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Sample records for promote t-cell binding

  1. IL-15 prolongs CD154 expression on human CD4 T cells via STAT5 binding to the CD154 transcriptional promoter

    PubMed Central

    Lowe, RM; Genin, A; Orgun, N; Cron, RQ

    2014-01-01

    Activation induced CD154 expression on CD4 T cells is prolonged in systemic lupus erythematosus but the mechanism(s) for its dysregulation are unknown. The studies reported herein demonstrate that IL-15 is capable of prolonging CD154 expression on PHA activated CD4 T cells. Since IL-15 signals through STAT5, predicted STAT5 binding sites in the human CD154 transcriptional promoter were identified, and STAT5 binding to the proximal CD154 promoter in vitro and in vivo following primary CD4 T cell activation was demonstrated. Moreover, overexpression of wild-type(WT) STAT5 in primary human CD4 T cells augmented CD154 transcription, whereas overexpression of a dominant negative (DN) STAT5 protein inhibited CD154 transcription. Mutation of the most proximal STAT5 binding site in the CD154 promoter resulted in diminished DNA binding and reduced CD154 transcriptional activity. Interestingly, STAT5-specific siRNA inhibited CD154 surface expression at 48 but not 24 hours after T cell activation. Thus, these findings provide some of the first evidence to support a possible mechanistic link to explain how the overexpression of IL-15 observed in lupus patients may be involved in the prolonged expression of CD154 that has also been observed on lupus CD4 T cells. PMID:24500400

  2. The regulation of TIM-3 transcription in T cells involves c-Jun binding but not CpG methylation at the TIM-3 promoter.

    PubMed

    Yun, Su Jin; Jun, Ka-Jung; Komori, Kuniharu; Lee, Mi Jin; Kwon, Myung-Hee; Chwae, Yong-Joon; Kim, Kyongmin; Shin, Ho-Joon; Park, Sun

    2016-07-01

    Tim-3 is an immunomodulatory protein that is expressed constitutively on monocytes but is induced in activated T cells. The mechanisms involved in the regulation of TIM-3 transcription are poorly understood. In the present study, we investigated whether methylation of the TIM-3 promoter is involved in regulatingTIM-3 transcription in T cells, and identified a transcription factor that regulates TIM-3 transcription by associating with the TIM-3 minimal promoter region. Pyrosequencing of the TIM-3 promoter up to -1440bp revealed 11 hypermethylated CpG sites and 4 hypomethylated CpG sites in human CD4(+) T cells as well as in CD11b(+) cells. Dimethylation of histone H3 lysine 4 (H3K4), a mark of transcriptional activation, was predominantly found in the proximal TIM-3 promoter -954 to -34bp region, whereas trimethylation of H3K9 and H3K27, which are markers of transcriptional suppression, were mostly observed in the distal promoter -1549 to -1048bp region in human CD4(+) T cells and CD11b(+) cells. However, no change in the methylation status of CpG sites and the histone H3 in the TIM-3 promoter was found during induction of TIM-3 transcription in T cells. Finally, AP-1 involvement in TIM-3 transcription was shown in relation with the TIM-3 minimal promoter -146 to +144bp region. The present study defines the minimal TIM-3 promoter region and demonstrates its interaction with c-Jun during TIM-3 transcription in CD4(+) T cells.

  3. PI3Kδ promotes CD4+ T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1

    PubMed Central

    Garçon, Fabien; Okkenhaug, Klaus

    2016-01-01

    Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice. PMID:26740009

  4. Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning.

    PubMed

    Paensuwan, Pussadee; Hartl, Frederike A; Yousefi, O Sascha; Ngoenkam, Jatuporn; Wipa, Piyamaporn; Beck-Garcia, Esmeralda; Dopfer, Elaine P; Khamsri, Boonruang; Sanguansermsri, Donruedee; Minguet, Susana; Schamel, Wolfgang W; Pongcharoen, Sutatip

    2016-01-01

    Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning. PMID:26590318

  5. Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses.

    PubMed

    Chervin, A S; Stone, J D; Soto, C M; Engels, B; Schreiber, H; Roy, E J; Kranz, D M

    2013-06-01

    Adoptive T-cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, which introduces antigen-specific T-cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T-cell repertoires. Studies have suggested that use of higher-affinity TCRs against class I major histocompatibility complex antigens could drive the activity of both CD4(+) and CD8(+) T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here, we describe a high-throughput platform of 'reverse biochemistry' whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes (TILs) or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8(+) T cells expressing the highest-affinity TCR variants were deleted in both the TIL population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4(+) T cells in the tumor, suggesting they had a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR-binding properties that promote peripheral T-cell survival and tumor elimination.

  6. Ethanol promotes T cell apoptosis through the mitochondrial pathway

    PubMed Central

    Kapasi, Aditi A; Patel, Geeta; Goenka, Anuj; Nahar, Nilay; Modi, Neeraj; Bhaskaran, Madhu; Reddy, Krishna; Franki, Nicholas; Patel, Jaimita; Singhal, Pravin C

    2003-01-01

    Clinical reports suggest that acute ethanol intoxication is often associated with lymphopenia. Previously, ethanol was reported to invoke thymocyte apoptosis. We studied the effect of ethanol on T cell apoptosis. In addition, we evaluated the molecular mechanism of ethanol-induced T cell apoptosis. Human T cells harvested from healthy subjects after an alcohol drinking binge showed enhanced T cell apoptosis (before, 0·4 ± 0·2% versus after, 19·6 ± 2·5% apoptotic lymphocytes/field; P < 0·001). In in vitro studies, ethanol in a concentration of 50 mm and higher enhanced the apoptosis of Jurkat cells. DNA isolated from ethanol-treated Jurkat cells displayed integer multiples of 180 base pairs. Ethanol decreased Jurkat cell expression of Bcl-2, whereas ethanol increased Jurkat cell expression of Bax. Jurkat cells treated with ethanol also showed translocation of cytochrome C into cytosol. Moreover, a caspase-9 inhibitor partially inhibited ethanol-induced Jurkat cell apoptosis. In in vivo studies, after binge drinking, T cell expression of Bcl-2 also decreased. In addition, binge drinking induced the cleavage of caspase-3, suggesting activation of caspase-3 in T cells. These results suggest that ethanol promotes T cell apoptosis through the activation of intrinsic or mitochondrial pathway. PMID:12603597

  7. KIR3DL2 binds to HLA-B27 dimers and free heavy chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis

    PubMed Central

    Wong-Baeza, Isabel; Ridley, Anna; Shaw, Jackie; Hatano, Hiroko; Rysnik, Oliwia; McHugh, Kirsty; Piper, Christopher; Brackenbridge, Simon; Fernandes, Ricardo; Chan, Anthoni; Bowness, Paul; Kollnberger, Simon

    2013-01-01

    1Abstract The Human Leukocyte Antigen HLA-B27(B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of antigen presenting cells (APC) as both classical β2m-associated B27 and as B27 free heavy chain forms (FHC) including disulphide-bonded heavy chain homodimers (termed B272). B27 FHC forms but not classical B27 bind to KIR3DL2. HLA-A3 which is not associated with spondyloarthritis (SpA) is also a ligand for KIR3DL2. Here we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2 transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric and monomeric free heavy chains from HLA-B27 expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε–transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFNγ secretion and promoted greater survival of KIR3DL2+CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+SpA patients proliferated more in response to antigen presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC. PMID:23440420

  8. Activated human T cells accomplish MHC class II expression through T cell-specific occupation of class II transactivator promoter III.

    PubMed

    Holling, Tjadine M; van der Stoep, Nienke; Quinten, Edwin; van den Elsen, Peter J

    2002-01-15

    Activated human T cells express HLA-DR, HLA-DQ, and HLA-DP on their surface, but the regulation and functioning of MHC class II molecules in T lymphocytes are poorly understood. Because the MHC class II transactivator (CIITA) is essential for MHC class II expression, we have investigated transcriptional activation of CIITA in activated T cells. In this study, we show that in human activated CD4(+) T cells, CIITA promoter III (CIITA-PIII) drives the expression of CIITA. The in vivo genomic footprint analysis revealed activated T cell-specific occupation of CIITA-PIII. Subsequent EMSA analysis of several promoter regions showed differences in banding pattern among activated T cells, naive T cells, primary B cells, and Raji B cells. Activating response element (ARE)-1 is shown to interact with the acute myeloid leukemia 2 transcription factor in nuclear extracts derived from both T and B cells. Interestingly, the acute myeloid leukemia 3 transcription factor was bound in nuclear extracts of T cells only. The ARE-2 sequence is able to bind CREB/activating transcription factor family members in both T and B cells. In addition, a yet unidentified Ets family member was found to interact with site C in activated T cells, whereas in B cells site C was bound by PU.1 and Pip/IFN regulatory factor 4/IFN consensus sequence binding protein for activated T cells. In Jurkat T cells, both ARE-1 and ARE-2 are crucial for CIITA-PIII activity, similar to Raji B cells. The differential banding pattern in in vivo genomic footprinting and transcription factor binding at the ARE-1 and site C between T cells and B cells probably reflects differences in CIITA-PIII activation pathways employed by these cell types. PMID:11777970

  9. Computational Reprogramming of T Cell Antigen Receptor Binding Properties.

    PubMed

    Riley, Timothy P; Singh, Nishant K; Pierce, Brian G; Baker, Brian M; Weng, Zhiping

    2016-01-01

    T-cell receptor (TCR) binding to peptide/MHC is key to antigen-specific cellular immunity, and there has been considerable interest in modulating TCR affinity and specificity for the development of therapeutics and imaging reagents. While in vitro engineering efforts using molecular evolution have yielded remarkable improvements in TCR affinity, such approaches do not offer structural control and can adversely affect receptor specificity, particularly if the attraction towards the MHC is enhanced independently of the peptide. Here we describe an approach to computational design that begins with structural information and offers the potential for more controlled manipulation of binding properties. Our design process models point mutations in selected regions of the TCR and ranks the resulting change in binding energy. Consideration is given to designing optimized scoring functions tuned to particular TCR-peptide/MHC interfaces. Validation of highly ranked predictions can be used to refine the modeling methodology and scoring functions, improving the design process. Our approach results in a strong correlation between predicted and measured changes in binding energy, as well as good agreement between modeled and experimental structures. PMID:27094299

  10. Plasmacytoid dendritic cells and type 1 interferon promote peripheral expansion of forkhead box protein 3(+) regulatory T cells specific for the ubiquitous RNA-binding nuclear antigen La/Sjögren's syndrome (SS)-B.

    PubMed

    Pan, Z-J; Horton, C G; Lawrence, C; Farris, A D

    2016-10-01

    RNA-binding nuclear antigens are a major class of self-antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)-B (La), is controlled by CD4(+) T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa-specific CD4(+) T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte-replete recipient mice expressing hLa as a neo-self-antigen. After initial antigen-specific cell division, hLa-specific donor CD4(+) T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)-10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3-negative donor cells demonstrated that accumulation of hLa-specific regulatory T cells (Treg ) was due primarily to expansion of small numbers of donor Treg . Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3(+) donor Treg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co-stimulatory and co-inhibitory molecules than B cells. Adoptive transfer of hLa peptide-loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa-specific Treg . Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa-specific T cells impaired FoxP3(+) donor T cell accumulation. Therefore, peripheral expansion of Treg specific for an RNA-binding nuclear antigen is mediated by antigen-presenting pDC in a type 1 IFN-dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA-binding nuclear antigens.

  11. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals

    PubMed Central

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2015-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  12. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

    PubMed

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2014-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  13. T cell metabolism. The protein LEM promotes CD8⁺ T cell immunity through effects on mitochondrial respiration.

    PubMed

    Okoye, Isobel; Wang, Lihui; Pallmer, Katharina; Richter, Kirsten; Ichimura, Takahuru; Haas, Robert; Crouse, Josh; Choi, Onjee; Heathcote, Dean; Lovo, Elena; Mauro, Claudio; Abdi, Reza; Oxenius, Annette; Rutschmann, Sophie; Ashton-Rickardt, Philip G

    2015-05-29

    Protective CD8(+) T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named lymphocyte expansion molecule (LEM) that promoted antigen-dependent CD8(+) T cell proliferation, effector function, and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results. Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration, resulting in the production of pro-proliferative mitochondrial reactive oxygen species (mROS). LEM provides a link between immune activation and the expansion of protective CD8(+) T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified cytotoxic CD8(+) T cells.

  14. β-Catenin promotes colitis and colon cancer through imprinting of proinflammatory properties in T cells.

    PubMed

    Keerthivasan, Shilpa; Aghajani, Katayoun; Dose, Marei; Molinero, Luciana; Khan, Mohammad W; Venkateswaran, Vysak; Weber, Christopher; Emmanuel, Akinola Olumide; Sun, Tianjao; Bentrem, David J; Mulcahy, Mary; Keshavarzian, Ali; Ramos, Elena M; Blatner, Nichole; Khazaie, Khashayarsha; Gounari, Fotini

    2014-02-26

    The density and type of lymphocytes that infiltrate colon tumors are predictive of the clinical outcome of colon cancer. High densities of T helper 17 (T(H)17) cells and inflammation predict poor outcome, whereas infiltration by T regulatory cells (Tregs) that naturally suppress inflammation is associated with longer patient survival. However, the role of Tregs in cancer remains controversial. We recently reported that Tregs in colon cancer patients can become proinflammatory and tumor-promoting. These properties were directly linked with their expression of RORγt (retinoic acid-related orphan receptor-γt), the signature transcription factor of T(H)17 cells. We report that Wnt/β-catenin signaling in T cells promotes expression of RORγt. Expression of β-catenin was elevated in T cells, including Tregs, of patients with colon cancer. Genetically engineered activation of β-catenin in mouse T cells resulted in enhanced chromatin accessibility in the proximity of T cell factor-1 (Tcf-1) binding sites genome-wide, induced expression of T(H)17 signature genes including RORγt, and promoted T(H)17-mediated inflammation. Strikingly, the mice had inflammation of small intestine and colon and developed lesions indistinguishable from colitis-induced cancer. Activation of β-catenin only in Tregs was sufficient to produce inflammation and initiate cancer. On the basis of these findings, we conclude that activation of Wnt/β-catenin signaling in effector T cells and/or Tregs is causatively linked with the imprinting of proinflammatory properties and the promotion of colon cancer.

  15. β-Catenin promotes colitis and colon cancer through imprinting of proinflammatory properties in T cells.

    PubMed

    Keerthivasan, Shilpa; Aghajani, Katayoun; Dose, Marei; Molinero, Luciana; Khan, Mohammad W; Venkateswaran, Vysak; Weber, Christopher; Emmanuel, Akinola Olumide; Sun, Tianjao; Bentrem, David J; Mulcahy, Mary; Keshavarzian, Ali; Ramos, Elena M; Blatner, Nichole; Khazaie, Khashayarsha; Gounari, Fotini

    2014-02-26

    The density and type of lymphocytes that infiltrate colon tumors are predictive of the clinical outcome of colon cancer. High densities of T helper 17 (T(H)17) cells and inflammation predict poor outcome, whereas infiltration by T regulatory cells (Tregs) that naturally suppress inflammation is associated with longer patient survival. However, the role of Tregs in cancer remains controversial. We recently reported that Tregs in colon cancer patients can become proinflammatory and tumor-promoting. These properties were directly linked with their expression of RORγt (retinoic acid-related orphan receptor-γt), the signature transcription factor of T(H)17 cells. We report that Wnt/β-catenin signaling in T cells promotes expression of RORγt. Expression of β-catenin was elevated in T cells, including Tregs, of patients with colon cancer. Genetically engineered activation of β-catenin in mouse T cells resulted in enhanced chromatin accessibility in the proximity of T cell factor-1 (Tcf-1) binding sites genome-wide, induced expression of T(H)17 signature genes including RORγt, and promoted T(H)17-mediated inflammation. Strikingly, the mice had inflammation of small intestine and colon and developed lesions indistinguishable from colitis-induced cancer. Activation of β-catenin only in Tregs was sufficient to produce inflammation and initiate cancer. On the basis of these findings, we conclude that activation of Wnt/β-catenin signaling in effector T cells and/or Tregs is causatively linked with the imprinting of proinflammatory properties and the promotion of colon cancer. PMID:24574339

  16. TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy.

    PubMed

    Matsuzawa, Yu; Oshima, Shigeru; Takahara, Masahiro; Maeyashiki, Chiaki; Nemoto, Yasuhiro; Kobayashi, Masanori; Nibe, Yoichi; Nozaki, Kengo; Nagaishi, Takashi; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Ma, Averil; Watanabe, Mamoru

    2015-01-01

    Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.

  17. TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy

    PubMed Central

    Matsuzawa, Yu; Oshima, Shigeru; Takahara, Masahiro; Maeyashiki, Chiaki; Nemoto, Yasuhiro; Kobayashi, Masanori; Nibe, Yoichi; Nozaki, Kengo; Nagaishi, Takashi; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Ma, Averil; Watanabe, Mamoru

    2015-01-01

    Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells. PMID:26043155

  18. End-binding protein 1 controls signal propagation from the T cell receptor

    PubMed Central

    Martín-Cófreces, Noa B; Baixauli, Francesc; López, María J; Gil, Diana; Monjas, Alicia; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2012-01-01

    The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome. PMID:22922463

  19. Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization

    PubMed Central

    Myers, Linda K; Cullins, David L; Park, Jeoung-Eun; Yi, Ae-Kyung; Brand, David D; Rosloniec, Edward F; Stuart, John M; Kang, Andrew H

    2015-01-01

    Factors that drive T cells to signal through differing pathways remain unclear. We have shown that an altered peptide ligand (A9) activates T cells to utilize an alternate signaling pathway which is dependent upon FcRγ and Syk. However, it remains unknown whether the affinity of peptide binding to MHC drives this selection. To answer this question we developed a panel of peptides designed so that amino acids interacting with the p6 and p9 predicted MHC binding pockets were altered. Analogs were tested for binding to I-Aq using a competitive binding assay and selected analogs were administered to arthritic mice. Using the collagen-induced arthritis (CIA) model, arthritis severity was correlated with T cell cytokine production and molecular T cell signaling responses. We establish that reduced affinity of interaction with the MHC correlates with T cell signaling through the alternative pathway, leading ultimately to secretion of suppressive cytokine and attenuation of arthritis. PMID:25982319

  20. Multivalent Antigens for Promoting B and T Cell Activation

    PubMed Central

    Bennett, Nitasha R.; Zwick, Daniel B.; Courtney, Adam H.; Kiessling, Laura L.

    2015-01-01

    Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B–T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using 19F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines. PMID:25970017

  1. Spatial congregation of STAT binding directs selective nuclear architecture during T-cell functional differentiation

    PubMed Central

    Hakim, Ofir; Sung, Myong-Hee; Nakayamada, Shingo; Voss, Ty C.; Baek, Songjoon; Hager, Gordon L.

    2013-01-01

    Higher-order genome organization shows tissue-specific patterns. However, functional relevance and the mechanisms shaping the genome architecture are poorly understood. Here we report a profound shift from promiscuous to highly selective genome organization that accompanies the effector lineage choice of differentiating T cells. As multipotent naive cells receive antigenic signals and commit to a T helper (Th) pathway, the genome-wide contacts of a lineage-specific cytokine locus are preferentially enriched for functionally relevant genes. Despite the establishment of divergent interactomes and global reprogramming of transcription in Th1 versus Th2, the overall expression status of the contact genes is surprisingly similar between the two lineages. Importantly, during differentiation, the genomic contacts are retained and strengthened precisely at DNA binding sites of the specific lineage-determining STAT transcription factor. In cells from the specific STAT knock-out mouse, the signature cytokine locus is unable to shed the promiscuous contacts established in the naive T cells, indicating the importance of genomic STAT binding. Altogether, the global aggregation of STAT binding loci from genic and nongenic regions highlights a new role for differentiation-promoting transcription factors in direct specification of higher-order nuclear architecture through interacting with regulatory regions. Such subnuclear environments have significant implications for efficient functioning of the mature effector lymphocytes. PMID:23212947

  2. Unusual features of Self-Peptide/MHC Binding by Autoimmune T Cell Receptors

    SciTech Connect

    Nicholson,M.; Hahn, M.; Wucherpfennig, K.

    2005-01-01

    Structural studies on T cell receptors (TCRs) specific for foreign antigens demonstrated a remarkably similar topology characterized by a central, diagonal TCR binding mode that maximizes interactions with the MHC bound peptide. However, three recent structures involving autoimmune TCRs demonstrated unusual interactions with self-peptide/MHC complexes. Two TCRs from multiple sclerosis patients bind with unconventional topologies, and both TCRs are shifted toward the peptide N terminus and the MHC class II {beta} chain helix. A TCR from the experimental autoimmune encephalomyelitis (EAE) model binds in a conventional orientation, but the structure is unusual because the self-peptide only partially fills the binding site. For all three TCRs, interaction with the MHC bound self-peptide is suboptimal, and only two or three TCR loops contact the peptide. Optimal TCR binding modes confer a competitive advantage for antimicrobial T cells during an infection, whereas altered binding properties may permit survival of a subset of autoreactive T cells during thymic selection.

  3. A new effect of IL-4 on human γδ T cells: promoting regulatory Vδ1 T cells via IL-10 production and inhibiting function of Vδ2 T cells

    PubMed Central

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

    2016-01-01

    Interleukin 4 (IL-4) has a variety of immune functions, including helper T-cell (Th-cell) differentiation and innate immune-response processes. However, the impact of IL-4 on gamma delta (γδ) T cells remains unclear. In this study, we investigate the effects of IL-4 on the activation and proliferation of γδ T cells and the balance between variable delta 1 (Vδ1) and Vδ2 T cells in humans. The results show that IL-4 inhibits the activation of γδ T cells in the presence of γδ T-cell receptor (TCR) stimulation in a STAT6-dependent manner. IL-4 promoted the growth of activated γδ T cells and increased the levels of Vδ1 T cells, which in turn inhibited Vδ2 T-cell growth via significant IL-10 secretion. Vδ1 T cells secreted significantly less interferon gamma (IFNγ) and more IL-10 relative to Vδ2. Furthermore, Vδ1 T cells showed relatively low levels of Natural Killer Group 2D (NKG2D) expression in the presence of IL-4, suggesting that Vδ1 T cells weaken the γδ T cell-mediated anti-tumor immune response. For the first time, our findings demonstrate a negative regulatory role of IL-4 in γδ T cell-mediated anti-tumor immunity. PMID:25942601

  4. Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

    PubMed

    Besseyrias, Valerie; Fiorini, Emma; Strobl, Lothar J; Zimber-Strobl, Ursula; Dumortier, Alexis; Koch, Ute; Arcangeli, Marie-Laure; Ezine, Sophie; Macdonald, H Robson; Radtke, Freddy

    2007-02-19

    Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.

  5. Itk Signals Promote Neuroinflammation by Regulating CD4+ T-Cell Activation and Trafficking

    PubMed Central

    Kannan, Arun K.; Kim, Do-Geun

    2015-01-01

    Here we demonstrate that interleukin-2-inducible T-cell kinase (Itk) signaling in cluster of differentiation 4-positive (CD4+) T cells promotes experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). We show that Itk−/− mice exhibit reduced disease severity, and transfer of Itk−/− CD4+ T cells into T cell-deficient recipients results in lower disease severity. We observed a significant reduction of CD4+ T cells in the CNS of Itk−/− mice or recipients of Itk−/− CD4+ T cells during EAE, which is consistent with attenuated disease. Itk−/− CD4+ T cells exhibit defective response to myelin antigen stimulation attributable to displacement of filamentous actin from the CD4+ coreceptor. This results in inadequate transmigration of Itk−/− CD4+ T cells into the CNS and across brain endothelial barriers in vitro. Finally, Itk−/− CD4+ T cells show significant reduction in production of T-helper 1 (Th1) and Th17 cytokines and exhibit skewed T effector/T regulatory cell ratios. These results indicate that signaling by Itk promotes autoimmunity and CNS inflammation, suggesting that it may be a viable target for treatment of MS. PMID:25568116

  6. R-Ras Regulates Murine T Cell Migration and Intercellular Adhesion Molecule-1 Binding.

    PubMed

    Yan, Xiaocai; Yan, Mingfei; Guo, Yihe; Singh, Gobind; Chen, Yuhong; Yu, Mei; Wang, Demin; Hillery, Cheryl A; Chan, Andrew M

    2015-01-01

    The trafficking of T-lymphocytes to peripheral draining lymph nodes is crucial for mounting an adaptive immune response. The role of chemokines in the activation of integrins via Ras-related small GTPases has been well established. R-Ras is a member of the Ras-subfamily of small guanosine-5'-triphosphate-binding proteins and its role in T cell trafficking has been investigated in R-Ras null mice (Rras-/-). An examination of the lymphoid organs of Rras-/- mice revealed a 40% reduction in the cellularity of the peripheral lymph nodes. Morphologically, the high endothelial venules of Rras-/- mice were more disorganized and less mature than those of wild-type mice. Furthermore, CD4+ and CD8+ T cells from Rras-/- mice had approximately 42% lower surface expression of L-selectin/CD62L. These aberrant peripheral lymph node phenotypes were associated with proliferative and trafficking defects in Rras-/- T cells. Furthermore, R-Ras could be activated by the chemokine, CCL21. Indeed, Rras-/- T cells had approximately 14.5% attenuation in binding to intercellular adhesion molecule 1 upon CCL21 stimulation. Finally, in a graft-versus host disease model, recipient mice that were transfused with Rras-/- T cells showed a significant reduction in disease severity when compared with mice transplanted with wild-type T cells. These findings implicate a role for R-Ras in T cell trafficking in the high endothelial venules during an effective immune response. PMID:26710069

  7. PTD-mediated intracellular delivery of mutant NFAT minimum DNA binding domain inhibited the proliferation of T cells.

    PubMed

    Liu, Xia; Zhao, Qianqian; Peng, Xin; Xia, Sheng; Shen, Weihong; Zong, Yangyong; Cheng, Jing; Wu, Weijiang; Zhang, Miaomiao; Du, Fengyi; Xu, Wenrong; Qian, Hui; Shao, Qixiang

    2014-03-01

    The nuclear factor of activated T cell (NFAT) family of calcium-regulated transcription factors plays a key role in the development and function of the immune system. Calcineurin, a protein phosphatase, activates NFAT by dephosphorylation. The activated NFAT is translocated into the nucleus, where it up-regulates the expression of interleukin 2 (IL-2) and other target genes. Calcineurin inhibitors such as cyclosporine A (CsA) and FK506 are effective immunosuppressant drugs and dramatically increase the success rate of organ transplantation procedures. However, since calcineurin is expressed in most tissues in the body and calcineurin inhibition alters many cellular processes besides immune cell activation, the therapeutic use of calcineurin inhibitors is limited by serious side effects. Thus inhibiting NFAT by other mechanisms such as blocking its binding to DNA could be a more selective and safer approach to target NFAT for therapeutic applications. In peripheral T cells, productive immune responses are dependent upon the cooperative binding of the NFAT/AP-1 transcriptional complex to the promoter regions of genes such as interleukin-2 (IL-2), while NFAT in the absence of AP-1 leads to T cell anergy. Protein transduction domains (PTDs) are able to penetrate cell membranes and can be used to transport exogenous proteins across the cell and nuclear membranes. In this study, we constructed a fusion protein of PTD and a minimum DNA binding domain of human NFAT1 (PTD-ΔNFATminiDBD), which contains two mutations (R466A and T533G) in the AP-1 binding sites. The delivery and functions of this fusion protein in T cells were investigated. The results indicated that PTD-ΔNFATminiDBD could be effectively delivered into T cells and transported into the nucleus. PTD-ΔNFATminiDBD attenuated IL-2 production in T cells and then inhibited T cell proliferation, likely through competing against endogenous NFAT for binding to the IL-2 gene promoter. These results demonstrated that

  8. LPA Promotes T Cell Recruitment through Synthesis of CXCL13

    PubMed Central

    Hui, Weili; Zhao, Chenqi; Bourgoin, Sylvain G.

    2015-01-01

    Lysophosphatidic acid (LPA) is a bioactive phospholipid playing an important role in various inflammatory diseases by inducing expression and secretion of many inflammatory cytokines/chemokines. Here we report in a murine air pouch model of inflammation that LPA induced CXCL13 secretion in a time-dependent manner and with exacerbation of the response when LPA was administered after a pretreatment with TNF-α, a key inflammatory cytokine. LPA mediates recruitment of leukocytes, including that of CD3+ cells into unprimed and TNF-α-primed air pouches. CXCL13 neutralization using a blocking antibody injected into air pouches prior to administration of LPA into TNF-α-primed air pouches decreased CD3+ cell influx. Our data highlight that LPA-mediated CXCL13 secretion plays a role in T cell recruitment and participates in regulation of the inflammatory response. PMID:26339130

  9. IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

    PubMed Central

    Younes, Souheil-Antoine; Freeman, Michael L.; Mudd, Joseph C.; Shive, Carey L.; Reynaldi, Arnold; Estes, Jacob D.; Deleage, Claire; Lucero, Carissa; Anderson, Jodi; Schacker, Timothy W.; Davenport, Miles P.; McCune, Joseph M.; Hunt, Peter W.; Lee, Sulggi A.; Debernardo, Robert L.; Jacobson, Jeffrey M.; Canaday, David H.; Sekaly, Rafick-Pierre; Sieg, Scott F.; Lederman, Michael M.

    2016-01-01

    In HIV-1–infected patients, increased numbers of circulating CD8+ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B+, CD8+ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B+ phenotype in CD8+ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8+ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8+ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8+ T cell expansion that is linked to increased morbidity and mortality in treated patients. PMID:27322062

  10. Intrinsic TGF-β signaling promotes age-dependent CD8+ T cell polyfunctionality attrition

    PubMed Central

    Bhadra, Rajarshi; Moretto, Magali M.; Castillo, Julio C.; Petrovas, Constantinos; Ferrando-Martinez, Sara; Shokal, Upasana; Leal, Manuel; Koup, Richard A.; Eleftherianos, Ioannis; Khan, Imtiaz A.

    2014-01-01

    Advanced age is associated with immune system deficits that result in an increased susceptibility to infectious diseases; however, specific mediators of age-dependent immune dysfunction have not been fully elucidated. Here we demonstrated that aged mice exhibit poor effector CD8+ T cell polyfunctionality, primarily due to CD8+ T cell–extrinsic deficits, and that reduced CD8+ T cell polyfunctionality correlates with increased susceptibility to pathogenic diseases. In aged animals challenged with the parasite Encephalitozoon cuniculi, effector CD8+ T cell survival and polyfunctionality were suppressed by highly elevated TGF-β1. Furthermore, TGF-β depletion reduced effector CD8+ T cell apoptosis in both young and aged mice and enhanced effector CD8+ T cell polyfunctionality in aged mice. Surprisingly, intrinsic blockade of TGF-β signaling in CD8+ T cells was sufficient to rescue polyfunctionality in aged animals. Together, these data demonstrate that low levels of TGF-β1 promote apoptosis of CD8+ effector T cells and high TGF-β1 levels associated with age result in both CD8+ T cell apoptosis and an altered transcriptional profile, which correlates with loss of polyfunctionality. Furthermore, elevated TGF-β levels are observed in the elderly human population and in aged Drosophila, suggesting that TGF-β represents an evolutionarily conserved negative regulator of the immune response in aging organisms. PMID:24762437

  11. Mechanism of DNA-binding enhancement by the human T-cell leukaemia virus transactivator Tax.

    PubMed

    Baranger, A M; Palmer, C R; Hamm, M K; Giebler, H A; Brauweiler, A; Nyborg, J K; Schepartz, A

    1995-08-17

    Tax protein activates transcription of the human T-cell leukaemia virus type I (HTLV-I) genome through three imperfect cyclic AMP-responsive element (CRE) target sites located within the viral promoter. Previous work has shown that Tax interacts with the bZIP element of proteins that bind the CRE target site to promote peptide dimerization, suggesting an association between Tax and bZIP coiled coil. Here we show that the site of interaction with Tax is not the coiled coil, but the basic segment. This interaction increases the stability of the GCN4 bZIP dimer by 1.7 kcal mol-1 and the DNA affinity of the dimer by 1.9 kcal mol-1. The differential effect of Tax on several bZip-DNA complexes that differ in peptide sequence or DNA conformation suggests a model for Tax action based on stabilization of a distinct DNA-bound protein structure. This model may explain how Tax interacts with transcription factors of considerable sequence diversity to alter patterns of gene expression.

  12. R-Ras Regulates Murine T Cell Migration and Intercellular Adhesion Molecule-1 Binding

    PubMed Central

    Yan, Xiaocai; Yan, Mingfei; Guo, Yihe; Singh, Gobind; Chen, Yuhong; Yu, Mei; Wang, Demin; Hillery, Cheryl A.; Chan, Andrew M.

    2015-01-01

    The trafficking of T-lymphocytes to peripheral draining lymph nodes is crucial for mounting an adaptive immune response. The role of chemokines in the activation of integrins via Ras-related small GTPases has been well established. R-Ras is a member of the Ras-subfamily of small guanosine-5’-triphosphate-binding proteins and its role in T cell trafficking has been investigated in R-Ras null mice (Rras−/−). An examination of the lymphoid organs of Rras−/− mice revealed a 40% reduction in the cellularity of the peripheral lymph nodes. Morphologically, the high endothelial venules of Rras−/− mice were more disorganized and less mature than those of wild-type mice. Furthermore, CD4+ and CD8+ T cells from Rras−/− mice had approximately 42% lower surface expression of L-selectin/CD62L. These aberrant peripheral lymph node phenotypes were associated with proliferative and trafficking defects in Rras−/− T cells. Furthermore, R-Ras could be activated by the chemokine, CCL21. Indeed, Rras−/− T cells had approximately 14.5% attenuation in binding to intercellular adhesion molecule 1 upon CCL21 stimulation. Finally, in a graft-versus host disease model, recipient mice that were transfused with Rras−/− T cells showed a significant reduction in disease severity when compared with mice transplanted with wild-type T cells. These findings implicate a role for R-Ras in T cell trafficking in the high endothelial venules during an effective immune response. PMID:26710069

  13. Loss of PTEN promotes resistance to T cell-mediated immunotherapy

    PubMed Central

    Peng, Weiyi; Chen, Jie Qing; Liu, Chengwen; Malu, Shruti; Creasy, Caitlin; Tetzlaff, Michael T; Xu, Chunyu; McKenzie, Jodi A; Zhang, Chunlei; Liang, Xiaoxuan; Williams, Leila J; Deng, Wanleng; Chen, Guo; Mbofung, Rina; Lazar, Alexander J; Torres-Cabala, Carlos A; Cooper, Zachary A; Chen, Pei-Ling; Tieu, Trang N; Spranger, Stefani; Yu, Xiaoxing; Bernatchez, Chantale; Forget, Marie-Andree; Haymaker, Cara; Amaria, Rodabe; McQuade, Jennifer L; Glitza, Isabella C; Cascone, Tina; Li, Haiyan S; Kwong, Lawrence N; Heffernan, Timothy P; Hu, Jianhua; Bassett, Roland L; Bosenberg, Marcus W; Woodman, Scott E; Overwijk, Willem W; Lizée, Gregory; Roszik, Jason; Gajewski, Thomas F; Wargo, Jennifer A; Gershenwald, Jeffrey E; Radvanyi, Laszlo; Davies, Michael A; Hwu, Patrick

    2015-01-01

    T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T cell trafficking into tumors. In patients, PTEN loss correlates with decreased T cell infiltration at tumor sites, reduced likelihood of successful T cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA4 antibodies in murine models. Together these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. PMID:26645196

  14. Adoptive T cell therapy promotes the emergence of genomically altered tumor escape variants.

    PubMed

    Kaluza, Karen M; Thompson, Jill M; Kottke, Timothy J; Flynn Gilmer, Heather C; Knutson, Darlene L; Vile, Richard G

    2012-08-15

    Adoptive T cell therapy has been proven effective against melanoma in mice and humans. However, because most responses are incomplete or transient, cures remain rare. To maximize the efficacy of this therapy, it will be essential to gain a better understanding of the processes which result in tumor relapse. We studied these processes using B16ova murine melanoma and adoptive transfer of OT-I T cells. Transfer of T cells as a single therapy provided a significant survival benefit for mice with established subcutaneous tumors. However, tumors which initially regressed often recurred. By analyzing tumors which emerged in the presence of a potent OT-I response, we identified a novel tumor escape mechanism in which tumor cells evaded T cell pressure by undergoing major genomic changes involving loss of the gene encoding the target tumor antigen. Furthermore, we show that these in vivo processes can be recapitulated in vitro using T cell/tumor cell co-cultures. A single round of in vitro co-culture led to significant loss of the ova gene and a tumor cell population with rapidly induced and diverse karyotypic changes. Although these current studies focus on the model OVA antigen, the finding that T cells can directly promote genomic instability has important implications for the development of adoptive T cell therapies.

  15. Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

    PubMed Central

    Cole, David K.; Bulek, Anna M.; Dolton, Garry; Schauenberg, Andrea J.; Szomolay, Barbara; Trimby, Andrew; Jothikumar, Prithiviraj; Fuller, Anna; Skowera, Ania; Rossjohn, Jamie; Zhu, Cheng; Miles, John J.; Wooldridge, Linda; Rizkallah, Pierre J.; Sewell, Andrew K.

    2016-01-01

    The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. PMID:27183389

  16. Progesterone and HMOX-1 promote fetal growth by CD8+ T cell modulation

    PubMed Central

    Solano, María Emilia; Kowal, Mirka Katharina; O’Rourke, Greta Eugenia; Horst, Andrea Kristina; Modest, Kathrin; Plösch, Torsten; Barikbin, Roja; Remus, Chressen Catharina; Berger, Robert G.; Jago, Caitlin; Ho, Hoang; Sass, Gabriele; Parker, Victoria J.; Lydon, John P.; DeMayo, Francesco J.; Hecher, Kurt; Karimi, Khalil; Arck, Petra Clara

    2015-01-01

    Intrauterine growth restriction (IUGR) affects up to 10% of pregnancies in Western societies. IUGR is a strong predictor of reduced short-term neonatal survival and impairs long-term health in children. Placental insufficiency is often associated with IUGR; however, the molecular mechanisms involved in the pathogenesis of placental insufficiency and IUGR are largely unknown. Here, we developed a mouse model of fetal-growth restriction and placental insufficiency that is induced by a midgestational stress challenge. Compared with control animals, pregnant dams subjected to gestational stress exhibited reduced progesterone levels and placental heme oxygenase 1 (Hmox1) expression and increased methylation at distinct regions of the placental Hmox1 promoter. These stress-triggered changes were accompanied by an altered CD8+ T cell response, as evidenced by a reduction of tolerogenic CD8+CD122+ T cells and an increase of cytotoxic CD8+ T cells. Using progesterone receptor– or Hmox1-deficient mice, we identified progesterone as an upstream modulator of placental Hmox1 expression. Supplementation of progesterone or depletion of CD8+ T cells revealed that progesterone suppresses CD8+ T cell cytotoxicity, whereas the generation of CD8+CD122+ T cells is supported by Hmox1 and ameliorates fetal-growth restriction in Hmox1 deficiency. These observations in mice could promote the identification of pregnancies at risk for IUGR and the generation of clinical interventional strategies. PMID:25774501

  17. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity

    PubMed Central

    Ayres, Cory M.; Scott, Daniel R.; Corcelli, Steven A.; Baker, Brian M.

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  18. DNA demethylation of the TIM-3 promoter is critical for its stable expression on T cells.

    PubMed

    Chou, F-C; Kuo, C-C; Chen, H-Y; Chen, H-H; Sytwu, H-K

    2016-04-01

    The T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is selectively expressed on terminally differentiated T helper 1 (Th1) cells and acts as a negative regulator that terminates Th1 responses. The dysregulation of TIM-3 expression on T cells is associated with several autoimmune phenotypes and with chronic viral infections; however, the mechanism of this regulation is unclear. In this study, we investigated the effect of DNA methylation on the expression of TIM-3. By analyzing the sequences of TIM-3 promoter regions in human and mouse, we identified a CpG island within the TIM-3 promoter and demonstrated that the promoter activity was controlled by DNA methylation. Furthermore, treatment with 5-aza-2'-deoxycytidine enhanced TIM-3 expression on mouse primary CD4(+) T cells under Th0-, Th1- or Th2-polarizing conditions. Finally, pyrosequencing analysis revealed that the methylation level of the TIM-3 promoter gradually decreased after each round of T-cell polarization, and this decrease was inversely correlated with TIM-3 expression. These data suggest that the DNA methylation of the TIM-3 promoter cooperates with lineage-specific transcription factors in the control of Th-cell development. In conclusion, DNA methylation-based regulation of TIM-3 may provide novel insights into understanding the dysregulation of TIM-3 expression under pathogenic conditions.

