repertoires indicated that the observed V-J combination overlap was already present before TCR-MHC selection in the thymus. Altogether, our results show that half of the T-cell repertoire combinatorial diversity in humans is genetically determined. PMID:22105329

  13. IL-12 Delivered Intratumorally by Multilamellar Liposomes Reactivates Memory T Cells in Human Tumor Microenvironments

    PubMed Central

    Simpson-Abelson, Michelle R.; Purohit, Vivek S.; Pang, Wing Man; Iyer, Vandana; Odunsi, Kunle; Demmy, Todd L; Yokota, Sandra J.; Loyall, Jenni L.; Kelleher, Raymond J.; Balu-Iyer, Sathy; Bankert, Richard B.

    2009-01-01

    Using a novel loading technique, IL-12 is reported here to be efficiently encapsulated within large multilamellar liposomes. The preclinical efficacy of the cytokine loaded liposomes to deliver IL-12 into human tumors and to reactive tumor-associated T cells in situ is tested using a human tumor xenograft model. IL-12 is released in vivo from these liposomes in a biologically active form when injected into tumor xenografts that are established by the subcutaneous implantation of non-disrupted pieces of human lung, breast or ovarian tumors into immunodeficient mice. The histological architecture of the original tumor tissue, including tumor-associated leukocytes, tumor cells and stromal cells is preserved anatomically and the cells remain functionally responsive to cytokines in these xenografts. The local and sustained release of IL-12 into the tumor microenvironment reactivates tumor-associated quiescent effector memory T cells to proliferate, produce and release IFN-γ resulting in the killing of tumor cells in situ. Very little IL-12 is detected in the serum of mice for up to 5 days after an intratumoral injection of the IL-12 liposomes. We conclude that IL-12 loaded large multilamellar liposomes provide a safe method for the local and sustained delivery of IL-12 to tumors and a therapeutically effective way of reactivating existing tumor-associated T cells in human solid tumor microenvironments. The potential of this local in situ T cell re-stimulation to induce a systemic anti-tumor immunity is discussed. PMID:19395317

  14. Timely and spatially regulated maturation of B and T cell repertoire during human fetal development.

    PubMed

    Rechavi, Erez; Lev, Atar; Lee, Yu Nee; Simon, Amos J; Yinon, Yoav; Lipitz, Schlomo; Amariglio, Ninette; Weisz, Boaz; Notarangelo, Luigi D; Somech, Raz

    2015-02-25

    Insights into the ontogeny of the human fetal adaptive immune system are of great value for understanding immunocompetence of the developing fetus. However, to date, this has remained largely uncharted territory, in large part because blood samples from healthy, early gestation fetuses have been hard to come by. In a comprehensive study, we analyzed levels of T cell receptor excision circles (TRECs), signal-joint κ receptor excision circles (sjKRECs), and intron recombination signal sequence-K-deleting element (iRSS-Kde) rearrangement, and T and B lymphocyte repertoire clonality in human fetuses from 12 to 26 weeks of gestational age. Using next-generation sequencing, we analyzed the diversity and complexity of T cell receptor β (TRB) and immunoglobulin heavy chain (IGH) repertoires in four fetuses at 12, 13, 22, and 26 weeks of gestation and in healthy full-term infants. We report the progressive increase of TREC, sjKREC, and iRSS-Kde levels over time and confirm that B cell development precedes T cell development in the human fetus. Temporally and spatially regulated maturation of B and T cell repertoire diversity and complexity during human fetal development was observed, including evidence that immunoglobulin somatic hypermutation and class switch recombination occur already during intrauterine life. Our results help define physiological levels of immunodeficiency in premature infants and may serve as a reference for future studies aimed at investigating the impact of intrauterine pathologies on fetal immune development and function. PMID:25717098

  15. Human T cell responses to Japanese encephalitis virus in health and disease.

    PubMed

    Turtle, Lance; Bali, Tanushka; Buxton, Gemma; Chib, Savita; Chan, Sajesh; Soni, Mohammed; Hussain, Mohammed; Isenman, Heather; Fadnis, Prachi; Venkataswamy, Manjunatha M; Satishkumar, Vishali; Lewthwaite, Penny; Kurioka, Ayako; Krishna, Srinivasa; Shankar, M Veera; Ahmed, Riyaz; Begum, Ashia; Ravi, Vasanthapuram; Desai, Anita; Yoksan, Sutee; Fernandez, Stefan; Willberg, Christian B; Kloverpris, Henrik N; Conlon, Christopher; Klenerman, Paul; Satchidanandam, Vijaya; Solomon, Tom

    2016-06-27

    Japanese encephalitis (JE) virus (JEV) is an important cause of encephalitis in children of South and Southeast Asia. However, the majority of individuals exposed to JEV only develop mild symptoms associated with long-lasting adaptive immunity. The related flavivirus dengue virus (DENV) cocirculates in many JEV-endemic areas, and clinical data suggest cross-protection between DENV and JEV. To address the role of T cell responses in protection against JEV, we conducted the first full-breadth analysis of the human memory T cell response using a synthetic peptide library. Ex vivo interferon-γ (IFN-γ) responses to JEV in healthy JEV-exposed donors were mostly CD8(+) and targeted nonstructural (NS) proteins, whereas IFN-γ responses in recovered JE patients were mostly CD4(+) and targeted structural proteins and the secreted protein NS1. Among patients, a high quality, polyfunctional CD4(+) T cell response was associated with complete recovery from JE. T cell responses from healthy donors showed a high degree of cross-reactivity to DENV that was less apparent in recovered JE patients despite equal exposure. These data reveal divergent functional CD4(+) and CD8(+) T cell responses linked to different clinical outcomes of JEV infection, associated with distinct targeting and broad flavivirus cross-reactivity including epitopes from DENV, West Nile, and Zika virus. PMID:27242166

  16. SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling.

    PubMed

    Hauck, Fabian; Blumenthal, Britta; Fuchs, Sebastian; Lenoir, Christelle; Martin, Emmanuel; Speckmann, Carsten; Vraetz, Thomas; Mannhardt-Laakmann, Wilma; Lambert, Nathalie; Gil, Marine; Borte, Stephan; Audrain, Marie; Schwarz, Klaus; Lim, Annick; Schamel, Wolfgang W; Fischer, Alain; Ehl, Stephan; Rensing-Ehl, Anne; Picard, Capucine; Latour, Sylvain

    2015-12-01

    Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells. PMID:26187144

  17. Human CD8(+) T Cells Target Multiple Epitopes in Respiratory Syncytial Virus Polymerase.

    PubMed

    Burbulla, Daniel; Günther, Patrick S; Peper, Janet K; Jahn, Gerhard; Dennehy, Kevin M

    2016-06-01

    Respiratory syncytial virus (RSV) infection is a serious health problem in young children, immunocompromised patients, and the elderly. The development of novel prevention strategies, such as a vaccine to RSV, is a high priority. One strategy is to design a peptide-based vaccine that activates appropriate CD8(+) T-cell responses. However, this approach is limited by the low number of RSV peptide epitopes defined to date that activate CD8(+) T cells. We aimed to identify peptide epitopes that are presented by common human leukocyte antigen types (HLA-A*01, -A*02, and -B*07). We identify one novel HLA-A*02-restricted and two novel HLA-A*01-restricted peptide epitopes from RSV polymerase. Peptide-HLA multimer staining of specific T cells from healthy donor peripheral blood mononuclear cell, the memory phenotype of such peptide-specific T cells ex vivo, and functional IFNγ responses in short-term stimulation assays suggest that these peptides are recognized during RSV infection. Such peptides are candidates for inclusion into a peptide-based RSV vaccine designed to stimulate defined CD8(+) T-cell responses. PMID:27070377