  19. Interplay between T Cell Receptor Binding Kinetics and the Level of Cognate Peptide Presented by Major Histocompatibility Complexes Governs CD8+ T Cell Responsiveness*

    PubMed Central

    Irving, Melita; Zoete, Vincent; Hebeisen, Michael; Schmid, Daphné; Baumgartner, Petra; Guillaume, Philippe; Romero, Pedro; Speiser, Daniel; Luescher, Immanuel; Rufer, Nathalie; Michielin, Olivier

    2012-01-01

    Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1157–165-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8+ T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8+ T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca2+ flux for CD8+ T cells expressing TCR within a dissociation constant (KD) range of ∼1–5 μm. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with KD < ∼1 μm, irrespective of CD8 co-engagement and of half-life (t1/2 = ln 2/koff) values. With increased peptide concentration, however, the activity levels of CD8+ T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8+ T cells, as well as for peptide vaccination strategies. PMID:22549784

  20. T cell Immunoglobulin Mucin Protein (TIM)-4 binds phosphatidylserine and mediates uptake of apoptotic cells

    PubMed Central

    Kobayashi, Norimoto; Karisola, Piia; Peña-Cruz, Victor; Dorfman, David M.; Jinushi, Masahisa; Umetsu, Sarah E.; Butte, Manish J.; Nagumo, Haruo; Chernova, Irene; Zhu, Baogong; Sharpe, Arlene H.; Ito, Susumu; Dranoff, Glenn; Kaplan, Gerardo G.; Casasnovas, Jose M.; Umetsu, Dale T.; DeKruyff, Rosemarie H.; Freeman, Gordon J.

    2009-01-01

    Summary The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Both TIM-4, expressed on human and mouse macrophages and dendritic cells, and TIM-1 specifically bind to phosphatidylserine (PS) on the surface of apoptotic cells and do not bind to any other phospholipid tested. TIM-4+ peritoneal macrophages, TIM-1+ kidney cells, as well as TIM-4 or TIM-1 transfected cells efficiently phagocytose apoptotic cells and phagocytosis can be blocked by TIM-4 or TIM-1 mAbs. TIM proteins have a unique binding cavity made by an unusual conformation of the CC′ and FG loops of the TIM IgV domain and mutations in this cavity eliminated PS binding and phagocytosis. TIM-4 mAbs that block PS binding and phagocytosis map to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors that recognize PS, critical for the efficient clearance of apoptotic cells and prevention of autoimmunity. PMID:18082433

  1. Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells.

    PubMed

    Perdicchio, Maurizio; Cornelissen, Lenneke A M; Streng-Ouwehand, Ingeborg; Engels, Steef; Verstege, Marleen I; Boon, Louis; Geerts, Dirk; van Kooyk, Yvette; Unger, Wendy W J

    2016-02-23

    The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created "sialic acid low" tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing "sialic acid low" tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack. PMID:26741508

  2. Myd88 Initiates Early Innate Immune Responses and Promotes CD4 T Cells during Coronavirus Encephalomyelitis

    PubMed Central

    Butchi, Niranjan; Kapil, Parul; Puntambekar, Shweta; Stohlman, Stephen A.; Hinton, David R.

    2015-01-01

    ABSTRACT Myd88 signaling is critical to the control of numerous central nervous system (CNS) infections by promoting both innate and adaptive immune responses. Nevertheless, the extent to which Myd88 regulates type I interferon (IFN) versus proinflammatory factors and T cell function, as well as the anatomical site of action, varies extensively with the pathogen. CNS infection by neurotropic coronavirus with replication confined to the brain and spinal cord induces protective IFN-α/β via Myd88-independent activation of melanoma differentiation-associated gene 5 (MDA5). However, a contribution of Myd88-dependent signals to CNS pathogenesis has not been assessed. Infected Myd88−/− mice failed to control virus, exhibited enhanced clinical disease coincident with increased demyelination, and succumbed to infection within 3 weeks. The induction of IFN-α/β, as well as of proinflammatory cytokines and chemokines, was impaired early during infection. However, defects in both IFN-α/β and select proinflammatory factors were rapidly overcome prior to T cell recruitment. Myd88 deficiency also specifically blunted myeloid and CD4 T cell recruitment into the CNS without affecting CD8 T cells. Moreover, CD4 T cells but not CD8 T cells were impaired in IFN-γ production. Ineffective virus control indeed correlated most prominently with reduced antiviral IFN-γ in the CNS of Myd88−/− mice. The results demonstrate a crucial role for Myd88 both in early induction of innate immune responses during coronavirus-induced encephalomyelitis and in specifically promoting protective CD4 T cell activation. In the absence of these responses, functional CD8 T cells are insufficient to control viral spread within the CNS, resulting in severe demyelination. IMPORTANCE During central nervous system (CNS) infections, signaling through the adaptor protein Myd88 promotes both innate and adaptive immune responses. The extent to which Myd88 regulates antiviral type I IFN, proinflammatory

  3. LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding

    PubMed Central

    Layer, Justin H.; Alford, Catherine E.; McDonald, W. Hayes

    2015-01-01

    LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R320LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens. PMID:26598604

  4. Regulatory T-cells are essential to promote proper CD4 T-cell priming upon mucosal infection

    PubMed Central

    Soerens, Andrew G.; Da Costa, Andreia; Lund, Jennifer M.

    2016-01-01

    Regulatory T-cells (Tregs) limit autoimmunity and immunopathology using a variety of suppressive mechanisms, but their roles during pathogen-directed immune responses remain unclear. Following Herpes Simplex virus-2 (HSV-2) infection, mice lacking Tregs fail to control viral replication, pointing to a role for Tregs in facilitating productive immune responses. Using adoptive transfer of TCR transgenic CD4 T-cells into Treg-sufficient or Treg-depleted mice prior to HSV-2 infection, we found that Tregs are required for timely accumulation of HSV-2-specific CD4 T-cells within the infected tissues. Further, Tregs are critical for appropriate trafficking of dendritic cells (DCs) from the vaginal mucosa to the dLN, which results in fully effective CD4 T-cell priming, activation, and ultimately migration to the infected tissues. Using CTLA-4 conditional knockout mice, we demonstrate that Tregs impact DC migration through a CTLA-4-mediated mechanism. Together, our data highlight the critical role of Tregs in proper potentiation of adaptive immune responses to microbial infection. PMID:27007674

  5. Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration.

    PubMed

    Kuropka, Benno; Witte, Amelie; Sticht, Jana; Waldt, Natalie; Majkut, Paul; Hackenberger, Christian P R; Schraven, Burkhart; Krause, Eberhard; Kliche, Stefanie; Freund, Christian

    2015-11-01

    Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

  6. IL-15 promotes the survival of naive and memory phenotype CD8+ T cells.

    PubMed

    Berard, Marion; Brandt, Katja; Bulfone-Paus, Silvia; Tough, David F

    2003-05-15

    IL-15 stimulates the proliferation of memory phenotype CD44(high)CD8(+) T cells and is thought to play a key role in regulating the turnover of these cells in vivo. We have investigated whether IL-15 also has the capacity to affect the life span of naive phenotype (CD44(low)) CD8(+) T cells. We report that IL-15 promotes the survival of both CD44(low) and CD44(high) CD8(+) T cells, doing so at much lower concentrations than required to induce proliferation of CD44(high) cells. Rescue from apoptosis was associated with the up-regulation of Bcl-2 in both cell types, whereas elevated expression of Bcl-x(L) was observed among CD44(high) but not CD44(low) CD8(+) cells. An investigation into the role of IL-15R subunits in mediating the effects of IL-15 revealed distinct contributions of the alpha- and beta- and gamma-chains. Most strikingly, IL-15R alpha was not essential for either induction of proliferation or promotion of survival by IL-15, but did greatly enhance the sensitivity of cells to low concentrations of IL-15. By contrast, the beta- and gamma-chains of the IL-15R were absolutely required for the proliferative and pro-survival effects of IL-15, although it was not necessary for CD44(high)CD8(+) cells to express higher levels of IL-15R beta than CD44(low) cells to proliferate in response to IL-15. These results show that IL-15 has multiple effects on CD8 T cells and possesses the potential to regulate the life span of naive as well as memory CD8(+) T cells. PMID:12734346

  7. 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

  8. T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

    PubMed Central

    Carel, S; Bron, C; Corradin, G

    1983-01-01

    T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2. Images PMID:6192442

  9. Amplification of the polyclonal activation of human T cells. I. Null-cell products promote the polyclonal proliferation of T cells.

    PubMed Central

    Caraux, J; Klein, B; Thierry, C; Serrou, B

    1982-01-01

    Synergy can be observed in the proliferative response to mitogens of cultures containing human T and Null cells when compared with those containing only highly purified cells of those two types. This synergy was analysed (i) by evaluation of the proliferative response at each step of the purification process leading to separation of T and Null cells; (ii) by back-mixing T and Nul cells at different rations; and (iii) by evaluation of the proliferative response of free suspension cultures of T cells overlaying a semi-solid layer containing Null cells, or of free suspension cultures of Null cells over a semi-solid culture layer of T cells. The following conclusions were reached: (i) purified Null cells are unresponsive to mitogen when cultured alone or in the presence of diffusible T-cell products; (ii) the T cells are less responsive when cultured alone than in the presence of Null cells or diffusible Null cell products. Thus the synergistic effect observed between T and Null cells is not due to the promotion of Null-cell proliferation by T -cell products but can be accounted for by diffusible Null-cell products enhancing the process of T lymphocyte activation by mitogens. PMID:6977479

  10. C3 Promotes Expansion of CD8+ and CD4+ T cells in a Listeria monocytogenes infection

    PubMed Central

    Nakayama, Yumi; Kim, Shin-Il; Kim, Eui Ho; Lambris, John D.; Sandor, Matyas; Suresh, M.

    2009-01-01

    It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of antigen-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of DCs or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild type mice showed that these T cells do not have intrinsic proliferative defects and paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on antigen-presenting cells. Based on these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens. PMID:19648268

  11. T Cell Immunoglobulin Mucin-3 Crystal Structure Reveals a Galectin-9-Independent Ligand-Binding Surface

    SciTech Connect

    Cao,E.; Zang, X.; Ramagopal, U.; Mukhopadhaya, A.; Fedorov, A.; Fedorov, E.; Zencheck, W.; Lary, J.; Cole, J.; et al.

    2007-01-01

    The T cell immunoglobulin mucin (Tim) family of receptors regulates effector CD4+ T cell functions and is implicated in autoimmune and allergic diseases. Tim-3 induces immunological tolerance, and engagement of the Tim-3 immunoglobulin variable (IgV) domain by galectin-9 is important for appropriate termination of T helper 1-immune responses. The 2 {angstrom} crystal structure of the Tim-3 IgV domain demonstrated that four cysteines, which are invariant within the Tim family, form two noncanonical disulfide bonds, resulting in a surface not present in other immunoglobulin superfamily members. Biochemical and biophysical studies demonstrated that this unique structural feature mediates a previously unidentified galectin-9-independent binding process and suggested that this structural feature is conserved within the entire Tim family. The current work provided a graphic example of the relationship between sequence, structure, and function and suggested that the interplay between multiple Tim-3-binding activities contributes to the regulated assembly of signaling complexes required for effective Th1-mediated immunity.

  12. Class I T-cell epitope prediction: improvements using a combination of proteasome cleavage, TAP affinity, and MHC binding.

    PubMed

    Doytchinova, Irini A; Flower, Darren R

    2006-05-01

    Cleavage by the proteasome is responsible for generating the C terminus of T-cell epitopes. Modeling the process of proteasome cleavage as part of a multi-step algorithm for T-cell epitope prediction will reduce the number of non-binders and increase the overall accuracy of the predictive algorithm. Quantitative matrix-based models for prediction of the proteasome cleavage sites in a protein were developed using a training set of 489 naturally processed T-cell epitopes (nonamer peptides) associated with HLA-A and HLA-B molecules. The models were validated using an external test set of 227 T-cell epitopes. The performance of the models was good, identifying 76% of the C-termini correctly. The best model of proteasome cleavage was incorporated as the first step in a three-step algorithm for T-cell epitope prediction, where subsequent steps predicted TAP affinity and MHC binding using previously derived models. PMID:16524630

  13. Galectin-8 promotes regulatory T cell differentiation by modulating IL-2 and TGFβ signaling

    PubMed Central

    Sampson, James F.; Suryawanshi, Amol; Chen, Wei-Sheng; Rabinovich, Gabriel A.; Panjwani, Noorjahan

    2015-01-01

    Galectins have emerged as potent immunoregulatory molecules that control chronic inflammation through distinct mechanisms. Galectin-8 (Gal-8), a tandem-repeat type galectin with unique preference for α2,3-sialylated glycans, is ubiquitously expressed, but little is known about its role in T cell differentiation. Here, we report that Gal-8 promotes the polyclonal differentiation of primary mouse Treg cells in vitro. We further show that Gal-8 also facilitates antigen-specific differentiation of regulatory T (Treg) cells, and that Treg cells polarized in the presence of Gal-8 express cytotoxic T lymphocyte antigen-4 (CTLA-4) and IL-10 at a higher frequency than control Treg cells, and efficiently inhibit proliferation of activated T cells in vitro. Investigation of the mechanism by which Gal-8 promotes Treg conversion revealed that Gal-8 activates TGFβ signaling and promotes sustained IL-2R signaling. Taken together, these data suggest that Gal-8 promotes the differentiation of highly suppressive Treg cells, which has implications for the treatment of inflammatory and autoimmune diseases. PMID:26282995

  14. Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell responses

    PubMed Central

    Tripathi, P; Koss, B; Opferman, J T; Hildeman, D A

    2013-01-01

    Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4+ and CD8+ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4+ and CD8+ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim. PMID:23558951

  15. The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility

    PubMed Central

    Comrie, William A.; Li, Shuixing; Boyle, Sarah

    2015-01-01

    Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse. PMID:25666808

  16. Depletion of CD4+ CD25+ Regulatory T Cells Promotes CCL21-Mediated Antitumor Immunity

    PubMed Central

    Zhou, Shuang; Tao, Huihong; Zhen, Zhiwei; Chen, Haixia; Chen, Guolin; Yang, Yaoqin

    2013-01-01

    CCL21 is known to attract dendritic cells (DCs) and T cells that may reverse tumor-mediated immune suppression. The massive infiltration of tumors by regulatory T cells (Tregs) prevents the development of a successful helper immune response. In this study, we investigated whether elimination of CD4+ CD25+ Tregs in the tumor microenvironment using anti-CD25 monoclonal antibodies (mAbs) was capable of enhancing CCL21-mediated antitumor immunity in a mouse hepatocellular carcinoma (HCC) model. We found that CCL21 in combination with anti-CD25 mAbs (PC61) resulted in improved antitumor efficacy and prolonged survival, not only inhibited tumor angiogenesis and cell proliferation, but also led to significant increases in the frequency of CD4+, CD8+ T cells and CD11c+ DCs within the tumor, coincident with marked induction of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) at the local tumor site. The intratumoral immune responses were accompanied by the enhanced elaboration of IL-12 and IFN-γ, but reduced release of the immunosuppressive mediators IL-10 and TGF-β1. The results indicated that depletion of Tregs in the tumor microenvironment could enhance CCL21-mediated antitumor immunity, and CCL21 combined with anti-CD25 mAbs may be a more effective immunotherapy to promote tumor rejection. PMID:24023916

  17. Regulatory T cells-derived IL-35 promotes the growth of adult acute myeloid leukemia blasts.

    PubMed

    Tao, Qianshan; Pan, Ying; Wang, Yiping; Wang, Huiping; Xiong, Shudao; Li, Qing; Wang, Jia; Tao, Lili; Wang, Zhitao; Wu, Fan; Zhang, Rui; Zhai, Zhimin

    2015-11-15

    Tumor immune escape mechanism mediated by CD4+CD25+regulatory T cells (Tregs) is a key factor in the pathogenesis of acute myeloid leukemia (AML). IL-35, as a novel inhibitory cytokine, is produced by Tregs specially and regulates functions of Tregs in murine. However, IL-35 expression of Tregs in human is still disputed, and its role in AML is yet to be elucidated. In this study, we found that IL-35 was expressed highly in peripheral blood plasma of adult patients with AML and significantly correlated with the clinical stages of malignancy. Tregs-derived from adult AML patients produced IL-35 in a stimulation-dependent manner. IL-35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD4+CD25-effector T cells (Teffs). Furthermore, IL-35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL-35 has an important role in the pathogenesis of AML.

  18. Immune activation promotes evolutionary conservation of T-cell epitopes in HIV-1.

    PubMed

    Sanjuán, Rafael; Nebot, Miguel R; Peris, Joan B; Alcamí, José

    2013-01-01

    The immune system should constitute a strong selective pressure promoting viral genetic diversity and evolution. However, HIV shows lower sequence variability at T-cell epitopes than elsewhere in the genome, in contrast with other human RNA viruses. Here, we propose that epitope conservation is a consequence of the particular interactions established between HIV and the immune system. On one hand, epitope recognition triggers an anti-HIV response mediated by cytotoxic T-lymphocytes (CTLs), but on the other hand, activation of CD4(+) helper T lymphocytes (TH cells) promotes HIV replication. Mathematical modeling of these opposite selective forces revealed that selection at the intrapatient level can promote either T-cell epitope conservation or escape. We predict greater conservation for epitopes contributing significantly to total immune activation levels (immunodominance), and when TH cell infection is concomitant to epitope recognition (trans-infection). We suggest that HIV-driven immune activation in the lymph nodes during the chronic stage of the disease may offer a favorable scenario for epitope conservation. Our results also support the view that some pathogens draw benefits from the immune response and suggest that vaccination strategies based on conserved TH epitopes may be counterproductive.

  19. Promotion of regulatory T cell induction by immunomodulatory herbal medicine licorice and its two constituents.

    PubMed

    Guo, Ao; He, Dongming; Xu, Hong-Bo; Geng, Chang-An; Zhao, Jian

    2015-01-01

    Regulatory T cells (Treg) play a critical role to control immune responses and to prevent autoimmunity, thus selective increase of Treg cells in vivo has broad therapeutic implications for autoimmune and inflammatory diseases. Licorice is a well-known herbal medicine used worldwide for over thousands of years, and accumulating evidence has shown its immunomodulatory potential. However, it is not clear whether licorice could regulate the induction and function of Treg cells. Here we found licorice extract could promote Treg cell induction, and then we used a rational approach to isolate its functional fractions and constituents. The results showed that two constituents, isoliquiritigenin and naringenin, promoted Treg cell induction both in vitro and in vivo. The effective fractions and two constituents of licorice also enhanced immune suppression of Treg cells, and they further reduced severity of DSS-induced colitis in mice. This study suggested that promotion of regulatory T cell induction could be an underlying mechanism of the historically and widely used herbal medicine licorice, providing its two effective molecules against autoimmune and inflammatory diseases.

  20. Stimulation of T-cells with OKT3 antibodies increases forskolin binding and cyclic AMP accumulation.

    PubMed

    Kvanta, A; Gerwins, P; Jondal, M; Fredholm, B B

    1990-01-01

    It has recently been shown that elevation of cAMP by adenosine receptor stimulation may be potentiated by stimulation of the T-cell receptor/CD3 complex on human T-cells with the monoclonal antibody OKT3, and that this is mimicked by activation of protein kinase C [Kvanta, A. et al. (1989) Naunyn-Schmeideberg's Arch. Pharmac. 340, 715-717]. In this study the diterpene forskolin, which binds to and activates the adenylate cyclase, has been used to examine further how the CD3 complex may influence the adenylate cyclase pathway. Stimulation with OKT3 alone was found to cause a small dose-dependent increase in basal cAMP accumulation. When combining OKT3 with a concentration of forskolin (10 microM), which by itself had little effect on the cyclase activity, the cAMP accumulation was markedly potentiated. This potentiation was paralleled by an increase in [3H]forskolin binding to saponine permeabilized Jurkat cells from 24 to 41 fmol/10(6) cells. The OKT3 effect on cAMP was blocked by chelating extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA and also by W-7, an inhibitor of calmodulin, but was unaffected by H-7, an inhibitor of protein kinase C. Even though OKT3 caused an increase in inositolphosphate turnover, and activated protein kinase C, neither phorbol 12,13 dibutyrate (PDBu) nor the Ca2(+)-ionophore A23187 could mimic the OKT3 effect, whereas a combination of PDBu and A23187 at high concentrations could potentiate forskolin stimulated cyclase activity. Together, these results indicated that stimulation of the CD3 complex could influence the adenylate cyclase by two different mechanisms, one involving activation of protein kinase C and another which does not. PMID:2177619

  1. Wnt/β-catenin signaling in T-cells drives epigenetic imprinting of pro-inflammatory properties and promotes colitis and colon cancer

    PubMed Central

    Keerthivasan, Shilpa; Aghajani, Katayoun; Dose, Marei; Molinero, Luciana; Khan, Mohammad W.; Venkatesvaran, Vysak; Weber, Christopher; Emmanuel, Akinola Olumide; Sun, Tianjao; Ramos, Elena M.; Keshavarzian, Ali; Mulcahy, Mary; Blatner, Nichole; Khazaie, Khashayarsha; Gounari, Fotini

    2014-01-01

    The density and type of lymphocytes that infiltrate colon tumors are predictive of the clinical outcome of colon cancer. High densities of TH17 cells and inflammation predict poor outcome, while infiltration by Tregs that naturally suppress inflammation is associated with longer patient survival. However, the role of Tregs in cancer remains controversial. We recently reported that Tregs in colon cancer patients can become pro-inflammatory and tumor promoting. These properties were directly linked with their expression of RORγt, the signature transcription factor of TH17 cells. Here, we report that Wnt/β-catenin signaling in T-cells promotes expression of RORγt. Expression of β-catenin was elevated in T-cells and Tregs of patients with colitis and colon cancer. Genetically engineered activation of β-catenin in mouse T-cells resulted in enhanced chromatin accessibility in the proximity of Tcf-1 binding sites genome-wide, induced expression of TH17 signature genes including RORγt, and promoted TH17-mediated inflammation. Strikingly, the mice had inflammation of intestine and colon and developed lesions indistinguishable from colitis-induced cancer. Activation of β-catenin only in Tregs was sufficient to produce inflammation and initiate cancer. Based on these findings we conclude that activation of Wnt/β-catenin signaling in T-cells and/or Tregs is causatively linked with the imprinting of pro-inflammatory properties and the promotion of colon cancer. PMID:24574339

  2. Identification of tribbles-1 as a novel binding partner of Foxp3 in regulatory T cells.

    PubMed

    Dugast, Emilie; Kiss-Toth, Endre; Docherty, Louise; Danger, Richard; Chesneau, Mélanie; Pichard, Virginie; Judor, Jean-Paul; Pettré, Ségolène; Conchon, Sophie; Soulillou, Jean-Paul; Brouard, Sophie; Ashton-Chess, Joanna

    2013-04-01

    In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4(+)CD25(hi)CD127(-) Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4(+)CD25(-) counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4(+)CD25(-) T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs. PMID:23417677

  3. Activation‐Induced Killer Cell Immunoglobulin‐like Receptor 3DL2 Binding to HLA–B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis

    PubMed Central

    Ridley, Anna; Hatano, Hiroko; Wong‐Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K.; Al‐Mossawi, Hussein; Ladell, Kristin; Price, David A.; Bowness, Paul

    2016-01-01

    Objective In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin‐like receptor 3DL2 (KIR‐3DL2). The aim of this study was to determine the factors that induce KIR‐3DL2 expression, and to characterize the relationship between HLA–B27 and the phenotype and function of KIR‐3DL2–expressing CD4+ T cells in SpA. Methods In total, 34 B27+ patients with SpA, 28 age‐ and sex‐matched healthy controls (20 B27− and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template‐switch anchored reverse transcription–polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme‐linked immunosorbent assay. Results Cellular activation induced KIR‐3DL2 expression on both naive and effector CD4+ T cells. KIR‐3DL2 binding to B27+ cells promoted expression of KIR‐3DL2, the Th17‐specific transcription factor retinoic acid receptor–related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR‐3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen‐presenting cells, KIR‐3DL2+CD4+ T cells produced less interleukin‐2 (IL‐2) but more IL‐17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR‐3DL2 to B27 heavy chains. Conclusion KIR‐3DL2 binding to HLA–B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA–B27–KIR‐3DL2 interactions for the treatment of B27+ patients with SpA. PMID:26841353

  4. Defect density in multiwalled carbon nanotubes influences ovalbumin adsorption and promotes macrophage activation and CD4+ T-cell proliferation

    PubMed Central

    Bai, Wei; Raghavendra, Achyut; Podila, Ramakrishna; Brown, Jared M

    2016-01-01

    Carbon nanotubes (CNTs) are of great interest for the development of drugs and vaccines due to their unique physicochemical properties. The high surface area to volume ratio and delocalized pi-electron cloud of CNTs promote binding of proteins to the surface forming a protein corona. This unique feature of CNTs has been recognized for potential delivery of antigens for strong and long-lasting antigen-specific immune responses. Based on an earlier study that demonstrated increased protein binding, we propose that carboxylated multiwalled CNTs (MWCNTs) can function as an improved carrier to deliver antigens such as ovalbumin (OVA). To test this hypothesis, we coated carboxylated MWCNTs with OVA and measured uptake and activation of antigen-presenting cells (macrophages) and their ability to stimulate CD4+ T-cell proliferation. We employed two types of carboxylated MWCNTs with different surface areas and defects (MWCNT-2 and MWCNT-30). MWCNT-2 and MWCNT-30 have surface areas of ~215 m2/g and 94 m2/g, respectively. The ratios of D- to G-band areas (ID/IG) were 0.97 and 1.37 for MWCNT-2 and MWCNT-30, respectively, samples showing that MWCNT-30 contained more defects. The increase in defects in MWCNT-30 led to increased binding of OVA as compared to MWCNT-2 (1,066±182 μg/mL vs 582±41 μg/mL, respectively). Both types of MWCNTs, along with MWCNT–OVA complexes, showed no observable toxicity to bone-marrow-derived macrophages up to 5 days. Surprisingly, we found that MWCNT–OVA complex significantly increased the expression of major histocompatibility complex class II on macrophages and production of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin 6), while MWCNTs without OVA protein corona did not. The coculture of MWCNT–OVA-complex-treated macrophages and OVA-specific CD4+ T-cells isolated from OT-II mice demonstrated robust proliferation of CD4+ T-cells. This study provides strong evidence for a role for defects in carboxylated MWCNTs and

  5. Defect density in multiwalled carbon nanotubes influences ovalbumin adsorption and promotes macrophage activation and CD4+ T-cell proliferation

    PubMed Central

    Bai, Wei; Raghavendra, Achyut; Podila, Ramakrishna; Brown, Jared M

    2016-01-01

    Carbon nanotubes (CNTs) are of great interest for the development of drugs and vaccines due to their unique physicochemical properties. The high surface area to volume ratio and delocalized pi-electron cloud of CNTs promote binding of proteins to the surface forming a protein corona. This unique feature of CNTs has been recognized for potential delivery of antigens for strong and long-lasting antigen-specific immune responses. Based on an earlier study that demonstrated increased protein binding, we propose that carboxylated multiwalled CNTs (MWCNTs) can function as an improved carrier to deliver antigens such as ovalbumin (OVA). To test this hypothesis, we coated carboxylated MWCNTs with OVA and measured uptake and activation of antigen-presenting cells (macrophages) and their ability to stimulate CD4+ T-cell proliferation. We employed two types of carboxylated MWCNTs with different surface areas and defects (MWCNT-2 and MWCNT-30). MWCNT-2 and MWCNT-30 have surface areas of ~215 m2/g and 94 m2/g, respectively. The ratios of D- to G-band areas (ID/IG) were 0.97 and 1.37 for MWCNT-2 and MWCNT-30, respectively, samples showing that MWCNT-30 contained more defects. The increase in defects in MWCNT-30 led to increased binding of OVA as compared to MWCNT-2 (1,066±182 μg/mL vs 582±41 μg/mL, respectively). Both types of MWCNTs, along with MWCNT–OVA complexes, showed no observable toxicity to bone-marrow-derived macrophages up to 5 days. Surprisingly, we found that MWCNT–OVA complex significantly increased the expression of major histocompatibility complex class II on macrophages and production of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin 6), while MWCNTs without OVA protein corona did not. The coculture of MWCNT–OVA-complex-treated macrophages and OVA-specific CD4+ T-cells isolated from OT-II mice demonstrated robust proliferation of CD4+ T-cells. This study provides strong evidence for a role for defects in carboxylated MWCNTs and

  6. Defect density in multiwalled carbon nanotubes influences ovalbumin adsorption and promotes macrophage activation and CD4(+) T-cell proliferation.

    PubMed

    Bai, Wei; Raghavendra, Achyut; Podila, Ramakrishna; Brown, Jared M

    2016-01-01

    Carbon nanotubes (CNTs) are of great interest for the development of drugs and vaccines due to their unique physicochemical properties. The high surface area to volume ratio and delocalized pi-electron cloud of CNTs promote binding of proteins to the surface forming a protein corona. This unique feature of CNTs has been recognized for potential delivery of antigens for strong and long-lasting antigen-specific immune responses. Based on an earlier study that demonstrated increased protein binding, we propose that carboxylated multiwalled CNTs (MWCNTs) can function as an improved carrier to deliver antigens such as ovalbumin (OVA). To test this hypothesis, we coated carboxylated MWCNTs with OVA and measured uptake and activation of antigen-presenting cells (macrophages) and their ability to stimulate CD4(+) T-cell proliferation. We employed two types of carboxylated MWCNTs with different surface areas and defects (MWCNT-2 and MWCNT-30). MWCNT-2 and MWCNT-30 have surface areas of ~215 m(2)/g and 94 m(2)/g, respectively. The ratios of D- to G-band areas (I D/I G) were 0.97 and 1.37 for MWCNT-2 and MWCNT-30, respectively, samples showing that MWCNT-30 contained more defects. The increase in defects in MWCNT-30 led to increased binding of OVA as compared to MWCNT-2 (1,066±182 μg/mL vs 582±41 μg/mL, respectively). Both types of MWCNTs, along with MWCNT-OVA complexes, showed no observable toxicity to bone-marrow-derived macrophages up to 5 days. Surprisingly, we found that MWCNT-OVA complex significantly increased the expression of major histocompatibility complex class II on macrophages and production of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin 6), while MWCNTs without OVA protein corona did not. The coculture of MWCNT-OVA-complex-treated macrophages and OVA-specific CD4(+) T-cells isolated from OT-II mice demonstrated robust proliferation of CD4(+) T-cells. This study provides strong evidence for a role for defects in carboxylated MWCNTs

  7. V{delta}1 T cell receptor binds specifically to MHC I chain related A: Molecular and biochemical evidences

    SciTech Connect

    Zhao Jianqing; Huang Jie; Chen Hui; Cui Lianxian; He Wei . E-mail: heweiimu@public.bta.net.cn

    2006-01-06

    Human MHC class I chain-related A (MICA) is a tumor-associated antigen that can be recognized by V{delta}1 subset of tumor-infiltrating {gamma}{delta} T cells. We previously reported that immobilized recombinant MICA protein could induce the proliferation of tumor-infiltrating V{delta}1 {gamma}{delta} T cells in vitro. But there has been no direct evidence showing the engagement of {gamma}{delta} T cell receptors (TCR) of the induced cells with MICA. In the current investigation, we show that MICA induces specific cytolytic activity of the expanded {gamma}{delta} T cells. We expressed the coupled V domains from the MICA-induced T cells as a single polypeptide chain V{delta}V{gamma} TCR ({gamma}{delta} scTCR). Such scTCR can specifically bind MICA of HeLa cells. Direct interaction of {gamma}{delta} scTCRs with in vitro expressed MICA was monitored using an IAsys biosensor. We found that the V{delta}1 scTCR can specifically bind to immobilized MICA molecule and MICA{alpha}1{alpha}2 domains are responsible for the binding reaction.

  8. Interleukin-21-Producing CD4(+) T Cells Promote Type 2 Immunity to House Dust Mites.

    PubMed

    Coquet, Jonathan M; Schuijs, Martijn J; Smyth, Mark J; Deswarte, Kim; Beyaert, Rudi; Braun, Harald; Boon, Louis; Karlsson Hedestam, Gunilla B; Nutt, Steven L; Hammad, Hamida; Lambrecht, Bart N

    2015-08-18

    Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.

  9. Binding of Soluble Natural Ligands to a Soluble Human T-Cell Receptor Fragment Produced in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Hilyard, Katherine L.; Reyburn, Hugh; Chung, Shan; Bell, John I.; Strominger, Jack L.

    1994-09-01

    An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

  10. A lectin-binding, protease-resistant mycobacterial ligand specifically activates V gamma 9+ human gamma delta T cells.

    PubMed

    Pfeffer, K; Schoel, B; Plesnila, N; Lipford, G B; Kromer, S; Deusch, K; Wagner, H

    1992-01-15

    Bacterial (exogeneous) superantigens have been defined as bifunctional proteinaceous molecules. They bind to class II MHC molecules of presenting cells and engage with particular TCR-V beta gene elements, thereby activating alpha beta T cells in a V beta-oriented fashion. In previous studies we have elucidated that gamma delta T cells exhibit a propensity to vigorously respond toward mycobacterial Ag. Intrigued by this finding we now analyzed whether mycobacteria express a superantigen for a subset of human gamma delta T cells definable by the selective use of TCR-V gene elements. Here we describe that a protease-resistant, low m.w. (1 to 3 kDa) component of mycobacteria selectively activates gamma delta T cells expressing TCR-V gamma 9 gene segments. Contained in mycobacterial lysates it stimulates TCR-V gamma 9-positive gamma delta T cells at a frequency of 1/6. Stimulation is critically dependent on the presence of class II MHC-positive presenting cells, the important structure being HLA-DR molecules. The fine specificity of the V gamma 9 seeking mycobacterial ligand differs from the gamma delta T cell-stimulating structures expressed by Daudi cells. In addition, the mycobacterial, V gamma 9-seeking ligand is bound selectively to lectins such as UEAI, SBA, and DBA. We conclude that mycobacteria contain a component that acts as a superantigen for human gamma delta T cells and we believe it is this property that explains the vigorous participation of gamma delta T cells in mycobacterial infections.

  11. TIM-1 glycoprotein binds the adhesion receptor P-selectin and mediates T cell trafficking during inflammation and autoimmunity.

    PubMed

    Angiari, Stefano; Donnarumma, Tiziano; Rossi, Barbara; Dusi, Silvia; Pietronigro, Enrica; Zenaro, Elena; Della Bianca, Vittorina; Toffali, Lara; Piacentino, Gennj; Budui, Simona; Rennert, Paul; Xiao, Sheng; Laudanna, Carlo; Casasnovas, Jose M; Kuchroo, Vijay K; Constantin, Gabriela

    2014-04-17

    Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease.

  12. TIM-1 glycoprotein binds the adhesion receptor P-selectin and mediates T cell trafficking during inflammation and autoimmunity.

    PubMed

    Angiari, Stefano; Donnarumma, Tiziano; Rossi, Barbara; Dusi, Silvia; Pietronigro, Enrica; Zenaro, Elena; Della Bianca, Vittorina; Toffali, Lara; Piacentino, Gennj; Budui, Simona; Rennert, Paul; Xiao, Sheng; Laudanna, Carlo; Casasnovas, Jose M; Kuchroo, Vijay K; Constantin, Gabriela

    2014-04-17

    Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease. PMID:24703780

  13. TIM-1 glycoprotein binds the adhesion receptor P-selectin and mediates T cell trafficking during inflammation and autoimmunity

    PubMed Central

    Angiari, Stefano; Donnarumma, Tiziano; Rossi, Barbara; Dusi, Silvia; Pietronigro, Enrica; Zenaro, Elena; Della Bianca, Vittorina; Toffali, Lara; Piacentino, Gennj; Budui, Simona; Rennert, Paul; Xiao, Sheng; Laudanna, Carlo; Casasnovas, Jose M.; Kuchroo, Vijay K.; Constantin, Gabriela

    2014-01-01

    SUMMARY Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper-1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 IgV domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease. PMID:24703780

  14. Expression of CD3-associated antigen-binding receptors on suppressor T cells.

    PubMed Central

    Kuchroo, V K; Steele, J K; Billings, P R; Selvaraj, P; Dorf, M E

    1988-01-01

    Three suppressor T (Ts)-cell hybridomas specific for 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were selected for surface expression of cluster determinant 3 (CD3) by using antibody (anti-CD3) or antigen (NP-bovine serum albumin) panning procedures followed by cloning at limiting dilution. The CD3-selected Ts hybridomas showed a 1-2 logarithmic enrichment in suppressor activity when compared to the parent lines; they also specifically bound NP-coupled sheep red blood cells in rosette assays. This antigen-binding ability could be down-modulated by anti-CD3 antibody. Similarly, surface expression of CD3 was specifically down-modulated by preincubation of these hybridomas with antigen. Anti-CD3 monoclonal antibody under reducing conditions coprecipitated a broad band of 38-50 kDa associated with two CD3 (25 and 16 kDa) bands. T-cell receptor, anti-alpha-specific monoclonal antibody also immunoprecipitated a broad band in the 41 to 49-kDa region. The combined results suggest that, like helper and cytotoxic T lymphocytes, Ts cells also bear antigen-specific receptors associated with CD3 molecules. Images PMID:2973609

  15. Targeting STAT3 in adoptively transferred T cells promotes their in vivo expansion and antitumor effects

    PubMed Central

    Kujawski, Maciej; Zhang, Chunyan; Herrmann, Andreas; Reckamp, Karen; Scuto, Anna; Jensen, Michael; Deng, Jiehui; Forman, Stephen; Figlin, Robert; Yu, Hua

    2010-01-01

    Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However, the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study, we investigated the possibility to circumvent these limitations by engineering Stat3-deficient CD8+ T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3 in CD8+ T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation, resulting in increased tumor antigen-specific T cell activity and tumor growth inhibition. For potential clinical translation, we combined adoptive T cell therapy with an FDA-approved tyrosine kinase inhibitor, sunitinib, in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells, reduced conversion of transferred Foxp3− T cells to tumor-associated T regulatory cells while increasing transferred CD8+ T cell infiltration and activation at the tumor site, leading to inhibition of primary tumor growth. These data demonstrate that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3 silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors. PMID:21118964

  16. PKCθ and HIV-1 Transcriptional Regulator Tat Co-exist at the LTR Promoter in CD4(+) T Cells.

    PubMed

    López-Huertas, María Rosa; Li, Jasmine; Zafar, Anjum; Rodríguez-Mora, Sara; García-Domínguez, Carlota; Mateos, Elena; Alcamí, José; Rao, Sudha; Coiras, Mayte

    2016-01-01

    PKCθ is essential for the activation of CD4(+) T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4(+) T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T(538) residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4(+) T cells and could be

  17. PKCθ and HIV-1 Transcriptional Regulator Tat Co-exist at the LTR Promoter in CD4+ T Cells

    PubMed Central

    López-Huertas, María Rosa; Li, Jasmine; Zafar, Anjum; Rodríguez-Mora, Sara; García-Domínguez, Carlota; Mateos, Elena; Alcamí, José; Rao, Sudha; Coiras, Mayte

    2016-01-01

    PKCθ is essential for the activation of CD4+ T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4+ T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T538 residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4+ T cells and could be used as a

  18. Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection

    PubMed Central

    Kim, Hwan Keun; Falugi, Fabiana; Missiakas, Dominique M.; Schneewind, Olaf

    2016-01-01

    A hallmark of Staphylococcus aureus disease in humans is persistent infections without development of protective immune responses. Infected patients generate VH3 plasmablast expansions and increased VH3 idiotype Ig; however, the mechanisms for staphylococcal modification of immune responses are not known. We report here that S. aureus-infected mice generate VH3 antibody expansions via a mechanism requiring MHC-restricted antigen presentation to CD4+ T cells and staphylococcal protein A (SpA), a cell wall-anchored surface molecule that binds Fcγ and VH3 variant heavy chains of Ig. VH3 expansion occurred with peptidoglycan-linked SpA from the bacterial envelope but not with recombinant SpA, and optimally required five tandem repeats of its Ig-binding domains. Signaling via receptor-interacting serine/threonine protein kinase 2 (RIPK2) was essential for implementing peptidoglycan-linked SpA superantigen activity. VH3 clan IgG from S. aureus-infected or SpA-treated animals was not pathogen-specific, suggesting that SpA cross-linking of VH3 idiotype B-cell receptors and activation via attached peptidoglycan are the determinants of staphylococcal escape from adaptive immune responses. PMID:27140614

  19. The alarmin IL-33 promotes regulatory T-cell function in the intestine.

    PubMed

    Schiering, Chris; Krausgruber, Thomas; Chomka, Agnieszka; Fröhlich, Anja; Adelmann, Krista; Wohlfert, Elizabeth A; Pott, Johanna; Griseri, Thibault; Bollrath, Julia; Hegazy, Ahmed N; Harrison, Oliver J; Owens, Benjamin M J; Löhning, Max; Belkaid, Yasmine; Fallon, Padraic G; Powrie, Fiona

    2014-09-25

    FOXP3(+) regulatory T cells (Treg cells) are abundant in the intestine, where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells acquire tissue-specific adaptations that facilitate their survival and function; however, key host factors controlling the Treg response in the intestine are poorly understood. The interleukin (IL)-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites, where it functions as an endogenous danger signal, or alarmin, in response to tissue damage. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease patients, suggesting a role for this cytokine in disease pathogenesis. In the intestine, both protective and pathological roles for IL-33 have been described in murine models of acute colitis, but its contribution to chronic inflammation remains ill defined. Here we show in mice that the IL-33 receptor ST2 is preferentially expressed on colonic Treg cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signalling in T cells stimulates Treg responses in several ways. First, it enhances transforming growth factor (TGF)-β1-mediated differentiation of Treg cells and, second, it provides a necessary signal for Treg-cell accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of inflammatory bowel disease, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.

  20. Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

    PubMed

    Nagashima, Ryuichi; Kawakami, Fumitaka; Takahashi, Shinichiro; Obata, Fumiya; Kubo, Makoto

    2014-08-01

    CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

  1. Arsenite-mediated promotion of anchorage-independent growth of HaCaT cells through placental growth factor.

    PubMed

    Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Ohgami, Nobutaka; Naito, Hisao; Shekhar, Hossain U; Omata, Yasuhiro; Kato, Masashi

    2015-04-01

    Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organization's guideline value of 10 μg l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer. PMID:25493652

  2. Arsenite-mediated promotion of anchorage-independent growth of HaCaT cells through placental growth factor.

    PubMed

    Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Ohgami, Nobutaka; Naito, Hisao; Shekhar, Hossain U; Omata, Yasuhiro; Kato, Masashi

    2015-04-01

    Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organization's guideline value of 10 μg l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer.

  3. CD8 T-cell recognition of acquired alloantigen promotes acute allograft rejection

    PubMed Central

    Harper, Simon J. F.; Ali, Jason M.; Wlodek, Elizabeth; Negus, Marg C.; Harper, Ines G.; Chhabra, Manu; Qureshi, M. Saeed; Mallik, Mekhola; Bolton, Eleanor; Bradley, J. Andrew; Pettigrew, Gavin J.

    2015-01-01

    Adaptive CD8 T-cell immunity is the principal arm of the cellular alloimmune response, but its development requires help. This can be provided by CD4 T cells that recognize alloantigen “indirectly,” as self-restricted allopeptide, but this process remains unexplained, because the target epitopes for CD4 and CD8 T-cell recognition are “unlinked” on different cells (recipient and donor antigen presenting cells (APCs), respectively). Here, we test the hypothesis that the presentation of intact and processed MHC class I alloantigen by recipient dendritic cells (DCs) (the “semidirect” pathway) allows linked help to be delivered by indirect-pathway CD4 T cells for generating destructive cytotoxic CD8 T-cell alloresponses. We show that CD8 T-cell–mediated rejection of murine heart allografts that lack hematopoietic APCs requires host secondary lymphoid tissue (SLT). SLT is necessary because within it, recipient dendritic cells can acquire MHC from graft parenchymal cells and simultaneously present it as intact protein to alloreactive CD8 T cells and as processed peptide alloantigen for recognition by indirect-pathway CD4 T cells. This enables delivery of essential help for generating cytotoxic CD8 T-cell responses that cause rapid allograft rejection. In demonstrating the functional relevance of the semidirect pathway to transplant rejection, our findings provide a solution to a long-standing conundrum as to why SLT is required for CD8 T-cell allorecognition of graft parenchymal cells and suggest a mechanism by which indirect-pathway CD4 T cells provide help for generating effector cytotoxic CD8 T-cell alloresponses at late time points after transplantation. PMID:26420874

  4. Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity.

    PubMed

    Andersen, Aram Nikolai; Landsverk, Ole Jørgen; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre; Øynebråten, Inger

    2016-01-01

    Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4(+) and CD8(+) T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8(+) T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans. PMID:27242780

  5. Id1 expression promotes peripheral CD4{sup +} T cell proliferation and survival upon TCR activation without co-stimulation

    SciTech Connect

    Liu, Chen; Jin, Rong; Wang, Hong-Cheng; Tang, Hui; Liu, Yuan-Feng; Qian, Xiao-Ping; Sun, Xiu-Yuan; Ge, Qing; Sun, Xiao-Hong; Zhang, Yu

    2013-06-21

    Highlights: •Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. •Id1 expression facilitates T cells survival when stimulated with anti-CD3. •Elevation of IL-2 production by Id1 contributes increased proliferation and survival. •Id1 potentiates NF-κB activation by anti-CD3 stimulation. -- Abstract: Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naïve CD4{sup +} cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-κB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity.

  6. Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity

    PubMed Central

    Andersen, Aram Nikolai; Landsverk, Ole Jørgen; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre; Øynebråten, Inger

    2016-01-01

    Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans. PMID:27242780

  7. Recombinant human interleukin-7 (CYT107) promotes T-cell recovery after allogeneic stem cell transplantation

    PubMed Central

    Goldberg, Jenna D.; Yuan, Jianda; Koehne, Guenther; Lechner, Lauren; Papadopoulos, Esperanza B.; Young, James W.; Jakubowski, Ann A.; Zaidi, Bushra; Gallardo, Humilidad; Liu, Cailian; Rasalan, Teresa; Wolchok, Jedd D.; Croughs, Therese; Morre, Michel; Devlin, Sean M.; van den Brink, Marcel R. M.

    2012-01-01

    Delays in immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are associated with increased risks of infection and relapse. IL-7 has a central role in T-cell development and survival and enhances immune recovery in murine models of allo-HSCT. We performed a phase 1 trial of r-hIL-7 (CYT107) in recipients of T-cell depleted allo-HSCTs. Twelve patients were treated with escalating doses of r-hIL-7 administered weekly for 3 weeks. The study drug was well tolerated with only one patient developing acute skin GVHD. At baseline, patients were profoundly lymphopenic. CYT107 induced a doubling in CD4+ and CD8+ T cells. The main effect of IL-7 was an expansion of effector memory T cells, the predominant subset identified in our patients. There was no significant effect on CD4+CD25+FoxP3+ T cells, NK, or B cells. Importantly, we not only saw quantitative increases in T cells after a short course of IL-7 but also demonstrated an increase in functional T cells, including viral-specific T cells that recognize CMV. Enhanced TCR diversity was also observed after treatment. Our results indicate that r-hIL-7 can enhance immune recovery after a T cell–depleted allo-HSCT without causing significant GVHD or other serious toxicity (www.clinicaltrials.gov; NCT00684008). PMID:23012326

  8. Fluorine substitutions in an antigenic peptide selectively modulate T-cell receptor binding in a minimally perturbing manner

    SciTech Connect

    Piepenbrink, Kurt H.; Borbulevych, Oleg Y.; Sommese, Ruth F.; Clemens, John; Armstrong, Kathryn M.; Desmond, Clare; Do, Priscilla; Baker, Brian M.

    2010-08-17

    TCR (T-cell receptor) recognition of antigenic peptides bound and presented by MHC (major histocompatibility complex) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide - MHC complexes weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Furthermore, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. In the present study, we examined how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the Tax peptide [HTLV (human T-cell lymphotropic virus)-1 Tax] enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with the A6 TCR independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, we probed the mechanism of fluorine's effect on TCR binding and we conclude that our results were most consistent with a 'polar hydrophobicity' mechanism, rather than a purely hydrophobic- or electrostatic-based mechanism. This finding should have an impact on other attempts to alter molecular recognition with fluorine.

  9. The RNA-Binding Protein, Polypyrimidine Tract-Binding Protein 1 (PTBP1) Is a Key Regulator of CD4 T Cell Activation

    PubMed Central

    Valentín-Acevedo, Aníbal

    2016-01-01

    We have previously shown that the RNA binding protein, polypyrimidine tract-binding protein (PTBP1) plays a critical role in regulating the expression of CD40L in activated CD4 T cells. This is achieved mechanistically through message stabilization at late times of activation as well as by altered distribution of CD40L mRNA within distinct cellular compartments. PTBP1 has been implicated in many different processes, however whether PTBP1 plays a broader role in CD4 T cell activation is not known. To examine this question, experiments were designed to introduce shRNA into primary human CD4 T cells to achieve decreased, but not complete ablation of PTBP1 expression. Analyses of shPTB-expressing CD4 T cells revealed multiple processes including cell proliferation, activation-induced cell death and expression of activation markers and cytokines that were regulated in part by PTBP1 expression. Although there was an overall decrease in the steady-state level of several activation genes, only IL-2 and CD40L appeared to be regulated by PTBP1 at the level of RNA decay suggesting that PTBP1 is critical at different regulatory steps of expression that is gene-specific. Importantly, even though the IL-2 protein levels were reduced in cells with lowered PTBP1, the steady-state level of IL-2 mRNA was significantly higher in these cells suggesting a block at the translational level. Evaluation of T cell activation in shPTB-expressing T cells revealed that PTBP1 was linked primarily to the activation of the PLCγ1/ERK1/2 and the NF-κB pathways. Overall, our results reveal the importance of this critical RNA binding protein in multiple steps of T cell activation. PMID:27513449

  10. CD8 T Cell-Initiated Vascular Endothelial Growth Factor Expression Promotes Central Nervous System Vascular Permeability under Neuroinflammatory Conditions

    PubMed Central

    Suidan, Georgette L.; Dickerson, Jonathan W.; Chen, Yi; McDole, Jeremiah R.; Tripathi, Pulak; Pirko, Istvan; Seroogy, Kim B.; Johnson, Aaron J.

    2010-01-01

    Dysregulation of the blood-brain barrier (BBB) is a hallmark feature of numerous neurologic disorders as diverse as multiple sclerosis, stroke, epilepsy, viral hemorrhagic fevers, cerebral malaria, and acute hemorrhagic leukoencephalitis. CD8 T cells are one immune cell type that have been implicated in promoting vascular permeability in these conditions. Our laboratory has created a murine model of CD8 T cell-mediated CNS vascular permeability using a variation of the Theiler’s murine encephalomyelitis virus system traditionally used to study multiple sclerosis. Previously, we demonstrated that CD8 T cells have the capacity to initiate astrocyte activation, cerebral endothelial cell tight junction protein alterations and CNS vascular permeability through a perforin-dependent process. To address the downstream mechanism by which CD8 T cells promote BBB dysregulation, in this study, we assess the role of vascular endothelial growth factor (VEGF) expression in this model. We demonstrate that neuronal expression of VEGF is significantly upregulated prior to, and coinciding with, CNS vascular permeability. Phosphorylation of fetal liver kinase-1 is significantly increased early in this process indicating activation of this receptor. Specific inhibition of neuropilin-1 significantly reduced CNS vascular permeability and fetal liver kinase-1 activation, and preserved levels of the cerebral endothelial cell tight junction protein occludin. Our data demonstrate that CD8 T cells initiate neuronal expression of VEGF in the CNS under neuroinflammatory conditions, and that VEGF may be a viable therapeutic target in neurologic disease characterized by inflammation-induced BBB disruption. PMID:20008293

  11. Investigating potential exogenous tumor initiating and promoting factors for Cutaneous T-Cell Lymphomas (CTCL), a rare skin malignancy.

    PubMed

    Litvinov, Ivan V; Shtreis, Anna; Kobayashi, Kenneth; Glassman, Steven; Tsang, Matthew; Woetmann, Anders; Sasseville, Denis; Ødum, Niels; Duvic, Madeleine

    2016-07-01

    Most skin malignancies are caused by external and often preventable environmental agents. Multiple reports demonstrated that cutaneous T-cell lymphomas (CTCL) can occur in married couples and cluster in families. Furthermore, recent studies document geographic clustering of this malignancy in Texas as well as in other areas of the United States. Multiple infectious, occupational, and medication causes have been proposed as triggers or promoters of this malignancy including hydrochlorothiazide diuretics, Staphylococcus aureus, dermatophytes, Mycobacterium leprae, Chlamydia pneumoniae, human T-Cell lymphotropic virus type 1 (HTLV1), Epstein-Barr virus (EBV), and herpes simplex virus (HSV). In this report, we review recent evidence evaluating the involvement of these agents in cancer initiation/progression. Most importantly, recent molecular experimental evidence documented for the first time that S. aureus can activate oncogenic STAT3 signaling in malignant T cells. Specifically, S. aureus Enterotoxin type A (SEA) was recently shown to trigger non-malignant infiltrating T cells to release IL-2 and other cytokines. These signals upon binging to their cognate receptors on malignant T cells are then able to activate STAT3 and STAT5 oncogenic signaling and promote cancer progression and IL-17 secretion. In light of these findings, it might be important for patients with exacerbation of their CTCL symptoms to maintain high index of suspicion and treat these individuals for S. aureus colonization and/or sepsis with topical and systemic antibiotics. PMID:27622024

  12. Regulation of T-plastin expression by promoter hypomethylation in primary cutaneous T-cell lymphoma.

    PubMed

    Jones, Christine L; Ferreira, Silvia; McKenzie, Robert C T; Tosi, Isabella; Caesar, Jacqueline A; Bagot, Martine; Whittaker, Sean J; Mitchell, Tracey J

    2012-08-01

    T-plastin (PLS3) is an actin-bundling protein normally expressed in epithelial cells but absent in cells of hematopoietic origin. Aberrant PLS3 expression has been demonstrated in lymphocytes from Sézary syndrome (SS) patients and has been proposed as a biomarker for SS; however, the mechanism underlying dysregulation of PLS3 has not been determined. In this study, PLS3 mRNA expression was demonstrated in 21/35 (60%) SS patients and in 3/8 (38%) mycosis fungoides patients, all of whom had clonal blood involvement. No evidence for PLS3 mutations within coding or promoter regions was found, but significant hypomethylation of CpG dinucleotides 95-99 within the PLS3 CpG island was observed and this was restricted to the PLS3+ population. A polyclonal antibody specific to PLS3 was raised to examine coexpression of PLS3 with a panel of T-cell differentiation markers. All PLS3+ cells were CD3+CD4+ and CD26-, suggesting that loss of CD26 is consistently associated with gain of PLS3, whereas all other markers were distributed heterogeneously. However, a patient-specific TCR copy number assay also demonstrated heterogeneity in PLS3 expression in tumor cell populations. Importantly, our findings demonstrate PLS3 expression in the majority of SS patients and provide insight into the molecular regulation of PLS3 expression in CTCL.

  13. Regulatory T cell transfer ameliorates lymphedema and promotes lymphatic vessel function

    PubMed Central

    Gousopoulos, Epameinondas; Proulx, Steven T.; Bachmann, Samia B.; Scholl, Jeannette; Dionyssiou, Dimitris; Demiri, Efterpi; Halin, Cornelia; Dieterich, Lothar C.

    2016-01-01

    Secondary lymphedema is a common postcancer treatment complication, but the underlying pathological processes are poorly understood and no curative treatment exists. To investigate lymphedema pathomechanisms, a top-down approach was applied, using genomic data and validating the role of a single target. RNA sequencing of lymphedematous mouse skin indicated upregulation of many T cell–related networks, and indeed depletion of CD4+ cells attenuated lymphedema. The significant upregulation of Foxp3, a transcription factor specifically expressed by regulatory T cells (Tregs), along with other Treg-related genes, implied a potential role of Tregs in lymphedema. Indeed, increased infiltration of Tregs was identified in mouse lymphedematous skin and in human lymphedema specimens. To investigate the role of Tregs during disease progression, loss-of-function and gain-of-function studies were performed. Depletion of Tregs in transgenic mice with Tregs expressing the primate diphtheria toxin receptor and green fluorescent protein (Foxp3-DTR-GFP) mice led to exacerbated edema, concomitant with increased infiltration of immune cells and a mixed TH1/TH2 cytokine profile. Conversely, expansion of Tregs using IL-2/anti–IL-2 mAb complexes significantly reduced lymphedema development. Therapeutic application of adoptively transferred Tregs upon lymphedema establishment reversed all of the major hallmarks of lymphedema, including edema, inflammation, and fibrosis, and also promoted lymphatic drainage function. Collectively, our results reveal that Treg application constitutes a potential new curative treatment modality for lymphedema. PMID:27734032

  14. The contribution of major histocompatibility complex contacts to the affinity and kinetics of T cell receptor binding

    PubMed Central

    Zhang, Hao; Lim, Hong-Sheng; Knapp, Berhard; Deane, Charlotte M.; Aleksic, Milos; Dushek, Omer; van der Merwe, P. Anton

    2016-01-01

    The interaction between the T cell antigen receptor (TCR) and antigenic peptide in complex with major histocompatibility complex (MHC) molecules is a crucial step in T cell activation. The relative contributions of TCR:peptide and TCR:MHC contacts to the overall binding energy remain unclear. This has important implications for our understanding of T cell development and function. In this study we used site directed mutagenesis to estimate the contribution of HLA-A2 side-chains to the binding of four TCRs. Our results show that these TCRs have very different energetic ‘footprints’ on HLA-A2, with no residues contributing to all TCR interactions. The estimated overall contribution of MHC side-chains to the total interaction energy was variable, with lower limits ranging from 11% to 50%. Kinetic analysis suggested a minor and variable contribution of MHC side-chains to the transition state complex, arguing against a two-step mechanism for TCR binding. PMID:27734930

  15. Production of IL-10 by CD4+ regulatory T cells during the resolution of infection promotes the maturation of memory CD8+ T cells

    PubMed Central

    Laidlaw, Brian J; Cui, Weiguo; Amezquita, Robert A; Gray, Simon M; Guan, Tianxia; Lu, Yisi; Kobayashi, Yasushi; Flavell, Richard A; Kleinstein, Steven H; Craft, Joe; Kaech, Susan M

    2016-01-01

    Memory CD8+ T cells are critical for host defense upon reexposure to intracellular pathogens. We found that interleukin 10 (IL-10) derived from CD4+ regulatory T cells (Treg cells) was necessary for the maturation of memory CD8+ T cells following acute infection with lymphocytic choriomeningitis virus (LCMV). Treg cell–derived IL-10 was most important during the resolution phase, calming inflammation and the activation state of dendritic cells. Adoptive transfer of IL-10-sufficient Treg cells during the resolution phase ‘restored’ the maturation of memory CD8+ T cells in IL-10-deficient mice. Our data indicate that Treg cell–derived IL-10 is needed to insulate CD8+ T cells from inflammatory signals, and reveal that the resolution phase of infection is a critical period that influences the quality and function of developing memory CD8+ T cells. PMID:26147684

  16. Characterization of the genome-wide TLX1 binding profile in T-cell acute lymphoblastic leukemia.

    PubMed

    Durinck, K; Van Loocke, W; Van der Meulen, J; Van de Walle, I; Ongenaert, M; Rondou, P; Wallaert, A; de Bock, C E; Van Roy, N; Poppe, B; Cools, J; Soulier, J; Taghon, T; Speleman, F; Van Vlierberghe, P

    2015-12-01

    The TLX1 transcription factor is critically involved in the multi-step pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and often cooperates with NOTCH1 activation during malignant T-cell transformation. However, the exact molecular mechanism by which these T-cell specific oncogenes cooperate during transformation remains to be established. Here, we used chromatin immunoprecipitation followed by sequencing to establish the genome-wide binding pattern of TLX1 in human T-ALL. This integrative genomics approach showed that ectopic TLX1 expression drives repression of T cell-specific enhancers and mediates an unexpected transcriptional antagonism with NOTCH1 at critical target genes, including IL7R and NOTCH3. These phenomena coordinately trigger a TLX1-driven pre-leukemic phenotype in human thymic precursor cells, reminiscent of the thymus regression observed in murine TLX1 tumor models, and create a strong genetic pressure for acquiring activating NOTCH1 mutations as a prerequisite for full leukemic transformation. In conclusion, our results uncover a functional antagonism between cooperative oncogenes during the earliest phases of tumor development and provide novel insights in the multi-step pathogenesis of TLX1-driven human leukemia. PMID:26108691

  17. Endothelial-binding, Proinflammatory T Cells Identified by MCAM (CD146) Expression: Characterization and Role in Human Autoimmune Diseases

    PubMed Central

    Dagur, Pradeep K; McCoy, J Philip

    2015-01-01

    A subset of T cells defined by the cell surface expression of MCAM (CD146) has been identified in the peripheral circulation of healthy individuals. These cells comprise approximately 3% of the pool of circulating T cells, have an effector memory phenotype, and are capable of producing several cytokines. Notably, the MCAM positive cells are enhanced for IL-17 production compared to MCAM negative effector memory T cells. These call are committed for IL-17 production and do not require in vitro polarization with exogenous cytokines. MCAM positive T cells also demonstrate an increased ability to bind to endothelial monolayers. In numerous autoimmune diseases these cells are found at increased proportions in the peripheral circulation, and at the sites of active inflammation in patients with autoimmune disease, these cells appear in large numbers are major contributors to IL-17 production. Studies to date have been performed with human subjects and it is uncertain if appropriate mouse models exist for this cells type. These cells could represent early components of the adaptive immune response and serve as targets of therapy in these diseases, although much work remains to be performed in order to discern the exact nature and function of these cells. PMID:25595133

  18. 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 ...

  19. Adhesion- and Degranulation-Promoting Adapter Protein Promotes CD8 T Cell Differentiation and Resident Memory Formation and Function during an Acute Infection.

    PubMed

    Fiege, Jessica K; Beura, Lalit K; Burbach, Brandon J; Shimizu, Yoji

    2016-09-15

    During acute infections, naive Ag-specific CD8 T cells are activated and differentiate into effector T cells, most of which undergo contraction after pathogen clearance. A small population of CD8 T cells persists as memory to protect against future infections. We investigated the role of adhesion- and degranulation-promoting adapter protein (ADAP) in promoting CD8 T cell responses to a systemic infection. Naive Ag-specific CD8 T cells lacking ADAP exhibited a modest expansion defect early after Listeria monocytogenes or vesicular stomatitis virus infection but comparable cytolytic function at the peak of response. However, reduced numbers of ADAP-deficient CD8 T cells were present in the spleen after the peak of the response. ADAP deficiency resulted in a greater frequency of CD127(+) CD8 memory precursors in secondary lymphoid organs during the contraction phase. Reduced numbers of ADAP-deficient killer cell lectin-like receptor G1(-) CD8 resident memory T (TRM) cell precursors were present in a variety of nonlymphoid tissues at the peak of the immune response, and consequently the total numbers of ADAP-deficient TRM cells were reduced at memory time points. TRM cells that did form in the absence of ADAP were defective in effector molecule expression. ADAP-deficient TRM cells exhibited impaired effector function after Ag rechallenge, correlating with defects in their ability to form T cell-APC conjugates. However, ADAP-deficient TRM cells responded to TGF-β signals and recruited circulating memory CD8 T cells. Thus, ADAP regulates CD8 T cell differentiation events following acute pathogen challenge that are critical for the formation and selected functions of TRM cells in nonlymphoid tissues. PMID:27521337

  20. Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells.

    PubMed

    Odorizzi, Pamela M; Pauken, Kristen E; Paley, Michael A; Sharpe, Arlene; Wherry, E John

    2015-06-29

    Programmed Death-1 (PD-1) has received considerable attention as a key regulator of CD8(+) T cell exhaustion during chronic infection and cancer because blockade of this pathway partially reverses T cell dysfunction. Although the PD-1 pathway is critical in regulating established "exhausted" CD8(+) T cells (TEX cells), it is unclear whether PD-1 directly causes T cell exhaustion. We show that PD-1 is not required for the induction of exhaustion in mice with chronic lymphocytic choriomeningitis virus (LCMV) infection. In fact, some aspects of exhaustion are more severe with genetic deletion of PD-1 from the onset of infection. Increased proliferation between days 8 and 14 postinfection is associated with subsequent decreased CD8(+) T cell survival and disruption of a critical proliferative hierarchy necessary to maintain exhausted populations long term. Ultimately, the absence of PD-1 leads to the accumulation of more cytotoxic, but terminally differentiated, CD8(+) TEX cells. These results demonstrate that CD8(+) T cell exhaustion can occur in the absence of PD-1. They also highlight a novel role for PD-1 in preserving TEX cell populations from overstimulation, excessive proliferation, and terminal differentiation. PMID:26034050

  1. Necroptosis of Dendritic Cells Promotes Activation of γδ T Cells.

    PubMed

    Collins, Cheryl C; Bashant, Kathleen; Erikson, Cuixia; Thwe, Phyu Myat; Fortner, Karen A; Wang, Hong; Morita, Craig T; Budd, Ralph C

    2016-01-01

    γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress. PMID:27431410

  2. Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells

    PubMed Central

    Ghai, Rajesh; Tello-Lafoz, Maria; Norwood, Suzanne J.; Yang, Zhe; Clairfeuille, Thomas; Teasdale, Rohan D.; Mérida, Isabel; Collins, Brett M.

    2015-01-01

    ABSTRACT Sorting nexin 27 (SNX27) controls the endosomal-to-cell-surface recycling of diverse transmembrane protein cargos. Crucial to this function is the recruitment of SNX27 to endosomes which is mediated by the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by its phox homology (PX) domain. In T-cells, SNX27 localizes to the immunological synapse in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide-lipid-binding capabilities of full-length SNX27, and discovered a new PtdInsP-binding site within the C-terminal 4.1, ezrin, radixin, moesin (FERM) domain. This binding site showed a clear preference for bi- and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the immunological synapse of activated T-cells, cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters the SNX27 distribution in both endosomal recycling compartments and PtdIns(3,4,5)P3-enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during immunological synapse assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking. PMID:25472716

  3. Localized expression of GITR-L in the tumor microenvironment promotes CD8+ T cell dependent anti-tumor immunity

    PubMed Central

    Cho, John S.; Hsu, Jeffrey V.; Morrison, Sherie L.

    2009-01-01

    The systemic administration of an agonist antibody against glucocorticoid-induced tumor necrosis factor receptor related (GITR) protein has been shown to be effective in overcoming immune tolerance and promoting tumor rejection in a variety of murine tumor models. However, little is known regarding the functional consequence of ligation of GITR with its natural ligand (GITR-L) in the context of regulatory T cell (Treg) suppression in vivo. To determine the mechanism of GITR-L action in vivo, we generated a panel of tumor cell clones that express varying levels of GITR-L. The ectopic expression of GITR-L on the tumor cell surface was sufficient to enhance anti-tumor immunity and delay tumor growth in syngeneic BALB/c mice. Within the range examined, the extent of anti-tumor activity in vivo did not correlate with the level of GITR-L expression, as all clones tested exhibited a similar delay in tumor growth. The localized expression of GITR-L on the tumor cells led to a significant increase in CD8+ T cell infiltration into the tumor compared to control tumors. The increased proportion of CD8+ T cells was only observed locally at the tumor site and was not seen in the tumor draining lymph node. Depletion studies showed that CD8+ T cells, but not CD4+ T cells, were required for GITR-L mediated protection against tumor growth. These studies demonstrate that signaling between GITR-L and GITR in the tumor microenvironment promotes the infiltration of CD8+ T cells, which are essential for controlling tumor growth. PMID:19018533

  4. ICOS Promotes the Function of CD4+ Effector T Cells during Anti-OX40-Mediated Tumor Rejection.

    PubMed

    Metzger, Todd C; Long, Hua; Potluri, Shobha; Pertel, Thomas; Bailey-Bucktrout, Samantha L; Lin, John C; Fu, Tihui; Sharma, Padmanee; Allison, James P; Feldman, Reid M R

    2016-07-01

    ICOS is a T-cell coregulatory receptor that provides a costimulatory signal to T cells during antigen-mediated activation. Antitumor immunity can be improved by ICOS-targeting therapies, but their mechanism of action remains unclear. Here, we define the role of ICOS signaling in antitumor immunity using a blocking, nondepleting antibody against ICOS ligand (ICOS-L). ICOS signaling provided critical support for the effector function of CD4(+) Foxp3(-) T cells during anti-OX40-driven tumor immune responses. By itself, ICOS-L blockade reduced accumulation of intratumoral T regulatory cells (Treg), but it was insufficient to substantially inhibit tumor growth. Furthermore, it did not impede antitumor responses mediated by anti-4-1BB-driven CD8(+) T cells. We found that anti-OX40 efficacy, which is based on Treg depletion and to a large degree on CD4(+) effector T cell (Teff) responses, was impaired with ICOS-L blockade. In contrast, the provision of additional ICOS signaling through direct ICOS-L expression by tumor cells enhanced tumor rejection and survival when administered along with anti-OX40 therapy. Taken together, our results showed that ICOS signaling during antitumor responses acts on both Teff and Treg cells, which have opposing roles in promoting immune activation. Thus, effective therapies targeting the ICOS pathway should seek to promote ICOS signaling specifically in effector CD4(+) T cells by combining ICOS agonism and Treg depletion. Cancer Res; 76(13); 3684-9. ©2016 AACR. PMID:27197182

  5. Daratumumab depletes CD38+ immune regulatory cells, promotes T-cell expansion, and skews T-cell repertoire in multiple myeloma

    PubMed Central

    Krejcik, Jakub; Casneuf, Tineke; Nijhof, Inger S.; Verbist, Bie; Bald, Jaime; Plesner, Torben; Syed, Khaja; Liu, Kevin; van de Donk, Niels W. C. J.; Weiss, Brendan M.; Ahmadi, Tahamtan; Lokhorst, Henk M.; Mutis, Tuna

    2016-01-01

    Daratumumab targets CD38-expressing myeloma cells through a variety of immune-mediated mechanisms (complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis) and direct apoptosis with crosslinking. These mechanisms may also target nonplasma cells that express CD38, which prompted evaluation of daratumumab’s effects on CD38-positive immune subpopulations. Peripheral blood (PB) and bone marrow (BM) from patients with relapsed/refractory myeloma from 2 daratumumab monotherapy studies were analyzed before and during therapy and at relapse. Regulatory B cells and myeloid-derived suppressor cells, previously shown to express CD38, were evaluated for immunosuppressive activity and daratumumab sensitivity in the myeloma setting. A novel subpopulation of regulatory T cells (Tregs) expressing CD38 was identified. These Tregs were more immunosuppressive in vitro than CD38-negative Tregs and were reduced in daratumumab-treated patients. In parallel, daratumumab induced robust increases in helper and cytotoxic T-cell absolute counts. In PB and BM, daratumumab induced significant increases in CD8+:CD4+ and CD8+:Treg ratios, and increased memory T cells while decreasing naïve T cells. The majority of patients demonstrated these broad T-cell changes, although patients with a partial response or better showed greater maximum effector and helper T-cell increases, elevated antiviral and alloreactive functional responses, and significantly greater increases in T-cell clonality as measured by T-cell receptor (TCR) sequencing. Increased TCR clonality positively correlated with increased CD8+ PB T-cell counts. Depletion of CD38+ immunosuppressive cells, which is associated with an increase in T-helper cells, cytotoxic T cells, T-cell functional response, and TCR clonality, represents possible additional mechanisms of action for daratumumab and deserves further exploration. PMID:27222480

  6. Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration.

    PubMed

    Xu, Xiaolu; Wang, Xinxin; Todd, Elizabeth M; Jaeger, Emily R; Vella, Jennifer L; Mooren, Olivia L; Feng, Yunfeng; Hu, Jiancheng; Cooper, John A; Morley, Sharon Celeste; Huang, Yina H

    2016-09-01

    Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration. PMID:27465533

  7. The Regulatory T Cell Lineage Factor Foxp3 Regulates Gene Expression through Several Distinct Mechanisms Mostly Independent of Direct DNA Binding

    PubMed Central

    Andersen, Kristian G.; Hebenstreit, Daniel; Teichmann, Sarah A.; Betz, Alexander G.

    2015-01-01

    The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3’s function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression. PMID:26107960

  8. CIP2A Promotes T-Cell Activation and Immune Response to Listeria monocytogenes Infection.

    PubMed

    Côme, Christophe; Cvrljevic, Anna; Khan, Mohd Moin; Treise, Irina; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Au-Yeung, Byron; Sittig, Eleonora; Laajala, Teemu Daniel; Chen, Yiling; Oeder, Sebastian; Calzada-Wack, Julia; Horsch, Marion; Aittokallio, Tero; Busch, Dirk H; Ollert, Markus W; Neff, Frauke; Beckers, Johannes; Gailus-Durner, Valerie; Fuchs, Helmut; Hrabě de Angelis, Martin; Chen, Zhi; Lahesmaa, Riitta; Westermarck, Jukka

    2016-01-01

    The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.

  9. CIP2A Promotes T-Cell Activation and Immune Response to Listeria monocytogenes Infection

    PubMed Central

    Cvrljevic, Anna; Khan, Mohd Moin; Treise, Irina; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Au-Yeung, Byron; Sittig, Eleonora; Laajala, Teemu Daniel; Chen, Yiling; Oeder, Sebastian; Calzada-Wack, Julia; Horsch, Marion; Aittokallio, Tero; Busch, Dirk H.; Ollert, Markus W.; Neff, Frauke; Beckers, Johannes; Gailus-Durner, Valerie; Fuchs, Helmut; de Angelis, Martin Hrabě; Chen, Zhi; Lahesmaa, Riitta; Westermarck, Jukka

    2016-01-01

    The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects. PMID:27100879

  10. Identification of two nuclear factor of activated T-cells (NFAT)-response elements in the 5'-upstream regulatory region of the ET-1 promoter.

    PubMed

    Strait, Kevin A; Stricklett, Peter K; Kohan, Rachel M; Kohan, Donald E

    2010-09-10

    Collecting duct-derived ET-1 regulates salt excretion and blood pressure. We have reported the presence of an inner medullary collecting duct (IMCD)-specific enhancer region in the 5'-upstream ET-1 promoter (Strait, K. A., Stricklett, P. K., Kohan, J. L., Miller, M. B., and Kohan, D. E. (2007) Am. J. Physiol. Renal Physiol. 293, F601-F606). The current studies provide further characterization of the ET-1 5'-upstream distal promoter to identify the IMCD-specific enhancer elements. Deletion studies identified two regions of the 5'-upstream ET-1 promoter, -1725 to -1319 bp and -1319 to -1026 bp, which were required for maximal promoter activity in transfected rat IMCD cells. Transcription factor binding site analysis of these regions identified two consensus nuclear factor of activated T-cells (NFAT) binding sites at -1263 and -1563. EMSA analysis using nuclear extracts from IMCD cells showed that both the -1263 and the -1563 NFAT sites in the ET-1 distal promoter competed for NFAT binding to previously identified NFAT sites in the IL-2 and TNF genes. Gel supershift analysis showed that each of the NFAT binding sites in the ET-1 promoter bound NFAT proteins derived from IMCD nuclear extracts, but they selectively bound different NFAT isoforms; ET-1263 bound NFATc1, whereas ET-1563 bound NFATc3. Site-directed mutagenesis of either the ET-1263 or the ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity. Thus, NFAT appears to be an important regulator of ET-1 transcription in IMCD cells, and thus, it may play a role in controlling blood pressure through ET-1 regulation of renal salt excretion.

  11. Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation.

    PubMed

    Ke, Xing; Zhang, Shuping; Xu, Jian; Liu, Genyan; Zhang, Lixia; Xie, Erfu; Gao, Li; Li, Daqian; Sun, Ruihong; Wang, Fang; Pan, Shiyang

    2016-05-01

    Patients with non-small-cell lung cancer (NSCLC) have immune defects that are poorly understood. Forkhead box protein P3 (Foxp3) is crucial for immunosuppression by CD4(+) regulatory T cells (Tregs). It is not well known how NSCLC induces Foxp3 expression and causes immunosuppression in tumor-bearing patients. Our study found a higher percentage of CD4(+) Tregs in the peripheral blood of NSCLC compared with healthy donors. NSCLC patients showed demethylation of eight CpG sites within the Foxp3 promoter with methylation ratios negatively correlated with CD4(+)CD25(+)Foxp3(+) T levels. Foxp3 expression in CD4(+) Tregs was directly regulated by Foxp3 promoter demethylation and was involved in immunosuppression by NSCLC. To verify the effect of tumor cells on the phenotype and function of CD4(+) Tregs, we established a coculture system using NSCLC cell line and healthy CD4(+) T cells and showed that SPC-A1 induced IL-10 and TGF-β1 secretion by affecting the function of CD4(+) Tregs. The activity of DNA methyltransferases from CD4(+) T was decreased during this process. Furthermore, eight CpG sites within the Foxp3 promoter also appeared to have undergone demethylation. Foxp3 is highly expressed in CD4(+) T cells, and this may be caused by gene promoter demethylation. These induced Tregs are highly immunosuppressive and dramatically inhibit the proliferative activity of naïve CD4(+) T cells. Our study provides one possible mechanism describing Foxp3 promoter demethylation changes by which NSCLC down-regulates immune responses and contributes to tumor progression. Foxp3 represents an important target for NSCLC anti-tumor immunotherapy.

  12. CD4+ T Cells Promote Antibody Production but Not Sustained Affinity Maturation during Borrelia burgdorferi Infection

    PubMed Central

    Elsner, Rebecca A.; Hastey, Christine J.