  18. Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

    PubMed

    Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M

    2015-11-15

    The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis. PMID:26213183

  19. Human Dendritic Cells Activated by TSLP and CD40L Induce Proallergic Cytotoxic T Cells

    PubMed Central

    Gilliet, Michel; Soumelis, Vassili; Watanabe, Norihiko; Hanabuchi, Shino; Antonenko, Svetlana; de Waal-Malefyt, Rene; Liu, Yong-Jun

    2003-01-01

    Human thymic stromal lymphopoietin (TSLP) is a novel epithelial cell–derived cytokine, which induces dendritic cell (DC)-mediated CD4+ T cell responses with a proallergic phenotype. Although the participation of CD8+ T cells in allergic inflammation is well documented, their functional properties as well as the pathways leading to their generation remain poorly understood. Here, we show that TSLP-activated CD11c+ DCs potently activate and expand naive CD8+ T cells, and induce their differentiation into interleukin (IL)-5 and IL-13–producing effectors exhibiting poor cytolytic activity. Additional CD40L triggering of TSLP-activated DCs induced CD8+ T cells with potent cytolytic activity, producing large amounts of interferon (IFN)-γ, while retaining their capacity to produce IL-5 and IL-13. These data further support the role of TSLP as initial trigger of allergic T cell responses and suggest that CD40L-expressing cells may act in combination with TSLP to amplify and sustain pro-allergic responses and cause tissue damage by promoting the generation of IFN-γ–producing cytotoxic effectors. PMID:12707303

  20. Early innate responses to pathogens: pattern recognition by unconventional human T-cells

    PubMed Central

    Liuzzi, Anna Rita; McLaren, James E; Price, David A; Eberl, Matthias

    2015-01-01

    Although typically viewed as a feature of innate immune responses, microbial pattern recognition is increasingly acknowledged as a function of particular cells nominally categorized within the adaptive immune system. Groundbreaking research over the past three years has shown how unconventional human T-cells carrying invariant or semi-invariant TCRs that are not restricted by classical MHC molecules sense microbial compounds via entirely novel antigen presenting pathways. This review will focus on the innate-like recognition of non-self metabolites by Vγ9/Vδ2 T-cells, mucosal-associated invariant T (MAIT) cells and germline-encoded mycolyl-reactive (GEM) T-cells, with an emphasis on early immune responses in acute infection. PMID:26182978

  1. Cutting Edge: AhR Is a Molecular Target of Calcitriol in Human T Cells.

    PubMed

    Takami, Mariko; Fujimaki, Kotaro; Nishimura, Michael I; Iwashima, Makio

    2015-09-15

    The immunoregulatory functions of vitamin D have been well documented in various immunological disorders, including multiple sclerosis, arthritis, and asthma. IL-10 is considered a chief effector molecule that promotes the vitamin D-induced immunosuppressive states of T cells and accessory cells. In this article, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (calcitriol), has a profound inhibitory effect on the development of human Th9, a CD4 T cell subset that is highly associated with asthma, in an IL-10-independent manner. Our data show that calcitriol represses the expression of BATF, a transcription factor essential for Th9, via suppressing the expression of aryl hydrocarbon receptor, without an increase in IL-10. The data show a novel link between vitamin D and two key transcription factors involved in T cell differentiation. PMID:26276877

  2. CD4+ T Cell Depletion in Human Immunodeficiency Virus (HIV) Infection: Role of Apoptosis

    PubMed Central

    Février, Michèle; Dorgham, Karim; Rebollo, Angelita

    2011-01-01

    Human immunodeficiency virus (HIV) infection is principally a mucosal disease and the gastrointestinal (GI) tract is the major site of HIV replication. Loss of CD4+ T cells and systemic immune hyperactivation are the hallmarks of HIV infection. The end of acute infection is associated with the emergence of specific CD4+ and CD8+ T cell responses and the establishment of a chronic phase of infection. Abnormal levels of immune activation and inflammation persist despite a low steady state level of viremia. Although the causes of persistent immune hyperactivation remain incompletely characterized, physiological alterations of gastrointestinal tract probably play a major role. Failure to restore Th17 cells in gut-associated lymphoid tissues (GALT) might impair the recovery of the gut mucosal barrier. This review discusses recent advances on understanding the contribution of CD4+ T cell depletion to HIV pathogenesis. PMID:21994747

  3. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.

    PubMed

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S; Jones, David R; Sadelain, Michel; Adusumilli, Prasad S

    2016-08-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1-mediated (PD-1-mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB-based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

  4. Double Positive CD4CD8 αβ T Cells: A New Tumor-Reactive Population in Human Melanomas

    PubMed Central

    Desfrançois, Juliette; Moreau-Aubry, Agnès; Vignard, Virginie; Godet, Yann; Khammari, Amir; Dréno, Brigitte; Jotereau, Francine; Gervois, Nadine

    2010-01-01

    Background Double positive (DP) CD4CD8 Tαβ cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated. We recently reported the increased frequency of DP Tαβ cells in human breast pleural effusions. This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas. Methodology/Principal Findings We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry. Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples. Interestingly, a high proportion of these cells produced TNF-α in response to autologous melanoma cell lines. Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells. In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node. This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen. However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells. Conclusions/Significance In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo. Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of

  5. Human Memory CD4+ T Cell Immune Responses against Giardia lamblia.

    PubMed

    Saghaug, Christina Skår; Sørnes, Steinar; Peirasmaki, Dimitra; Svärd, Staffan; Langeland, Nina; Hanevik, Kurt

    2016-01-01

    The intestinal protozoan parasite Giardia lamblia may cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response to Giardia infection, and memory responses may last years. Recently, TH17 responses have been found in three animal studies of Giardia infection. The aim of this study was to characterize the human CD4(+) T cell responses to Giardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulated in vitro with Giardia lamblia proteins. Production of tumor necrosis factor alpha (TNF-α), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4(+) effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4(+) EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-α, to be significantly elevated in the Giardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated with Giardia antigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4(+) T cell activation and proliferation, to be significantly elevated in the Giardia-exposed individuals. We conclude that symptomatic Giardia infection in humans induces a CD4(+) EM T cell response of which IL-17A production seems to be an important component. PMID:26376930

  6. Human Memory CD4+ T Cell Immune Responses against Giardia lamblia

    PubMed Central

    Sørnes, Steinar; Peirasmaki, Dimitra; Svärd, Staffan; Langeland, Nina

    2015-01-01

    The intestinal protozoan parasite Giardia lamblia may cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response to Giardia infection, and memory responses may last years. Recently, TH17 responses have been found in three animal studies of Giardia infection. The aim of this study was to characterize the human CD4+ T cell responses to Giardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulated in vitro with Giardia lamblia proteins. Production of tumor necrosis factor alpha (TNF-α), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4+ effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4+ EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-α, to be significantly elevated in the Giardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated with Giardia antigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4+ T cell activation and proliferation, to be significantly elevated in the Giardia-exposed individuals. We conclude that symptomatic Giardia infection in humans induces a CD4+ EM T cell response of which IL-17A production seems to be an important component. PMID:26376930

  7. Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation.