    2014-01-01

    CD4 T cells are crucial for enhancing B cell-mediated immunity, supporting the induction of high-affinity, class-switched antibody responses, long-lived plasma cells, and memory B cells. Previous studies showed that the immune response to Borrelia burgdorferi appears to lack robust T-dependent B cell responses, as neither long-lived plasma cells nor memory B cells form for months after infection, and nonswitched IgM antibodies are produced continuously during this chronic disease. These data prompted us to evaluate the induction and functionality of B. burgdorferi infection-induced CD4 TFH cells. We report that CD4 T cells were effectively primed and TFH cells induced after B. burgdorferi infection. These CD4 T cells contributed to the control of B. burgdorferi burden and supported the induction of B. burgdorferi-specific IgG responses. However, while affinity maturation of antibodies against a prototypic T-dependent B. burgdorferi protein, Arthritis-related protein (Arp), were initiated, these increases were reversed later, coinciding with the previously observed involution of germinal centers. The cessation of affinity maturation was not due to the appearance of inhibitory or exhausted CD4 T cells or a strong induction of regulatory T cells. In vitro T-B cocultures demonstrated that T cells isolated from B. burgdorferi-infected but not B. burgdorferi-immunized mice supported the rapid differentiation of B cells into antibody-secreting plasma cells rather than continued proliferation, mirroring the induction of rapid short-lived instead of long-lived T-dependent antibody responses in vivo. The data further suggest that B. burgdorferi infection drives the humoral response away from protective, high-affinity, and long-lived antibody responses and toward the rapid induction of strongly induced, short-lived antibodies of limited efficacy. PMID:25312948

  13. Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells tolerized by peptide immunotherapy

    PubMed Central

    McPherson, Rhoanne C; Konkel, Joanne E; Prendergast, Catriona T; Thomson, John P; Ottaviano, Raffaele; Leech, Melanie D; Kay, Oliver; Zandee, Stephanie E J; Sweenie, Claire H; Wraith, David C; Meehan, Richard R; Drake, Amanda J; Anderton, Stephen M

    2014-01-01

    Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4+ autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation. DOI: http://dx.doi.org/10.7554/eLife.03416.001 PMID:25546306

  14. Cyclosporin A sensitivity of the NF-kappa B site of the IL2R alpha promoter in untransformed murine T cells.

    PubMed Central

    McCaffrey, P G; Kim, P K; Valge-Archer, V E; Sen, R; Rao, A

    1994-01-01

    We have investigated the characteristics of IL2R alpha gene induction in untransformed murine T cells. Induction of IL2R alpha mRNA by TCR/CD3 ligands in a murine T cell clone and in short-term splenic T cell cultures was inhibited by protein synthesis inhibitors and by CsA. This result was contrary to previous observations in JURKAT T leukemia cells and human peripheral blood T cells, suggesting a difference in the mechanisms of IL2R alpha gene induction in these different cell types. The CsA sensitivity of IL2R alpha mRNA induction represented a direct effect on the TCR/CD3 response, and was not due to CsA-sensitive release of the lymphokines IL2 or tumour necrosis factor alpha (TNF alpha) and consequent lymphokine-mediated induction of IL2R alpha mRNA. The NF-kappa B site of the IL2R alpha promoter was essential for gene induction through the TCR/CD3 complex, and the induction of reporter plasmids containing multimers of this site was significantly inhibited by CsA. Northern blotting analysis indicated that while the p65 subunit of NF-kappa B was constitutively expressed and not appreciably induced upon T cell activation, mRNA for the p105 precursor of p50 NF-kappa B was induced in response to TCR/CD3 stimulation and this induction was sensitive to CsA. Electrophoretic mobility shift assays and antiserum against the p50 subunit of NF-kappa B indicated that p50 was a component of the inducible nuclear complex that bound to the IL2R alpha kappa B site. Appearance of the kB-binding proteins was insensitive to CsA at early times after activation (approximately 15 min), but was partially sensitive to CsA at later times. Based on these results, we propose that the NF-kappa B site of the IL2R alpha promoter mediates at least part of the CsA sensitivity of IL2R alpha gene induction in untransformed T cells, possibly because de novo synthesis of p105 NF-kappa B is required for sustained IL2R alpha expression. Images PMID:8029023

  15. Hyper-IL-15 suppresses metastatic and autochthonous liver cancer by promoting tumour-specific CD8+ T cell responses

    PubMed Central

    Cheng, Liang; Du, Xuexiang; Wang, Zheng; Ju, Jianqi; Jia, Mingming; Huang, Qibin; Xing, Qiao; Xu, Meng; Tan, Yi; Liu, Mingyue; Du, Peishuang; Su, Lishan; Wang, Shengdian

    2015-01-01

    Background & Aims Liver cancer has a very dismal prognosis due to lack of effective therapy. Here, we studied the therapeutic effects of hyper-interleukin15 (hyper-IL-15), which is composed of IL-15 and the sushi domain of the IL-15 receptor α chain, on metastatic and autochthonous liver cancers. Methods Liver metastatic tumour models were established by intraportally injecting syngeneic mice with murine CT26 colon carcinoma cells or B16-OVA melanoma cells. Primary hepatocellular carcinoma (HCC) was induced by diethylnitrosamine (DEN). A hydrodynamics-based gene delivery method was used to achieve sustained hyper-IL-15 expression in the liver. Results Liver gene delivery of hyper-IL-15 robustly expanded CD8+ T and NK cells, leading to a long-term (more than 40 days) accumulation of CD8+ T cells in vivo, especially in the liver. Hyper-IL-15 treatment exerted remarkable therapeutic effects on well-established liver metastatic tumours and even on DEN-induced autochthonous HCC, and these effects were abolished by depletion of CD8+ T cells but not NK cells. Hyper-IL-15 triggered IL-12 and interferon-γ production and reduced the expression of co-inhibitory molecules on dendritic cells in the liver. Adoptive transfer of T cell receptor (TCR) transgenic OT-1 cells showed that hyper-IL-15 preferentially expanded tumour-specific CD8+ T cells and promoted their interferon-γ synthesis and cytotoxicity. Conclusions Liver delivery of hyper-IL-15 provides an effective therapy against well-established metastatic and autochthonous liver cancers in mouse models by preferentially expanding tumour-specific CD8+ T cells and promoting their anti-tumour effects. PMID:25016226

  16. Tumor-associated macrophages promote tumor cell proliferation in nasopharyngeal NK/T-cell lymphoma

    PubMed Central

    Liu, Yixiong; Fan, Linni; Wang, Yingmei; Li, Peifeng; Zhu, Jin; Wang, Lu; Zhang, Weichen; Zhang, Yuehua; Huang, Gaosheng

    2014-01-01

    Objective: To explore the relationship between the number of tumor-associated macrophages (TAMs) and proliferative activity of tumor cells and the relationship between two macrophage biomarkers CD68 and CD163 in nasopharyngeal NK/T-cell lymphoma. Methods: Immunohistochemistry was used to reconfirm the diagnosis of nasal NK/T-cell lymphoma and detect the numbers of TAMs and the ki-67 label index of the tumor cells in all 31 cases. In addition, 12 cases of inflammatory cases were collected as controls, for which the immunostaining of CD68 and CD163 were done as well. Then staining results were analyzed with Pearson correlation and t test. Results: The number of TAMs was positively correlated with tumor proliferative activity (P = 0.024) in nasopharyngeal NK/T-cell lymphoma. The expression of CD68 and CD163 was closely related (P = 0.009), and the positive rate of CD68 was generally higher than CD163, however there is no statistical significance. Conclusion: The increase in numbers of TAMs in nasopharyngeal NK/T-cell lymphoma is related to higher proliferative index, indicating the TAMs play an important role in tumor proliferation. Meanwhile both CD68 and CD163 might be the markers for TAMs but CD163 would be the better one. PMID:25337185

  17. Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

    PubMed Central

    Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

    2016-01-01

    Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

  18. Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection*

    PubMed Central

    Salim, Mahboob; Knowles, Timothy J.; Hart, Rosie; Mohammed, Fiyaz; Woodward, Martin J.; Willcox, Carrie R.; Overduin, Michael; Hayday, Adrian C.; Willcox, Benjamin E.

    2016-01-01

    Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection. PMID:26917727

  19. Molecular analysis of tumor-promoting CD8+ T cells in two-stage cutaneous chemical carcinogenesis.

    PubMed

    Kwong, Bernice Y; Roberts, Scott J; Silberzahn, Tobias; Filler, Renata B; Neustadter, Jason H; Galan, Anjela; Reddy, Swapna; Lin, William M; Ellis, Peter D; Langford, Cordelia F; Hayday, Adrian C; Girardi, Michael

    2010-06-01

    T-pro are tumor-infiltrating TCRalphabeta(+)CD8(+) cells of reduced cytotoxic potential that promote experimental two-stage chemical cutaneous carcinogenesis. Toward understanding their mechanism of action, this study uses whole-genome expression analysis to compare T-pro with systemic CD8(+) T cells from multiple groups of tumor-bearing mice. T-pro show an overt T helper 17-like profile (high retinoic acid-related orphan receptor-(ROR)gammat, IL-17A, IL-17F; low T-bet and eomesodermin), regulatory potential (high FoxP3, IL-10, Tim-3), and transcripts encoding epithelial growth factors (amphiregulin, Gro-1, Gro-2). Tricolor flow cytometry subsequently confirmed the presence of TCRbeta(+) CD8(+) IL-17(+) T cells among tumor-infiltrating lymphocytes (TILs). Moreover, a time-course analysis of independent TIL isolates from papillomas versus carcinomas exposed a clear association of the "T-pro phenotype" with malignant progression. This molecular characterization of T-pro builds a foundation for elucidating the contributions of inflammation to cutaneous carcinogenesis, and may provide useful biomarkers for cancer immunotherapy in which the widely advocated use of tumor-specific CD8(+) cytolytic T cells should perhaps accommodate the cells' potential corruption toward the T-pro phenotype. The data are also likely germane to psoriasis, in which the epidermis may be infiltrated by CD8(+) IL-17-producing T cells.

  20. The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells

    PubMed Central

    Ichiyama, Kenji; Chen, Tingting; Wang, Xiaohu; Yan, Xiaowei; Kim, Byung-Seok; Tanaka, Shinya; Ndiaye-Lobry, Delphine; Deng, Yuhua; Zou, Yanli; Zheng, Pan; Tian, Qiang; Aifantis, Iannis; Wei, Lai; Dong, Chen

    2015-01-01

    Summary Epigenetic regulation of lineage-specific genes is important for the differentiation and function of T cell. Ten-eleven translocation (Tet) proteins catalyze 5-methylcytosine (5mC) conversion to 5-hydroxymethylcytosine (5hmC) to mediate DNA demethylation. However, the roles of Tet proteins in the immune response are unknown. Here, we characterized the genome-wide distribution of 5hmC in CD4+ T cells and found 5hmC marks putative regulatory elements in signature genes associated with effector cell differentiation. Moreover, Tet2 protein was recruited to 5hmC-containing regions, dependent on lineage-specific transcription factors. Deletion of the Tet2 gene in T cells decreased their cytokine expression, associated with reduced p300 recruitment. In vivo, Tet2 plays a critical role in the control of cytokine gene expression in autoimmune disease. Collectively, our findings suggest that Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells. PMID:25862091

  1. Vitamin D Regulates Cytokine Patterns Secreted by Dendritic Cells to Promote Differentiation of IL-22-Producing T Cells

    PubMed Central

    Sommer, Andrea; Fabri, Mario

    2015-01-01

    One central mechanism, by which vitamin D regulates human immune responses, is the direct modulation of dendritic cells (DCs). However, the effect of vitamin D on several key DC functions, such as the secretion of central inflammatory cytokines, remains controversial. Moreover, whether vitamin D treatment of DCs regulates their ability to promote differentiation of IL-17-/IL-22-producing T cell subsets, such as Th17 and Th22 cell, is not known. Here, we report that vitamin D treatment during differentiation of monocytes into DCs markedly enhanced their ability to secrete TNF-α, IL-6, IL-1β and IL-23. Cytokines secreted by vitamin D-treated DC were significantly more potent in driving differentiation of IL-22-producing T cells, but not IL-17-producing T cells, as compared to secreted cytokines of not-vitamin D-treated DCs. Finally, we found that the differentiation of IL-22-producing T cells mediated by supernatants of vitamin D-treated DCs was dependent on TNF-α IL-6 and IL-23. In summary, our study suggests a novel role of vitamin D in regulating DC-mediated immune responses in humans. PMID:26107738

  2. Pediatric T-cell lymphoblastic leukemia evolves into relapse by clonal selection, acquisition of mutations and promoter hypomethylation

    PubMed Central

    Kunz, Joachim B.; Rausch, Tobias; Bandapalli, Obul R.; Eilers, Juliane; Pechanska, Paulina; Schuessele, Stephanie; Assenov, Yassen; Stütz, Adrian M.; Kirschner-Schwabe, Renate; Hof, Jana; Eckert, Cornelia; von Stackelberg, Arend; Schrappe, Martin; Stanulla, Martin; Koehler, Rolf; Avigad, Smadar; Elitzur, Sarah; Handgretinger, Rupert; Benes, Vladimir; Weischenfeldt, Joachim; Korbel, Jan O.; Muckenthaler, Martina U.; Kulozik, Andreas E.

    2015-01-01

    Relapsed precursor T-cell acute lymphoblastic leukemia is characterized by resistance against chemotherapy and is frequently fatal. We aimed at understanding the molecular mechanisms resulting in relapse of T-cell acute lymphoblastic leukemia and analyzed 13 patients at first diagnosis, remission and relapse by whole exome sequencing, targeted ultra-deep sequencing, multiplex ligation dependent probe amplification and DNA methylation array. Compared to primary T-cell acute lymphoblastic leukemia, in relapse the number of single nucleotide variants and small insertions and deletions approximately doubled from 11.5 to 26. Targeted ultra-deep sequencing sensitively detected subclones that were selected for in relapse. The mutational pattern defined two types of relapses. While both are characterized by selection of subclones and acquisition of novel mutations, ‘type 1’ relapse derives from the primary leukemia whereas ‘type 2’ relapse originates from a common pre-leukemic ancestor. Relapse-specific changes included activation of the nucleotidase NT5C2 resulting in resistance to chemotherapy and mutations of epigenetic modulators, exemplified by SUZ12, WHSC1 and SMARCA4. While mutations present in primary leukemia and in relapse were enriched for known drivers of leukemia, relapse-specific changes revealed an association with general cancer-promoting mechanisms. This study thus identifies mechanisms that drive progression of pediatric T-cell acute lymphoblastic leukemia to relapse and may explain the characteristic treatment resistance of this condition. PMID:26294725

  3. A THEMIS:SHP1 complex promotes T-cell survival.

    PubMed

    Paster, Wolfgang; Bruger, Annika M; Katsch, Kristin; Grégoire, Claude; Roncagalli, Romain; Fu, Guo; Gascoigne, Nicholas R J; Nika, Konstantina; Cohnen, Andre; Feller, Stephan M; Simister, Philip C; Molder, Kelly C; Cordoba, Shaun-Paul; Dushek, Omer; Malissen, Bernard; Acuto, Oreste

    2015-02-01

    THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation. PMID:25535246

  4. A THEMIS:SHP1 complex promotes T-cell survival

    PubMed Central

    Paster, Wolfgang; Bruger, Annika M; Katsch, Kristin; Grégoire, Claude; Roncagalli, Romain; Fu, Guo; Gascoigne, Nicholas RJ; Nika, Konstantina; Cohnen, Andre; Feller, Stephan M; Simister, Philip C; Molder, Kelly C; Cordoba, Shaun-Paul; Dushek, Omer; Malissen, Bernard; Acuto, Oreste

    2015-01-01

    THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation. PMID:25535246

  5. Keratinocytes-associated chemokines and enzymatically quiescent heparanase induce the binding of resting CD4+ T cells.

    PubMed

    Hershkoviz, R; Marikovsky, M; Gilat, D; Lider, O

    1996-02-01

    Whether the chemokines macrophage inflammatory protein-1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted (RANTES), which interact specifically with glycosaminoglycans and thus mediate the recruitment, attachment, and migration of leukocytes to vascular endothelia and extracellular matrix, are also involved in interactions between CD4+ murine T lymphocytes and keratinocytes was examined. We have previously observed that depending on the local pH, a mammalian extracellular matrix-degrading enzyme, endo-beta-D glucuronidase (heparanase), which cleaves heparin sulfate proteoglycans, can function wither as an enzyme or as an adhesion molecule for CD4+ T lymphocytes. Herein, the involvement of heparanase in T cell-keratinocyte interactions was also probed. At 37 degree C and pH 7.2, radioactively labeled MIP-1 beta, RANTES, and heparanase bound to confluent layers of resting keratinocytes in a saturable and an heparan sulfate- or heparin-dependent manner, and thereby induced the adhesion of resting CD4+ T cells to keratinocytes. At a relatively acidic pH characteristic of inflammatory milieu, enzymatically active heparanase did not bind to the keratinocytes but, rather, inhibited the binding of MIP-1beta, RANTES, and the enzymatically quiescent heparanase to keratinocytes. These results suggest that certain chemokines and heparanase may function to restrict passing leukocytes, notable T lymphocytes, in the cutaneous micro-environment, a site which is continuously challenged with antigens. These keratinocyte-bound lymphocytes can serve as a reservoir of immediate responders to immunological stimuli. PMID:8601723

  6. Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

    SciTech Connect

    Sigalov, Alexander B.; Hendricks, Gregory M.

    2009-11-13

    Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including {zeta}{sub cyt} and CD3{epsilon}{sub cyt} all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein-lipid interactions studies.

  7. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes.

    PubMed

    Li, Bing; Schmidt, Nathan W

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  8. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes

    PubMed Central

    Li, Bing; Schmidt, Nathan W.

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  9. Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

    PubMed

    Inoue, D; Santiago, P; Horne, W C; Baron, R

    1997-10-01

    Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

  10. Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity.

    PubMed

    Fisher, Timothy S; Kamperschroer, Cris; Oliphant, Theodore; Love, Victoria A; Lira, Paul D; Doyonnas, Regis; Bergqvist, Simon; Baxi, Sangita M; Rohner, Allison; Shen, Amy C; Huang, Chunli; Sokolowski, Sharon A; Sharp, Leslie L

    2012-10-01

    4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies. PMID:22406983

  11. The adaptor protein LAD/TSAd mediates laminin-dependent T cell migration via association with the 67 kDa laminin binding protein

    PubMed Central

    Park, Eunkyung; Choi, Youngbong; Ahn, Eunseon; Park, Inyoung

    2009-01-01

    The adaptor protein, LAD/TSAd, plays essential roles in T cell activation. To further understand the functions of this protein, we performed yeast two-hybrid screening using TSAd as bait and identified 67 kDa laminin binding protein (LBP) as the interacting partner. Subsequently, TSAd-LBP interaction was confirmed in D1.1 T cell line. Upon costimulation by T cell receptor (TCR) plus laminin crosslinking or TCR plus integrin α6 crosslinking, LBP was coimmunoprecipitated with TSAd. Moreover, TCR plus laminin costimulation-dependent T cell migration was enhanced in D1.1 T cells overexpressing TSAd but was disrupted in D1.1 cells overexpressing dominant negative form of TSAd or TSAd shRNA. These data show that, upon TCR plus integrin costimulation, TSAd associates with LBP and mediates T lymphocyte migration. PMID:19561400

  12. Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding.

    PubMed

    Willcox, B E; Gao, G F; Wyer, J R; O'Callaghan, C A; Boulter, J M; Jones, E Y; van der Merwe, P A; Bell, J I; Jakobsen, B K

    1999-11-01

    A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

  13. Structural requirements for binding of an immunodominant myelin basic protein peptide to DR2 isotypes and for its recognition by human T cell clones

    PubMed Central

    1994-01-01

    Immunodominant T cell epitopes of myelin basic protein (MBP) may be target antigens for major histocompatibility complex class II- restricted, autoreactive T cells in multiple sclerosis (MS). Since susceptibility to MS is associated with the DR2 haplotype, the binding and presentation of the immunodominant MBP(84-102) peptide by DR2 antigens were examined. The immunodominant MBP(84-102) peptide was found to bind with high affinity to DRB1*1501 and DRB5*0101 molecules of the disease-associated DR2 haplotype. Overlapping but distinct peptide segments were critical for binding to these molecules; hydrophobic residues (Val189 and Phe92) in the MBP(88-95) segment were critical for peptide binding to DRB1*1501 molecules, whereas hydrophobic and charged residues (Phe92, Lys93) in the MBP(89-101/102) sequence contributed to DRB5*0101 binding. The different registers for peptide binding made different peptide side chains available for interaction with the T cell receptor. Although the peptide was bound with high affinity by both DRB1 and DRB5 molecules, only DRB1 (DRB1*1501 and 1602) but not DRB5 molecules served as restriction elements for a panel of T cell clones generated from two MS patients suggesting that the complex of MBP(84-102) and DRB1 molecules is more immunogenic for MBP reactive T cells. The minimal MBP peptide epitope for several T cell clones and the residues important for binding to DRB1*1501 molecules and for T cell stimulation have been defined. PMID:7505801

  14. B Cell-Intrinsic Expression of the HuR RNA-Binding Protein Is Required for the T Cell-Dependent Immune Response In Vivo.

    PubMed

    DeMicco, Amy; Naradikian, Martin S; Sindhava, Vishal J; Yoon, Je-Hyun; Gorospe, Myriam; Wertheim, Gerald B; Cancro, Michael P; Bassing, Craig H

    2015-10-01

    The HuR RNA-binding protein posttranscriptionally controls expression of genes involved in cellular survival, proliferation, and differentiation. To determine roles of HuR in B cell development and function, we analyzed mice with B lineage-specific deletion of the HuR gene. These HuRΔ/Δ mice have reduced numbers of immature bone marrow and mature splenic B cells, with only the former rescued by p53 inactivation, indicating that HuR supports B lineage cells through developmental stage-specific mechanisms. Upon in vitro activation, HuRΔ/Δ B cells have a mild proliferation defect and impaired ability to produce mRNAs that encode IgH chains of secreted Abs, but no deficiencies in survival, isotype switching, or expression of germinal center (GC) markers. In contrast, HuRΔ/Δ mice have minimal serum titers of all Ab isotypes, decreased numbers of GC and plasma B cells, and few peritoneal B-1 B cells. Moreover, HuRΔ/Δ mice have severely decreased GCs, T follicular helper cells, and high-affinity Abs after immunization with a T cell-dependent Ag. This failure of HuRΔ/Δ mice to mount a T cell-dependent Ab response contrasts with the ability of HuRΔ/Δ B cells to become GC-like in vitro, indicating that HuR is essential for aspects of B cell activation unique to the in vivo environment. Consistent with this notion, we find in vitro stimulated HuRΔ/Δ B cells exhibit modestly reduced surface expression of costimulatory molecules whose expression is similarly decreased in humans with common variable immunodeficiency. HuRΔ/Δ mice provide a model to identify B cell-intrinsic factors that promote T cell-dependent immune responses in vivo.

  15. B Cell-Intrinsic Expression of the HuR RNA-Binding Protein Is Required for the T Cell-Dependent Immune Response In Vivo.

    PubMed

    DeMicco, Amy; Naradikian, Martin S; Sindhava, Vishal J; Yoon, Je-Hyun; Gorospe, Myriam; Wertheim, Gerald B; Cancro, Michael P; Bassing, Craig H

    2015-10-01

    The HuR RNA-binding protein posttranscriptionally controls expression of genes involved in cellular survival, proliferation, and differentiation. To determine roles of HuR in B cell development and function, we analyzed mice with B lineage-specific deletion of the HuR gene. These HuRΔ/Δ mice have reduced numbers of immature bone marrow and mature splenic B cells, with only the former rescued by p53 inactivation, indicating that HuR supports B lineage cells through developmental stage-specific mechanisms. Upon in vitro activation, HuRΔ/Δ B cells have a mild proliferation defect and impaired ability to produce mRNAs that encode IgH chains of secreted Abs, but no deficiencies in survival, isotype switching, or expression of germinal center (GC) markers. In contrast, HuRΔ/Δ mice have minimal serum titers of all Ab isotypes, decreased numbers of GC and plasma B cells, and few peritoneal B-1 B cells. Moreover, HuRΔ/Δ mice have severely decreased GCs, T follicular helper cells, and high-affinity Abs after immunization with a T cell-dependent Ag. This failure of HuRΔ/Δ mice to mount a T cell-dependent Ab response contrasts with the ability of HuRΔ/Δ B cells to become GC-like in vitro, indicating that HuR is essential for aspects of B cell activation unique to the in vivo environment. Consistent with this notion, we find in vitro stimulated HuRΔ/Δ B cells exhibit modestly reduced surface expression of costimulatory molecules whose expression is similarly decreased in humans with common variable immunodeficiency. HuRΔ/Δ mice provide a model to identify B cell-intrinsic factors that promote T cell-dependent immune responses in vivo. PMID:26320247

  16. CD4+ T Cell-derived IL-10 Promotes Brucella abortus Persistence via Modulation of Macrophage Function

    PubMed Central

    Xavier, Mariana N.; Winter, Maria G.; Spees, Alanna M.; Nguyen, Kim; Atluri, Vidya L.; Silva, Teane M. A.; Bäumler, Andreas J.; Müller, Werner; Santos, Renato L.; Tsolis, Renée M.

    2013-01-01

    Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4+CD25+ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25+CD4+ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection. PMID:23818855

  17. Alefacept promotes immunosuppression-free renal allograft survival in nonhuman primates via depletion of recipient memory T cells.

    PubMed

    Lee, S; Yamada, Y; Tonsho, M; Boskovic, S; Nadazdin, O; Schoenfeld, D; Cappetta, K; Atif, M; Smith, R-N; Cosimi, A B; Benichou, G; Kawai, T

    2013-12-01

    Renal allograft tolerance has been achieved in MHC-mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow transplantation (DBMT). To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel-conditioning regimen, the "delayed protocol" in which donor bone marrow (DBM) is transplanted several months after kidney transplantation. However, activation/expansion of donor-reactive CD8(+) memory T cells (TMEM) occurring during the interval between kidney and DBM transplantation impaired tolerance induction using this strategy. In the current study, we tested whether, Alefacept, a fusion protein which targets LFA-3/CD2 interactions and selectively depletes CD2(high) CD8(+) effector memory T cells (TEM) could similarly induce long-term immunosuppression-free renal allograft survival but avoid the deleterious effects of anti-CD8 mAb treatment. We found that Alefacept significantly delayed the expansion of CD2(high) cells including CD8(+) TEM while sparing naïve CD8(+) T and NK cells and achieved mixed chimerism and long-term immunosuppression-free renal allograft survival. In conclusion, elimination of CD2(high) T cells represents a promising approach to prevent electively the expansion/activation of donor-reactive TEM and promotes tolerance induction via the delayed protocol mixed chimerism approach.

  18. An Engineered Switch in T Cell Receptor Specificity Leads to an Unusual but Functional Binding Geometry.

    PubMed

    Harris, Daniel T; Singh, Nishant K; Cai, Qi; Smith, Sheena N; Vander Kooi, Craig W; Procko, Erik; Kranz, David M; Baker, Brian M

    2016-07-01

    Utilizing a diverse binding site, T cell receptors (TCRs) specifically recognize a composite ligand comprised of a foreign peptide and a major histocompatibility complex protein (MHC). To help understand the determinants of TCR specificity, we studied a parental and engineered receptor whose peptide specificity had been switched via molecular evolution. Altered specificity was associated with a significant change in TCR-binding geometry, but this did not impact the ability of the TCR to signal in an antigen-specific manner. The determinants of binding and specificity were distributed among contact and non-contact residues in germline and hypervariable loops, and included disruption of key TCR-MHC interactions that bias αβ TCRs toward particular binding modes. Sequence-fitness landscapes identified additional mutations that further enhanced specificity. Our results demonstrate that TCR specificity arises from the distributed action of numerous sites throughout the interface, with significant implications for engineering therapeutic TCRs with novel and functional recognition properties. PMID:27238970

  19. [Lipopolysaccharide promotes the proliferation of CD4(+) T cells by modulating the survive and cytokine secretion of dendritic cells].

    PubMed

    Chen, Song; Lu, Lisha; Wang, Weiqiang; Xue, Ting; Yu, Juan; Sun, Zhina; Zhao, Chunxiao; Liao, Fang

    2016-09-01

    Objective To explore the mechanism modulating the function of dendritic cells (DCs) and promoting the T cell response by lipopolysaccharide (LPS). Methods Splenic DCs were purified with anti-CD11c immunomagnetic beads. After DCs were stimulated with LPS, the expressions of co-stimulatory molecules CD80 and CD86 on the DCs were detected by flow cytometry. The protein levels of pro-inflammatory cytokines interleukin 4 (IL-4), IL-5, IL-6, IL-12p40, IL-12p70 and tumor necrosis factor alpha (TNF-α) in the culture supernatant were measured by ELISA. The apoptotic levels of DCs which were labeled with annexinV-FITC/PI were determined by flow cytometry. The phosphorylation level of nuclear factor κB P65 (NF-κB P65) was assessed by phos-flow. The mRNA levels of variable genes in microarray were determined by real-time PCR. The proliferation of CD4(+) T cells which were co-cultured with OVA323-329-treated DCs was analyzed by flow cytometry. Results The purity of DCs reached over 93% after isolation. LPS up-regulated the expressions of CD80 and CD86 and enhanced DCs-mediated proliferation of CD4(+) T cells. In addition, LPS increased the protein levels of IL-12p40, TNF-α and IL-6, and inhibited the apoptosis of DCs through the NF-κB signaling pathway. Conclusion LPS could enhance DC-mediated proliferation of CD4(+) T cells by modulating the DCs survival and cytokine secretion. PMID:27609566

  20. Tanshinone IIA inhibits the dihydrotestosterone-induced secretion of lipids and activation of sterol regulatory element binding protein-1 in HaCaT cells

    PubMed Central

    SONG, DONG-YAN; HUANG, QIU-HONG; ZHOU, BING-RONG; XU, YANG; YIN, ZHI-QIANG; PERMATASARI, FELICIA; LUO, DAN

    2012-01-01

    To study the effects and mechanisms of Tanshinone IIA (Tan IIA) on the dihydrotestosterone (DHT)-induced expression of sterol regulatory element binding protein-1 (SREBP-1), the synthesis and secretion of lipids in HaCaT cells were examined. HaCaT cells were treated with DHT and Tan IIA at different concentrations. Real-time PCR was used to detect the expression of SREBP-1c, fatty acid synthase (FAS), acyl-CoA synthetase (ACS), stearoyl-CoA desaturase (SCD) and HMG-CoA reductase (HMGCR) mRNA in HaCaT cells. Western blotting was used to analyze the protein expression of SREBP-1 and phosphorylation of Akt. Flow cytometry and Nile red staining were used to detect the synthesis and secretion of lipids in HaCaT cells. We observed that Tan IIA inhibited the DHT-induced expression of SREBP-1 and p-AKT in HaCaT cells, which produced an effect similar to that of LY294002. Tan IIA significantly inhibited the transcription of lipid synthesis-related genes and decreased lipid secretion in HaCaT cells. In conclusion, Tan IIA downregulates the expression of lipid synthesis-related genes and decreases lipid secretion in HaCaT cells, which is correlated with the inhibitory effect on the DHT-induced mRNA and protein expression of SREBP-1 in HaCaT cells. PMID:23139722

  1. Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

    PubMed

    Gerriets, Valerie A; Danzaki, Keiko; Kishton, Rigel J; Eisner, William; Nichols, Amanda G; Saucillo, Donte C; Shinohara, Mari L; MacIver, Nancie J

    2016-08-01

    Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, glucose metabolism, and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff-cell glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells.

  2. Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

    PubMed

    Gerriets, Valerie A; Danzaki, Keiko; Kishton, Rigel J; Eisner, William; Nichols, Amanda G; Saucillo, Donte C; Shinohara, Mari L; MacIver, Nancie J

    2016-08-01

    Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, glucose metabolism, and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff-cell glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells. PMID:27222115

  3. Disparate Degrees of Hypervariable Loop Flexibility Control T-Cell Receptor Cross-Reactivity, Specificity, and Binding Mechanism

    SciTech Connect

    Scott, Daniel R.; Borbulevych, Oleg Y.; Piepenbrink, Kurt H.; Corcelli, Steven A.; Baker, Brian M.

    2012-06-19

    {alpha}{beta} T-cell receptors (TCRs) recognize multiple antigenic peptides bound and presented by major histocompatibility complex molecules. TCR cross-reactivity has been attributed in part to the flexibility of TCR complementarity-determining region (CDR) loops, yet there have been limited direct studies of loop dynamics to determine the extent of its role. Here we studied the flexibility of the binding loops of the {alpha}{beta} TCR A6 using crystallographic, spectroscopic, and computational methods. A significant role for flexibility in binding and cross-reactivity was indicated only for the CDR3{alpha} and CDR3{beta} hypervariable loops. Examination of the energy landscapes of these two loops indicated that CDR3{beta} possesses a broad, smooth energy landscape, leading to rapid sampling in the free TCR of a range of conformations compatible with different ligands. The landscape for CDR3{alpha} is more rugged, resulting in more limited conformational sampling that leads to specificity for a reduced set of peptides as well as the major histocompatibility complex protein. In addition to informing on the mechanisms of cross-reactivity and specificity, the energy landscapes of the two loops indicate a complex mechanism for TCR binding, incorporating elements of both conformational selection and induced fit in a manner that blends features of popular models for TCR recognition.

  4. Cell surface-expressed moesin-like receptor regulates T cell interactions with tissue components and binds an adhesion-modulating IL-2 peptide generated by elastase.

    PubMed

    Ariel, A; Hershkoviz, R; Altbaum-Weiss, I; Ganor, S; Lider, O

    2001-03-01

    The adhesion of leukocytes to the extracellular matrix (ECM) depends on their responses to variations in the chemotactic signals in their milieu, as well as on the functioning of cytoskeletal and context-specific receptors. Ezrin, radixin, and moesin constitute a family of proteins that link the plasma membrane to the actin cytoskeleton. The surface expression of moesin on T cells and its role in cell adhesion has not been fully elucidated. Recently, we found that IL-2 peptides generated by elastase modified the adhesion of activated T cells to ECM ligands. Here, we further examined the adhesion regulatory effects of EFLNRWIT, one of the IL-2 peptides, as well as the existence and putative function of its receptor on T cells. We found that when presented to T cells in the absence of another activator, the EFLNRWIT peptide induced cell adhesion to vessel wall and ECM components. Binding of a radiolabeled peptide to T cells, precipitation with the immobilized peptide, and amino acid sequencing of the precipitated protein revealed that EFLNRWIT exerts its function via a cell surface-expressed moesin-like moiety, whose constitutive expression on T cells was increased after activation. This notion was further supported by our findings that: 1) anti-moesin mAb inhibited the binding of T cells to the immobilized EFLNRWIT peptide, 2) immobilized recombinant moesin bound the IL-2 peptide, and 3) soluble moesin inhibited the EFLNRWIT-induced T cell adhesion to fibronectin. Interestingly, moesin appears to be generally involved in T cell responses to adhesion-regulating signals. Thus, the IL-2 peptide EFLNRWIT appears to exert its modulating capacities via an adhesion-regulating moesin-like receptor. PMID:11207255

  5. Plasticity in the contribution of T cell receptor variable region residues to binding of peptide-HLA-A2 complexes.

    PubMed

    Smith, Sheena N; Sommermeyer, Daniel; Piepenbrink, Kurt H; Blevins, Sydney J; Bernhard, Helga; Uckert, Wolfgang; Baker, Brian M; Kranz, David M

    2013-11-15

    One hypothesis accounting for major histocompatibility complex (MHC) restriction by T cell receptors (TCRs) holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. While this "germline codon" hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα region and recognize the same MHC allele (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key germline codon residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. With the use of single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type germline codon residues, but several alternative residues could also accommodate binding and, hence, MHC restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e., MHC restriction), there is significant plasticity in requirements for particular side chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs. PMID:23954306

  6. Plasticity in the contribution of T cell receptor variable region residues to binding of peptide-HLA-A2 complexes

    PubMed Central

    Smith, Sheena N.; Sommermeyer, Daniel; Piepenbrink, Kurt H.; Blevins, Sydney J.; Bernhard, Helga; Uckert, Wolfgang; Baker, Brian M.; Kranz, David M.

    2013-01-01

    One hypothesis to account for MHC-restriction by T cell receptors (TCRs) holds that there are several evolutionary-conserved residues in TCR variable regions that contact MHC. While this ‘germline-codon’ hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key ‘germline codon” residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. Using single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type ‘germline codon’ residues, but several alternative residues could also accommodate binding and hence, MHC-restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e. MHC-restriction), there is significant plasticity in requirements for particular side-chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs. PMID:23954306

  7. Gr-1⁺CD11b⁺ immature myeloid cells (IMC) promote resistance of pro-inflammatory T cells to suppression by regulatory T cells in atherosclerotic Apo E- deficient mice.

    PubMed

    Chen, Yulin; Jian, Ying; Liu, Minjie; Zhong, Liang; Zhang, Fang; Yang, Weifeng; Xu, Zhao; Chen, Guofan; Liu, Yuhua

    2014-01-01

    Accumulating evidence indicates that both defects in Treg numbers and/or function as well as resistance of effector T cells to suppression may contribute to the development of human chronic inflammatory diseases. However, which mechanism involved in the progression of atherosclerosis remains unclear. In this study, we evaluated the production and function of CD4⁺ inflammatory and regulatory T cells in atherosclerosis-prone mice. We found that the hyperactivity and unresponsiveness to Treg-mediated suppression of inflammatory CD4⁺ T cells occurred in the progression of atherosclerosis, though Treg cells were present in very large numbers and fully functional. We further found that Gr-1⁺CD11b⁺ immature myeloid cells were significantly accumulated in atherosclerotic Apo E⁻/⁻ mice, and they promoted resistance of inflammatory CD4⁺ T cells to Treg-mediated suppression in vitro and in vivo. we further confirmed that Gr-1⁺CD11b⁺ immature myeloid cells produced high level of interleukin 6 which was at least partially responsible for inducing unresponsiveness of inflammatory CD4⁺ T cells to suppression via activation of Jak/Stat signaling pathway. Taken together, these findings might provide new insights to explore potential targets for immune therapeutic intervention in atherosclerosis.

  8. PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion.

    PubMed

    Tinoco, Roberto; Carrette, Florent; Barraza, Monique L; Otero, Dennis C; Magaña, Jonathan; Bosenberg, Marcus W; Swain, Susan L; Bradley, Linda M

    2016-05-17

    Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment. PMID:27192578

  9. Physical and functional interaction between the human T-cell lymphotropic virus type 1 Tax1 protein and the CCAAT binding protein NF-Y.

    PubMed Central

    Pise-Masison, C A; Dittmer, J; Clemens, K E; Brady, J N

    1997-01-01

    Tax1, a potent activator of human T-cell lymphotropic virus type 1 (HTLV-1) transcription, has been shown to modulate expression of many cellular genes. Tax1 does not bind DNA directly but regulates transcription through protein-protein interactions with sequence-specific transcription factors. Using the yeast two-hybrid system to screen for proteins which interact with Tax1, we isolated the B subunit of the CCAAT binding protein NF-Y from a HeLa cDNA library. The interaction of Tax1 with NF-YB was specific in that NF-YB did not interact with a variety of other transcription factors, including human immunodeficiency virus Tat, human papillomavirus E6, and Bicoid, or with the M7 (amino acids 29CP-AS) Tax1 mutant. However, NF-YB did interact with the C-terminal Tax1 mutants M22 (130TL-AS) and M47 (319LL-RS). We also show that in vitro-translated NF-YB specifically bound to a glutathione S-transferase-Tax1 fusion protein. Further, Tax1 coimmunoprecipitated with NF-Y from nuclear extracts of HTLV-1-transformed cells, providing evidence for in vivo interaction of Tax1 and NF-YB. We further demonstrate that Tax1 specifically activated the NF-Y-responsive DQbeta promoter, as well as a minimal promoter which contains only the Y-box element. In addition, mutation of the Y-box element alone abrogated Tax1-mediated activation. Taken together, these data indicate that Tax1 interacts with NF-Y through the B subunit and that this interaction results in activation of the major histocompatibility complex class II promoter. Through activation of this and other NF-Y driven promoters, the Tax1-NF-Y interaction may play a critical role in causing cellular transformation and HTLV-1 pathogenesis. PMID:9032250

  10. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion.

    PubMed

    Oliphant, Christopher J; Hwang, You Yi; Walker, Jennifer A; Salimi, Maryam; Wong, See Heng; Brewer, James M; Englezakis, Alexandros; Barlow, Jillian L; Hams, Emily; Scanlon, Seth T; Ogg, Graham S; Fallon, Padraic G; McKenzie, Andrew N J

    2014-08-21

    Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.

  11. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT.

    PubMed

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I Jolanda M; Adema, Gosse J; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  12. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT

    PubMed Central

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I. Jolanda M.; Adema, Gosse J.; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280–288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  13. Human thymus medullary epithelial cells promote regulatory T-cell generation by stimulating interleukin-2 production via ICOS ligand.

    PubMed

    Nazzal, D; Gradolatto, A; Truffault, F; Bismuth, J; Berrih-Aknin, S

    2014-09-11

    Natural thymic T regulatory (tTreg) cells maintain tolerance to self-antigen. These cells are generated in the thymus, but how this generation occurs is still controversial. Furthermore, the contribution of thymus epithelial cells to this process is still unclear, especially in humans. Using an exceptional panel of human thymic samples, we demonstrated that medullary thymus epithelial cells (mTECs) promote the generation of tTreg cells and favor their function. These effects were mediated through soluble factors and were mTEC specific since other cell types had no such effect. By evaluating the effects of mTECs on the absolute number of Treg cells and their state of proliferation or cell death, we conclude that mTECs promote the proliferation of newly generated CD25+ cells from CD4+CD25- cells and protect Treg cells from cell death. This observation implicates Bcl-2 and mitochondrial membrane potential changes, indicating that the intrinsic cell death pathway is involved in Treg protection by mTECs. Interestingly, when the mTECs were cultured directly with purified Treg cells, they were able to promote their phenotype but not their expansion, suggesting that CD4+CD25- cells have a role in the expansion process. To explore the mechanisms involved, several neutralizing antibodies were tested. The effects of mTECs on Treg cells were essentially due to interleukin (IL)-2 overproduction by thymus CD4+ T cells. We then searched for a soluble factor produced by mTECs able to increase IL-2 production by CD4+ cells and could identify the inducible T-cell costimulator ligand (ICOSL). Our data strongly suggest a « ménage à trois »: mTEC cells (via ICOSL) induce overproduction of IL-2 by CD25- T cells leading to the expansion of tTreg cells. Altogether, these results demonstrate for the first time a role of mTECs in promoting Treg cell expansion in the human thymus and implicate IL-2 and ICOSL in this process.