    PubMed

    O'Boyle, Graeme; Fox, Christopher R J; Walden, Hannah R; Willet, Joseph D P; Mavin, Emily R; Hine, Dominic W; Palmer, Jeremy M; Barker, Catriona E; Lamb, Christopher A; Ali, Simi; Kirby, John A

    2012-03-20

    The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis. PMID:22392992

  8. CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus-transformed B cells.

    PubMed

    Long, Heather M; Zuo, Jianmin; Leese, Alison M; Gudgeon, Nancy H; Jia, Hui; Taylor, Graham S; Rickinson, Alan B

    2009-07-23

    Epstein-Barr virus (EBV)-specific T-cell preparations, generated by stimulating immune donor lymphocytes with the autologous virus-transformed B-lymphoblastoid cell line (LCL) in vitro, can be used to target EBV-positive malignancies. Although these preparations are enriched for EBV antigen-specific CD8(+) T cells, most also contain a CD4(+) T-cell population whose specificity is unknown. Here, we show that, although CD4(+) T-cell clones derived from such cultures recognize HLA class II-matched LCLs but not mitogen-activated B lymphoblasts, many (1) do not map to any known EBV antigen, (2) can be raised from EBV-naive as well as EBV-immune persons, and (3) can recognize a broad range of human B lymphoma-derived cell lines irrespective of EBV genome status, providing those lines to express the relevant HLA class II-restricting allele. Importantly, such CD4(+) clones not only produce IFNgamma but are also cytotoxic and can control the outgrowth of HLA-matched lymphoma cells in cocultivation assays. We infer that such CD4(+) T cells recognize cellular antigens that are preferentially up-regulated in EBV-transformed but not mitogen-activated B lymphoblasts and that are also expressed in a range of B-cell malignancies. Such antigens are therefore of potential value as targets for CD4(+) T cell-based immunotherapy. PMID:19443664

  9. γδ T cells: first line of defense and beyond.

    PubMed

    Chien, Yueh-hsiu; Meyer, Christina; Bonneville, Marc

    2014-01-01

    γδ T cells, αβ T cells, and B cells are present together in all but the most primitive vertebrates, suggesting that each population contributes to host immune competence uniquely and that all three are necessary for maintaining immune competence. Functional and molecular analyses indicate that in infections, γδ T cells respond earlier than αβ T cells do and that they emerge late after pathogen numbers start to decline. Thus, these cells may be involved in both establishing and regulating the inflammatory response. Moreover, γδ T cells and αβ T cells are clearly distinct in their antigen recognition and activation requirements as well as in the development of their antigen-specific repertoire and effector function. These aspects allow γδ T cells to occupy unique temporal and functional niches in host immune defense. We review these and other advances in γδ T cell biology in the context of their being the major initial IL-17 producers in acute infection. PMID:24387714

  10. Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells

    PubMed Central

    Mansour, Salah; Tocheva, Anna S.; Cave-Ayland, Chris; Machelett, Moritz M.; Sander, Barbara; Lissin, Nikolai M.; Molloy, Peter E.; Baird, Mark S.; Stübs, Gunthard; Schröder, Nicolas W. J.; Schumann, Ralf R.; Rademann, Jörg; Postle, Anthony D.; Jakobsen, Bent K.; Marshall, Ben G.; Gosain, Rajendra; Elkington, Paul T.; Elliott, Tim; Skylaris, Chris-Kriton; Essex, Jonathan W.; Tews, Ivo; Gadola, Stephan D.

    2016-01-01

    Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors. PMID:26884207

  11. Human peripheral gamma delta T cells recognize hsp60 molecules on Daudi Burkitt's lymphoma cells.

    PubMed

    Kaur, I; Voss, S D; Gupta, R S; Schell, K; Fisch, P; Sondel, P M

    1993-03-01

    Studies with the use of polyclonal rabbit antiserum reactive with 60-kDa heat shock proteins (hsp60) suggested that hsp60-related molecules could be found on the surface of Daudi cells and were involved in their recognition by certain human gamma delta T cells. The present study confirms this finding by using a mAb specifically recognizing hsp60. This mAb can block outgrowth of human gamma delta T cells in response to stimulation with Daudi and in response to an extract of the mycobacteria H37Ra. This anti-hsp60 mAb stains the surface of Daudi cells, but does not stain either Raji or EBV-transformed B cells, cells which do not stimulate gamma delta T cell outgrowth. Anti-hsp60 mAb could immunoprecipitate a 60-kDa molecule from the H37Ra extract but was unable to precipitate this 60-kDa molecule if the mAb was first absorbed on Daudi cells. This mAb also precipitated a 60-kDa molecule from the surface of Daudi cells which shows an electrophoretic mobility pattern consistent with hsp60. These experiments demonstrate that human gamma delta T cells recognize hsp60-related epitopes on the surface of Daudi cells and within mycobacterial extracts. PMID:8094731

  12. CD4(+)CD25 (+)CD127 (low/-) T cells: a more specific Treg population in human peripheral blood.

    PubMed

    Yu, Ning; Li, Xiaomei; Song, Weiya; Li, Dongmei; Yu, Daliang; Zeng, Xiaofeng; Li, Mengtao; Leng, Xiaomei; Li, Xiangpei

    2012-12-01

    The quantitative identification and enrichment of viable regulatory T cells (Treg) requires reliable surface markers that are selectively expressed on Treg. Foxp3 is the accepted marker of nTreg, but it cannot be used to isolate cells for functional studies. In this study, we compared four staining profiles of Treg, including CD4(+)CD25(high) T cells, CD4(+)CD39(+) T cells, CD4(+)CD73(+) T cells, and CD4(+)CD25(+)CD127(low/-) T cells. We found that CD4(+)CD25(+)CD127(low/-) T cells expressed the highest level of Foxp3 and had the strongest correlation with CD4(+)CD25(+)Foxp3(+) T cells, the accepted identifying characteristics for "real" nTreg cells. Moreover, functional data showed that CD4(+)CD25(+)CD127(low/-) T cells could effectively suppress the proliferation of CD4(+)CD25(-) T cells, suggesting that compared with the other three populations, CD4(+)CD25(+)CD127(low/-) T cells best fit the definition of naturally occurring regulatory T cells in human peripheral blood. Finally, we showed that CD4(+)CD25(+)CD127(low/-) can be used to quantitate Treg cells in individuals with systemic lupus erythematosus supporting the use of CD4(+)CD25(+)CD127(low/-) to identify human Treg cells. PMID:22752562

  13. Cyclic dinucleotides modulate human T-cell response through monocyte cell death.

    PubMed

    Tosolini, Marie; Pont, Frédéric; Verhoeyen, Els; Fournié, Jean-Jacques

    2015-12-01

    Cyclic dinucleotides, a class of microbial messengers, have been recently identified in bacteria, but their activity in humans remains largely unknown. Here, we have studied the function of cyclic dinucleotides in humans. We found that c-di-AMP and cGAMP, two adenosine-based cyclic dinucleotides, activated T lymphocytes in an unusual manner through monocyte cell death. c-di-AMP and cGAMP induced the selective apoptosis of human monocytes, and T lymphocytes were activated by the direct contact with these dying monocytes. The ensuing T-cell response comprised cell-cycle exit, phenotypic maturation into effector memory cells and proliferation arrest, but not cell death. This quiescence was transient since T cells remained fully responsive to further restimulation. Together, our results depict a novel activation pattern for human T lymphocytes: a transient quiescence induced by c-di-AMP- or cGAMP-primed apoptotic monocytes. PMID:26460927

  14. On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments

    PubMed Central

    Toor, Amir A.; Toor, Abdullah A.; Rahmani, Mohamed; Manjili, Masoud H.

    2016-01-01

    The human T-cell repertoire is complex and is generated by the rearrangement of variable (V), diversity (D) and joining (J) segments on the T-cell receptor (TCR) loci. The T-cell repertoire demonstrates self-similarity in terms clonal frequencies when defined by V, D and J gene segment usage; therefore to determine whether the structural ordering of these gene segments on the TCR loci contributes to the observed clonal frequencies, the TCR loci were examined for self-similarity and periodicity in terms of gene segment organization. Logarithmic transformation of numeric sequence order demonstrated that the V and J gene segments for both T-cell receptor α (TRA) and β (TRB) loci are arranged in a self-similar manner when the spacing between the adjacent segments was considered as a function of the size of the neighbouring gene segment, with an average fractal dimension of approximately 1.5. Accounting for the gene segments occurring on helical DNA molecules with a logarithmic distribution, sine and cosine functions of the log-transformed angular coordinates of the start and stop nucleotides of successive TCR gene segments showed an ordered progression from the 5′ to the 3′ end of the locus, supporting a log-periodic organization. T-cell clonal frequency estimates, based on V and J segment usage, from normal stem cell donors were plotted against the V and J segment on TRB locus and demonstrated a periodic distribution. We hypothesize that this quasi-periodic variation in gene-segment representation in the T-cell clonal repertoire may be influenced by the location of the gene segments on the periodic-logarithmically scaled TCR loci. Interactions between the two strands of DNA in the double helix may influence the probability of gene segment usage by means of either constructive or destructive interference resulting from the superposition of the two helices. PMID:26763333

  15. Human CD1-restricted T cell recognition of lipids from pollens.

    PubMed

    Agea, Elisabetta; Russano, Anna; Bistoni, Onelia; Mannucci, Roberta; Nicoletti, Ildo; Corazzi, Lanfranco; Postle, Anthony D; De Libero, Gennaro; Porcelli, Steven A; Spinozzi, Fabrizio

    2005-07-18

    Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids. PMID:16009719

  16. GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate.

    PubMed

    Van de Walle, Inge; Dolens, Anne-Catherine; Durinck, Kaat; De Mulder, Katrien; Van Loocke, Wouter; Damle, Sagar; Waegemans, Els; De Medts, Jelle; Velghe, Imke; De Smedt, Magda; Vandekerckhove, Bart; Kerre, Tessa; Plum, Jean; Leclercq, Georges; Rothenberg, Ellen V; Van Vlierberghe, Pieter; Speleman, Frank; Taghon, Tom

    2016-01-01

    The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment. PMID:27048872

  17. GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate

    PubMed Central

    Van de Walle, Inge; Dolens, Anne-Catherine; Durinck, Kaat; De Mulder, Katrien; Van Loocke, Wouter; Damle, Sagar; Waegemans, Els; De Medts, Jelle; Velghe, Imke; De Smedt, Magda; Vandekerckhove, Bart; Kerre, Tessa; Plum, Jean; Leclercq, Georges; Rothenberg, Ellen V.; Van Vlierberghe, Pieter; Speleman, Frank; Taghon, Tom

    2016-01-01

    The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment. PMID:27048872

  18. Chromogranin A is a T Cell Antigen in Human Type 1 Diabetes

    PubMed Central

    Gottlieb, Peter A; Delong, Thomas; Baker, Rocky L.; Fitzgerald-Miller, Lisa; Wagner, Rebecca; Cook, Gabrielle; Rewers, Marian R.; Michels, Aaron; Haskins, Kathryn

    2013-01-01

    Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n=27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n=31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n=11) and control subjects (n=11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease. PMID:24239002

  19. Screening of promising chemotherapeutic candidates from plants against human adult T-cell leukemia/lymphoma (III).

    PubMed

    Nakano, Daisuke; Ishitsuka, Kenji; Kamikawa, Mio; Matsuda, Michika; Tsuchihashi, Ryota; Okawa, Masafumi; Okabe, Hikaru; Tamura, Kazuo; Kinjo, Junei

    2013-10-01

    Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature peripheral T lymphocytes caused by human T-cell lymphotropic virus type I (HTLV-I). In our previous paper, 214 extracts from 162 plants were screened to elucidate the anti-proliferative principles against HTLV-I-infected T-cell lines. In this study, 245 extracts from 182 plants belonging to 61 families were further tested against two HTLV-I-infected T-cell lines (MT-1 and MT-2). Potent anti-proliferative effects were exhibited against MT-1 and MT-2 cells by 52 and 60 of the 245 extracts tested, respectively. Of these, two extracts showed strong inhibitory activity (EC₅₀ values 0.1-1 μg/mL; +++) against both cells, 7 extracts showed moderate inhibitory activity (EC5₅₀ values 1-10 μg/mL; ++), and 43 extracts showed weak inhibitory activity (EC₅₀ values 10-100 μg/mL; +), whereas the remaining extracts did not show any activity (EC₅₀ values >100 μg/mL; -) against MT-1 cells. On the other hand, 10 extracts showed moderate inhibitory activit and, 48 extracts showed weak inhibitory activity, whereas the remaining extracts did not show any activity against MT-2 cells. Extracts from the aerial parts of Annona reticulata and A. squamosa showed the most potent inhibitory activity and three aporphine alkaloids were isolated from their extracts as the active principles by activity-guided fractionation. PMID:23397239

  20. L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice.

    PubMed

    Hong, Hao; Brown, Christine E; Ostberg, Julie R; Priceman, Saul J; Chang, Wen-Chung; Weng, Lihong; Lin, Paul; Wakabayashi, Mark T; Jensen, Michael C; Forman, Stephen J

    2016-01-01

    New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (TCM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R+ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R+ TCM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R+ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer. PMID:26761817

  1. Differentiation between cutaneous form of adult T cell leukemia/lymphoma and cutaneous T cell lymphoma by in situ hybridization using a human T cell leukemia virus-1 DNA probe.

    PubMed Central

    Arai, E.; Chow, K. C.; Li, C. Y.; Tokunaga, M.; Katayama, I.

    1994-01-01

    Adult T cell leukemia/lymphoma (ATLL) shares overlapping clinicopathological features with cutaneous T cell lymphoma (CTCL), requiring detection of monoclonal integration of proviral DNA of type 1 human T cell leukemia virus for its differential diagnosis from the latter. We applied in situ hybridization (ISH) and polymerase chain reaction (PCR) to paraffin sections from 63 Japanese autopsy cases that had been diagnosed as CTCL in earlier years when ATLL was still not widely known. Eleven and two cases with confirmed diagnoses of ATLL and CTCL served as positive and negative controls, respectively. It was found that ISH was positive in 7 of 63 test cases and 10 of 11 positive controls, whereas PCR was positive in none of the test cases and eight of the positive control cases. Two negative controls were negative for both ISH and PCR. We conclude that ISH is superior to PCR for detecting type 1 human T cell leukemia virus proviral DNA on paraffin sections and that the ISH method is useful for differentiating CTCL from the cutaneous form of ATLL. Images Figure 1 PMID:8291605

  2. Cernunnos deficiency reduces thymocyte life span and alters the T cell repertoire in mice and humans.

    PubMed

    Vera, Gabriella; Rivera-Munoz, Paola; Abramowski, Vincent; Malivert, Laurent; Lim, Annick; Bole-Feysot, Christine; Martin, Christelle; Florkin, Benoit; Latour, Sylvain; Revy, Patrick; de Villartay, Jean-Pierre

    2013-02-01

    Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice. PMID:23207905

  3. T Cells

    MedlinePlus

    ... or turn off the immune response. Cytotoxic or “killer” T cells directly attack and destroy cells bearing ... involve selective activation of helper T cells and killer T cells, with a corresponding decrease in regulatory ...