  14. IL-17/Th17 promotes type 1 T cell immunity against pulmonary intracellular bacterial infection through modulating dendritic cell function.

    PubMed

    Bai, Hong; Cheng, Jianjun; Gao, Xiaoling; Joyee, Antony George; Fan, Yijun; Wang, Shuhe; Jiao, Lei; Yao, Zhi; Yang, Xi

    2009-11-01

    Although their contribution to host defense against extracellular infections has been well defined, IL-17 and Th17 are generally thought to have limited impact on intracellular infections. In this study, we investigated the role and mechanisms of IL-17/Th17 in host defense against Chlamydia muridarum, an obligate intracellular bacterium, lung infection. Our data showed rapid increase in IL-17 production and expansion of Th17 cells following C. muridarum infection and significant detrimental impact of in vivo IL-17 neutralization by anti-IL-17 mAb on disease course, immune response, and dendritic cell (DC) function. Specifically, IL-17-neutralized mice exhibited significantly greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with sham-treated control mice. Immunological analysis showed that IL-17 neutralization significantly reduced Chlamydia-specific Th1 responses, but increased Th2 responses. Interestingly, the DC isolated from IL-17-neutralized mice showed lower CD40 and MHC II expression and IL-12 production, but higher IL-10 production compared with those from sham-treated mice. In two DC-T cell coculture systems, DC isolated from IL-17-neutralized mice induced higher IL-4, but lower IFN-gamma production by Ag-specific T cells than those from sham-treated mice in cell priming and reaction settings. Adoptive transfer of DC isolated from IL-17-neutralized mice, unlike those from sham-treated mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that IL-17/Th17 plays an important role in host defense against intracellular bacterial infection, and suggest that IL-17/Th17 can promote type 1 T cell immunity through modulating DC function.

  15. IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells.

    PubMed

    Curran, Terry-Ann; Jalili, Reza Baradar; Farrokhi, Ali; Ghahary, Aziz

    2014-01-01

    Regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs) can be induced and expanded by dendritic cells (DCs) in the presence of the enzyme indoleamine 2,3-dioxygenase (IDO). Here we report that a possible alternative to DCs are IDO expressing dermal fibroblasts (DFs), which are easier to isolate and sustain in culture compared to DCs. When mouse splenocytes were co-cultured with IDO expressing DFs, a significant increase in frequency and the number of Tregs was found compared to those of control group (13.16%±1.8 vs. 5.53%±1.2, p<0.05). Despite observing a higher total number of dead CD4(+) cells in the IDO group, there was a more abundant live CD4(+)CD25(+) subpopulation in this group. Further analysis reveales that these CD4(+) CD25(+) cells have the capacity to expand in the presence of IDO expressing DFs. Greater number of CTLA-4(+) cells and high expression of TGF-β and IL-10 were found in CD4(+) cells of the IDO group compared to those of the controls. This finding confirmed a suppressive functionality of the expanded Tregs. Furthermore, CD4(+) CD25(+) cells isolated from the IDO group showed an alloantigen specific suppressive effect in a mixed lymphocyte reaction assay. These results confirm that IDO expressing dermal fibroblasts can expand a population of suppressive antigen specific Tregs. In conclusion, IDO expressing dermal fibroblasts have the capacity to stimulate the expansion of a subset of Tregs which can be used to generate antigen-specific immune tolerance.

  16. Activation of decidual invariant natural killer T cells promotes lipopolysaccharide-induced preterm birth.

    PubMed

    Li, Liping; Yang, Jing; Jiang, Yao; Tu, Jiaoqin; Schust, Danny J

    2015-04-01

    Invariant natural killer T (iNKT) cells are crucial for host defense against a variety of microbial pathogens, but the underlying mechanisms of iNKT cells activation by microbes are not fully explained. In this study, we investigated the molecular mechanisms of iNKT cell activation in lipopolysaccharide (LPS)-stimulated preterm birth using an adoptive transfer system and diverse neutralizing antibodies (Abs) and inhibitors. We found that adoptive transfer of decidual iNKT cells to LPS-stimulated iNKT cell deficient Jα18(-/-) mice that lack invariant Vα14Jα281T cell receptor (TCR) expression significantly decreased the time to delivery and increased the percentage of decidual iNKT cells. Neutralizing Abs against Toll-like receptor 4 (TLR-4), CD1d, interleukin (IL)-12 and IL-18, and inhibitors blocking the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK) significantly reduced in vivo percentages of decidual iNKT cells, their intracellular interferon (IFN)-γ production and surface CD69 expression. In vitro, in the presence of the same Abs and inhibitors used as in vivo, decidual iNKT cells co-cultured with LPS-pulsed dendritic cells (DCs) showed significantly decreased extracellular and intracellular IFN-γ secretion and surface CD69 expression. Our data demonstrate that the activation of decidual iNKT cells plays an important role in inflammation-induced preterm birth. Activation of decidual iNKT cells also requires TLR4-mediated NF-κB, MAPK p38 and ERK pathways, the proinflammatory cytokines IL-12 and IL-18, and endogenous glycolipid antigens presented by CD1d.

  17. MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

    SciTech Connect

    Mirlekar, Bhalchandra; Patil, Sachin; Bopanna, Ramanamurthy; Chattopadhyay, Samit

    2015-08-21

    T{sub reg} cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T{sub reg} cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T{sub reg} phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T{sub reg} cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T{sub reg} cells in SMAR1{sup −/−} mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T{sub reg} cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T{sub reg} physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T{sub reg} cells. • SMAR1{sup −/−} T{sub reg} cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1{sup −/−} mice. • Both Foxp3 and SMAR1 maintain T{sub reg} phenotype that controls colitis.

  18. Human CD1 dimeric proteins as indispensable tools for research on CD1-binding lipids and CD1-restricted T cells

    PubMed Central

    Shiratsuchi, Takayuki; Schneck, Jonathan; Kawamura, Akira; Tsuji, Moriya

    2009-01-01

    Antigen presenting molecules play an important role in both innate and adoptive immune responses by priming and activating T cells. Among them, CD1 molecules have been identified to present both exogenous and endogenous lipid antigens to CD1-restricted T cells. The involvement of CD1-restricted T cells in autoimmune diseases and in defense against infectious diseases, however, remains largely unknown. Identifying novel antigenic lipids that bind to CD1 molecules and understanding the role of CD1-restricted T cells should lead to the successful development of vaccines, because the lipids can be used as antigens and also as adjuvants. In this paper, we have constructed functional recombinant human CD1 dimeric proteins and established a competitive ELISA assay to measure the lipid binding to CD1 molecules using the CD1 dimers. By using the competitive ELISA assay, we were able to show that the lipid extracts from murine malaria parasites can actually be loaded onto CD1 molecules. In addition, we have demonstrated that artificial antigen-presenting cells, which consist of magnetic beads coated with CD1d dimer and anti-CD28 antibody, stimulated and expanded human invariant NKT cells as efficiently as autologous immature DCs. A set of the tools presented in the current study should be valuable for screening various CD1 molecule-binding lipid antigens and for isolating CD1-restricted T cells. PMID:19374905

  19. Decidual vascular endothelial cells promote maternal-fetal immune tolerance by inducing regulatory T cells through canonical Notch1 signaling.

    PubMed

    Yao, Yanyi; Song, Jieping; Wang, Weipeng; Liu, Nian

    2016-05-01

    Adaptation of the maternal immune response to accommodate the semiallogeneic fetus is necessary for pregnancy success. However, the mechanisms by which the fetus avoids rejection despite expression of paternal alloantigens remain incompletely understood. Regulatory T cells (Treg cells) are pivotal for maintaining immune homeostasis, preventing autoimmune disease and fetus rejection. In this study, we found that maternal decidual vascular endothelial cells (DVECs) sustained Foxp3 expression in resting Treg cells in vitro. Moreover, under in vitro Treg cell induction condition with agonistic antibodies and transforming growth factor (TGF)-β, DVECs promoted Treg cell differentiation from non-Treg conventional T cells. Consistent with the promotion of Treg cell maintenance and differentiation, Treg cell-associated gene expression such as TGF-β, Epstein-Barr-induced gene-3, CD39 and glucocorticoid-induced tumor necrosis factor receptor was also increased in the presence of DVECs. Further study revealed that DVECs expressed Notch ligands such as Jagged-1, Delta-like protein 1 (DLL-1) and DLL-4, while Treg cells expressed Notch1 on their surface. The effects of DVECs on Treg cells was inhibited by siRNA-induced knockdown of expression of Jagged-1 and DLL-1 in DVECs. Downregulation of Notch1 in Treg cells using lentiviral shRNA transduction decreased Foxp3 expression in Treg cells. Adoptive transfer of Notch1-deficient Treg cells increased abortion rate in a murine semiallogeneic pregnancy model. Taken together, our study suggests that maternal DVECs are able to maintain decidual Treg cell identity and promote Treg cell differentiation through activation of Notch1 signal pathway in Treg cells and subsequently inhibit the immune response against semiallogeneic fetuses and preventing spontaneous abortion. PMID:26714886

  20. Selective immunosuppression by administration of major histocompatibility complex (MHC) class II-binding peptides. I. Evidence for in vivo MHC blockade preventing T cell activation

    PubMed Central

    1992-01-01

    Draining lymph node cells (LNC) from mice immunized with hen egg white lysozyme (HEL) display at their surface antigen-MHC complexes able to stimulate, in the absence of any further antigen addition, HEL peptide- specific, class II-restricted T cell hybridomas. Chloroquine addition to these LNC cultures fails to inhibit antigen presentation, indicating that antigenic complexes of class II molecules and HEL peptides are formed in vivo. MHC class II restriction of antigen presentation by LNC from HEL-primed mice was verified by the use of anti-class II monoclonal antibodies. Coinjection of HEL and the I-Ak-binding peptide HEL 112-129 in mice of H-2k haplotype inhibits the ability of LNC to stimulate I-Ak-restricted, HEL 46-61-specific T cell hybridomas. Similar results are obtained in mice coinjected with the HEL peptides 46-61 and 112-129. Inhibition of T hybridoma activation can also be observed using as antigen-presenting cells irradiated, T cell-depleted LNC from mice coinjected with HEL 46-61 and HEL 112-129, ruling out the possible role of either specific or nonspecific suppressor T cells. Inhibition of T cell proliferation is associated with MHC-specific inhibition of antigen presentation and with occupancy by the competitor of class II binding sites, as measured by activation of peptide- specific T cell hybridomas. These results demonstrate that administration of MHC class II binding peptide competitors selectively inhibits antigen presentation to class II-restricted T cells, indicating competitive blockade of class II molecules in vivo. PMID:1569402

  1. Solid-state capture and real-time analysis of individual T cell activation via self-assembly of binding multimeric proteins on functionalized materials surfaces.

    PubMed

    Diener, Kerrilyn R; Christo, Susan N; Griesser, Stefani S; Sarvestani, Ghafar T; Vasilev, Krasimir; Griesser, Hans J; Hayball, John D

    2012-01-01

    Polyfunctional T cell responses are increasingly underpinning new and improved vaccination regimens. Studies of the nature and extent of these T cell responses may be facilitated if specific T cell populations can be assessed from mixed populations by ligand-mediated capture in a solid-state assay format. Accordingly, we report here the development of a novel strategy for the solid-state capture and real-time activation analyses of individual cognate T cells which utilizes a spontaneous self-assembly process for generating multimers of biotinylated class I major histocompatibility-peptide complex (MHCp) directly on the solid-state assay surface while also ensuring stability by covalent interfacial binding. The capture surface was constructed by the fabrication of multilayer coatings onto standard slides. The first layer was a thin polymer coating with surface aldehyde groups, onto which streptavidin was covalently immobilized, followed by the docking of multimers of biotinylated MHCp or biotinylated anti-CD45.1 monoclonal antibody. The high binding strength at each step of this immobilization sequence aims to ensure that artefacts such as (partial) detachment, or displacement by proteins from solution, would not interfere with the intended biological assays. The multilayer coating steps were monitored by X-ray photoelectron spectroscopy; data indicated that the MHCp proteins self-assembled in a multimeric form onto the streptavidin surface. Immobilized multimeric MHCp demonstrated the capacity to bind and retain antigen-specific T cells from mixed populations of cells onto the solid carrier. Furthermore, real-time confocal microscopic detection and quantification of subsequent calcium flux using paired fluorescent ratiometric probes facilitated the analysis of individual T cell response profiles, as well as population analyses using a combination of individual T cell events. PMID:21945827

  2. Lectin interactions with the Jurkat leukemic T-cell line: quantitative binding studies and interleukin-2 production

    SciTech Connect

    Dupuis, G.; Bastin, B.

    1988-03-01

    Phytohemagglutinin (PHA), concanavalin A (Con A), pea lectin, and wheat germ agglutinin (WGA) have been used to investigate their binding properties to Jurkat 77 6.8 leukemic human T cells and their ability to induce these cells to produce interleukin-2 (IL-2). Binding studies showed that the Jurkat cells fixed 0.82 +/- 0.11 microgram pea lectin, 2.02 +/- 0.17 micrograms Con A, 1.85 +/- 0.07 micrograms PHA and 8.88 +/- 0.61 micrograms WGA. Scatchard plots were linear, indicating that the binding process was homogeneous with respect to the binding constant. PHA and Con A bound with the highest affinity (Kass (apparent) approximately equal to 9 x 10(9) M-1), followed by pea lectin and WGA (Kass (apparent) approximately equal to 3 x 10(9) M-1). The number of lectin binding sites was in agreement with the results of saturation experiments. We also evaluated the effect of the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the binding process. Results show that there were no gross alterations in the value of (apparent) Kass in the case of PHA and WGA. In contrast, the presence of TPA decreased the affinity of Con A and modified the Scatchard profile for pea lectin, which was curvilinear with a concavity turned upward. In this case, data were (apparent) K1 = 17.7 x 10(9) M-1 (high-affinity sites) and (apparent) K2 = 2.6 x 10(9) M-1 (low-affinity sites). The four lectins shared the ability to stimulate Jurkat 77 6.8 cells to secrete IL-2. Optimal lectin concentrations were 20 micrograms/ml (PHA) and 50 micrograms/ml (WGA and Con A). Pea lectin failed to display a dose-response relationship, and IL-2 production increased proportionally with lectin concentration. Con A was the most efficient stimulator (250 U/ml), followed by WGA (160 U/ml) and PHA (108 U/ml).

  3. Tuberous sclerosis complex 1 (Tsc1) enforces quiescence of naive T cells to promote immune homeostasis and function

    PubMed Central

    Yang, Kai; Neale, Geoffrey; Green, Douglas R.; He, Weifeng; Chi, Hongbo

    2011-01-01

    The mechanisms that regulate T cell quiescence are poorly understood. We report that tuberous sclerosis complex 1 (Tsc1) establishes a quiescence program in naive T cells by controlling cell size, cell cycle entry, and responses to T cell receptor stimulation. Loss of quiescence predisposed Tsc1-deficient T cells to apoptosis that resulted in loss of conventional T cells and invariant natural killer T cells. Loss of Tsc1 function dampened in vivo immune responses to bacterial infection. Tsc1-deficient T cells exhibited increased mTORC1 but diminished mTORC2 activities, with mTORC1 activation essential for the disruption of immune homeostasis. Therefore, Tsc1-dependent control of mTOR is crucial in establishing naive T cell quiescence to facilitate adaptive immune function. PMID:21765414

  4. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  5. Abnormal centrosome amplification in cells through the targeting of Ran-binding protein-1 by the human T cell leukemia virus type-1 Tax oncoprotein

    PubMed Central

    Peloponese, Jean-Marie; Haller, Kerstin; Miyazato, Akiko; Jeang, Kuan-Teh

    2005-01-01

    Human T cell leukemia virus type-1 (HTLV-1) is an oncogenic retrovirus etiologically causal of adult T cell leukemia. The virus encodes a Tax oncoprotein that functions in transcriptional regulation, cell cycle control, and transformation. Because adult T cell leukemia like many other human cancers is a disease of genomic instability with frequent gains and losses of chromosomes, to understand this disease it is important to comprehend how HTLV-1 engenders aneuploidy in host cells. In this regard, loss of cell cycle checkpoints permits tolerance of aneuploidy but does not explain how aneuploidy is created. We show here that HTLV-1 Tax causes abnormal centrosome fragmentation in the mitotic phase of the cell cycle. We report that Tax directly binds Ran and Ran-binding protein-1, locates to centrosomes/spindle poles, and causes supernumerary centrosomes. PMID:16365316

  6. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    NASA Astrophysics Data System (ADS)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  7. Regulatory CD4+CD25+ T Cells Dampen Inflammatory Disease in Murine Mycoplasma Pneumonia and Promote IL-17 and IFN-γ Responses

    PubMed Central

    Odeh, Adam N.; Simecka, Jerry W.

    2016-01-01

    Mycoplasmas cause respiratory diseases characterized by persistent infection and chronic airway inflammation. Mycoplasma lung disease is immunopathologic, with CD4+ Th cells determining both disease severity and resistance to infection. Th2 cell responses promote immunopathology, while Th1 cells confer resistance to infection. However, regulatory CD4+ T cells may also have a role in the pathogenesis of mycoplasma respiratory diseases. We hypothesized Treg cells control the severity of the inflammatory lesions and may also promote persistence of infection. To examine this, BALB/c mice were depleted of CD25+ cells, and had increased disease severity due to Mycoplasma pulmonis infection. Increases in mycoplasma antibody responses and lymphocyte infiltration into lungs also occurred after CD25+ cell depletion. CD4+CD25+ regulatory T cells promoted IFN-γ and IL-17 mycoplasma-specific CD4+ T cell responses in vitro and in vivo, while dampening IL-13+ Th responses. Neither IL-10 nor TGF-ß expression was detected in CD4+CD25+ T cells from lymph nodes. Thus, a regulatory T cell population plays an important role in controlling damaging immune responses in mycoplasma respiratory disease but does not contribute to persistence of infection. It appears that a regulatory T cell population preferentially dampens Th2 cell-mediated inflammatory responses to mycoplasma through a mechanism independent of IL-10 or TGF-ß characteristic of “classic” Treg cells. PMID:27175511

  8. T cell intrinsic USP15 deficiency promotes excessive IFN-γ production and an immunosuppressive tumor microenvironment in MCA-induced fibrosarcoma

    PubMed Central

    Zou, Qiang; Jin, Jin; Xiao, Yichuan; Zhou, Xiaofei; Hu, Hongbo; Cheng, Xuhong; Kazimi, Nasser; Ullrich, Stephen E; Sun, Shao-Cong

    2015-01-01

    USP15 is a deubiquitinase that negatively regulates activation of naïve CD4+ T cells and generation of IFN-γ-producing T helper 1 (Th1) cells. USP15 deficiency in mice promotes antitumor T cell responses in a transplantable cancer model; however, it has remained unclear how deregulated T cell activation impacts primary tumor development during the prolonged interplay between tumors and the immune system. Here, we find that the USP15-deficient mice are hypersensitive to methylcholantrene (MCA)-induced fibrosarcomas. Excessive IFN-γ production in USP15-deficient mice promotes expression of the immunosuppressive molecule PD-L1 and the chemokine CXCL12, causing accumulation of T-bet+ regulatory T cells and CD11b+Gr-1+ myeloid-derived suppressor cells at tumor site. Mixed bone marrow adoptive transfer studies further reveals a T cell-intrinsic role for USP15 in regulating IFN-γ production and tumor development. These findings suggest that T cell intrinsic USP15 deficiency causes excessive production of IFN-γ, which promotes an immunosuppressive tumor microenvironment, during MCA-induced primary tumorigenesis. PMID:26686633

  9. Monocytes promote tumor cell survival in T-cell lymphoproliferative disorders and are impaired in their ability to differentiate into mature dendritic cells

    PubMed Central

    Wilcox, Ryan A.; Wada, David A.; Ziesmer, Steven C.; Elsawa, Sherine F.; Comfere, Nneka I.; Dietz, Allan B.; Novak, Anne J.; Witzig, Thomas E.; Feldman, Andrew L.; Pittelkow, Mark R.

    2009-01-01

    A variety of nonmalignant cells present in the tumor microenvironment promotes tumorigenesis by stimulating tumor cell growth and metastasis or suppressing host immunity. The role of such stromal cells in T-cell lymphoproliferative disorders is incompletely understood. Monocyte-derived cells (MDCs), including professional antigen-presenting cells such as dendritic cells (DCs), play a central role in T-cell biology. Here, we provide evidence that monocytes promote the survival of malignant T cells and demonstrate that MDCs are abundant within the tumor microenvironment of T cell–derived lymphomas. Malignant T cells were observed to remain viable during in vitro culture with autologous monocytes, but cell death was significantly increased after monocyte depletion. Furthermore, monocytes prevent the induction of cell death in T-cell lymphoma lines in response to either serum starvation or doxorubicin, and promote the engraftment of these cells in nonobese diabetic/severe combined immunodeficient mice. Monocytes are actively recruited to the tumor microenvironment by CCL5 (RANTES), where their differentiation into mature DCs is impaired by tumor-derived interleukin-10. Collectively, the data presented demonstrate a previously undescribed role for monocytes in T-cell lymphoproliferative disorders. PMID:19671921

  10. The Length Distribution of Class I-Restricted T Cell Epitopes Is Determined by Both Peptide Supply and MHC Allele-Specific Binding Preference.

    PubMed

    Trolle, Thomas; McMurtrey, Curtis P; Sidney, John; Bardet, Wilfried; Osborn, Sean C; Kaever, Thomas; Sette, Alessandro; Hildebrand, William H; Nielsen, Morten; Peters, Bjoern

    2016-02-15

    HLA class I-binding predictions are widely used to identify candidate peptide targets of human CD8(+) T cell responses. Many such approaches focus exclusively on a limited range of peptide lengths, typically 9 aa and sometimes 9-10 aa, despite multiple examples of dominant epitopes of other lengths. In this study, we examined whether epitope predictions can be improved by incorporating the natural length distribution of HLA class I ligands. We found that, although different HLA alleles have diverse length-binding preferences, the length profiles of ligands that are naturally presented by these alleles are much more homogeneous. We hypothesized that this is due to a defined length profile of peptides available for HLA binding in the endoplasmic reticulum. Based on this, we created a model of HLA allele-specific ligand length profiles and demonstrate how this model, in combination with HLA-binding predictions, greatly improves comprehensive identification of CD8(+) T cell epitopes. PMID:26783342

  11. Lack of Phosphotyrosine Phosphatase SHP-1 Expression in Malignant T-Cell Lymphoma Cells Results from Methylation of the SHP-1 Promoter

    PubMed Central

    Zhang, Qian; Raghunath, Puthiyaveettil N.; Vonderheid, Eric; Ødum, Niels; Wasik, Mariusz A.

    2000-01-01

    SHP-1 is an important negative regulator of signaling by several receptors including receptors for interleukin-2 (IL-2R) and other cytokines. SHP-1 acts by dephosphorylating the receptors and receptor-associated kinases such as IL-2R-associated Jak3 kinase. We found that SHP-1 protein was not detectable or greatly diminished in most (six of seven) T cell lines derived from various types of T cell lymphomas and all (eight of eight) cutaneous T-cell lymphoma tissues with a transformed, large-cell morphology. All T-cell lymphoma lines tested (eight of eight) expressed diminished amounts or no detectable SHP-1 mRNA. These T cell lines did not, however, carry any mutations in the SHP-1 gene-coding, splice-junction, and promoter regions. Importantly, SHP-1 DNA promoter region in the T cell lines was resistant to digestion with three different methylation-sensitive restriction enzymes. This resistance was reversed by treatment of the cells with a demethylating agent, 5-deoxyazacytidine. The treatment resulted also in the expression of SHP-1 mRNA and, less frequently, SHP-1 protein. The expression of SHP-1 protein was associated with dephosphorylation of the Jak3 kinase. These results show that lack of SHP-1 expression is frequent in malignant T cells and results from methylation of the SHP-1 gene promoter. Furthermore, they indicate that SHP-1 loss may play a role in the pathogenesis of T cell lymphomas by permitting persistence of signals generated by IL-2R and, possibly, other receptor complexes. PMID:11021818

  12. CCAAT Enhancer Binding Protein and Nuclear Factor of Activated T Cells Regulate HIV-1 LTR via a Novel Conserved Downstream Site in Cells of the Monocyte-Macrophage Lineage

    PubMed Central

    Dahiya, Satinder; Liu, Yujie; Nonnemacher, Michael R.; Dampier, Will; Wigdahl, Brian

    2014-01-01

    Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the start site. In silico transcription factor binding analysis of the HIV-1 subtype B LTR sequences revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This binding site (+158 to+172), designated DS3, was found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed in terms of nucleotide sequence as well as physical location in the LTR. DS3 was found to be well represented in other subtypes as well. Interestingly, DS3 overlaps with a previously identified region that bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFATc2 exhibited a higher relative affinity for DS3 as compared with members of the C/EBP family (C/EBP α and β). DS3 was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBPα binding. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1 cells. A binding knockout of DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions only in cells of the monocyte-macrophage lineage cells and not in cells of T-cell origin. Thus, the events at DS3 positively regulate the HIV-1 promoter in cells of the monocyte-macrophage lineage. PMID:24551078

  13. Galectin-9 Protein Is Up-regulated in Astrocytes by Tumor Necrosis Factor and Promotes Encephalitogenic T-cell Apoptosis*

    PubMed Central

    Steelman, Andrew J.; Smith, Roger; Welsh, C. Jane; Li, Jianrong

    2013-01-01

    Demyelination and axonal damage in multiple sclerosis (MS) are thought to be a consequence of inflammatory processes that are perpetuated by activated glia and infiltrating leukocytes. Galectin-9 is a β-galactoside binding lectin capable of modulating immune responses and appears to be up-regulated in MS. However, its role in the pathogenesis of MS has yet to be determined. Here, we report that proinflammatory cytokines induce galectin-9 (Gal-9) expression in primary astrocytes and the mechanism by which TNF up-regulates Gal-9. Astrocytes did not express Gal-9 under basal conditions nor did IL-6, IL-10, or IL-13 trigger Gal-9 expression. In contrast, IL-1β, IFN-γ, and particularly TNF up-regulated Gal-9 in astrocytes. TNF-induced Gal-9 expression was dependent on TNF receptor 1 (TNFR1) as TNF failed to induce Gal-9 in TNFR1−/− astrocytes. Blockade of the JNK MAP kinase pathway with the JNK inhibitor SP600125 abrogated TNF-induced Gal-9, whereas p38 and MEK inhibitors had minimal effects. Furthermore, specific knockdown of c-Jun via siRNA in astrocytes before TNF treatment greatly suppressed Gal-9 transcription, suggesting that TNF induces astroglial Gal-9 through the TNF/TNFR1/JNK/cJun signaling pathway. Finally, utilizing astrocytes from Lgals9 mutant (Gal-9−/−) mice as well as a myelin basic protein-specific Tim-3+ encephalitogenic T-cell clone (LCN-8), we found that conditioned medium from TNF-stimulated Gal-9+/+ but not Gal-9−/− astrocytes increased the percentage of apoptotic encephalitogenic T-cells. Together, our results suggest that Gal-9 is induced in astrocytes by TNF via the JNK/c-Jun pathway and that astrocyte-derived Gal-9 may function as an immunoregulatory protein in response to ongoing neuroinflammation. PMID:23836896

  14. CRTAM is negatively regulated by ZEB1 in T cells.

    PubMed

    Rojas-Marquez, C; Valle-Rios, R; Lopez-Bayghen, E; Ortiz-Navarrete, V

    2015-08-01

    T cell activation leads to the induction of genes that are required for appropriate immune responses. This includes CRTAM (Class-I MHC-restricted T cell associated molecule), a protein that plays a key role in T cell development, proliferation, and generating cell polarity during activation. We previously characterized the CRTAM promoter and described how AP-1 family members are important for inducing CRTAM expression upon antigenic activation. Here, we show that CRTAM is a molecular target for ZEB1 (zinc finger E-box-binding protein), a homeodomain/Zn finger transcription factor. Overexpression of ZEB1 repressed CRTAM promoter activity, as well as endogenous CRTAM levels in human T cells. ZEB1-mediated transcriptional repression was abolished when E-box-like elements in the CRTAM promoter are mutated. In summary, ZEB1 functions as a transcriptional repressor for the CRTAM gene in both non-stimulated and stimulated T cells, thereby modulating adaptive immune responses.

  15. IFNα/βR Signaling Promotes Regulatory T Cell Development and Function Under Stress Conditions

    PubMed Central

    Metidji, Amina; Rieder, Sadiye Amcaoglu; Glass, Deborah Dacek; Cremer, Isabelle; Punkosdy, George A.; Shevach, Ethan M.

    2015-01-01

    Type I IFNs are a family of cytokines with antiviral and immunomodulatory properties. While the antiviral effects of IFNs are well characterized, their immunomodulatory properties are less clear. To specifically address the effects of type I IFNs on Treg, we studied mixed bone morrow (BM) chimeras between wild-type (WT) and IFNα/βR (IFNAR) knockout (KO) mice, and heterozygous female mice expressing a Treg-specific deletion of the IFNAR. In these two models, IFNAR signaling promotes the development of the Treg lineage in the thymus and their survival in the periphery. IFNAR KO Treg had a higher expression of the pro-apoptotic gene Bim and higher frequency of active caspase positive cells. IFNAR KO Treg from chimeric mice displayed a more naïve phenotype, accompanied by lower levels of CD25 and phosphorylated STAT5. Therefore, in Treg IFNAR signaling may directly or indirectly affect phosphorylation of STAT5. In mixed chimeras with Scurfy fetal liver, Treg derived from IFNAR KO BM were unable to control T effector cell activation and tissue inflammation. Under stress conditions or in a competitive environment, IFNAR signaling may be required to maintain Treg homeostasis and function. PMID:25795758

  16. Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development.

    PubMed

    Klug, D B; Crouch, E; Carter, C; Coghlan, L; Conti, C J; Richie, E R

    2000-02-15

    We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.

  17. Transcription of the human T-cell lymphotropic virus type I promoter by an alpha-amanitin-resistant polymerase.

    PubMed Central

    Piras, G; Kashanchi, F; Radonovich, M F; Duvall, J F; Brady, J N

    1994-01-01

    The human T-lymphotropic virus type I (HTLV-I) promoter contains the structural features of a typical RNA polymerase II (pol II) template. The promoter contains a TATA box 30 bp upstream of the transcription initiation site and binding sites for several pol II transcription factors, and long poly(A)+ RNA is synthesized from the integrated HTLV-I proviral DNA in vivo. Consistent with these characteristics, HTLV-I transcription activity was reconstituted in vitro by using TATA-binding protein, TFIIA, recombinant TFIIB, TFIIE, and TFIIF, TFIIH, and pol II. Transcription of the HTLV-I promoter in the reconstituted system requires RNA pol II. In HeLa whole cell extracts, however, the HTLV-I long terminal repeat also contains an overlapping transcription unit (OTU). HTLV-I OTU transcription is initiated at the same nucleotide site as the RNA isolated from the HTLV-I-infected cell line MT-2 but was not inhibited by the presence of alpha-amanitin at concentrations which inhibited the adenovirus major late pol II promoter (6 micrograms/ml). HTLV-I transcription was inhibited when higher concentrations of alpha-amanitin (60 micrograms/ml) were used, in the range of a typical pol III promoter (VA-I). Neutralization and depletion experiments with three distinct pol II antibodies demonstrate that RNA pol II is not required for HTLV-I OTU transcription. Antibodies to basal transcription factors TATA-binding protein and TFIIB, but not TFIIIC, inhibited HTLV-I OTU transcription. These observations suggest that the HTLV-I long terminal repeat contains overlapping promoters, a typical pol II promoter and a unique pol III promoter which requires a distinct set of transcription factors. Images PMID:7521915

  18. Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

    PubMed Central

    1993-01-01

    The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In

  19. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  20. AZD1775 sensitizes T cell acute lymphoblastic leukemia cells to cytarabine by promoting apoptosis over DNA repair.

    PubMed

    Ford, James B; Baturin, Dmitry; Burleson, Tamara M; Van Linden, Annemie A; Kim, Yong-Mi; Porter, Christopher C

    2015-09-29

    While some children with acute lymphoblastic leukemia (ALL) have excellent prognoses, the prognosis for adults and children with T cell ALL is more guarded. Treatment for T-ALL is heavily dependent upon antimetabolite chemotherapeutics, including cytarabine. Targeted inhibition of WEE1 with AZD1775 has emerged as a strategy to sensitize cancer cells to cytarabine and other chemotherapeutics. We sought to determine if this strategy would be effective for T-ALL with clinically relevant anti-leukemia agents. We found that AZD1775 sensitizes T-ALL cells to several traditional anti-leukemia agents, acting synergistically with cytarabine by enhancing DNA damage and apoptosis. In addition to increased phosphorylation of H2AX at serine 139 (γH2AX), AZD1775 led to increased phosphorylation of H2AX at tyrosine 142, a signaling event associated with promotion of apoptosis over DNA repair. In a xenograft model of T-ALL, the addition of AZD1775 to cytarabine slowed leukemia progression and prolonged survival. Inhibition of WEE1 with AZD1775 sensitizes T-ALL to several anti-leukemia agents, particularly cytarabine and that mechanistically, AZD1775 promotes apoptosis over DNA repair in cells treated with cytarabine. These data support the development of clinical trials including AZD1775 in combination with conventional chemotherapy for acute leukemia. PMID:26334102

  1. XCR1+ dendritic cells promote memory CD8+ T cell recall upon secondary infections with Listeria monocytogenes or certain viruses

    PubMed Central

    Alexandre, Yannick O.; Ghilas, Sonia; Sanchez, Cindy; Le Bon, Agnès

    2016-01-01

    Naive CD8+ T cell priming during tumor development or many primary infections requires cross-presentation by XCR1+ dendritic cells (DCs). Memory CD8+ T lymphocytes (mCTLs) harbor a lower activation threshold as compared with naive cells. However, whether their recall responses depend on XCR1+ DCs is unknown. By using a new mouse model allowing fluorescent tracking and conditional depletion of XCR1+ DCs, we demonstrate a differential requirement of these cells for mCTL recall during secondary infections by different pathogens. XCR1+ DCs were instrumental to promote this function upon secondary challenges with Listeria monocytogenes, vesicular stomatitis virus, or Vaccinia virus, but dispensable in the case of mouse cytomegalovirus. We deciphered how XCR1+ DCs promote mCTL recall upon secondary infections with Listeria. By visualizing for the first time the in vivo choreography of XCR1+ DCs, NK cells and mCTLs during secondary immune responses, and by neutralizing in vivo candidate molecules, we demonstrate that, very early after infection, mCTLs are activated, and attracted in a CXCR3-dependent manner, by NK cell–boosted, IL-12–, and CXCL9-producing XCR1+ DCs. Hence, depending on the infectious agent, strong recall of mCTLs during secondary challenges can require cytokine- and chemokine-dependent cross-talk with XCR1+ DCs and NK cells. PMID:26694969

  2. Human herpes virus-6 increases HIV-1 expression in co-infected T cells via nuclear factors binding to the HIV-1 enhancer.

    PubMed Central

    Ensoli, B; Lusso, P; Schachter, F; Josephs, S F; Rappaport, J; Negro, F; Gallo, R C; Wong-Staal, F

    1989-01-01

    Human Herpes virus-6 (HHV-6) can co-infect with HIV-1 human CD4+ T-cells, leading to accelerated cell death, and factors in HHV-6-infected cells stimulate HIV-1 LTR directed gene expression. In this study, we have examined the mechanism of HIV-1 activation by HHV-6 and localized the cis-acting sequences of HIV-1 LTR responsive to trans-activation. Increased HIV-1 LTR directed gene expression is obtained in HIV-1 infected cells co-infected with HHV-6, or in HHV-6 infected cells co-transfected with the HIV-1 tat gene. Parallel increases of HIV-1-specific transcripts are seen by in situ hybridization in HHV-6/HIV-1 doubly infected cells as compared to single HIV-1 infection. Similarly, infection by HHV-6 increases the steady-state level of HIV-1 LTR mRNA that parallels CAT enzymatic activity, suggesting a transcriptional and/or post-transcriptional activation. Sequences necessary for HIV-1 LTR activation by HHV-6 are distinct from those required for that tat response and map to a region of the HIV-1 LTR from -103 to -48. The HIV-1 enhancer sequence (-105 to -80) is sufficient to confer HHV-6 inducibility to a heterologous promoter, and nuclear protein(s) activated or induced by HHV-6 infection specifically bind to the NF kappa B motifs of the HIV-1 enhancer region.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2573513

  3. Sterol Regulatory Element-Binding Protein 2 Couples HIV-1 Transcription to Cholesterol Homeostasis and T Cell Activation ▿§

    PubMed Central

    Taylor, Harry E.; Linde, Michael E.; Khatua, Atanu K.; Popik, Waldemar; Hildreth, James E. K.

    2011-01-01

    Cholesterol plays an essential role in the life cycle of several enveloped viruses. Many of these viruses manipulate host cholesterol metabolism to facilitate their replication. HIV-1 infection of CD4+ T cells activates the sterol regulatory element-binding protein 2 (SREBP2) transcriptional program, which includes genes involved in cholesterol homeostasis. However, the role of SREBP2-dependent transcription in HIV-1 biology has not been fully examined. Here, we identify TFII-I, a gene critical for HIV-1 transcription in activated T cells, as a novel SREBP2 target gene. We found TFII-I expression increased after HIV-1 infection or activation of human primary CD4+ T cells. We show that inhibition of SREBP2 activity reduced TFII-I induction in response to these stimuli. More importantly, small interfering RNA (siRNA)-mediated gene silencing of either SREBP2 or TFII-I significantly reduced HIV-1 production in CD4+ T cells. We also found that TFII-I potentiates Tat-dependent viral gene expression, consistent with a role at the level of HIV-1 transcription. Collectively, our results demonstrate for the first time that HIV-1 transcription in T cells is linked to cholesterol homeostasis through control of TFII-I expression by SREBP2. PMID:21613400

  4. Serum amyloid A binds specific extracellular matrix glycoproteins and induces the adhesion of resting CD4+ T cells.

    PubMed

    Preciado-Patt, L; Hershkoviz, R; Fridkin, M; Lider, O

    1996-02-01

    Serum amyloid A (SAA), a prototypic acute phase protein reactant, exists naturally in the serum of healthy individuals. However, the levels of SAA in serum and its presence in sites of inflammation increase during certain chronic diseases associated with a local elevation of cytokine concentrations. Although the chemical structure of SAA is defined, its putative immunologic role(s) is still obscure. Nevertheless, it has been shown that 1) SAA acts as a chemoattractant and regulator of the migration of monocytes, polymorphonuclear cells, and T lymphocytes through endothelial cell monolayers; and 2) SAA and its proteolytically degraded N-terminal amyloid A fragment contain an extracellular matrix (ECM)-related cell adhesion epitopes. Herein, we examined whether SAA can associate with specific ECM moieties, and whether immobilized SAA-ECM complexes affect T lymphocyte adhesion. Radiolabeled human rSAA ([125I]rSAA) interacted avidly (Kd = 10(-9) M) and transiently with intact ECM, laminin, and vitronectin, but not with fibronectin or collagen type II. The binding of [125I]rSAA to ECM and laminin was inhibited by unlabeled rSAA and by the AA fragment, but not by the C-terminal portion of SAA (amino acid residues 2-82 and 77-104, respectively). Upon interactions with SAA or amyloid A, immobilized ECM, laminin, and vitronectin induced the adhesion of resting human CD4+ T cells in an apparently beta 1-integrin-mediated manner. Thus, the ECM appears to serve as a temporary anchorage site for SAA and amyloid A, and these ECM-complexed molecules seem to be involved in regulating the recruitment and accumulation of immunocytes in extravascular inflammatory compartments. PMID:8557997

  5. Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy

    PubMed Central

    van der Waart, Anniek B.; van de Weem, Noortje M. P.; Maas, Frans; Kramer, Cynthia S. M.; Kester, Michel G. D.; Falkenburg, J. H. Frederik; Schaap, Nicolaas; Jansen, Joop H.; van der Voort, Robbert; Gattinoni, Luca; Hobo, Willemijn

    2014-01-01

    Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell–like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell–like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+ stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+ T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+ memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+ T-cell population containing a high proportion of stem cell–like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+ T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma–bearing mice. These findings provide a rationale for clinical exploitation of ex vivo–generated Akt-inhibited MiHA-specific CD8+ T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients. PMID:25336630

  6. Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy.