  4. Characterization of small spheres derived from various solid tumor cell lines: are they suitable targets for T cells?

    PubMed

    Busse, Antonia; Letsch, Anne; Fusi, Alberto; Nonnenmacher, Anika; Stather, David; Ochsenreither, Sebastian; Regenbrecht, Christian R A; Keilholz, Ulrich

    2013-08-01

    T cell based immunotherapy has been investigated in a variety of malignancies and analyses have been mostly founded on in vitro data with tumor cell monolayers. However, three-dimensional (3D) culture models might mimic more closely the 'in vivo' conditions than 2D monolayers. Therefore, we analyzed the expression of tumor-associated antigens (TAA) and of molecules involved in antigen processing and presentation (APM) in tumor spheres, which served as an in vitro model for micrometastasis which might be enriched in tumor propagating cancer stem cells. For enrichment of sphere cells 12 human solid tumor cell lines were cultured in serum-free medium. Expression of a variety of TAA and APM were analyzed by RT-PCR and/or flow cytometry and compared to expression in corresponding adherent bulk cells grown in regular growth medium. Compared to adherent cells, spheres showed equal or higher mRNA expression levels of LMP2, LMP7 and MECL-1, of TAP1 and TAP2 transporters and, surprisingly, also of TAA including differentiation antigens. However, downregulation or loss of HLA-I and HLA-II molecules in spheres was observed in 8 of 10 and 1 of 2 cell lines, respectively, and was unresponsive to stimulation with IFN-γ. Although tumor spheres express TAA and molecules of intracellular antigen processing, they are defective in antigen presentation due to downregulation of HLA surface expression which may lead to immune evasion. PMID:23519726

  5. Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

    PubMed Central

    Eruslanov, Evgeniy B.; Bhojnagarwala, Pratik S.; Quatromoni, Jon G.; Stephen, Tom Li; Ranganathan, Anjana; Deshpande, Charuhas; Akimova, Tatiana; Vachani, Anil; Litzky, Leslie; Hancock, Wayne W.; Conejo-Garcia, José R.; Feldman, Michael; Albelda, Steven M.; Singhal, Sunil

    2014-01-01

    Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%–25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62LloCD54hi) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses. PMID:25384214

  6. Isolation and characterization of human papillomavirus type 6-specific T cells infiltrating genital warts.

    PubMed Central

    Hong, K; Greer, C E; Ketter, N; Van Nest, G; Paliard, X

    1997-01-01

    The potential role of T cells in the control of human papillomavirus type 6 (HPV-6) infections is an appealing premise, but their actual role has been sparsely investigated. Since HPV-6 infections are confined to the epithelium, such an investigation should focus on the T cells present at the site of infection (i.e., the warts). Therefore, we isolated wart-infiltrating lymphocytes (WIL) from patients with clinically diagnosed anogenital warts. These WIL were characterized by their phenotype and their specificity for E7 and L1 proteins of HPV-6. The phenotype of WIL varied drastically from patient to patient, as determined by their expression of CD4, CD8, T-cell receptor alpha/beta chain (TCR alpha beta), and TCR gamma delta. Despite this heterogeneity in phenotype, HPV-6 E7 and/or L1-specific WIL, as determined by lymphoproliferation, could be isolated from more than 75% of the patients studied. Among all L1 peptides recognized by WIL, peptides 311-330 and 411-430 were the most consistently detected, with seven of nine patients for whom L1 peptide reactivity was observed responding to at least one of them. Moreover, the HPV-6 epitopic peptides recognized by WIL differed to some extent from those recognized by peripheral T cells. PMID:9261360

  7. Self-Reactive CFTR T Cells in Humans: Implications for Gene Therapy

    PubMed Central

    Calcedo, Roberto; Griesenbach, Uta; Dorgan, Daniel J.; Soussi, Samia; Boyd, A. Christopher; Davies, Jane C.; Higgins, Tracy E.; Hyde, Stephen C.; Gill, Deborah R.; Innes, J. Alastair; Porteous, David J.; Alton, Eric W.; Wilson, James M.

    2013-01-01

    Abstract Cystic fibrosis (CF) is one of the most common autosomal recessive lethal disorders affecting white populations of northern European ancestry. To date there is no cure for CF. Life-long treatments for CF are being developed and include gene therapy and the use of small-molecule drugs designed to target specific cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. Irrespective of the type of molecular therapy for CF, which may include gene replacement, exon skipping, nonsense suppression, or molecular correctors, because all of these modulate gene expression there is an inherent risk of activation of T cells against the wild-type version of CFTR. Here we report the validation of the human interferon-γ enzyme-linked immunospot assay and its application for the analysis of CFTR-specific T cell responses in patients with CF and in non-CF subjects. We found non-CF subjects with low levels of self-reactive CFTR-specific T cells in the United States and several patients with CF with low to high levels of self-reactive CFTR-specific T cells in both the United States and the United Kingdom. PMID:23790242

  8. Self-reactive CFTR T cells in humans: implications for gene therapy.

    PubMed

    Calcedo, Roberto; Griesenbach, Uta; Dorgan, Daniel J; Soussi, Samia; Boyd, A Christopher; Davies, Jane C; Higgins, Tracy E; Hyde, Stephen C; Gill, Deborah R; Innes, J Alastair; Porteous, David J; Alton, Eric W; Wilson, James M; Limberis, Maria P

    2013-09-01

    Cystic fibrosis (CF) is one of the most common autosomal recessive lethal disorders affecting white populations of northern European ancestry. To date there is no cure for CF. Life-long treatments for CF are being developed and include gene therapy and the use of small-molecule drugs designed to target specific cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. Irrespective of the type of molecular therapy for CF, which may include gene replacement, exon skipping, nonsense suppression, or molecular correctors, because all of these modulate gene expression there is an inherent risk of activation of T cells against the wild-type version of CFTR. Here we report the validation of the human interferon-γ enzyme-linked immunospot assay and its application for the analysis of CFTR-specific T cell responses in patients with CF and in non-CF subjects. We found non-CF subjects with low levels of self-reactive CFTR-specific T cells in the United States and several patients with CF with low to high levels of self-reactive CFTR-specific T cells in both the United States and the United Kingdom. PMID:23790242

  9. T-Cell Immunity to Infection with Dengue Virus in Humans

    PubMed Central

    Weiskopf, Daniela; Sette, Alessandro

    2014-01-01

    Dengue virus (DENV) is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans. Up to 400 million DENV infections occur every year, and severity can range from asymptomatic to an acute self-limiting febrile illness. In a small proportion of patients, the disease can exacerbate and progress to dengue hemorrhagic fever and/or dengue shock syndrome, characterized by severe vascular leakage, thrombocytopenia, and hemorrhagic manifestations. A unique challenge in vaccine development against DENV is the high degree of sequence variation, characteristically associated with RNA viruses. This is of particular relevance in the case of DENV since infection with one DENV serotype (primary infection) presumably affords life-long serotype-specific immunity but only partial and temporary immunity to other serotypes in secondary infection settings. The role of T cells in DENV infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing to severe disease. Our recent study has suggested an HLA-linked protective role for T cells. Herein, we will discuss the role of T cells in protection and pathogenesis from severe disease as well as the implications for vaccine design. PMID:24639680

  10. Inhibition of protein synthesis by the T cell receptor-inducible human TDAG51 gene product.

    PubMed

    Hinz, T; Flindt, S; Marx, A; Janssen, O; Kabelitz, D

    2001-05-01

    The T cell death associated gene 51 (TDAG51) was shown to be required for T cell receptor (TCR)-dependent induction of Fas/Apo1/CD95 expression in a murine T cell hybridoma. Despite the absence of a nuclear localization sequence and a nucleic acid binding domain, it was suggested to be localized in the nucleus and to function as a transcription factor regulating Fas-expression. However, we demonstrate that the human (h)TDAG51 protein is localized in the cytoplasm and the nucleoli, suggesting a role in ribosome biogenesis and/or translation regulation. Indeed, it strongly inhibited translation of a luciferase mRNA in a reticulocyte translational extract. Furthermore, cotransfection of hTDAG51 and the luciferase gene into 293T cells resulted in a strong inhibition of luciferase mRNA translation. Our findings were further strengthened by isolating in a yeast two-hybrid screen three proteins which are involved in the regulation of translation. We speculate that hTDAG51 couples TCR signaling to inhibition of protein biosynthesis in activated T lymphocytes. PMID:11369516

  11. Generation of a universal CD4 memory T cell recall peptide effective in humans, mice and non-human primates.

    PubMed

    Fraser, Christopher C; Altreuter, David H; Ilyinskii, Petr; Pittet, Lynnelle; LaMothe, Robert A; Keegan, Mark; Johnston, Lloyd; Kishimoto, Takashi Kei

    2014-05-19

    CD4T cells play a key role in humoral immunity by providing help to B cells, enabling effective antibody class switching and affinity maturation. Some vaccines may generate a poor response due to a lack of effective MHC class II epitopes, resulting in ineffective helper T cell activation and recall and consequently poor humoral immunity. It may be beneficial to provide a CD4T cell helper peptide with a vaccine particularly in the case of a poorly immunogenic antigen. Such a T cell helper peptide must be promiscuous in its ability to bind a broad range of MHC class II alleles due to broad allelic variation in the human population. We designed a chimeric MHC class II peptide (TpD) with epitopes from tetanus toxoid and diphtheria toxoid, separated by an internal cathepsin cleavage site. TpD was capable of inducing a memory recall response in peripheral blood mononuclear cells from 20/20 human donors. T cells responding to TpD showed a central memory phenotype. Immunization of mice with a synthetic nicotine nanoparticle vaccine containing TpD showed that the peptide was required for robust antibody production and resulted in a long term CD4 memory T cell recall response. As a pre-clinical model two non-human primate species, rhesus macaques and cynomolgus monkeys, were immunized with a nicotine nanoparticle vaccine and evaluated for an anti-nicotine antibody response and TpD specific memory T cells. We found that 4/4 rhesus monkeys had both sustained antibody production and TpD memory T cells for the duration of the experiment (119 days). In addition 30/30 cynomolgus monkeys dosed with nicotine vaccine nanoparticles showed dose-dependent antibody generation and T cell recall response compared to saline injected controls. In summary we have developed a potent universal memory T cell helper peptide (TpD) that is active in vitro in human PBMCs and in vivo in mice and non-human primates. PMID:24583006

  12. Human T-cell receptor v{beta} gene polymorphism and multiple sclerosis

    SciTech Connect

    Wei, S.; Charmley, P.; Birchfield, R.I.; Concannon, P.

    1995-04-01

    Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the {beta} chain of the T-cell receptor for antigen (RCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of {approximately}110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility. The TCRBV coding region-specific markers generated in these studies, as well as the approach of testing for associations with specific functionally relevant polymorphic sites within individual BV genes, should be useful in the evaluation of the many reported disease associations involving the human TCRB region. 22 refs., 1 fig., 3 tabs.

  13. Escin sodium induces apoptosis of human acute leukemia Jurkat T cells.

    PubMed

    Zhang, Zhenzhen; Gao, Jian; Cai, Xueting; Zhao, Youlong; Wang, Yafei; Lu, Wuguang; Gu, Zhenhua; Zhang, Shuangquan; Cao, Peng

    2011-12-01

    Escin sodium has been used in the clinic as an antioedematous, antiexudative and vasoprotective agent for many years and has shown excellent tolerability. However, little is known about its anticancer activity. This is a report for the first time that escin sodium exerts a cytotoxic effect on human acute leukemia Jurkat T cells via the induction of apoptosis rather than cell cycle arrest. Escin sodium activated the initiator caspase-8, -9, and the effector caspase-3, degraded poly (ADP-ribose) polymerase (PARP) and attenuated the expression of Bcl-2. In addition, escin sodium inhibited the growth of cancer cells in a selective manner with Jurkat cells most sensitive to it. Taken together, the data show that escin sodium possesses potent apoptogenic activity toward human acute leukemia Jurkat T cells. PMID:21452372

  14. Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence

    PubMed Central

    Carpentier, Alexandre; Barez, Pierre-Yves; Hamaidia, Malik; Gazon, Hélène; de Brogniez, Alix; Perike, Srikanth; Gillet, Nicolas; Willems, Luc

    2015-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans. PMID:26198240

  15. Tissue resident regulatory T cells: novel therapeutic targets for human disease

    PubMed Central

    Zhou, Xiaohui; Tang, Jiayou; Cao, Hao; Fan, Huimin; Li, Bin

    2015-01-01

    Over the past decade, the ability of regulatory T cells (Tregs) to suppress multiple types of immune cells has received tremendous attention. Mounting evidence has revealed that tissue resident Tregs control non-immunological processes of their target tissues and contribute to a plethora of human diseases. The identification of novel tissue-specific Tregs has highlighted their heterogeneity and complexity. This review summarizes the recent findings for visceral adipose tissue CD4+Foxp3+ regulatory T cells (VAT Tregs), muscle Tregs, bone Tregs and skin memory Tregs, with a focus on their unique functions in local tissues. This interpretation of the roles of tissue-specific Tregs and of their involvement in disease progression provides new insight into the discovery of potential therapeutic targets of human diseases. PMID:25891216

  16. Characterization of HLA-DR-restricted T-cell epitopes derived from human proteinase 3.

    PubMed

    Piesche, Matthias; Hildebrandt, York; Chapuy, Björn; Wulf, Gerald G; Trümper, Lorenz; Schroers, Roland

    2009-07-23

    Human proteinase 3 (PRTN3) is a leukemia-associated antigen specifically recognized by CD8+ cytotoxic T-lymphocytes (CTL). PRTN3 also has been shown to elicit both antibody responses and T-cell proliferation in patients with Wegener's granulomatosis. In order to improve current vaccines that aim to stimulate CTL without inducing harmful autoimmune disease, it is necessary to study the role of PRTN3-specific CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Since both TH and Treg cells recognize antigens in the context of HLA-class-II-molecules, identification of HLA-class-II-associated peptide-epitopes from self-antigens such as PRTN3 is required. Here, we analyzed T-cell responses against proteinase 3 using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. We first screened a panel of ten epitope peptide candidates selected with the TEPITOPE program and found that nine out of ten peptides induced PRTN3 peptide-specific proliferation of T-cells with precursor frequencies of 0-1.1 x 10(-6). For one peptide-epitope, PRTN3(235), T-cell-clones were demonstrated to be capable of recognizing naturally processed protein antigen in a HLA-DR-restricted fashion. PRTN3(235)-specific T-cells could be stimulated from the blood of healthy individuals with multiple HLA-DR-genotypes. In summary, the identified PRTN3(235)-epitope can be used to study the role of CD4+ TH- and Treg-cells in immune responses against PRTN3 in leukemia patients and patients with Wegener's disease. PMID:19446593

  17. Lethal cutaneous disease in transgenic mice conditionally expressing type I human T cell leukemia virus Tax.

    PubMed

    Kwon, Hakju; Ogle, Louise; Benitez, Bobby; Bohuslav, Jan; Montano, Mauricio; Felsher, Dean W; Greene, Warner C

    2005-10-21

    Type I human T cell leukemia virus (HTLV-I) is etiologically linked with adult T cell leukemia, an aggressive and usually fatal expansion of activated CD4+ T lymphocytes that frequently traffic to skin. T cell transformation induced by HTLV-I involves the action of the 40-kDa viral Tax transactivator protein. Tax both stimulates the HTLV-I long terminal repeat and deregulates the expression of select cellular genes by altering the activity of specific host transcription factors, including cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor, NF-kappaB/Rel, and serum response factor. To study initiating events involved in HTLV-I Tax-induced T cell transformation, we generated "Tet-off" transgenic mice conditionally expressing in a lymphocyte-restricted manner (EmuSR alpha promoter-enhancer) either wild-type Tax or mutant forms of Tax that selectively compromise the NF-kappaB (M22) or CREB/activating transcription factor (M47) activation pathways. Wild-type Tax and M47 Tax-expressing mice, but not M22-Tax expressing mice, developed progressive alopecia, hyperkeratosis, and skin lesions containing profuse activated CD4 T cell infiltrates with evidence of deregulated inflammatory cytokine production. In addition, these animals displayed systemic lymphadenopathy and splenomegaly. These findings suggest that Tax-mediated activation of NF-kappaB plays a key role in the development of this aggressive skin disease that shares several features in common with the skin disease occurring during the preleukemic stage in HTLV-I-infected patients. Of note, this skin disease completely resolved when Tax transgene expression was suppressed by administration of doxycycline, emphasizing the key role played by this viral oncoprotein in the observed pathology. PMID:16105841