    PubMed

    van der Waart, Anniek B; van de Weem, Noortje M P; Maas, Frans; Kramer, Cynthia S M; Kester, Michel G D; Falkenburg, J H Frederik; Schaap, Nicolaas; Jansen, Joop H; van der Voort, Robbert; Gattinoni, Luca; Hobo, Willemijn; Dolstra, Harry

    2014-11-27

    Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell-like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell-like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+ stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+ T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+ memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+ T-cell population containing a high proportion of stem cell-like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+ T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma-bearing mice. These findings provide a rationale for clinical exploitation of ex vivo-generated Akt-inhibited MiHA-specific CD8+ T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients. PMID:25336630

  7. Regulatory T Cells Promote β-Catenin–Mediated Epithelium-to-Mesenchyme Transition During Radiation-Induced Pulmonary Fibrosis

    SciTech Connect

    Xiong, Shanshan; Pan, Xiujie; Xu, Long; Yang, Zhihua; Guo, Renfeng; Gu, Yongqing; Li, Ruoxi; Wang, Qianjun; Xiao, Fengjun; Du, Li; Zhou, Pingkun; Zhu, Maoxiang

    2015-10-01

    Purpose: Radiation-induced pulmonary fibrosis results from thoracic radiation therapy and severely limits radiation therapy approaches. CD4{sup +}CD25{sup +}FoxP3{sup +} regulatory T cells (Tregs) as well as epithelium-to-mesenchyme transition (EMT) cells are involved in pulmonary fibrosis induced by multiple factors. However, the mechanisms of Tregs and EMT cells in irradiation-induced pulmonary fibrosis remain unclear. In the present study, we investigated the influence of Tregs on EMT in radiation-induced pulmonary fibrosis. Methods and Materials: Mice thoraxes were irradiated (20 Gy), and Tregs were depleted by intraperitoneal injection of a monoclonal anti-CD25 antibody 2 hours after irradiation and every 7 days thereafter. Mice were treated on days 3, 7, and 14 and 1, 3, and 6 months post irradiation. The effectiveness of Treg depletion was assayed via flow cytometry. EMT and β-catenin in lung tissues were detected by immunohistochemistry. Tregs isolated from murine spleens were cultured with mouse lung epithelial (MLE) 12 cells, and short interfering RNA (siRNA) knockdown of β-catenin in MLE 12 cells was used to explore the effects of Tregs on EMT and β-catenin via flow cytometry and Western blotting. Results: Anti-CD25 antibody treatment depleted Tregs efficiently, attenuated the process of radiation-induced pulmonary fibrosis, hindered EMT, and reduced β-catenin accumulation in lung epithelial cells in vivo. The coculture of Tregs with irradiated MLE 12 cells showed that Tregs could promote EMT in MLE 12 cells and that the effect of Tregs on EMT was partially abrogated by β-catenin knockdown in vitro. Conclusions: Tregs can promote EMT in accelerating radiation-induced pulmonary fibrosis. This process is partially mediated through β-catenin. Our study suggests a new mechanism for EMT, promoted by Tregs, that accelerates radiation-induced pulmonary fibrosis.

  8. Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice.

    PubMed

    Linch, Stefanie N; Kasiewicz, Melissa J; McNamara, Michael J; Hilgart-Martiszus, Ian F; Farhad, Mohammad; Redmond, William L

    2016-01-19

    Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti-cytotoxic T-lymphocyte-associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti-DEC-205 (dendritic and epithelial cells, 205 kDa)-HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival.

  9. Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice.

    PubMed

    Linch, Stefanie N; Kasiewicz, Melissa J; McNamara, Michael J; Hilgart-Martiszus, Ian F; Farhad, Mohammad; Redmond, William L

    2016-01-19

    Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti-cytotoxic T-lymphocyte-associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti-DEC-205 (dendritic and epithelial cells, 205 kDa)-HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival. PMID:26729864

  10. A canonical Vγ4Vδ4+ γδ T cell population with distinct stimulation requirements which promotes the Th17 response.

    PubMed

    Roark, Christina L; Huang, Yafei; Jin, Niyun; Aydintug, M Kemal; Casper, Tamara; Sun, Deming; Born, Willi K; O'Brien, Rebecca L

    2013-03-01

    We previously reported a subset of γδ T cells in mice which preferentially responds following intradermal immunization with collagen in complete Freund's adjuvant (CFA). These cells express a nearly invariant "canonical" Vγ4Vδ4+ TCR. They are potent producers of IL-17A and promote the development of collagen-induced arthritis. In this study, we report that CFA emulsified with PBS alone (without collagen) is sufficient to induce a strong response of Vγ4Vδ4+ cells in the draining lymph nodes of DBA/1 and C57BL/6 mice and that the TCRs of the elicited Vγ4Vδ4+ cells in both strains heavily favor the canonical sequence. However, although both CFA and incomplete Freund's adjuvant (which lacks the killed mycobacteria present in CFA) induced Vγ4Vδ4+ γδ T cell to expand, only CFA stimulated them to express IL-17A. The route of immunization was also critical, since intraperitoneal CFA induced only a weak response by these cells, whereas intradermal or subcutaneous CFA strongly stimulated them, suggesting that the canonical CFA-elicited Vγ4Vδ4+ cells are recruited from Vγ4+ γδ T cells normally found in the dermis. Their IL-17A response requires the toll-like receptor adapter protein MyD88, and their activation is enhanced by IFNγ, although αβ T cells need not be present. The CFA-elicited Vγ4Vδ4+ γδ T cells show a cytokine profile different from that of other previously described IL-17-producing γδ T cells. Finally, the Vγ4Vδ4+ subset appears to promote the Th17 αβ T cell response, suggesting its importance in mounting an effective immune response against certain pathogens.

  11. Kaposi's-sarcoma-associated-herpesvirus-activated dendritic cells promote HIV-1 trans-infection and suppress CD4{sup +} T cell proliferation

    SciTech Connect

    Liu, Wan; Qin, Yan; Bai, Lei; Lan, Ke; Wang, Jian-Hua

    2013-06-05

    Infection of Kaposi's sarcoma-associated herpesvirus (KSHV) is commonly occurred in AIDS patients. KSHV and HIV-1 act cooperatively in regulating infection with each other and in human carcinogenesis. Dendritic cells (DCs), as the pivotal cells in host immunity, may be modulated by both viruses, for immunoevasion and dissemination, therefore, the interaction between DCs and each virus has been a prior focus for pathogenesis elucidation. Here, we assessed the potential effect of KSHV on DC–HIV-1 interaction. We found that KSHV stimulation could promote maturation of monocyte-derived DCs (MDDCs) and impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells, demonstrating the immunosuppression induced by KSHV. More importantly, KSHV-stimulated MDDCs could capture more HIV-1 and efficiently transferred these infectious viruses to Hut/CCR5 T cell line. Our results reveal the novel modulation of DC-mediated HIV-1 dissemination by KSHV, and highlight the importance of studying DC–HIV-1 interaction to elucidate HIV/AIDS pathogenesis. - Highlights: ► KSHV impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells. ► KSHV stimulation matured MDDCs and enhanced HIV-1 endocytosis. ► KSHV stimulated MDDCs increased ICAM-1 expression and tighten contact with T cells. ► KSHV-stimulated MDDCs promoted HIV-1 trans-infection of CD4{sup +} T cells.

  12. Neisseria gonorrhoeae triggers the PGE2/IL-23 pathway and promotes IL-17 production by human memory T cells.

    PubMed

    Stefanelli, Paola; Teloni, Raffaela; Carannante, Anna; Mariotti, Sabrina; Nisini, Roberto; Gagliardi, Maria Cristina

    2012-10-01

    PGE2 is a potent modulator of the T helper (Th)17 immune response that plays a critical role in the host defense against bacterial, fungal and viral infections. We recently showed high serum levels of interleukin (IL)-17 in patients with gonococcal infection and we hypothesized that Neisseria gonorrhoeae could exploit a PGE2 mediated mechanism to promote IL-17 production. Here we show that N. gonorrhoeae induces human dendritic cell (DC) maturation, secretion of prostaglandin E2 and proinflammatory cytokines, including the pro-Th17 cytokine IL-23. Blocking PGE2 endogenous synthesis selectively reduces IL-23 production by DC in response to gonococcal stimulation, confirming recent data on PGE2/IL-23 crosstalk. N. gonorrhoeae stimulated DC induce a robust IL-17 production by memory CD4(+) T cells and this function correlates with PGE2 production. Our findings delineate a previously unknown role for PGE2 in the immune response to N. gonorrhoeae, suggesting its contribute via Th17 cell expansion. PMID:22542425

  13. Tn Glycosylation of the MUC6 Protein Modulates Its Immunogenicity and Promotes the Induction of Th17-biased T Cell Responses*

    PubMed Central

    Freire, Teresa; Lo-Man, Richard; Bay, Sylvie; Leclerc, Claude

    2011-01-01

    The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4+ hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells. PMID:21193402

  14. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

    PubMed

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S

    2016-04-15

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

  15. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    PubMed Central

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  16. Essential role for retinoic acid in the promotion of CD4+ T cell effector responses via retinoic acid receptor alpha

    PubMed Central

    Hall, J.A.; Cannons, J.L.; Grainger, J.R.; Santos, L.M. Dos; Hand, T.W.; Naik, S.; Wohlfert, E.A.; Chou, D.B.; Oldenhove, G.; Robinson, M.; Grigg, M.E.; Kastenmayer, R.; Schwartzberg, P.L.; Belkaid, Y.

    2012-01-01

    SUMMARY Vitamin A and its metabolite, retinoic acid (RA), have recently been implicated in the regulation of immune homeostasis via the peripheral induction of regulatory T cells. Here we show that RA is also required to elicit proinflammatory CD4+ helper T cell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) is the critical mediator of these effects. Strikingly, antagonism of RAR signaling and deficiency in RARα(Rara−/−) results in a cell autonomous CD4+ T cell activation defect. Altogether, these findings reveal a fundamental role for the RA/RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive T cell responses. PMID:21419664

  17. Exosomes from red blood cell units bind to monocytes and induce proinflammatory cytokines, boosting T-cell responses in vitro

    PubMed Central

    Danesh, Ali; Inglis, Heather C.; Jackman, Rachael P.; Wu, Shiquan; Deng, Xutao; Muench, Marcus O.; Heitman, John W.

    2014-01-01

    Extracellular vesicles (EVs) are small, double membrane vesicles derived from leukocytes, platelets, and cells of other tissues under physiological or pathological conditions. Generation of EVs in stored blood is thought to be associated with adverse effects and potentially immunosuppression in blood transfusion recipients. We measured the quantity and cells of origin for EVs isolated from stored red blood cell (RBC) units and tested whether they had any effects on T-cell–mediated immune responses. Mixing peripheral blood mononuclear cells (PBMCs) with EVs resulted in secretion of proinflammatory cytokines and chemokines and increased survival of unstimulated PBMCs. EVs augmented mitogen-induced CD4+ and CD8+ T-cell proliferation in an antigen-presenting cell (APC)-dependent manner. We demonstrated that EVs interacted primarily with monocytes and induced proinflammatory cytokine secretion. We also showed that the exosome fraction of EVs and not larger microvesicles was responsible for induction of TNF-α production by monocytes. Furthermore, blockade of CD40 or CD40L accessory molecules largely neutralized the EV augmentation of T-cell responses, implying a role for cell-cell interaction between T cells and EV-activated monocytes. Contrary to our hypothesis, the data demonstrate that EVs isolated from RBC units increase the potency of APCs and boost mitogen-driven T-cell proliferative responses. PMID:24335232

  18. Mapping an antibody-binding site and a T-cell-stimulating site on the 1A protein of respiratory syncytial virus.

    PubMed Central

    Nicholas, J A; Mitchell, M A; Levely, M E; Rubino, K L; Kinner, J H; Harn, N K; Smith, C W

    1988-01-01

    A synthetic peptide modeled on residues 45 to 60 of the 1A protein of respiratory syncytial (RS) virus [1A(45-60)] was constructed and used for immunization of mice and rabbits. The immunoglobulin G fraction of the resulting rabbit antibody, purified on protein A-Sepharose, immunoprecipitated from RS-infected HEp-2 cells a protein with a molecular size of approximately 9.5 kilodaltons, which corresponds to the previously published molecular size of the 1A protein (Y. T. Huang, P. L. Collins, and G. W. Wertz, Virus Res. 2:157-173, 1985). To investigate the T-cell-inducing properties of 1A(45-60), six strains of mice were immunized and their popliteal lymph node cells were tested for proliferation upon restimulation with peptide in vitro. The lymph node cells of all six strains of mice were responsive to restimulation with 1A(45-60) and showed high- and low-responder strain variation. These peptide-primed lymph node cells also proliferated upon in vitro restimulation with RS virus-infected cells. Correlation of proliferation with interleukin 2 production suggested that the responding lymphocytes were T-helper cells. The antibody-binding and T-cell-stimulating sites of 1A were mapped by constructing a series of overlapping synthetic peptides and testing each for ability to react with antiserum prepared by immunization of BALB/C mice with free peptide 1A(45-60) or for ability to restimulate proliferation in 1A(45-60)-primed lymph node cells of BALB/C mice. Human antibody, obtained during confirmed RS virus infection, was similarly tested with the truncated peptides. Antibody-binding activity was reduced after truncation from the carboxy terminus, and a binding site was mapped to residues 51 through 60, the smallest peptide tested. T-cell-stimulating activity in mice was relatively resistant to truncation from the carboxy terminus and sensitive to truncation from the amino terminus. The smallest region which retained significant T-cell-stimulating activity mapped to

  19. CD27 stimulation promotes the frequency of IL-7R expressing memory precursors and prevents IL-12 mediated loss of CD8+ T cell memory in the absence of CD4+ T cell help

    PubMed Central

    Dong, Han; Franklin, Nathan. A.; Roberts, Drew J.; Yagita, Hideo; Glennie, Martin J.; Bullock, Timothy N.J.

    2012-01-01

    Fully functional CD8+ T cell memory is highly dependent upon CD4+ T cell support. CD4+ T cells play a critical role in inducing the expression of CD70, the ligand for CD27, on dendritic cells. Here we demonstrate that CD27 stimulation during primary CD8+ T cell responses regulates the ability to mount secondary CD8+ T cell responses. CD27 stimulation during vaccinia and dendritic cell immunization controls the expression of the IL-7 receptor (CD127), which has been shown to be necessary for memory CD8+ T cell survival. Further, CD27 stimulation during primary CD8+ T cell responses to vaccinia virus restrained the late expression on memory precursor cells of cytokine receptors that support terminal differentiation. The formation of CD8+ T cell memory precursors and secondary CD8+ T cell responses were restored in the absence of CD27 costimulation when endogenous IL-12 was not available. Similarly, the lesion in CD8+ T cell memory that occurs in the absence ofCD4+ T cells did not occur in mice lacking IL-12. These data indicate that CD4+ T cell help and, by extension, CD27 stimulation supports CD8+ T cell memory by modulating the expression of cytokine receptors that influence the differentiation and survival of memory CD8+ T cells. PMID:22422886

  20. RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

    PubMed Central

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

    2013-01-01

    T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

  1. Extracellular matrix secreted by senescent fibroblasts induced by UVB promotes cell proliferation in HaCaT cells through PI3K/AKT and ERK signaling pathways.

    PubMed

    Kang, Jian; Chen, Wenqi; Xia, Jiping; Li, Yanhua; Yang, Bo; Chen, Bin; Sun, Weiling; Song, Xiuzu; Xiang, Wenzhong; Wang, Xiaoyong; Wang, Fei; Bi, Zhigang; Wan, Yinsheng

    2008-06-01

    Chronic exposure to solar ultraviolet radiation (UV) induces photoaging, and ultimately photocarcinogenesis. Senescent human skin fibroblasts (HSFs) in UVB stress-induced premature senescence (UVB-SIPS) share a similar extracellular matrix (ECM) phenotype with other types of senescent fibroblast. ECM from senescent fibroblasts induced by a variety of stresses has been shown to promote preneoplastic and neoplastic epithelial cell growth, a potential mechanism in carcinogenesis. We undertook this study to explore whether the extracellular matrices from UVB-induced senescent fibroblasts have any effect on the proliferation of HaCaT cells. The results showed that ECM secreted from HSFs in UVB-SIPS has 13.15 and 29.27% more stimulatory effect on proliferation than ECM secreted from presenescent HSFs and non-ECM, respectively. ECM from fibroblasts in UVB-SIPS activates FAK, ERK, and AKT in HaCaT cells. ERK and PI3K/AKT inhibitors inhibit ECM-induced ERK, AKT activation and cell proliferation. Cytochalasin D, a destructive agent of the cytoskeleton, inhibits ECM-induced FAK activation and cell proliferation in HaCaT cells. Collectively, we conclude that ECM secreted from HSFs in UVB-SIPS promotes cell proliferation via ERK and PI3K/AKT pathways and modulation of FAK and cytoskeletal proteins in HaCaT cells. Pharmacological manipulation of those signaling components may lead to the prevention and treatment of skin cancer induced by chronic solar exposure.

  2. Reduced Epithelial Na+/H+ Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis

    PubMed Central

    Midura-Kiela, Monica T.; Ramalingam, Rajalakshmy; Larmonier, Claire B.; Chase, John H.; Caporaso, J. Gregory; Besselsen, David G.; Ghishan, Fayez K.; Kiela, Pawel R.

    2016-01-01

    Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na+/H+ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na+/H+ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na+/H+ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2-/- and NHE3-/-/Rag2-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25-FoxP3+ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community. PMID:27050757

  3. MicroRNA-184 Promotes Proliferation and Inhibits Apoptosis in HaCaT Cells: An In Vitro Study.

    PubMed

    Bi, Xiaodong; Cao, Yu; Chen, Rixin; Liu, Chengyin; Chen, Jinghong; Min, Dongfang

    2016-01-01

    BACKGROUND This study aimed to investigate the role of miR-184 in the proliferation and apoptosis of keratinocyte (HaCaT cells). MATERIAL AND METHODS HaCaT cells were cultured in a growth medium. The miR-184 was transfected with siRNA, then cell viability and apoptosis were assayed by MTT and flow cytometry, respectively. The colony-forming efficacy of HaCaT cells were detected as well. mRNA expressions of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β1 were measured with RT-PCR. The expressions of apoptosis-related proteins caspase-3 and Bcl-x in HaCaT cells were determined by Western blot. RESULTS After miR-184 was transfected with siRNA, cell viability and colony forming ability decreased significantly, and apoptosis was significantly increased. The expressions of growth factors TGF-β1 and bFGF mRNAs, as well as apoptosis-related proteins Bcl-x, in HaCaT cells declined significantly after miR-184 was transfected with siRNA. In addition, the expression of pro-apoptotic protein caspase-3 increased significantly. CONCLUSIONS Our results suggest distinct roles of miR-184 during the growth, proliferation, and apoptosis of keratinocytes. PMID:27571235

  4. B cells promote induction of experimental autoimmune encephalomyelitis by facilitating reactivation of T cells in the central nervous system.

    PubMed

    Pierson, Emily R; Stromnes, Ingunn M; Goverman, Joan M

    2014-02-01

    The efficacy of rituximab treatment in multiple sclerosis has renewed interest in the role of B cells in CNS autoimmunity. In this study, we show that B cells are the predominant MHC class II(+) subset in the naive CNS in mice, and they constitutively express proinflammatory cytokines. Incidence of experimental autoimmune encephalomyelitis induced by adoptive transfer was significantly reduced in C3HeB/Fej μMT (B cell-deficient) mice, suggesting an important role for CNS B cells in initiating inflammatory responses. Initial T cell infiltration of the CNS occurred normally in μMT mice; however, lack of production of T cell cytokines and other immune mediators indicated impaired T cell reactivation. Subsequent recruitment of immune cells from the periphery driven by this initial T cell reactivation did not occur in μMT mice. B cells required exogenous IL-1β to reactivate Th17 but not Th1 cells in vitro. Similarly, reactivation of Th1 cells infiltrating the CNS was selectively impaired compared with Th17 cells in μMT mice, causing an increased Th17/Th1 ratio in the CNS at experimental autoimmune encephalomyelitis onset and enhanced brain inflammation. These studies reveal an important role for B cells within the CNS in reactivating T cells and influencing the clinical manifestation of disease.

  5. MicroRNA-184 Promotes Proliferation and Inhibits Apoptosis in HaCaT Cells: An In Vitro Study

    PubMed Central

    Bi, Xiaodong; Cao, Yu; Chen, Rixin; Liu, Chengyin; Chen, Jinghong; Min, Dongfang

    2016-01-01

    Background This study aimed to investigate the role of miR-184 in the proliferation and apoptosis of keratinocyte (HaCaT cells). Material/Methods HaCaT cells were cultured in a growth medium. The miR-184 was transfected with siRNA, then cell viability and apoptosis were assayed by MTT and flow cytometry, respectively. The colony-forming efficacy of HaCaT cells were detected as well. mRNA expressions of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β1 were measured with RT-PCR. The expressions of apoptosis-related proteins caspase-3 and Bcl-x in HaCaT cells were determined by Western blot. Results After miR-184 was transfected with siRNA, cell viability and colony forming ability decreased significantly, and apoptosis was significantly increased. The expressions of growth factors TGF-β1 and bFGF mRNAs, as well as apoptosis-related proteins Bcl-x, in HaCaT cells declined significantly after miR-184 was transfected with siRNA. In addition, the expression of pro-apoptotic protein caspase-3 increased significantly. Conclusions Our results suggest distinct roles of miR-184 during the growth, proliferation, and apoptosis of keratinocytes. PMID:27571235

  6. The RNA-binding protein hnRNPLL induces a T cell alternative splicing program delineated by differential intron retention in polyadenylated RNA

    PubMed Central

    2014-01-01

    Background Retention of a subset of introns in spliced polyadenylated mRNA is emerging as a frequent, unexplained finding from RNA deep sequencing in mammalian cells. Results Here we analyze intron retention in T lymphocytes by deep sequencing polyadenylated RNA. We show a developmentally regulated RNA-binding protein, hnRNPLL, induces retention of specific introns by sequencing RNA from T cells with an inactivating Hnrpll mutation and from B lymphocytes that physiologically downregulate Hnrpll during their differentiation. In Ptprc mRNA encoding the tyrosine phosphatase CD45, hnRNPLL induces selective retention of introns flanking exons 4 to 6; these correspond to the cassette exons containing hnRNPLL binding sites that are skipped in cells with normal, but not mutant or low, hnRNPLL. We identify similar patterns of hnRNPLL-induced differential intron retention flanking alternative exons in 14 other genes, representing novel elements of the hnRNPLL-induced splicing program in T cells. Retroviral expression of a normally spliced cDNA for one of these targets, Senp2, partially corrects the survival defect of Hnrpll-mutant T cells. We find that integrating a number of computational methods to detect genes with differentially retained introns provides a strategy to enrich for alternatively spliced exons in mammalian RNA-seq data, when complemented by RNA-seq analysis of purified cells with experimentally perturbed RNA-binding proteins. Conclusions Our findings demonstrate that intron retention in mRNA is induced by specific RNA-binding proteins and suggest a biological significance for this process in marking exons that are poised for alternative splicing. PMID:24476532

  7. The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires.

    PubMed

    Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F; Sette, Alessandro

    2015-11-01

    Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.

  8. The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires

    PubMed Central

    Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M.; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F.; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F.; Sette, Alessandro

    2016-01-01

    Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif. PMID:26399241

  9. Suppressors of cytokine signaling promote Fas-induced apoptosis through downregulation of NF-κB and mitochondrial Bfl-1 in leukemic T cells.

    PubMed

    Oh, Jiyoung; Kim, Seol-Hee; Ahn, Sinae; Lee, Choong-Eun

    2012-12-15

    Suppressors of cytokine signaling (SOCS) are known as negative regulators of cytokine- and growth factor-induced signal transduction. Recently they have emerged as multifunctional proteins with regulatory roles in inflammation, autoimmunity, and cancer. We have recently reported that SOCS1 has antiapoptotic functions against the TNF-α- and the hydrogen peroxide-induced T cell apoptosis through the induction of thioredoxin, which protects protein tyrosine phosphatases and attenuates Jaks. In this study, we report that SOCS, on the contrary, promote death receptor Fas-mediated T cell apoptosis. The proapoptotic effect of SOCS1 was manifested with increases in Fas-induced caspase-8 activation, truncated Bid production, and mitochondrial dysfunctions. Both caspase-8 inhibitor c-Flip and mitochondrial antiapoptotic factor Bfl-1 were significantly reduced by SOCS1. These proapoptotic responses were not associated with changes in Jak or p38/Jnk activities but were accompanied with downregulation of NF-κB and NF-κB-dependent reporter gene expression. Indeed, p65 degradation via ubiquitination was accelerated in SOCS1 overexpressing cells, whereas it was attenuated in SOCS1 knockdown cells. With high NF-κB levels, the SOCS1-ablated cells displayed resistance against Fas-induced apoptosis, which was abrogated upon siBfl-1 transfection. The results indicate that the suppression of NF-κB-dependent induction of prosurvival factors, such as Bfl-1 and c-Flip, may serve as a mechanism for SOCS action to promote Fas-mediated T cell apoptosis. SOCS3 exhibited a similar proapoptotic function. Because both SOCS1 and SOCS3 are induced upon TCR stimulation, SOCS would play a role in activation-induced cell death by sensitizing activated T cells toward Fas-mediated apoptosis to maintain T cell homeostasis.

  10. Interleukin-21 is associated with the severity of psoriasis vulgaris through promoting CD4+ T cells to differentiate into Th17 cells

    PubMed Central

    Wang, Ying; Wang, Li-Li; Yang, Hao-Yu; Wang, Fei-Fei; Zhang, Xue-Xiu; Bai, Yan-Ping

    2016-01-01

    Interleukin-21 (IL-21) and T helper 17 (Th17) cells are known to be involved in the pathogenesis of psoriasis, but little is known about their relationship in psoriasis. Herein, we investigated whether IL-21 could regulate Th17 cell induction in patients with psoriasis vulgaris. 32 patients with psoriasis vulgaris and 13 healthy controls were recruited. Flow cytometry was used to detect the frequencies of cells mainly secreting IL-21 (including IL-21+CD4+ T and IL-21+ Th17 cells) and Th17 cells. An enzyme-linked immunosorbent assay (ELISA) was used to determine the serum content of IL-21. Severity of the psoriasis was evaluated by a Psoriasis Area and Severity Index (PASI) score. In addition, the differentiation of CD4+ T cells with IL-21 and the different frequencies of IL-21+CD4+ T cells, IL-21+ Th17 cells and Th17 cells were assessed, as were serum levels of IL-21 in patients with moderate to severe psoriasis before and after treatment. Our results showed that the levels of IL-21, IL-21+CD4+ T cells, IL-21+ Th17 cells and Th17 cells were significantly increased in patients and positively associated with PASI score (P < 0.01). Moreover, the levels of IL-21, IL-21+CD4+ T cells and IL-21+ Th17 cells were positively correlated with the frequency of Th17 cells (P < 0.01). In vitro experiments demonstrated that IL-21 could promote CD4+ T cells to differentiate into Th17 cells. After a 4-week treatment of acitretin and a topical therapy, all the immune markers observed in patients decreased significantly (P < 0.01), but the levels remained higher than those in healthy controls (P < 0.01). These findings indicate that IL-21 might promote Th17 cell induction in psoriasis and might be a potential immune marker for targeting this disease. PMID:27508040

  11. Interleukin-21 is associated with the severity of psoriasis vulgaris through promoting CD4+ T cells to differentiate into Th17 cells.

    PubMed

    Wang, Ying; Wang, Li-Li; Yang, Hao-Yu; Wang, Fei-Fei; Zhang, Xue-Xiu; Bai, Yan-Ping

    2016-01-01

    Interleukin-21 (IL-21) and T helper 17 (Th17) cells are known to be involved in the pathogenesis of psoriasis, but little is known about their relationship in psoriasis. Herein, we investigated whether IL-21 could regulate Th17 cell induction in patients with psoriasis vulgaris. 32 patients with psoriasis vulgaris and 13 healthy controls were recruited. Flow cytometry was used to detect the frequencies of cells mainly secreting IL-21 (including IL-21+CD4+ T and IL-21+ Th17 cells) and Th17 cells. An enzyme-linked immunosorbent assay (ELISA) was used to determine the serum content of IL-21. Severity of the psoriasis was evaluated by a Psoriasis Area and Severity Index (PASI) score. In addition, the differentiation of CD4+ T cells with IL-21 and the different frequencies of IL-21+CD4+ T cells, IL-21+ Th17 cells and Th17 cells were assessed, as were serum levels of IL-21 in patients with moderate to severe psoriasis before and after treatment. Our results showed that the levels of IL-21, IL-21+CD4+ T cells, IL-21+ Th17 cells and Th17 cells were significantly increased in patients and positively associated with PASI score (P < 0.01). Moreover, the levels of IL-21, IL-21+CD4+ T cells and IL-21+ Th17 cells were positively correlated with the frequency of Th17 cells (P < 0.01). In vitro experiments demonstrated that IL-21 could promote CD4+ T cells to differentiate into Th17 cells. After a 4-week treatment of acitretin and a topical therapy, all the immune markers observed in patients decreased significantly (P < 0.01), but the levels remained higher than those in healthy controls (P < 0.01). These findings indicate that IL-21 might promote Th17 cell induction in psoriasis and might be a potential immune marker for targeting this disease.

  12. Biphasic function of TLR3 adjuvant on tumor and spleen dendritic cells promotes tumor T cell infiltration and regression in a vaccine therapy.

    PubMed

    Azuma, Masahiro; Takeda, Yohei; Nakajima, Hiroko; Sugiyama, Haruo; Ebihara, Takashi; Oshiumi, Hiroyuki; Matsumoto, Misako; Seya, Tsukasa

    2016-08-01

    Successful cancer immunotherapy necessitates T cell proliferation and infiltration into tumor without exhaustion, a process closely links optimal maturation of dendritic cells (DC), and adjuvant promotes this process as an essential prerequisite. Poly(I:C) has contributed to adjuvant immunotherapy that evokes an antitumor response through the Toll-loke receptor 3 (TLR3)/TICAM-1 pathway in DC. However, the mechanism whereby Poly(I:C) acts on DC for T cell proliferation and migration remains undetermined. Subcutaneous injection of Poly(I:C) regressed implant tumors (WT1-C1498 or OVA-EG7) in C57BL/6 mice, which coincided with tumor-infiltration of CD8(+) T cells. Epitope-specific cytotoxic T lymphocytes (CTLs) were increased in spleen by challenge with Poly(I:C)+Db126 WT-1 peptide but not Poly(I:C) alone, suggesting the need of an exogenous Ag density for cross-priming. In tumor, CXCR3 ligands were upregulated by Poly(I:C), which facilitated recruitment of CTL to the tumor. Thus, Poly(I:C) acts on splenic CD8α(+) DC to cross-prime T cells and on intratumor cells to attract CTLs. Besides CD8(+) T cell cross-priming, T cell recruitment into tumor was significantly dampened in Batf3 (-/-) mice, reflecting the importance of tumor Batf3-dependent DC rather than macrophages in T cell recruitment. Poly(I:C)-induced XCR1(hi) CD8α(+) DC with high TLR3 levels were markedly decreased in Batf3 (-/-) mice, which hampered the production of IL-12 and IL-12-mediated CD4(+)/CD8(+) T cell proliferation. Subcutaneous administration of Poly(I:C) and adoptive transfer of wild-type CD8α(+) DC largely recovered antitumor response in those Batf3 (-/-) mice. Collectively, Poly(I:C) tunes up proper maturation of CD8α(+) DC to establish TLR3-mediated IL-12 function and cross-presentation in spleen and lymphocyte-attractive antitumor microenvironment in tumor. PMID:27622060

  13. Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111-129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire.

    PubMed Central

    Muraro, P A; Vergelli, M; Kalbus, M; Banks, D E; Nagle, J W; Tranquill, L R; Nepom, G T; Biddison, W E; McFarland, H F; Martin, R

    1997-01-01

    The pathogenesis of multiple sclerosis (MS) is currently ascribed in part to a T cell-mediated process targeting myelin components. The T cell response to one candidate autoantigen, myelin basic protein (MBP), in the context of HLA-DR15Dw2, has been previously studied in detail. However, the characteristics of cellular immunity in the context of other MS-associated HLA-DR haplotypes are scarcely known. MBP-specific T cell lines (TCL) were generated from HLA-DR4 (B1*0401)-positive MS subjects. Out of 275 MBP-specific TCL, 178 (64. 7%) specifically recognized region MBP(111-129), predominantly in the context of DRB1*0401. The major T cell epitope for MBP recognition corresponded to residues MBP(116-123). These TCL expressed disparate profiles of cytokine secretion and cytotoxicity. T cell receptor analysis, on the other hand, revealed a strikingly limited heterogeneity of rearrangements. In contrast to MBP(81-99), which binds with high affinity to HLA-DR15 and is recognized by a diverse T cell repertoire, MBP(111-129) binds weakly to DRB1*0401, suggesting that only high affinity T cell receptors might be able to efficiently engage such unstable MHC/peptide complexes, thus accounting for the T cell receptor restriction we observed. This study provides new insight about MBP recognition and proposes an alternative mechanism for immunodominance of self-antigen T cell epitopes in humans. PMID:9218510

  14. Gamma delta T cells promote inflammation and insulin resistance during high fat diet-induced obesity in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gamma delta T cells are resident in adipose tissue and increase during diet-induced obesity. Their possible contribution to the inflammatory response that accompanies diet-induced obesity was investigated in mice after a 5-10 week high milk fat diet. The high milk fat diet resulted in significant in...

  15. Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line

    SciTech Connect

    Tsukahara, Tomonori; Agawa, Hideyuki; Matsumoto, Sayori; Matsuda, Mizuho; Ueno, Shuichi; Yamashita, Yuki; Yamada, Koichiro; Tanaka, Nobuyuki; Kojima, Katsuhiko; Takeshita, Toshikazu . E-mail: takesit@sch.md.shinshu-u.ac.jp

    2006-07-07

    Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established. The data showed that MLV preferred integration near the transcription start sites ({+-}5 kb), near CpG islands ({+-}1 kb), and within the first intron of RefSeq genes. We also identified MLV integration hot spots that contained three or more integrations within a 100 kb region. RT-PCR revealed that mRNA-levels of T-cell clones that contained MLV integrations near transcription start sites or introns were dysregulated compared to the uninfected cells. These studies help define the profile of MLV integration in T-cells and the risks associated with MLV-based gene therapy.

  16. Conditional PDK1 Ablation Promotes Epidermal and T-Cell-Mediated Dysfunctions Leading to Inflammatory Skin Disease.

    PubMed

    Yu, Minjun; Owens, David M; Ghosh, Sankar; Farber, Donna L

    2015-11-01

    Phosphoinositide-dependent kinase-1 (PDK1) is a key signaling molecule downstream of the phosphatidylinositol 3-kinase pathway and is a master regulator of multiple kinases in cells of epithelial and hematopoietic lineages. The physiological role of PDK1 in regulating skin and immune homeostasis is not known. Here we developed a mouse model in which PDK1 is conditionally ablated in activated CD4 T cells, regulatory T cells, and mature keratinocytes through OX40-Cre recombinase expression. The resultant mice (PDK1-CKO) spontaneously developed severe dermatitis, skin fibrosis, and systemic T helper type 2 immunity, succumbing by 11 weeks of age. Through a series of T-cell transfers, bone marrow reconstitutions, and crossing to lymphocyte-deficient backgrounds, we demonstrate that ablation of PDK1 in keratinocytes is the major driver of disease pathogenesis. PDK1-deficient keratinocytes exhibit intrinsic defects in the expression of key structural proteins including cytokeratin-10 and loricrin, resulting in increased keratinocyte turnover, which in turn triggers inflammation, T-cell recruitment, and immune-mediated destruction. Our results reveal PDK1 as a central regulator of keratinocyte homeostasis that prevents skin immune infiltration and inflammation. PMID:26099023

  17. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    SciTech Connect

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N.

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

  18. Splenic Long-Lived Plasma Cells Promote the Development of Follicular Helper T Cells during Autoimmune Responses.

    PubMed

    Jang, Eunkyeong; Cho, Wang Sik; Oh, Yeon-Kyung; Cho, Mi-La; Kim, Jung Mogg; Paik, Doo-Jin; Youn, Jeehee

    2016-02-01

    Long-lived plasma cells (LLPCs) develop under the help of follicular helper T (Tfh) cells and reside mainly in the bone marrow. However, these cells are unusually abundant in the spleen of several autoimmune models including K/BxNsf mice, yet their pathogenic impact remains unknown. To investigate a previously unappreciated role of splenic LLPCs, we sorted splenic plasma cells (PCs) from K/BxNsf and K/BxN mice, corresponding to LLPCs and conventional short-lived PCs, respectively, and compared their phenotypes and ability to prime and induce the differentiation of naive CD4(+) T cells into effector cells in vitro and in vivo. We found that K/BxNsf PCs had lower levels of the Ag presentation machinery and costimulators than K/BxN PCs, and also a lower CD4(+) T cell priming capacity. Autoantigen-pulsed K/BxNsf PCs selectively polarized cognate CD4(+) T cells toward the expression of molecules necessary for Tfh development and function. As a result, the K/BxNsf PC-primed CD4(+) T cells were more effective in stimulating B cells to produce autoantigen-specific IgGs than K/BxN PCs or even dendritic cells. Adoptive transfer of K/BxNsf PCs, but not K/BxN PCs, to K/BxN mice increased numbers of Tfh cells in draining lymph nodes. These results propose that abnormal accumulation of LLPCs in the spleen of autoimmune models drives the differentiation of autoantigen-primed CD4(+) T cells to Tfh cells. This positive feedback loop between splenic LLPCs and Tfh cells may contribute to the persistence of humoral autoimmunity. PMID:26729802

  19. A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells

    PubMed Central

    Chakraborty, Paramita; Ganguly, Avishek; Adhikary, Arghya; Mukhopadhyay, Debanjan; Ganguli, Sudipta; Banerjee, Rajdeep; Ashraf, Mohammad; Biswas, Jaydip; Das, Pradeep K.; Sa, Gourisankar; Chatterjee, Mitali; Das, Tanya; Choudhuri, Soumitra Kumar

    2009-01-01

    Background At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis. Methodology/Principal Findings The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-γ but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-β producing CD4+CD25+ T cells toward IFN-γ producing T cells. Conclusion/Significance Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth. PMID:19756150

  20. γδ T cells promote inflammation and insulin resistance during high fat diet-induced obesity in mice.

    PubMed

    Mehta, Pooja; Nuotio-Antar, Alli Martina; Smith, C Wayne

    2015-01-01

    γδ T cells are resident in AT and increase during diet-induced obesity. Their possible contribution to the inflammatory response that accompanies diet-induced obesity was investigated in mice after a 5 to 10 week milk HFD. The HFD resulted in significant increases in CD44(hi), CD62L(lo), and TNF-α(+) γδ T cells in eAT of WT mice. Mice deficient in all γδ T cells (TCRδ(-/-)) or only Vγ4 and Vγ6 subsets (Vγ4/6(-/-)) were compared with WT mice with regard to proinflammatory cytokine production and macrophage accumulation in eAT. Obesity among these mouse strains did not differ, but obese TCRδ(-/-) and Vγ4/6(-/-) mice had significantly reduced eAT expression of F4/80, a macrophage marker, and inflammatory mediators CCL2 and IL-6 compared with WT mice. Obese TCRδ(-/-) mice had significantly reduced CD11c(+) and TNF-α(+) macrophage accumulation in eAT after 5 and 10 weeks on the HFD, and obese Vγ4/6(-/-) mice had significantly increased CD206(+) macrophages in eAT after 5 weeks on the diet and significantly reduced macrophages after 10 weeks. Obese TCRδ(-/-) mice had significant reductions in systemic insulin resistance and inflammation in liver and skeletal muscle after longer-term HFD feeding (10 and 24 weeks). In vitro studies revealed that isolated γδ T cells directly stimulated RAW264.7 macrophage TNF-α expression but did not stimulate inflammatory mediator expression in 3T3-L1 adipocytes. These findings are consistent with a role for γδ T cells in the proinflammatory response that accompanies diet-induced obesity.