  18. Immunomodulatory effect of procainamide in man. Inhibition of human suppressor T-cell activity in vitro.

    PubMed Central

    Ochi, T; Goldings, E A; Lipsky, P E; Ziff, M

    1983-01-01

    Procainamide (PA) induces the production of a number of autoantibodies in a high proportion of treated individuals and in some a syndrome closely resembling systemic lupus erythematosus. The mechanism underlying this action of PA is unclear. To examine the possibility that PA might induce autoantibody formation by altering normal immunoregulatory mechanisms, the action of this drug on an in vitro model of antibody formation in man was examined. PA was found to augment the generation of immunoglobulin-secreting cells (ISC) from human peripheral blood mononuclear cells (PBM) in response to pokeweed mitogen but had no effect on pokeweed mitogen-induced tritiated thymidine incorporation. When purified populations of B and T cells were used, PA enhanced the generation of ISC in B-cell cultures supported by untreated T cells but not by T cells treated with mitomycin C. These results indicate that PA augmented B-cell responses by inhibiting suppressor T-cell activity and not by augmenting helper T-cell or B-cell function. N-Acetyl-procainamide had no effect on the generation of ISC in this system. The effect of PA on concanavalin A (Con A)-induced suppressor cell activity was also examined to determine whether PA altered the generation or expression of suppressor T-cell function. PBM were cultured with 30 microgram/ml of Con A for 48 h to generate suppressor cells. When these were co-cultured with fresh PBM, the number of ISC generated was decreased by 58.1 +/- 3.4% (mean +/- SEM, n = 6). Cells that had been similarly incubated without Con A were not inhibitory. The addition of PA to the Con A-stimulated cultures inhibited the generation of suppressor cells as indicated by the fact that the response of fresh cells co-cultured with the Con A-stimulated cells was diminished by only 27.2 +/- 4.3%. In this system too, N-acetyl-procaimamide had no effect. By contrast, adding PA only to the co-culture of Con A-stimulated cells with fresh PBM had a less marked effect on

  19. Early-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues.

    PubMed

    Thome, Joseph J C; Bickham, Kara L; Ohmura, Yoshiaki; Kubota, Masaru; Matsuoka, Nobuhide; Gordon, Claire; Granot, Tomer; Griesemer, Adam; Lerner, Harvey; Kato, Tomoaki; Farber, Donna L

    2016-01-01

    It is unclear how the immune response in early life becomes appropriately stimulated to provide protection while also avoiding excessive activation as a result of diverse new antigens. T cells are integral to adaptive immunity; mouse studies indicate that tissue localization of T cell subsets is important for both protective immunity and immunoregulation. In humans, however, the early development and function of T cells in tissues remain unexplored. We present here an analysis of lymphoid and mucosal tissue T cells derived from pediatric organ donors in the first two years of life, as compared to adult organ donors, revealing early compartmentalization of T cell differentiation and regulation. Whereas adult tissues contain a predominance of memory T cells, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (T(EM)) found only in the lungs and small intestine. Additionally, regulatory T (T(reg)) cells comprise a high proportion (30-40%) of CD4(+) T cells in pediatric tissues but are present at much lower frequencies (1-10%) in adult tissues. Pediatric tissue T(reg) cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest T(reg):T(EM) cell ratios, which suggests control in situ of immune responses in early life. PMID:26657141

  20. YM155 suppresses cell proliferation and induces cell death in human adult T-cell leukemia/lymphoma cells.

    PubMed

    Sasaki, Ryousei; Ito, Shigeki; Asahi, Maki; Ishida, Yoji

    2015-12-01

    Adult T-cell leukemia (ATL) is an aggressive malignancy of peripheral T cells infected with human T-cell leukemia virus type 1 (HTLV-1). The prognosis of patients with aggressive ATL remains poor because ATL cells acquire resistance to conventional cytotoxic agents. Therefore, development of novel agents is urgently needed. We examined the effects of YM155, sepantronium bromide, on cell proliferation and survival of ATL or HTLV-1-infected T-cell lines, S1T, MT-1, and MT-2. We found that YM155 suppressed cell proliferation in these cells and induced cell death in S1T and MT-1 cells. Both real-time quantitative polymerase chain reaction and immunoblot analyses showed suppression of survivin expression in S1T, MT-1, and MT-2 cells. In addition, we observed the cleavage of caspase-3 and poly(ADP-ribose) polymerase in YM155-treated S1T and MT-1 cells, indicating that YM155 induces caspase-dependent apoptosis in these cells. To clarify the mechanism of drug tolerance of MT-2 cells in terms of YM155-induced cell death, we examined intracellular signaling status in these cells. We found that STAT3, STAT5, and AKT were constitutively phosphorylated in MT-2 cells but not in S1T and MT-1 cells. Treatment with YM155 combined with the STAT3 inhibitor S3I-201 significantly suppressed cell proliferation compared to that with either YM155 or S3I-201 in MT-2 cells, indicating that STAT3 may play a role in tolerance of MT-2 cells to YM155 and that STAT3 may therefore be a therapeutic target for YM155-resistant ATL cells. These results suggest that YM155 presents potent antiproliferative and apoptotic effects via suppression of survivin in ATL cells in which STAT3 is not constitutively phosphorylated. YM155 merits further investigation as a potential chemotherapeutic agent for ATL. PMID:26547260

  1. B lymphoblastoid cell lines as efficient APC to elicit CD8+ T cell responses against a cytomegalovirus antigen.

    PubMed

    Sun, Q; Burton, R L; Dai, L J; Britt, W J; Lucas, K G

    2000-10-01

    Potent and readily accessible APC are critical for development of immunotherapy protocols to treat viral disease and cancer. We have shown that B lymphoblastoid cell lines (BLCL) that stably express CMV phosphoprotein 65 (BLCLpp65), as a result of retroviral transduction, can be used to generate ex vivo CTL cultures that possess cytotoxicity against CMV and EBV. In this report, we demonstrate that the EBV-specific cytotoxicity in the BLCLpp65-primed culture had a spectrum of EBV-Ag recognition similar to that of the BLCL-primed counterpart, suggesting that retroviral transduction and expression of the CMV Ag would not compromise the Ag-presenting capacity of BLCL. In addition, BLCLpp65 appeared to present multiple natural pp65 epitopes, because pp65-specific CTL, which recognized different CMV clinical isolates, were generated in BLCLpp65-primed cultures from individuals with various HLA backgrounds. Consistent with a polyclonal expansion of virus-specific CTL, T cell lines established from the BLCLpp65-primed CTL cultures expressed different TCR-Vbeta Although most of the virus-specific T cell isolates were CD8+, EBV-specific CD4+ lines were also established from BLCLpp65-primed cultures. Western blot analysis revealed that the CD8+ lines, but not the CD4+ line, expressed granzyme B, consistent with features of classic CTL. Thus, our results suggested that BLCL stably expressing a foreign Ag might be used as a practical APC to elicit CD8+ T cell responses. PMID:11034422

  2. In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

    PubMed Central

    Jung, Kyeong Cheon; Jeon, Yoon Kyung; Ban, Young Larn; Min, Hye Sook; Kim, Eun Ji; Kim, Ju Hyun; Kang, Byung Hyun; Bae, Youngmee; Yoon, Il-Hee; Kim, Yong-Hee; Lee, Jae-Il; Kim, Jung-Sik; Shin, Jun-Seop; Yang, Jaeseok; Kim, Sung Joo; Rostlund, Emily; Muller, William A.

    2011-01-01

    Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection. PMID:22025302

  3. Molecular Imprint of Exposure to Naturally Occurring Genetic Variants of Human Cytomegalovirus on the T cell Repertoire

    NASA Astrophysics Data System (ADS)

    Smith, Corey; Gras, Stephanie; Brennan, Rebekah M.; Bird, Nicola L.; Valkenburg, Sophie A.; Twist, Kelly-Anne; Burrows, Jacqueline M.; Miles, John J.; Chambers, Daniel; Bell, Scott; Campbell, Scott; Kedzierska, Katherine; Burrows, Scott R.; Rossjohn, Jamie; Khanna, Rajiv

    2014-02-01

    Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot-print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.

  4. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. PMID:20164855

  5. The Type of Responder T-Cell Has a Significant Impact in a Human In Vitro Suppression Assay

    PubMed Central

    Jana, Srikanta; Campbell, Hope; Woodliff, Jeffrey; Waukau, Jill; Jailwala, Parthav; Ghorai, Jugal; Ghosh, Soumitra; Glisic, Sanja

    2010-01-01

    Background In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4+CD25+high, or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4+CD25low T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease. Methods/Principal Findings We investigated human CD4+CD25low T cells and compared them to CD4+CD25- T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4+CD25low T cells divided more rapidly than CD4+CD25- T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25low compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25low T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively). Conclusions/Significance The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases. PMID:21151941

  6. Human immunodeficiency virus (HIV) type 1 Vpr induces differential regulation of T cell costimulatory molecules: Direct effect of Vpr on T cell activation and immune function

    SciTech Connect

    Venkatachari, Narasimhan J.; Majumder, Biswanath; Ayyavoo, Velpandi . E-mail: velpandi@pitt.edu

    2007-02-20

    Human immunodeficiency virus type 1 (HIV-1) viral proteins disrupt the normal host cellular immune pathways thus exploiting the cellular machinery for replication, survival and to escape host immune attack. Here we evaluated the direct effects of HIV-1 Vpr-mediated immune modulation of infected T cells. Vpr specifically downregulated the expression of CD28 and increased the expression of CTLA-4, whereas no significant difference in the expression of CD25 and HLA-DR was observed. Interferon gamma (IFN-{gamma}) production in T cells was evaluated as a measure of the downstream effector functions. Results indicate that Vpr significantly inhibited IFN-{gamma} production and this may, in part, due to Vpr's ability to inhibit the nuclear translocation of NF-{kappa}B, and its transcriptional regulation. Together these results support that HIV-1 Vpr selectively dysregulates the immune functions at multiple levels and exerts its inhibitory effects in the presence of other viral proteins.

  7. Adult T-cell leukemia-lymphoma in a pregnant woman diagnosed as a human T-cell lymphotropic virus type 1 carrier.

    PubMed

    Fuchi, Naoki; Miura, Kiyonori; Imaizumi, Yoshitaka; Hasegawa, Hiroo; Yanagihara, Katsunori; Miyazaki, Yasushi; Masuzaki, Hideaki

    2016-03-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia-lymphoma (ATL), which is difficult to cure. In Japan, a nationwide HTLV-1 screening test in pregnant women has been recommended since 2011. A 30-year-old woman was diagnosed as being an HTLV-1 carrier in her previous pregnancy. During the current pregnancy, she had persistent fever and cough. Although she had treatment with antibiotics, peripheral white blood cell count remained high, with an abnormal lymphocyte count. Given that she was an HTLV-1 carrier, she was diagnosed with unfavorable chronic ATL (aggressive ATL) at 12 weeks gestation. After pregnancy termination, her ATL status became favorable chronic ATL (indolent ATL). Therefore, watchful waiting was performed until disease progression. This is the first case report of chronic ATL in early pregnancy, in a woman already diagnosed as an HTLV-1 carrier on screening test. PMID:26663442

  8. A promising sword of tomorrow: Human γδ T cell strategies reconcile allo-HSCT complications.

    PubMed

    Hu, Yongxian; Cui, Qu; Luo, Chao; Luo, Yi; Shi, Jimin; Huang, He

    2016-05-01

    Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative therapeutic option for hematological malignancies. In clinical practice, transplantation associated complications greatly affected the final therapeutical outcomes. Currently, primary disease relapse, graft-versus-host disease (GVHD) and infections remain the three leading causes of a high morbidity and mortality in allo-HSCT patients. Various strategies have been investigated in the past several decades including human γδ T cell-based therapeutical regimens. In different microenvironments, human γδ T cells assume features reminiscent of classical Th1, Th2, Th17, NKT and regulatory T cells, showing diverse biological functions. The cytotoxic γδ T cells could be utilized to target relapsed malignancies, and recently regulatory γδ T cells are defined as a novel implement for GVHD management. In addition, human γδ Τ cells facilitate control of post-transplantation infections and participate in tissue regeneration and wound healing processes. These features potentiate γδ T cells a versatile therapeutical agent to target transplantation associated complications. This review focuses on insights of applicable potentials of human γδ T cells reconciling complications associated with allo-HSCT. We believe an improved understanding of pertinent γδ T cell functions would be further exploited in the design of innovative immunotherapeutic approaches in allo-HSCT, to reduce mortality and morbidity, as well as improve quality of life for patients after transplantation. PMID:26654098

  9. CD1d-restricted peripheral T cell lymphoma in mice and humans.

    PubMed

    Bachy, Emmanuel; Urb, Mirjam; Chandra, Shilpi; Robinot, Rémy; Bricard, Gabriel; de Bernard, Simon; Traverse-Glehen, Alexandra; Gazzo, Sophie; Blond, Olivier; Khurana, Archana; Baseggio, Lucile; Heavican, Tayla; Ffrench, Martine; Crispatzu, Giuliano; Mondière, Paul; Schrader, Alexandra; Taillardet, Morgan; Thaunat, Olivier; Martin, Nadine; Dalle, Stéphane; Le Garff-Tavernier, Magali; Salles, Gilles; Lachuer, Joel; Hermine, Olivier; Asnafi, Vahid; Roussel, Mikael; Lamy, Thierry; Herling, Marco; Iqbal, Javeed; Buffat, Laurent; Marche, Patrice N; Gaulard, Philippe; Kronenberg, Mitchell; Defrance, Thierry; Genestier, Laurent

    2016-05-01

    Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans. PMID:27069116

  10. Differential regulation of human T cell responsiveness by mucosal versus blood monocytes.

    PubMed

    Qiao, L; Braunstein, J; Golling, M; Schürmann, G; Autschbach, F; Möller, P; Meuer, S

    1996-04-01

    Human intestinal T lymphocytes are constantly exposed to a large number of foreign antigens without developing a systemic immune response. One crucial mechanisms leading to this intestinal hyporesponsiveness is based on impaired signal transduction through the T cell receptor/CD3 complex in lamina propria T lymphocytes (LP-T). In this study, we addressed the question whether a lack of co-stimulatory/progression signals might also contribute to LP-T hyporesponsiveness. To this end, isolated human monocyte populations from the intestinal lamina propria were obtained and their phenotypes as well as their capacity to promote T cell activation studied. Here, we demonstrate that lamina propria macrophages (LP-MO), in contrast to peripheral blood monocytes (PB-MO), do not support proliferation of either LP-T or PB-T. This may be due to the low expression of ligands (CD54, CD58, CD80) for the T cell accessory receptors CD11/18, CD2 and CD28/CTLA-4 on mucosal macrophages. Thus, down-regulation of both recognition/competence and co-stimulatory/progression signals contribute to intestinal hypo- or unresponsiveness. PMID:8625989