  1. L1CAM promotes enrichment of immunosuppressive T cells in human pancreatic cancer correlating with malignant progression.

    PubMed

    Grage-Griebenow, Evelin; Jerg, Elfi; Gorys, Artur; Wicklein, Daniel; Wesch, Daniela; Freitag-Wolf, Sandra; Goebel, Lisa; Vogel, Ilka; Becker, Thomas; Ebsen, Michael; Röcken, Christoph; Altevogt, Peter; Schumacher, Udo; Schäfer, Heiner; Sebens, Susanne

    2014-07-01

    Regulatory T cell (T-reg) enrichment in the tumor microenvironment is regarded as an important mechanism of tumor immune escape. Hence, the presence of T-regs in highly malignant pancreatic ductal adenocarcinoma (PDAC) is correlated with short survival. Likewise, the adhesion molecule L1CAM is upregulated during PDAC progression in the pancreatic ductal epithelium also being associated with poor prognosis. To investigate whether L1CAM contributes to enrichment of T-regs in PDAC, human CD4(+)CD25(+)CD127(-)CD49d(-) T-regs and CD4(+)CD25(-) T-effector cells (T-effs) were isolated by magnetic bead separation from blood of healthy donors. Their phenotype and functional behavior were analyzed in dependence on human premalignant (H6c7) or malignant (Panc1) pancreatic ductal epithelial cells, either exhibiting or lacking L1CAM expression. T cells derived from blood and tumors of PDAC patients were analyzed by flow cytometry and findings were correlated with clinical parameters. Predominantly T-regs but not T-effs showed an increased migration on L1CAM expressing H6c7 and Panc1 cells. Whereas proliferation of T-regs did not change in the presence of L1CAM, T-effs proliferated less, exhibited a decreased CD25 expression and an increased expression of CD69. Moreover, these T-effs exhibited a regulatory phenotype as they inhibited proliferation of autologous T cells. Accordingly, CD4(+)CD25(-)CD69(+) T cells were highly abundant in PDAC tissues compared to blood being associated with nodal invasion and higher grading in PDAC patients. Overall, these data point to an important role of L1CAM in the enrichment of immunosuppressive T cells in particular of a CD4(+)CD25(-)CD69(+)-phenotype in PDAC providing a novel mechanism of tumor immune escape which contributes to tumor progression. PMID:24746181

  2. LFA-1/ICAM-1 Ligation in Human T Cells Promotes Th1 Polarization through a GSK3β Signaling-Dependent Notch Pathway.

    PubMed

    Verma, Navin K; Fazil, M H U Turabe; Ong, Seow Theng; Chalasani, Madhavi Latha S; Low, Jian Hui; Kottaiswamy, Amuthavalli; P, Praseetha; Kizhakeyil, Atish; Kumar, Sunil; Panda, Aditya K; Freeley, Michael; Smith, Sinead M; Boehm, Bernhard O; Kelleher, Dermot

    2016-07-01

    In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4(+) T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4(+) T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1-mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1-induced inactivation of glycogen synthase kinase 3β (GSK3β), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3β. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK-GSK3β signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses. PMID:27206767

  3. Increased expression of TLR2 in CD4(+) T cells from SLE patients enhances immune reactivity and promotes IL-17 expression through histone modifications.

    PubMed

    Liu, Yu; Liao, Jieyue; Zhao, Ming; Wu, Haijing; Yung, Susan; Chan, Tak Mao; Yoshimura, Akihiko; Lu, Qianjin

    2015-09-01

    The innate immune system has been shown to play an important pathologic role in systemic lupus erythematosus (SLE). TLR2, a PRR, recognizes exogenous PAMPs, and endogenous damage-associated molecular patterns and has been implicated in the initiation and maintenance of the perpetuated inflammatory reactions in autoimmune diseases. Here, we report increased expression of TLR2 in CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes from SLE patients. Conventional treatment, such as hydroxychloroquine and corticosteroids, showed no effect on TLR2 expression in CD4(+) T cells from SLE patients. In vitro stimulation of TLR2 in CD4(+) T cells from SLE patients increased CD40L and CD70 expression, as well as secretion of IL-6, IL-17A, IL-17F, and TNF-α, while Foxp3 transcription decreased. This effect was reversed by TLR2 siRNA. Moreover, TLR2 activation upregulated H3K4 tri-methylation and H4 acetylation levels while downregulated H3K9 tri-methylation level in the IL-17A promoter region. In addition, it also increased H4 acetylation levels and decreased H3K9 tri-methylation levels in the IL-17F promoter region. In summary, our findings demonstrate that increased expression of TLR2 contributes to immune reactivity and promotes IL-17A and IL-17F expression through histone modifications in SLE.

  4. A Highly Tilted Binding Mode by a Self-Reactive T Cell Receptor Results in Altered Engagement of Peptide and MHC

    SciTech Connect

    D Sethi; D Schubert; A Anders; A Heroux; D Bonsor; C Thomas; E Sundberg; J Pyrdol; K Wucherpfennig

    2011-12-31

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  5. A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

    SciTech Connect

    Sethi, D.K.; Heroux, A.; Schubert, D. A.; Anders, A.-K.; Bonsor, D. A.; Thomas, C. P.; Sundberg, E. J.; Pyrdol, J.; Wucherpfennig, K. W.

    2011-01-17

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  6. Direct Binding of Hepatitis C Virus Core to gC1qR on CD4+ and CD8+ T Cells Leads to Impaired Activation of Lck and Akt

    PubMed Central

    Yao, Zhi Qiang; Eisen-Vandervelde, Audrey; Waggoner, Stephen N.; Cale, Evan M.; Hahn, Young S.

    2004-01-01

    Complement plays a pivotal role in the regulation of innate and adaptive immunity. It has been shown that the binding of C1q, a natural ligand of gC1qR, on T cells inhibits their proliferation. Here, we demonstrate that direct binding of the hepatitis C virus (HCV) core to gC1qR on T cells leads to impaired Lck/Akt activation and T-cell function. The HCV core associates with the surface of T cells specifically via gC1qR, as this binding is inhibited by the addition of either anti-gC1qR antibody or soluble gC1qR. The binding affinity constant of core protein for gC1qR, as determined by BIAcore analysis, is 3.8 × 10−7 M. The specificity of the HCV core-gC1qR interaction is confirmed by reduced core binding on Molt-4 T cells treated with gC1qR-silencing small interfering RNA and enhanced core binding on GPC-16 guinea pig cells transfected with human gC1qR. Interestingly, gC1qR is expressed at higher levels on CD8+ than on CD4+ T cells, resulting in more severe core-induced suppression of the CD8+-T-cell population. Importantly, T-cell receptor-mediated activation of the Src kinases Lck and ZAP-70 but not Fyn and the phosphorylation of Akt are impaired by the HCV core, suggesting that it inhibits the very early events of T-cell activation. PMID:15163734

  7. IL4RA on lymphatic endothelial cells promotes T cell egress during sclerodermatous graft versus host disease

    PubMed Central

    Urso, Katia; Alvarez, David; Cremasco, Viviana; Tsang, Kelly; Grauel, Angelo; Lafyatis, Robert; von Andrian, Ulrich H.; Ermann, Joerg; Aliprantis, Antonios O.

    2016-01-01

    Systemic sclerosis (SSc) is a potentially fatal autoimmune disorder with limited therapeutic options. Sclerodermatous graft versus host disease (sclGvHD), induced by transfer of B10.D2 splenocytes into BALB/c Rag2−/− mice, models an inflammatory subset of SSc characterized by a prominent IL13-induced gene expression signature in the skin. Host mice deficient in IL4RA, a subunit of the type II IL4/IL13 receptor, are protected from sclGvHD. While IL4RA has a well-established role in Th2 differentiation and alternative macrophage activation, we report here a previously unappreciated function for IL4RA in lymphatic endothelial cells (LECs): regulation of activated T cell egress. Seven days after splenocyte transfer, Il4ra−/− hosts had increased numbers of activated graft CD4+ T cells in skin draining lymph nodes (dLNs) but fewer T cells in efferent lymph, blood, and skin. Sphingosine-1 phosphate (S1P), master regulator of lymphocyte egress from LNs, was lower in dLNs of Il4ra−/− hosts with a corresponding decrease of S1P kinase 1 (Sphk1) expression in LECs. Bypassing the efferent lymphatics via i.v. injection of CD4+ T cells from dLNs of Il4ra−/− sclGvHD mice restored clinical GvHD in secondary Il4ra−/− recipients. These results identify a role for IL4RA and suggest that modulation of lymphocyte egress from LNs may be effective in SSc and GvHD. PMID:27547823

  8. IL4RA on lymphatic endothelial cells promotes T cell egress during sclerodermatous graft versus host disease

    PubMed Central

    Urso, Katia; Alvarez, David; Cremasco, Viviana; Tsang, Kelly; Grauel, Angelo; Lafyatis, Robert; von Andrian, Ulrich H.; Ermann, Joerg; Aliprantis, Antonios O.

    2016-01-01

    Systemic sclerosis (SSc) is a potentially fatal autoimmune disorder with limited therapeutic options. Sclerodermatous graft versus host disease (sclGvHD), induced by transfer of B10.D2 splenocytes into BALB/c Rag2–/– mice, models an inflammatory subset of SSc characterized by a prominent IL13-induced gene expression signature in the skin. Host mice deficient in IL4RA, a subunit of the type II IL4/IL13 receptor, are protected from sclGvHD. While IL4RA has a well-established role in Th2 differentiation and alternative macrophage activation, we report here a previously unappreciated function for IL4RA in lymphatic endothelial cells (LECs): regulation of activated T cell egress. Seven days after splenocyte transfer, Il4ra–/– hosts had increased numbers of activated graft CD4+ T cells in skin draining lymph nodes (dLNs) but fewer T cells in efferent lymph, blood, and skin. Sphingosine-1 phosphate (S1P), master regulator of lymphocyte egress from LNs, was lower in dLNs of Il4ra–/– hosts with a corresponding decrease of S1P kinase 1 (Sphk1) expression in LECs. Bypassing the efferent lymphatics via i.v. injection of CD4+ T cells from dLNs of Il4ra–/– sclGvHD mice restored clinical GvHD in secondary Il4ra–/– recipients. These results identify a role for IL4RA and suggest that modulation of lymphocyte egress from LNs may be effective in SSc and GvHD. PMID:27547823

  9. The dual targeting of immunosuppressive cells and oxidants promotes effector and memory T-cell functions against lung cancer

    PubMed Central

    Sawant, Anandi; Schafer, Cara C; Ponnazhagan, Selvarangan; Deshane, Jessy S

    2014-01-01

    We have recently demonstrated that the combination of gemcitabine and a superoxide dismutase mimetic protects mice against lung cancer by suppressing the functions of myeloid-derived suppressor cells and by activating memory CD8+ T-cell responses. Persistent memory cells exhibited a glycolytic metabolism, which may have directly enhanced their effector functions. This combinatorial therapeutic regimen may reduce the propensity of some cancer patients to relapse. PMID:24711958

  10. Interruption of dendritic cell-mediated TIM-4 signaling induces regulatory T cells and promotes skin allograft survival.

    PubMed

    Yeung, Melissa Y; McGrath, Martina M; Nakayama, Masafumi; Shimizu, Tetsunosuke; Boenisch, Olaf; Magee, Ciara N; Abdoli, Rozita; Akiba, Hisaya; Ueno, Takuya; Turka, Laurence A; Najafian, Nader

    2013-10-15

    Dendritic cells (DCs) are the central architects of the immune response, inducing inflammatory or tolerogenic immunity, dependent on their activation status. As such, DCs are highly attractive therapeutic targets and may hold the potential to control detrimental immune responses. TIM-4, expressed on APCs, has complex functions in vivo, acting both as a costimulatory molecule and a phosphatidylserine receptor. The effect of TIM-4 costimulation on T cell activation remains unclear. In this study, we demonstrate that Ab blockade of DC-expressed TIM-4 leads to increased induction of induced regulatory T cells (iTregs) from naive CD4(+) T cells, both in vitro and in vivo. iTreg induction occurs through suppression of IL-4/STAT6/Gata3-induced Th2 differentiation. In addition, blockade of TIM-4 on previously activated DCs still leads to increased iTreg induction. iTregs induced under TIM-4 blockade have equivalent potency to control and, upon adoptive transfer, significantly prolong skin allograft survival in vivo. In RAG(-/-) recipients of skin allografts adoptively transferred with CD4(+) T cells, we show that TIM-4 blockade in vivo is associated with a 3-fold prolongation in allograft survival. Furthermore, in this mouse model of skin transplantation, increased induction of allospecific iTregs and a reduction in T effector responses were observed, with decreased Th1 and Th2 responses. This enhanced allograft survival and protolerogenic skewing of the alloresponse is critically dependent on conversion of naive CD4(+) to Tregs in vivo. Collectively, these studies identify blockade of DC-expressed TIM-4 as a novel strategy that holds the capacity to induce regulatory immunity in vivo.

  11. Identification and characterization of an antigen binding receptor on an inducible suppressor T cell hybridoma specific for GAT

    SciTech Connect

    Sorensen, C.M.; Pierce, C.W.

    1986-03-05

    C57BL/10 spleen cells were incubated for 18 hrs with monoclonal GAT-TsF1 and soluble GAT, and fused with BW5147. Hybridomas that did not produce GAT-TsF2 constitutively but could be induced by GAT-TsF1 to do so were cloned. One such clone (1556A2.1) was shown to bind GAT via a cell surface structure having the properties of a classical receptor. Binding of /sup 125/I-GAT was saturable and maximal by 4 hrs at 4/sup 0/C. Scatchard analysis indicated 5.8 x 10/sup 4/ binding sites per cell with an avidity of 8 x 10/sup -14/M. Binding of /sup 125/I-GAT was specific because cold GAT, GT, and GLT competed for binding, whereas BSA and GA did not. Furthermore, monoclonal antibody against a shared anti-GAT idiotype also blocked binding of /sup 125/I-GAT. Finally, the antigen-binding receptor isolated from uninduced 1556A2.1 hybridoma failed to complement independently isolated I-J/sup +/ chain from TsF2 obtained from induced 1556A2.1. These data suggest the presence of a molecule on the Ts/sub 2/ cell capable of binding antigen which is related to, but functionally different from, the TsF2 antigen-binding chain.

  12. Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells.

    PubMed

    Serafini, Paolo; Mgebroff, Stephanie; Noonan, Kimberly; Borrello, Ivan

    2008-07-01

    Tumor-induced T-cell tolerance is a major mechanism that facilitates tumor progression and limits the efficacy of immune therapeutic interventions. Regulatory T cells (Treg) play a central role in the induction of tolerance to tumor antigens, yet the precise mechanisms regulating its induction in vivo remain to be elucidated. Using the A20 B-cell lymphoma model, here we identify myeloid-derived suppressor cells (MDSC) as the tolerogenic antigen presenting cells capable of antigen uptake and presentation to tumor-specific Tregs. MDSC-mediated Treg induction requires arginase but is transforming growth factor-beta independent. In vitro and in vivo inhibition of MDSC function, respectively, with NOHA or sildenafil abrogates Treg proliferation and tumor-induced tolerance in antigen-specific T cells. These findings establish a role for MDSCs in antigen-specific tolerance induction through preferential antigen uptake mediating the recruitment and expansion of Tregs. Furthermore, therapeutic interventions, such as in vivo phosphodiesterase 5-inhibition, which effectively abrogate the immunosuppressive role of MDSCs and reduce Treg numbers, may play a critical role in delaying and/or reversing tolerance induction. PMID:18593947

  13. Developmental exposure to trichloroethylene promotes CD4{sup +} T cell differentiation and hyperactivity in association with oxidative stress and neurobehavioral deficits in MRL+/+ mice

    SciTech Connect

    Blossom, Sarah J. Doss, Jason C.; Hennings, Leah J.; Jernigan, Stefanie; Melnyk, Stepan; James, S. Jill

    2008-09-15

    The non adult immune system is particularly sensitive to perinatal and early life exposures to environmental toxicants. The common environmental toxicant, trichloroethylene (TCE), was shown to increase CD4+ T cell production of the proinflammatory cytokine IFN-{gamma} following a period of prenatal and lifetime exposure in autoimmune-prone MRL+/+ mice. In the current study, MRL+/+ mice were used to further examine the impact of TCE on the immune system in the thymus and periphery. Since there is considerable cross-talk between the immune system and the brain during development, the potential relationship between TCE and neurobehavioral endpoints were also examined. MRL+/+ mice were exposed to 0.1 mg/ml TCE ({approx} 31 mg/kg/day) via maternal drinking water or direct exposure via the drinking water from gestation day 1 until postnatal day (PD) 42. TCE exposure did not impact gross motor skills but instead significantly altered social behaviors and promoted aggression associated with indicators of oxidative stress in brain tissues in male mice. The immunoregulatory effects of TCE involved a redox-associated promotion of T cell differentiation in the thymus that preceded the production of proinflammatory cytokines, IL-2, TNF-{alpha}, and IFN-{gamma} by mature CD4+ T cells. The results demonstrated that developmental and early life TCE exposure modulated immune function and may have important implications for neurodevelopmental disorders.

  14. Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived inducible factor/IL-22.

    PubMed

    Dumoutier, L; Lejeune, D; Colau, D; Renauld, J C

    2001-06-15

    The class II cytokine receptor family includes the receptors for IFN-alphabeta, IFN-gamma, IL-10, and IL-10-related T cell-derived inducible factor/IL-22. By screening genomic DNA databases, we identified a gene encoding a protein of 231 aa, showing 33 and 34% amino acid identity with the extracellular domains of the IL-22 receptor and of the IL-20R/cytokine receptor family 2-8, respectively, but lacking the transmembrane and cytoplasmic domains. A lower but significant sequence identity was found with other members of this family such as the IL-10R (29%), cytokine receptor family 2-4/IL-10Rbeta (30%), tissue factor (26%), and the four IFN receptor chains (23-25%). This gene is located on chromosome 6q24, at 35 kb from the IFNGR1 gene, and is expressed in various tissues with maximal expression in breast, lungs, and colon. The recombinant protein was found to bind IL-10-related T cell-derived inducible factor/IL-22, and to inhibit the activity of this cytokine on hepatocytes and intestinal epithelial cells. We propose to name this natural cytokine antagonist IL-22BP for IL-22 binding protein.

  15. Monoclonal rat anti-human lymphocyte antibody Campath-1 binds to T and B lymphocytes but effectively lyses only T cells.

    PubMed

    Gazitt, Y; Or, R; Mumcuoglu, M; Slavin, S

    1987-12-01

    Binding and human complement-mediated T and B lymphocyte lysis were investigated in bone marrow samples obtained from 15 normal donors and 12 patients with a variety of malignant disorders undergoing marrow cryopreservation prior to autologous bone marrow transplantation. All marrow samples were obtained during remission except for one patient with neuroblastoma. The mononuclear cell fractions were collected and the distribution of B cell restricted markers (surface Ig and GP-70) and T cell surface markers (Leu-1 and rosettes with sheep red blood cells) were studied before and after marrow purging with Campath-1, a monoclonal rat anti-human lymphocyte antibody, and autologous serum as complement. Effective lysis of T lymphocytes (greater than 99.5%) was documented in all cases. However, although effective binding of Campath-1 to B lymphocytes was uniform, no effective lysis occurred. The data suggest that effective lysis of mature T lymphocytes can be accomplished while leaving normal B lymphocytes intact.

  16. TGF-beta and IL-6 signals modulate chromatin binding and promoter occupancy by acetylated FOXP3.

    PubMed

    Samanta, Arabinda; Li, Bin; Song, Xiaomin; Bembas, Kathryn; Zhang, Geng; Katsumata, Makoto; Saouaf, Sandra J; Wang, Qiang; Hancock, Wayne W; Shen, Yuan; Greene, Mark I

    2008-09-16

    Expression of FOXP3, a potent gene-specific transcriptional repressor, in regulatory T cells is required to suppress autoreactive and alloreactive effector T cell function. Recent studies have shown that FOXP3 is an acetylated protein in a large nuclear complex and FOXP3 actively represses transcription by recruiting enzymatic corepressors, including histone modification enzymes. The mechanism by which extracellular stimuli regulate the FOXP3 complex ensemble is currently unknown. Although TGF-beta is known to induce murine FOXP3(+) Treg cells, TGF-beta in combination with IL-6 attenuates the induction of FOXP3 functional activities. Here we show that TCR stimuli and TGF-beta signals modulate the disposition of FOXP3 into different subnuclear compartments, leading to enhanced chromatin binding in human CD4(+)CD25(+) regulatory T cells. TGF-beta treatment increases the level of acetylated FOXP3 on chromatin and site-specific recruitment of FOXP3 on the human IL-2 promoter. However, the proinflammatory cytokine IL-6 down-regulates FOXP3 binding to chromatin in the presence of TGF-beta. Moreover, histone deacetylation inhibitor (HDACi) treatment abrogates the down-regulating effects of IL-6 and TGF-beta. These studies indicate that HDACi can enhance regulatory T cell function via promoting FOXP3 binding to chromatin even in a proinflammatory cellular microenvironment. Collectively, our data provide a framework of how different signals affect intranuclear redistribution, posttranslational modifications, and chromatin binding patterns of FOXP3. PMID:18779564

  17. TGF-β and IL-6 signals modulate chromatin binding and promoter occupancy by acetylated FOXP3

    PubMed Central

    Samanta, Arabinda; Li, Bin; Song, Xiaomin; Bembas, Kathryn; Zhang, Geng; Katsumata, Makoto; Saouaf, Sandra J.; Wang, Qiang; Hancock, Wayne W.; Shen, Yuan; Greene, Mark I.

    2008-01-01

    Expression of FOXP3, a potent gene-specific transcriptional repressor, in regulatory T cells is required to suppress autoreactive and alloreactive effector T cell function. Recent studies have shown that FOXP3 is an acetylated protein in a large nuclear complex and FOXP3 actively represses transcription by recruiting enzymatic corepressors, including histone modification enzymes. The mechanism by which extracellular stimuli regulate the FOXP3 complex ensemble is currently unknown. Although TGF-β is known to induce murine FOXP3+ Treg cells, TGF-β in combination with IL-6 attenuates the induction of FOXP3 functional activities. Here we show that TCR stimuli and TGF-β signals modulate the disposition of FOXP3 into different subnuclear compartments, leading to enhanced chromatin binding in human CD4+CD25+ regulatory T cells. TGF-β treatment increases the level of acetylated FOXP3 on chromatin and site-specific recruitment of FOXP3 on the human IL-2 promoter. However, the proinflammatory cytokine IL-6 down-regulates FOXP3 binding to chromatin in the presence of TGF-β. Moreover, histone deacetylation inhibitor (HDACi) treatment abrogates the down-regulating effects of IL-6 and TGF-β. These studies indicate that HDACi can enhance regulatory T cell function via promoting FOXP3 binding to chromatin even in a proinflammatory cellular microenvironment. Collectively, our data provide a framework of how different signals affect intranuclear redistribution, posttranslational modifications, and chromatin binding patterns of FOXP3. PMID:18779564

  18. INTERRUPTION OF DENDRITIC CELL-MEDIATED TIM-4 SIGNALING INDUCES REGULATORY T CELLS AND PROMOTES SKIN ALLOGRAFT SURVIVAL

    PubMed Central

    Yeung, Melissa Y.; McGrath, Martina M.; Nakayama, Masafumi; Shimizu, Tetsunosuke; Boenisch, Olaf; Magee, Ciara N.; Abdoli, Rozita; Akiba, Hisaya; Ueno, Takuya; Turka, Laurence A.; Najafian, Nader

    2013-01-01

    Dendritic cells (DCs) are the central architects of the immune response, inducing inflammatory or tolerogenic immunity, dependent upon their activation status. As such, DCs are highly attractive therapeutic targets and may hold the potential to control detrimental immune responses. TIM-4, expressed on antigen presenting cells, has complex functions in vivo, acting both as a costimulatory molecule and a phosphatidylserine (PS) receptor. The effect of TIM-4 costimulation on T cell activation remains unclear. Here, we demonstrate that antibody blockade of DC-expressed TIM-4 leads to increased induction of iTregs from naïve CD4+ T cells, both in vitro and in vivo. iTreg induction occurs through suppression of IL-4/STAT6/Gata3 induced Th2 differentiation. In addition, blockade of TIM-4 on previously activated DCs still leads to increased iTreg induction. iTregs induced under TIM-4 blockade have equivalent potency to control and upon adoptive transfer, significantly prolong skin allograft survival in vivo. In RAG−/− recipients of skin allografts adoptively transferred with CD4+ T cells, we show that TIM-4 blockade in vivo is associated with a three-fold prolongation in allograft survival. Furthermore, in this mouse model of skin transplantation, increased induction of allospecific iTregs and a reduction in T effector responses were observed, with decreased Th1 and Th2 responses. This enhanced allograft survival and pro-tolerogenic skewing of the alloresponse is critically dependent upon conversion of naïve CD4+ to Tregs in vivo. Collectively, these studies identify blockade of DC-expressed TIM-4 as a novel strategy which holds the capacity to induce regulatory immunity in vivo. PMID:24038092

  19. B7-H3 Promotes Pathogenesis of Autoimmune Disease and Inflammation by Regulating the Activity of Different T Cell Subsets

    PubMed Central

    Luo, Liqun; Zhu, Gefeng; Xu, Haiying; Yao, Sheng; Zhou, Gang; Zhu, Yuwen; Tamada, Koji; Huang, Lanqing; Flies, Andrew D.; Broadwater, Megan; Ruff, William; van Deursen, Jan M. A.; Melero, Ignacio; Zhu, Zhou; Chen, Lieping

    2015-01-01

    B7-H3 is a cell surface molecule in the immunoglobulin superfamily that is frequently upregulated in response to autoantigens and pathogens during host T cell immune responses. However, B7-H3's role in the differential regulation of T cell subsets remains largely unknown. Therefore, we constructed a new B7-H3 deficient mouse strain (B7-H3 KO) and evaluated the functions of B7-H3 in the regulation of Th1, Th2, and Th17 subsets in experimental autoimmune encephalomyelitis (EAE), experimental asthma, and collagen-induced arthritis (CIA); these mouse models were used to predict human immune responses in multiple sclerosis, asthma, and rheumatoid arthritis, respectively. Here, we demonstrate that B7-H3 KO mice have significantly less inflammation, decreased pathogenesis, and limited disease progression in both EAE and CIA mouse models when compared with littermates; these results were accompanied by a decrease in IFN-γ and IL-17 production. In sharp contrast, B7-H3 KO mice developed severe ovalbumin (OVA)-induced asthma with characteristic infiltrations of eosinophils in the lung, increased IL-5 and IL-13 in lavage fluid, and elevated IgE anti-OVA antibodies in the blood. Our results suggest B7-H3 has a costimulatory function on Th1/Th17 but a coinhibitory function on Th2 responses. Our studies reveal that B7-H3 could affect different T cell subsets which have important implications for regulating pathogenesis and disease progression in human autoimmune disease. PMID:26065426

  20. Deletion of 5-Lipoxygenase in the Tumor Microenvironment Promotes Lung Cancer Progression and Metastasis through Regulating T Cell Recruitment

    PubMed Central

    Poczobutt, Joanna M.; Nguyen, Teresa T.; Hanson, Dwight; Li, Howard; Sippel, Trisha R.; Weiser-Evans, Mary C. M.; Gijon, Miguel; Murphy, Robert C.

    2016-01-01

    Eicosanoids, including PGs, produced by cyclooxygenases (COX), and leukotrienes, produced by 5-lipoxygenase (5-LO) have been implicated in cancer progression. These molecules are produced by both cancer cells and the tumor microenvironment (TME). We previously reported that both COX and 5-LO metabolites increase during progression in an orthotopic immunocompetent model of lung cancer. Although PGs in the TME have been well studied, less is known regarding 5-LO products produced by the TME. We examined the role of 5-LO in the TME using a model in which Lewis lung carcinoma cells are directly implanted into the lungs of syngeneic WT mice or mice globally deficient in 5-LO (5-LO-KO). Unexpectedly, primary tumor volume and liver metastases were increased in 5-LO-KO mice. This was associated with an ablation of leukotriene (LT) production, consistent with production mainly mediated by the microenvironment. Increased tumor progression was partially reproduced in global LTC4 synthase KO or mice transplanted with LTA4 hydrolase-deficient bone marrow. Tumor-bearing lungs of 5-LO-KO had decreased numbers of CD4 and CD8 T cells compared with WT controls, as well as fewer dendritic cells. This was associated with lower levels of CCL20 and CXL9, which have been implicated in dendritic and T cell recruitment. Depletion of CD8 cells increased tumor growth and eliminated the differences between WT and 5-LO mice. These data reveal an antitumorigenic role for 5-LO products in the microenvironment during lung cancer progression through regulation of T cells and suggest that caution should be used in targeting this pathway in lung cancer. PMID:26663781

  1. N-3-(oxododecanoyl)-L-homoserine lactone promotes the induction of regulatory T-cells by preventing human dendritic cell maturation

    PubMed Central

    Li, Youqiang; Zhou, Huayou; Zhang, Yunyan; Huang, Bin; Qu, Pinghua; Zeng, Jianming; E, Shunmei; Zhang, Xuan; Liu, Jianping

    2015-01-01

    N-3-(Oxododecanoyl)-L-homoserine lactone (C12) is a small bacterial signaling molecule secreted by Pseudomonas aeruginosa (PA), which activates mammalian cells through TLR4-independent mechanisms. C12 acts as an immunosuppressant and it has been shown to modulate murine bone marrow-derived dendritic cell-mediated T-helper 2 (Th2) cell polarizations in vitro. In the present study, we initially examined the impact of C12 on the maturation of human monocyte-derived dendritic cells (Mo-DCs) and the induction of regulatory T-cells (iTregs) in culture. Our findings demonstrate that C12-treated Mo-DCs failed to undergo lipopolysaccharide (LPS)-induced maturation. At the molecular level, C12 blocked the upregulation of surface molecules, including CD11c, HLA-DR, CD40, and CD80, and it switched to an interleukin (IL)-10high, IL-12p70low phenotype. Moreover, C12 selectively inhibited the capacity of Mo-DCs to stimulate the proliferation of allogeneic CD4+ T-cells. Otherwise, the C12-treated Mo-DCs promoted the generation of CD4+CD25+Foxp3+-induced regulatory T-cells (iTregs) and enhanced their IL-10 and transforming growth factor (TGF)-β production associated with reduced interferon (IFN)-γ and IL-12p70 production. These findings provide new insights towards understanding the persistence of chronic inflammation in PA infection. PMID:25749498

  2. c-CBL E3 Ubiquitin Ligase is Over-Expressed in Cutaneous T-Cell Lymphoma: Its Inhibition Promotes Activation Induced Cell Death

    PubMed Central

    Wu, Jianqiang; Salva, Katrin A.; Wood, Gary S.

    2014-01-01

    Mycosis fungoides (MF) and Sezary syndrome (SS) are two major forms of cutaneous T-cell lymphoma (CTCL) characterized by resistance to apoptosis. A central pathway for T-cell apoptosis is activation-induced cell death (AICD) which is triggered through the T-cell receptor (TCR). This results in upregulation of FAS-ligand (FASL) and subsequent apoptosis through the FAS death receptor pathway. It has been known for more than a decade that TCR signaling is defective in CTCL; however, the underlying mechanism has not been apparent. In this report, we show that the E3 ubiquitin ligase, c-CBL, is over-expressed in CTCL and that its knockdown overcomes defective TCR signaling resulting in phosphorylation of PLCg1, calcium influx, ROS generation, up-regulation of FASL and extrinsic pathway apoptosis in CTCL cells expressing adequate FAS. In CTCL cells with suboptimal FAS expression, FAS can be upregulated epigenetically by derepression of the FAS promoter using methotrexate (MTX) which we showed previously has activity as a DNA methylation inhibitor. Using these combined strategies, FAS-low as well as FAS-high CTCL cells can be killed effectively. PMID:25140833

  3. Research Note Mesenchymal stem cells from skin lesions of psoriasis patients promote proliferation and inhibit apoptosis of HaCaT cells.

    PubMed

    Liu, R F; Wang, F; Wang, Q; Zhao, X C; Zhang, K M

    2015-12-22

    Psoriasis is an inflammatory skin disease characterized by excessive proliferation and abnormal differentiation and apoptosis of keratinocytes (KCs). Mesenchymal stem cells (MSCs) from skin lesions of psoriasis patients demonstrate abnormal cytokine secretion, which may affect KC proliferation and apoptosis. Here, we explored how MSCs from skin lesions of psoriasis patients affect HaCaT cell proliferation and apoptosis. First, flow cytometry and multipotent differentiation methods were used to identify skin MSCs, which were then co-cultured with HaCaT cells. HaCaT cell proliferation was analyzed in real-time, and cell cycle progression and apoptosis were assessed by flow cytometry. Cell morphologies and multipotencies of skin MSCs were similar between the psoriasis group and healthy control group, with high levels of CD29, CD44, CD73, CD90, and CD105 and limited expression of CD34, CD45, and HLA-DR. MSCs from skin lesions of psoriasis patients promote KC proliferation more potently and are less capable of inducing KC apoptosis. This may underlie KC proliferation and abnormal apoptosis in psoriasis skin lesions, which results in abnormal thickening of the epidermis.

  4. Loss of CAR promotes migration and proliferation of HaCaT cells, and accelerates wound healing in rats via Src-p38 MAPK pathway

    PubMed Central

    Su, Linlin; Fu, Lanqing; Li, Xiaodong; Zhang, Yue; Li, Zhenzhen; Wu, Xue; Li, Yan; Bai, Xiaozhi; Hu, Dahai

    2016-01-01

    The coxsackie and adenovirus receptor (CAR) is a cell adhesion molecule mostly localized to cell-cell contacts in epithelial and endothelial cells. CAR is known to regulate tumor progression, however, its physiological role in keratinocyte migration and proliferation, two essential steps in re-epithelialization during wound healing, has less been investigated. Here we showed that CAR was predominantly expressed in the epidermis of human skin, CAR knockdown by RNAi significantly accelerated HaCaT cell migration and proliferation. In addition, knockdown of CAR in vitro increased p-Src, p-p38, and p-JNK protein levels; however, Src inhibitor PP2 prevented the increase of p-Src and p-p38 induced by CAR RNAi, but not p-JNK, and decelerated cell migration and proliferation. More intriguingly, in vivo CAR RNAi on the skin area surrounding the wounds on rat back visually accelerated wound healing and re-epithelialization process, while treatment with PP2 or p38 inhibitor SB203580 obviously inhibited these effects. By contrast, overexpressing CAR in HaCaT cells significantly decelerated cell migration and proliferation. Above results demonstrate that suppression of CAR could accelerate HaCaT cell migration and proliferation, and promote wound healing in rat skin, probably via Src-p38 MAPK pathway. CAR thus might serve as a novel therapeutic target for facilitating wound healing. PMID:26804208

  5. The TLR7 agonist induces tumor regression both by promoting CD4+T cells proliferation and by reversing T regulatory cell-mediated suppression via dendritic cells

    PubMed Central

    Wang, Xiaofeng; Wu, Xiongyan; Chen, Xuehua; Li, Jianfang; Zhu, Zhenggang; Liu, Bingya; Su, Liping

    2015-01-01

    Treg-induced immunosuppression is now recognized as a key element in enabling tumors to escape immune-mediated destruction. Although topical TLR7 therapies such as imiquimod have been proved successful in the treatment of dermatological malignancy and a number of conditions beyond the FDA-approved indications, the mechanism behind the effect of TLR7 on effector T cell and Treg cell function in cancer immunosurveillance is still not well understood. Here, we found that Loxoribin, one of the TLR7 ligands, could inhibit tumor growth in xenograft models of colon cancer and lung cancer, and these anti-tumor effects of Loxoribin were mediated by promoting CD4+T cell proliferation and reversing Treg-mediated suppression via dendritic cells (DCs). However, deprivation of IL-6 using a neutralizing antibody abrogated the ability of Loxoribin-treated DCs, which reversed the Treg cell-mediated suppression. Furthermore, adoptive transfer of Loxoribin-treated DCs inhibited the tumor growth in vivo. Thus, this study links TLR7 signaling to the functional control of effector T cells and Treg cells and identifies Loxoribin as a new therapeutic strategy in cancer treatment, which may offer new opportunities to improve the outcome of cancer immunotherapy. PMID:25593198

  6. Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

    PubMed Central

    Wang, Shimin; Gao, Xiang; Shen, Guobo; Wang, Wei; Li, Jingyu; Zhao, Jingyi; Wei, Yu-Quan; Edwards, Carl K.

    2016-01-01

    Regulatory T cells (Tregs) expand in peripheral lymphoid organs and can produce immunosuppressive cytokines to support tumor growth. IL-10 abrogation efficiently induces Treg formation but dampens tumoral neuropilin-1 (Nrp-1) Treg signaling, which simultaneously augments Th1 and Th17 immunity. These effects are associated with the plasticity and stability of Tregs and effector T cell functions that can limit tumorigenesis. Within the tumor microenvironment, there appears to be a “mutual antagonism” between immunoenhancement and immunosuppression mechanisms, eventually leading to decreased metastasis. In contrast, tumor progression is paralleled by a reduction in Nrp-1-producing Tregs controlled by the IL-10 and TGF-β1 levels. However, Th1, Th17 and Treg immunity is primarily regulated by IL-10 or Nrp-1 and not TGF-β1 except when combined with IL-10. These results emphasize the important implications for the therapeutic use of Tregs. The number of Treg cells must be maintained in a healthy and dynamic homeostatic range to prevent malignant diseases. Moreover, Treg-mediated immunosuppression can be limited by reducing tumor-derived Treg Nrp-1 levels. PMID:27075020

  7. The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines.

    PubMed

    Paul, N L; Millet, I; Ruddle, N H

    1993-07-01

    The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B.

  8. Transactivation of the proenkephalin gene promoter by the Tax sub 1 protein of human T-cell lymphotropic virus type I

    SciTech Connect

    Joshi, J.B. ); Dave, H.P.G. )

    1992-02-01

    Human T-cell lymphotropic virus type I (HTLV-I), an etiologic agent for adult T-cell leukemia, is strongly associated with certain neurological diseases. The HTLV-I genome encodes a protein, Tax{sub 1}, that transactivates viral gene transcription. CD4-positive T helper lymphocytes express the proenkephalin gene, and enkephalins have been implicated as neuroimmunomodulators. The authors have investigated the effect of Tax{sub 1} on the proenkephalin gene promoter in C6 rat glioma cells and demonstrated its transactivation. Analysis using 5{prime} deletion mutants of the promoter region showed that sequences upstream of base pair - 190 are necessary for maximal transactivation. Forskolin, a cAMP modulator, synergistically increased Tax{sub 1}-mediated transactivation of the proenkephalin promoter. Neither Tax{sub 1} transactivation alone nor Tax{sub 1}/cAMP synergism exclusively involved cAMP-responsive elements. Endogenous proenkephalin gene expression increased in Tax{sub 1}-expressing C6 cells. Since HTLV-I infects lymphocytes, which express proenkephalin mRNA, Tax{sub 1} transregulation of proenkephalin expression may provide bidirectional communication between the nervous and immune systems in HTLV-I-related diseases.

  9. A cellulose-binding domain-fused recombinant human T cell connective tissue-activating peptide-III manifests heparanase activity.

    PubMed

    Rechter, M; Lider, O; Cahalon, L; Baharav, E; Dekel, M; Seigel, D; Vlodavsky, I; Aingorn, H; Cohen, I R; Shoseyov, O

    1999-02-24

    The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a lambdagt11 cDNA library of PHA-activated human CD4(+) T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators. PMID:10049766

  10. Rapid copper acquisition by developing murine mesothelioma: decreasing bioavailable copper slows tumor growth, normalizes vessels and promotes T cell infiltration.

    PubMed

    Crowe, Andrew; Jackaman, Connie; Beddoes, Katie M; Ricciardo, Belinda; Nelson, Delia J

    2013-01-01

    Copper, an essential trace element acquired through nutrition, is an important co-factor for pro-angiogenic factors including vascular endothelial growth factor (VEGF). Decreasing bioavailable copper has been used as an anti-angiogenic and anti-cancer strategy with promising results. However, the role of copper and its potential as a therapy in mesothelioma is not yet well understood. Therefore, we monitored copper levels in progressing murine mesothelioma tumors and analyzed the effects of lowering bioavailable copper. Copper levels in tumors and organs were assayed using atomic absorption spectrophotometry. Mesothelioma tumors rapidly sequestered copper at early stages of development, the copper was then dispersed throughout growing tumor tissues. These data imply that copper uptake may play an important role in early tumor development. Lowering bioavailable copper using the copper chelators, penicillamine, trientine or tetrathiomolybdate, slowed in vivo mesothelioma growth but did not provide any cures similar to using cisplatin chemotherapy or anti-VEGF receptor antibody therapy. The impact of copper lowering on tumor blood vessels and tumor infiltrating T cells was measured using flow cytometry and confocal microscopy. Copper lowering was associated with reduced tumor vessel diameter, reduced endothelial cell proliferation (reduced Ki67 expression) and lower surface ICAM/CD54 expression implying reduced endothelial cell activation, in a process similar to endothelial normalization. Copper lowering was also associated with a CD4(+) T cell infiltrate. In conclusion, these data suggest copper lowering is a potentially useful anti-mesothelioma treatment strategy that slows tumor growth to provide a window of opportunity for inclusion of other treatment modalities to improve patient outcomes.

  11. Lithium induces gene expression through lymphoid enhancer-binding factor/T-cell factor responsive element in rat PC12 cells.

    PubMed

    Bettini, Ezio; Magnani, Enrico; Terstappen, Georg C

    2002-01-01

    Lithium inhibits glycogen synthase kinase-3 (GSK-3), which leads to an increase of cytoplasmic beta-catenin levels. In some cell types, but not in others, activated beta-catenin interacts with members of the lymphoid enhancer-binding factor (LEF)/T-cell factor (TCF) family of transcription factors and induces gene expression. Lithium effect on LEF/TCF-mediated gene expression has never been evaluated in cells with a neuronal phenotype. We have constructed a LEF/TCF-dependent luciferase reporter gene to investigate lithium effects on transcription in PC12 cells. In transiently transfected PC12 cells, lithium induced a time-dependent increase in LEF/TCF-mediated luciferase activity. These results are consistent with the known inhibitory effects of lithium on GSK-3 and represent the first demonstration that a LEF/TCF responsive element also mediates lithium-induced gene expression in PC12 cells.

  12. Binding of the Galanthus nivalis agglutinin to thymocytes reveals alterations in surface glycosylation during T-cell development.

    PubMed

    Sinkora, J; Kolínská, J; Reháková, Z; Cerný, J; Doubravská, L

    2002-02-01

    Surface binding of the Galanthus nivalis agglutinin (GNA) to thymocyte subsets has been studied in pigs and rodents by multicolour flow cytometry. In all the species examined, analogous staining profiles have been recorded. Counter-staining with anti-CD3epsilon, anti-CD4 and anti-CD8 monoclonal antibodies (MoAb) revealed that a significant increase of the GNA targets on the cell surface occurred during early thymocyte differentiation and reached its maximum at the level of the CD3loCD4+CD8+ small cortical thymocyte. This was followed by a decrease in the GNA binding capacity upon terminal maturation to the single positive thymocytes. PAGE analysis has revealed a dominant GNA-binding glycoprotein (molar mass approx. 90 kDa) present on thymocyte plasma membranes and absent on the surface of splenic lymphocytes, although both the whole cell lysates from both organs contained GNA ligands of the same size. Our findings are in agreement with previous data showing that immature thymocytes differ from their mature counterparts and peripheral T lymphocytes in the surface glycosylation pattern, and support the hypothesis that lectin-glycoprotein interaction plays a significant role in the cell-to-cell crosstalk in the thymic cortex.

  13. T-cell count

    MedlinePlus

    Thymus derived lymphocyte count; T-lymphocyte count; T cell count ... T cells are a type of lymphocyte. Lymphocytes are white blood cells. They make up part of the immune system. T cells help the body fight diseases or harmful ...

  14. STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment.

    PubMed

    Koyama, Shohei; Akbay, Esra A; Li, Yvonne Y; Aref, Amir R; Skoulidis, Ferdinandos; Herter-Sprie, Grit S; Buczkowski, Kevin A; Liu, Yan; Awad, Mark M; Denning, Warren L; Diao, Lixia; Wang, Jing; Parra-Cuentas, Edwin R; Wistuba, Ignacio I; Soucheray, Margaret; Thai, Tran; Asahina, Hajime; Kitajima, Shunsuke; Altabef, Abigail; Cavanaugh, Jillian D; Rhee, Kevin; Gao, Peng; Zhang, Haikuo; Fecci, Peter E; Shimamura, Takeshi; Hellmann, Matthew D; Heymach, John V; Hodi, F Stephen; Freeman, Gordon J; Barbie, David A; Dranoff, Glenn; Hammerman, Peter S; Wong, Kwok-Kin

    2016-03-01

    STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T-cell-suppressive effects, along with a corresponding increase in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1-inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1-targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL6-neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1-mutated tumors with PD-1-targeting antibody therapies. PMID:26833127

  15. Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors.

    PubMed

    Gadola, Stephan D; Koch, Michael; Marles-Wright, Jon; Lissin, Nikolai M; Shepherd, Dawn; Matulis, Gediminas; Harlos, Karl; Villiger, Peter M; Stuart, David I; Jakobsen, Bent K; Cerundolo, Vincenzo; Jones, E Yvonne

    2006-03-20

    Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

  16. Promoter characterization of the novel human matrix metalloproteinase-26 gene: regulation by the T-cell factor-4 implies specific expression of the gene in cancer cells of epithelial origin.

    PubMed Central

    Marchenko, George N; Marchenko, Natalia D; Leng, Jay; Strongin, Alex Y

    2002-01-01

    A novel matrix metalloproteinase-26 (MMP-26) is known to be specifically expressed in epithelial carcinomas. To facilitate studies of MMP-26 transcriptional regulation, we have cloned and characterized a 1 kb 5'-flanking region of the human MMP-26 gene. Altogether, our findings indicate that the MMP-26 promoter has distinctive structural and functional features among MMP genes. An unusual polyadenylation site proximal to the transcription-factor-binding sites protects transcription of the MMP-26 gene from the upstream promoters and represents a part of the stringent transcriptional regulation of the gene. The MMP-26 gene has a consensus TATA-box and one transcriptional start site located 60 and 35 nucleotides upstream of the translational start site, respectively. The MMP-26 promoter was able to drive luciferase expression in human A549 lung carcinoma, HT1080 fibrosarcoma and HEK293 embryonic kidney cells. The basal transcription efficiency of the MMP-26 promoter is relatively low, thereby explaining the minute expression of the gene in most cells and tissues. When compared with other MMP genes, the MMP-26 promoter contains binding sites for a few transcription factors. Sequential deletion and mutation analysis, and electrophoretic mobility-shift assay have identified the T-cell factor-4 (Tcf-4) motif and the activator protein-1 site as the major regulatory elements of the MMP-26 promoter. Since previous studies have established that the Tcf-4 transcription factor is subjected exclusively to regulation through the beta-catenin/E(epithelial)-cadherin pathway, this implies the specific expression of MMP-26 in cancer cells of epithelial origin. PMID:11931652

  17. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape

    PubMed Central

    Woo, Seng-Ryong; Turnis, Meghan E.; Goldberg, Monica V.; Bankoti, Jaishree; Selby, Mark; Nirschl, Christopher J.; Bettini, Matthew L.; Gravano, David; Vogel, Peter; Liu, Chih Long; Tangsombatvisit, Stephanie; Grosso, Joseph F.; Netto, George; Smeltzer, Matthew P.; Chaux, Alcides; Utz, Paul J.; Workman, Creg J.; Pardoll, Drew M.; Korman, Alan J.; Drake, Charles G.; Vignali, Dario A.A.

    2012-01-01

    Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here we reveal extensive co-expression of PD-1 and LAG-3 on tumor-infiltrating CD4+ and CD8+ T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathological sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3−/−Pdcd1−/− mice demonstrated markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. Additionally, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer. PMID:22186141

  18. Invariant Natural Killer T cells in lupus patients promote IgG and IgG autoantibody production

    PubMed Central

    Shen, Lei; Zhang, Hong; Caimol, Maria; Benike, Claudia J.; Chakravarty, Eliza F.; Strober, Samuel; Engleman, Edgar G.

    2014-01-01

    IgG autoantibodies, including antibodies to double-stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus, but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG, in vitro. Despite the rarity of iNKT cells in lupus blood (0.002∼0.05% of CD3-positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti-dsDNA IgG by lupus PBMCs. Addition of anti-iNKT or anti-CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B cell maturation. Like fresh iNKT cells, expanded iNKT cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti-dsDNA. This activity was inhibited by anti-CD40L antibody, as well as anti-CD1d antibody, confirming a role for CD40L-CD40 and TCR-CD1d interactions in lupus iNKT mediated help. These results reveal a critical role for iNKT cells in B cell maturation and autoantibody production in patients with lupus. PMID:25352488

  19. IL-2 / α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment

    PubMed Central

    Mahr, Benedikt; Unger, Lukas; Hock, Karin; Pilat, Nina; Baranyi, Ulrike; Schwarz, Christoph; Maschke, Svenja; Farkas, Andreas Michael; Wekerle, Thomas

    2016-01-01

    Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p<0.05). Adding IL-2 complexes to an otherwise effective regimen involving recipient irradiation (1Gy) but no Treg transfer indeed actively triggered donor BM rejection at higher doses (0/8 with IL-2 complexes vs. 9/11 without, p<0.01) and had no detectable effect at two lower doses (3/5 vs. 9/11, p>0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM

  20. IL-2/α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment.

    PubMed

    Mahr, Benedikt; Unger, Lukas; Hock, Karin; Pilat, Nina; Baranyi, Ulrike; Schwarz, Christoph; Maschke, Svenja; Farkas, Andreas Michael; Wekerle, Thomas

    2016-01-01

    Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p<0.05). Adding IL-2 complexes to an otherwise effective regimen involving recipient irradiation (1Gy) but no Treg transfer indeed actively triggered donor BM rejection at higher doses (0/8 with IL-2 complexes vs. 9/11 without, p<0.01) and had no detectable effect at two lower doses (3/5 vs. 9/11, p>0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM

  1. H3K27 Demethylation at the Proviral Promoter Sensitizes Latent HIV to the Effects of Vorinostat in Ex Vivo Cultures of Resting CD4+ T Cells

    PubMed Central

    Tripathy, Manoj K.; McManamy, Mary E. M.; Burch, Brandon D.; Archin, Nancie M.

    2015-01-01

    infected primary resting CD4+ T cells is synergistically increased upon exposure to the histone deacetylase inhibitor vorinostat. Demethylation at H3K27 sensitizes the HIV promoter to the effects of an HDACi and provides a proof-of-concept for the testing of combination epigenetic approaches to disrupt latent HIV infection, a necessary step toward the eradication of HIV infection. PMID:26041287

  2. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    PubMed

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

  3. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    PubMed

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape. PMID:27427982

  4. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16).

    PubMed

    Spruyt, L L; Glennie, M J; Beyers, A D; Williams, A F

    1991-12-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.

  5. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16)

    PubMed Central

    1991-01-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i. PMID:1683892

  6. Liposomal vaccines incorporating molecular adjuvants and intrastructural T-cell help promote the immunogenicity of HIV membrane-proximal external region peptides

    PubMed Central

    Hanson, Melissa C.; Abraham, Wuhbet; Crespo, Monica P.; Chen, Stephanie H.; Liu, Haipeng; Szeto, Greg Lee; Kim, Mikyung; Reinherz, Ellis L.; Irvine, Darrell J.

    2015-01-01

    An HIV vaccine capable of inducing high and durable levels of broadly neutralizing antibodies has thus far proven elusive. A promising antigen is the membrane-proximal external region (MPER) from gp41, a segment of the viral envelope recognized by a number of broadly neutralizing antibodies. Though an attractive vaccine target due to the linear nature of the epitope and its highly conserved sequence, MPER peptides are poorly immunogenic and may require display on membranes to achieve a physiological conformation matching the native virus. Here we systematically explored how the structure and composition of liposomes displaying MPER peptides impacts the strength and durability of humoral responses to this antigen as well as helper T-cell responses in mice. Administration of MPER peptides anchored to the surface of liposomes induced MPER-specific antibodies whereas MPER administered in oil-based emulsion adjuvants or alum did not, even when combined with Toll like receptor agonists. High-titer IgG responses to liposomal MPER required the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced by incorporating high-Tm lipids in the vesicle bilayer and optimizing the MPER density to a mean distance of ~10–15 nm between peptides on the liposomes' surfaces. Encapsulation of helper epitopes within the vesicles allowed efficient “intrastructural” T-cell help, which promoted IgG responses to MPER while minimizing competing B-cell responses against the helper sequence. These results define several key properties of liposome formulations that promote durable, high-titer antibody responses against MPER peptides, which will be a prerequisite for a successful MPER-targeting vaccine. PMID:25559188

  7. Complement component C5a Promotes Expression of IL-22 and IL-17 from Human T cells and its Implication in Age-related Macular Degeneration

    PubMed Central

    2011-01-01

    Background Age related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH), the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a) have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD. Methods Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500. Results We show that C5a promotes interleukin (IL)-22 and IL-17 expression by human CD4+ T cells. This effect is dependent on B7, IL-1β and IL-6 expression from monocytes. We have also found that C5a could protect human CD4+ cells from undergoing apoptosis. Importantly, consistent with a role of C5a in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls. Conclusions Our results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD. PMID:21762495

  8. Cloning of two members of the SIRP alpha family of protein tyrosine phosphatase binding proteins in cattle that are expressed on monocytes and a subpopulation of dendritic cells and which mediate binding to CD4 T cells.

    PubMed

    Brooke, G P; Parsons, K R; Howard, C J

    1998-01-01

    Recent experimental studies have greatly clarified the function of cell surface molecules in the induction and modulation of T cell responses by antigen-presenting cells (APC). However, the differences in ability to stimulate T cells evident for different types and subpopulations of the same APC, such as dendritic cell subsets, is less well understood. This report details an investigation of an antigen expressed on monocytes that is also expressed on a subset of cattle afferent lymph veiled cells (ALVC). A cDNA library derived from cattle monocytes was screened with monoclonal antibodies (mAb) for expression in COS-7 cells. Using separate mAb for screening, two cDNA were cloned, the sequences of which showed a single long open reading frame encoding a predicted type I glycoprotein of 506 amino acids that contained three immunoglobulin superfamily domains and a long 112-amino acid cytoplasmic tail. We have termed this antigen MyD-1, reflecting its myeloid and dendritic cell distribution. Analysis of the EMBL database revealed that the molecule is a member of the recently described family of signal regulatory proteins (SIRP). The outeremost Ig domain was of the adhesion/receptor I-type, suggesting that MyD-1 might bind to a ligand on another cell. Evidence for this was subsequently obtained by demonstrating that COS-7 cells transfected with MyD-1 cDNA bound CD4 T cells and this binding was blocked by specific mAb. The potential importance of this interaction was supported by the finding that the proliferation of resting memory CD4 T cells to ovalbumin-pulsed monocytes was significantly reduced in the presence of mAb to MyD-1. A role for the molecule in the modulation of the monocyte/dendritic APC response is also predicted from the existence of multiple potential tyrosine phosphorylation sites in the cytoplasmic domain, including the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the observation that the SIRP alpha family members have been

  9. MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T cells Promotes Mucosal Tolerance and Enforces Commensalism

    PubMed Central

    Wang, Sen; Charbonnier, Louis-Marie; Rivas, Magali Noval; Georgiev, Peter; Li, Ning; Gerber, Georg; Bry, Lynn; Chatila, Talal A

    2015-01-01

    SUMMARY Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll like receptors (TLR). We report that Treg cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper-17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer’s patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA-responses. PMID:26231118

  10. MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism.

    PubMed

    Wang, Sen; Charbonnier, Louis-Marie; Noval Rivas, Magali; Georgiev, Peter; Li, Ning; Gerber, Georg; Bry, Lynn; Chatila, Talal A

    2015-08-18

    Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll-like receptors (TLRs). We report that Treg-cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper 17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer's patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA responses.

  11. Predicting promiscuous antigenic T cell epitopes of Mycobacterium tuberculosis mymA operon proteins binding to MHC Class I and Class II molecules.

    PubMed

    Saraav, Iti; Pandey, Kirti; Sharma, Monika; Singh, Swati; Dutta, Prasun; Bhardwaj, Anshu; Sharma, Sadhna

    2016-10-01

    Limited efficacy of Bacillus Calmette-Guérin vaccine has raised the need to explore other immunogenic candidates to develop an effective vaccine against Mycobacterium tuberculosis (Mtb). Both CD4+ and CD8+ T cells play a critical role in host immunity to Mtb. Infection of macrophages with Mtb results in upregulation of mymA operon genes thereby suggesting their importance as immune targets. In the present study, after exclusion of self-peptides mymA operon proteins of Mtb were analyzed in silico for the presence of Human Leukocyte Antigen (HLA) Class I and Class II binding peptides using Bioinformatics and molecular analysis section, NetMHC 3.4, ProPred and Immune epitope database software. Out of 56 promiscuous epitopes obtained, 41 epitopes were predicted to be antigenic for MHC Class I. In MHC Class II, out of 336 promiscuous epitopes obtained, 142 epitopes were predicted to be antigenic. The comparative bioinformatics analysis of mymA operon proteins found Rv3083 to be the best vaccine candidate. Molecular docking was performed with the most antigenic peptides of Rv3083 (LASGAASVV with alleles HLA-B51:01, HAATSGTLI with HLA-A02, IVTATGLNI and EKIHYGLKVNTA with HLA-DRB1_01:01) to study the structural basis for recognition of peptides by various HLA molecules. The software binding prediction was validated by the obtained molecular docking score of peptide-HLA complex. These peptides can be further investigated for their immunological relevance in patients of tuberculosis using major histocompatibility complex tetramer approach. PMID:27389362

  12. Evidence for protein kinase C-dependent and -independent activation of mitogen-activated protein kinase in T cells: potential role of additional diacylglycerol binding proteins.

    PubMed

    Puente, L G; Stone, J C; Ostergaard, H L

    2000-12-15

    Activation of mitogen-activated protein kinases (MAPK) is a critical signal transduction event for CTL activation, but the signaling mechanisms responsible are not fully characterized. Protein kinase C (PKC) is thought to contribute to MAPK activation following TCR stimulation. We have found that dependence on PKC varies with the method used to stimulate the T cells. Extracellular signal-regulated kinase (ERK) activation in CTL stimulated with soluble cross-linked anti-CD3 is completely inhibited by the PKC inhibitor bisindolylmaleimide (BIM). In contrast, only the later time points in the course of ERK activation are sensitive to BIM when CTL are stimulated with immobilized anti-CD3, a condition that stimulates CTL degranulation. Surprisingly, MAPK activation in response to immobilized anti-CD3 is strongly inhibited at all time points by the diacylglycerol (DAG)-binding domain inhibitor calphostin C implicating the contribution of a DAG-dependent but PKC-independent pathway in the activation of ERK in CTL clones. Chronic exposure to phorbol ester down-regulates the expression of DAG-responsive PKC isoforms; however, this treatment of CTL clones does not inhibit anti-CD3-induced activation of MAPK. Phorbol ester-treated cells have reduced expression of several isoforms of PKC but still express the recently described DAG-binding Ras guanylnucleotide-releasing protein. These results indicate that the late phase of MAPK activation in CTL clones in response to immobilized anti-CD3 stimulation requires PKC while the early phase requires a DAG-dependent, BIM-resistant component.

  13. Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L

    PubMed Central

    Ramello, M C; Tosello Boari, J; Canale, F P; Mena, H A; Negrotto, S; Gastman, B; Gruppi, A; Acosta Rodríguez, E V; Montes, C L

    2014-01-01

    Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1β and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer. PMID:25375372

  14. Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L.

    PubMed

    Ramello, M C; Tosello Boari, J; Canale, F P; Mena, H A; Negrotto, S; Gastman, B; Gruppi, A; Acosta Rodríguez, E V; Montes, C L

    2014-01-01

    Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1β and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer.

  15. Interleukin-12 is sufficient to promote antigen-independent interferon-γ production by CD8 T cells in old mice

    PubMed Central

    Rottinghaus, Erin K; Vesosky, Bridget; Turner, Joanne

    2009-01-01

    Numerous functional defects have been identified in naive T cells from aged mice, including deficiencies in proliferation, cytokine production and signal transduction. It is well documented that the ratio of naïve to memory T cells significantly decreases with age resulting in the majority of T cells from aged hosts expressing activated/memory T-cell markers (CD44hi), yet it is unclear whether T cells with a CD44hi phenotype in aged hosts are functionally equivalent to T cells with a similar phenotype in young hosts. We have identified a population of CD44hi CD8 T cells in old mice that are capable of secreting interferon-γ (IFN-γ) in response to interleukin-12 (IL-12) stimulation. This occurred in the absence of T-cell receptor engagement, a function that was not observed in CD8 T cells from young mice. This phenotype was associated with increased IL-12 receptor β2 gene expression and IL-12 induced signal transducer and activator of transcription 4 (STAT-4) activation, even when CD8 T-cell numbers from young and old mice were normalized for CD44hi expression. Furthermore, we demonstrate that IL-12-induced STAT-4 activation was required for T helper type 1 (Th1) cytokine-induced IFN-γ production in CD8 T cells. These data illustrate that old mice possess a specialized subset of CD44hi CD8 T cells with an enhanced responsiveness to IL-12, enabling these cells to produce substantial amounts of IFN-γ in response to Th1 cytokine stimulation. We have therefore identified a functional difference in the populations of CD44hi CD8 T cells from young and old mice, and believe that understanding age-associated immunological changes is essential for helping the elderly combat deadly diseases. PMID:19740329

  16. Oestradiol potentiates the suppressive function of human CD4+ CD25+ regulatory T cells by promoting their proliferation

    PubMed Central

    Prieto, G Aleph; Rosenstein, Yvonne

    2006-01-01

    CD4+ CD25+ regulatory T (Treg) cells play an important role in the control of the immune system by suppressing the proliferation of effector cells, thereby preventing autoreactive, unnecessary or inconvenient responses. Recently, it has been shown that the number of Treg cells increases during pregnancy, a period with high serum levels of female sex hormones. Oestrogen replacement therapy has been reported to alleviate the symptoms of autoimmune diseases, yet the cellular and molecular mechanisms involved are not fully understood. Here, we show that physiological doses of oestradiol (E2) found during pregnancy, combined with activation through CD3/CD28 engagement, promoted the proliferation of Treg cells without altering their suppressive phenotype. Enhanced suppression was detected when Treg cells were pretreated with the hormone as well as when both cell subpopulations (Treg and T effector) were exposed to E2 throughout the experiment. Together, these data suggest that when combined with an activating stimulus, E2 can modulate the function of human Treg cells by regulating their numbers, and highlight a potential use of E2, or its analogs, to manipulate Treg function. PMID:16630023

  17. Anti-inflammatory effects of interleukin-23 receptor cytokine-binding homology region rebalance T cell distribution in rodent collagen-induced arthritis

    PubMed Central

    Guo, Wei; Yu, Dongmei; Wang, Xin; Luo, Cheng; Chen, Yucong; Lei, Wen; Wang, Chen; Ge, Yaoyao; Xue, Wenyao; Tian, Qiqi; Gao, Xiangdong; Yao, Wenbing

    2016-01-01

    IL-23 is an important cytokine to regulate Th17 cell differentiation and promote the proliferation of inflammatory cells in Th17-mediated autoimmune diseases. The collagen-induced arthritis (CIA) in rat is a model of rheumatoid arthritis characterized by pronounced inflammatory auto-responses from B and T cells, especially Th17 cells in lesions. In the present study, we used rhIL23R-CHR to block the IL-23 signaling pathway to probe the importance of IL-23 in misbalancing the ratio of Th17/Th9/Treg cells in CIA rats. After treatments with rhIL23R-CHR, the CIA rats showed a significant decrease of secretions of IL-17 and IL-9, whereas FoxP3 was activated in the process, indicating that IL-23 can manipulate the balance of Th17/Th9/Treg cells. Similar to the animal model, IL-23 also possessed remarkable proinflammatory effects on human fibroblast-like synoviocyte cells (HFLS), showing synergetic outcomes with TNF-α. Together, IL-23 could act as a modulator to imbalance the ratio of Th17/Th9/Treg cells, and rhIL23R-CHR could serve as a potential therapeutic agent for RA patients. PMID:27177334

  18. Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

    PubMed Central

    Strauss, Laura; Czystowska, Malgorzata; Szajnik, Marta; Mandapathil, Magis; Whiteside, Theresa L.

    2009-01-01

    Background The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-γ chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. Methodology/Principal Findings CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway. Conclusions/Significance RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation. PMID:19543393

  19. miR-664 negatively regulates PLP2 and promotes cell proliferation and invasion in T-cell acute lymphoblastic leukaemia

    SciTech Connect

    Zhu, Hong; Miao, Mei-hua; Ji, Xue-qiang; Xue, Jun; Shao, Xue-jun

    2015-04-03

    MicroRNAs (miRNAs) play important roles in the pathogenesis of many types of cancers by negatively regulating gene expression at posttranscriptional level. However, the role of microRNAs in leukaemia, particularly T-cell acute lymphoblastic leukaemia (T-ALL), has remained elusive. Here, we identified miR-664 and its predicted target gene PLP2 were differentially expressed in T-ALL using bioinformatics methods. In T-ALL cell lines, CCK-8 proliferation assay indicated that the cell proliferation was promoted by miR-664, while miR-664 inhibitor could significantly inhibited the proliferation. Moreover, migration and invasion assay showed that overexpression of miR-664 could significantly promoted the migration and invasion of T-ALL cells, whereas miR-664 inhibitor could reduce cell migration and invasion. luciferase assays confirmed that miR-664 directly bound to the 3'untranslated region of PLP2, and western blotting showed that miR-664 suppressed the expression of PLP2 at the protein levels. This study indicated that miR-664 negatively regulates PLP2 and promotes proliferation and invasion of T-ALL cell lines. Thus, miR-664 may represent a potential therapeutic target for T-ALL intervention. - Highlights: • miR-664 mimics promote the proliferation and invasion of T-ALL cells. • miR-664 inhibitors inhibit the proliferation and invasion of T-ALL cells. • miR-664 targets 3′ UTR of PLP2 in T-ALL cells. • miR-664 negatively regulates PLP2 in T-ALL cells.

  20. Nab2 regulates secondary CD8+ T-cell responses through control of TRAIL expression

    PubMed Central

    Gerlach, Carmen; Arens, Ramon; Janssen, Edith M.; Fitzgerald, Patrick; Schumacher, Ton N.; Medema, Jan Paul; Green, Douglas R.; Schoenberger, Stephen P.

    2012-01-01

    CD4+ Th cells are pivotal for the generation and maintenance of CD8+ T-cell responses. “Helped” CD8+ T cells receive signals during priming that prevent the induction of the proapoptotic molecule TNF-related apoptosis-inducing ligand (TRAIL) during reactivation, thereby enabling robust secondary expansion. Conversely, “helpless” CD8+ T cells primed in the absence of Th induce TRAIL expression after restimulation and undergo activation-induced cell death. In the present study, we investigated the molecular basis for the differential regulation of TRAIL in helped versus helpless CD8+ T cells by comparing their transcriptional profiles, and have identified a transcriptional corepressor, NGFI-A binding protein 2 (Nab2), that is selectively induced in helped CD8+ T cells. Enforced expression of Nab2 prevents TRAIL induction after restimulation of primary helpless CD8+ T cells, and expression of a dominant-negative form of Nab2 in helped CD8+ T cells impairs their secondary proliferative response that is reversible by TRAIL blockade. Finally, we observe that the CD8+ T-cell autocrine growth factor IL-2 coordinately increases Nab2 expression and decreases TRAIL expression. These findings identify Nab2 as a mediator of Th-dependent CD8+ T-cell memory responses through the regulation of TRAIL and the promotion of secondary expansion, and suggest a mechanism through which this operates. PMID:22128144

  1. Loss of ABCG1 influences regulatory T cell differentiation and atherosclerosis.

    PubMed

    Cheng, Hsin-Yuan; Gaddis, Dalia E; Wu, Runpei; McSkimming, Chantel; Haynes, LaTeira D; Taylor, Angela M; McNamara, Coleen A; Sorci-Thomas, Mary; Hedrick, Catherine C

    2016-09-01

    ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol accumulation and alters T cell homeostasis, which may contribute to progression of atherosclerosis. Here, we investigated how the selective loss of ABCG1 in T cells impacts atherosclerosis in LDL receptor-deficient (LDLR-deficient) mice, a model of the disease. In LDLR-deficient mice fed a high-cholesterol diet, T cell-specific ABCG1 deficiency protected against atherosclerotic lesions. Furthermore, T cell-specific ABCG1 deficiency led to a 30% increase in Treg percentages in aorta and aorta-draining lymph nodes (LNs) of these mice compared with animals with only LDLR deficiency. When Abcg1 was selectively deleted in Tregs of LDLR-deficient mice, we observed a 30% increase in Treg percentages in aorta and aorta-draining LNs and reduced atherosclerosis. In the absence of ABCG1, intracellular cholesterol accumulation led to downregulation of the mTOR pathway, which increased the differentiation of naive CD4 T cells into Tregs. The increase in Tregs resulted in reduced T cell activation and increased IL-10 production by T cells. Last, we found that higher ABCG1 expression in Tregs was associated with a higher frequency of these cells in human blood samples. Our study indicates that ABCG1 regulates T cell differentiation into Tregs, highlighting a pathway by which cholesterol accumulation can influence T cell homeostasis in atherosclerosis. PMID:27482882

  2. Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains (Pag), a Novel Ubiquitously Expressed Transmembrane Adaptor Protein, Binds the Protein Tyrosine Kinase Csk and Is Involved in Regulation of T Cell Activation

    PubMed Central

    Brdic̆ka, Tomás̆; Pavlis̆tová, Dagmar; Leo, Albrecht; Bruyns, Eddy; Kor̆ínek, Vladimír; Angelisová, Pavla; Scherer, Jeanette; Shevchenko, Andrej; Shevchenko, Anna; Hilgert, Ivan; C̆erný, Jan; Drbal, Karel; Kuramitsu, Yasuhiro; Kornacker, Birgit; Hor̆ejs̆í, Václav; Schraven, Burkhart

    2000-01-01

    According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood α/β T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood α/β T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor–mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG–Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state. PMID:10790433

  3. Regulation of the tumor marker Fascin by the viral oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) depends on promoter activation and on a promoter-independent mechanism.

    PubMed

    Mohr, Caroline F; Gross, Christine; Bros, Matthias; Reske-Kunz, Angelika B; Biesinger, Brigitte; Thoma-Kress, Andrea K

    2015-11-01

    Adult T-cell leukemia/lymphoma is a highly infiltrative neoplasia of CD4(+) T-lymphocytes that occurs in about 5% of carriers infected with the deltaretrovirus human T-cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax perturbs cellular signaling pathways leading to upregulation of host cell factors, amongst them the actin-bundling protein Fascin, an invasion marker of several types of cancer. However, transcriptional regulation of Fascin by Tax is poorly understood. In this study, we identified a triple mode of transcriptional induction of Fascin by Tax, which requires (1) NF-κB-dependent promoter activation, (2) a Tax-responsive region in the Fascin promoter, and (3) a promoter-independent mechanism sensitive to the Src family kinase inhibitor PP2. Thus, Tax regulates Fascin by a multitude of signals. Beyond, using Tax-expressing and virus-transformed lymphocytes as a model system, our study is the first to identify the invasion marker Fascin as a novel target of PP2, an inhibitor of metastasis.

  4. Androgens Up-regulate Transcription of the Notch Inhibitor Numb in C2C12 Myoblasts via Wnt/β-Catenin Signaling to T Cell Factor Elements in the Numb Promoter*

    PubMed Central

    Liu, Xin-Hua; Wu, Yong; Yao, Shen; Levine, Alice C.; Kirschenbaum, Alexander; Collier, Lauren; Bauman, William A.; Cardozo, Christopher P.

    2013-01-01

    Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3β. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which β-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling. PMID:23649620

  5. Dexamethasone and Monophosphoryl Lipid A-Modulated Dendritic Cells Promote Antigen-Specific Tolerogenic Properties on Naive and Memory CD4+ T Cells

    PubMed Central

    Maggi, Jaxaira; Schinnerling, Katina; Pesce, Bárbara; Hilkens, Catharien M.; Catalán, Diego; Aguillón, Juan C.

    2016-01-01

    Tolerogenic dendritic cells (DCs) are a promising tool to control T cell-mediated autoimmunity. Here, we evaluate the ability of dexamethasone-modulated and monophosphoryl lipid A (MPLA)-activated DCs [MPLA-tolerogenic DCs (tDCs)] to exert immunomodulatory effects on naive and memory CD4+ T cells in an antigen-specific manner. For this purpose, MPLA-tDCs were loaded with purified protein derivative (PPD) as antigen and co-cultured with autologous naive or memory CD4+ T cells. Lymphocytes were re-challenged with autologous PPD-pulsed mature DCs (mDCs), evaluating proliferation and cytokine production by flow cytometry. On primed-naive CD4+ T cells, the expression of regulatory T cell markers was evaluated and their suppressive ability was assessed in autologous co-cultures with CD4+ effector T cells and PPD-pulsed mDCs. We detected that memory CD4+ T cells primed by MPLA-tDCs presented reduced proliferation and proinflammatory cytokine expression in response to PPD and were refractory to subsequent stimulation. Naive CD4+ T cells were instructed by MPLA-tDCs to be hyporesponsive to antigen-specific restimulation and to suppress the induction of T helper cell type 1 and 17 responses. In conclusion, MPLA-tDCs are able to modulate antigen-specific responses of both naive and memory CD4+ T cells and might be a promising strategy to “turn off” self-reactive CD4+ effector T cells in autoimmunity.

  6. Dexamethasone and Monophosphoryl Lipid A-Modulated Dendritic Cells Promote Antigen-Specific Tolerogenic Properties on Naive and Memory CD4+ T Cells

    PubMed Central

    Maggi, Jaxaira; Schinnerling, Katina; Pesce, Bárbara; Hilkens, Catharien M.; Catalán, Diego; Aguillón, Juan C.

    2016-01-01

    Tolerogenic dendritic cells (DCs) are a promising tool to control T cell-mediated autoimmunity. Here, we evaluate the ability of dexamethasone-modulated and monophosphoryl lipid A (MPLA)-activated DCs [MPLA-tolerogenic DCs (tDCs)] to exert immunomodulatory effects on naive and memory CD4+ T cells in an antigen-specific manner. For this purpose, MPLA-tDCs were loaded with purified protein derivative (PPD) as antigen and co-cultured with autologous naive or memory CD4+ T cells. Lymphocytes were re-challenged with autologous PPD-pulsed mature DCs (mDCs), evaluating proliferation and cytokine production by flow cytometry. On primed-naive CD4+ T cells, the expression of regulatory T cell markers was evaluated and their suppressive ability was assessed in autologous co-cultures with CD4+ effector T cells and PPD-pulsed mDCs. We detected that memory CD4+ T cells primed by MPLA-tDCs presented reduced proliferation and proinflammatory cytokine expression in response to PPD and were refractory to subsequent stimulation. Naive CD4+ T cells were instructed by MPLA-tDCs to be hyporesponsive to antigen-specific restimulation and to suppress the induction of T helper cell type 1 and 17 responses. In conclusion, MPLA-tDCs are able to modulate antigen-specific responses of both naive and memory CD4+ T cells and might be a promising strategy to “turn off” self-reactive CD4+ effector T cells in autoimmunity. PMID:27698654

  7. Genome-wide approaches reveal functional vascular endothelial growth factor (VEGF)-inducible nuclear factor of activated T cells (NFAT) c1 binding to angiogenesis-related genes in the endothelium.

    PubMed

    Suehiro, Jun-ichi; Kanki, Yasuharu; Makihara, Chihiro; Schadler, Keri; Miura, Mai; Manabe, Yuuka; Aburatani, Hiroyuki; Kodama, Tatsuhiko; Minami, Takashi

    2014-10-17

    VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.

  8. IL-21-Induced MHC Class II+ NK Cells Promote the Expansion of Human Uncommitted CD4+ Central Memory T Cells in a Macrophage Migration Inhibitory Factor-Dependent Manner.

    PubMed

    Loyon, Romain; Picard, Emilie; Mauvais, Olivier; Queiroz, Lise; Mougey, Virginie; Pallandre, Jean-René; Galaine, Jeanne; Mercier-Letondal, Patricia; Kellerman, Guillaume; Chaput, Nathalie; Wijdenes, John; Adotévi, Olivier; Ferrand, Christophe; Romero, Pedro; Godet, Yann; Borg, Christophe

    2016-07-01

    NK cells are critical for innate immunity-mediated protection. The main roles of NK cells rely on their cytotoxic functions or depend on the tuning of Th1 adaptive immunity by IFN-γ. However, the precise influence of inflammatory cytokines on NK cell and CD4 T lymphocyte interactions was never investigated. In this study, we provide evidence that IL-21, a cytokine produced during chronic inflammation or infectious diseases, promotes the differentiation of a specific subset of NK cells coexpressing CD86 and HLA-DR and lacking NKp44. More importantly, IL-21-propagated HLA-DR(+) NK cells produce macrophage migration inhibitory factor and provide costimulatory signaling during naive CD4(+) T cell priming inducing the differentiation of uncommitted central memory T cells. Central memory T cells expanded in the presence of HLA-DR(+) NK cells are CXCR3(+)CCR6(-)CCR4(-)CXCR5(-) and produce IL-2, as well as low levels of TNF-α. Costimulation of CD4(+) T cells by HLA-DR(+) NK cells prevents the acquisition of effector memory phenotype induced by IL-2. Moreover, we identified this population of NK HLA-DR(+) macrophage migration inhibitory factor(+) cells in inflammatory human appendix. Collectively, these results demonstrate a novel function for IL-21 in tuning NK and CD4(+) T cell interactions promoting a specific expansion of central memory lymphocytes. PMID:27233967

  9. Crosstalk between human DC subsets promotes antibacterial activity and CD8+ T-cell stimulation in response to bacille Calmette-Guérin

    PubMed Central

    Lozza, Laura; Farinacci, Maura; Faé, Kellen; Bechtle, Marina; Stäber, Manuela; Dorhoi, Anca; Bauer, Mario; Ganoza, Christian; Weber, Stephan; Kaufmann, Stefan HE

    2014-01-01

    To date, little is known about the unique contributions of specialized human DC subsets to protection against tuberculosis (TB). Here, we focus on the role of human plasmacytoid (p)DCs and myeloid (m)DCs in the immune response to the TB vaccine bacille Calmette-Guérin (BCG). Ex vivo DC subsets from human peripheral blood were purified and infected with BCG expressing GFP to distinguish between infected and noninfected cells. BDCA-1+ myeloid DCs were more susceptible than BDCA-3+ mDCs to BCG infection. Plasmacytoid DCs have poor phagocytic activity but are equipped with endocytic receptors and can be activated by bystander stimulation. Consequently, the mutual interaction of the two DC subsets in response to BCG was analyzed. We found that pDCs were activated by BCG-infected BDCA-1+ mDCs to upregulate maturation markers and to produce granzyme B, but not IFN-α. Reciprocally, the presence of activated pDCs enhanced mycobacterial growth control by infected mDCs and increased IL-1β availability. The synergy between the two DC subsets promoted BCG-specific CD8+ T-cell stimulation and the role of BCG-infected BDCA-1+ mDCs could not be efficiently replaced by infected BDCA-3+ mDCs in the crosstalk with pDCs. We conclude that mDC–pDC crosstalk should be exploited for rational design of next-generation TB vaccines. PMID:24114554

  10. Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4⁺ T cells to T-regulatory cells.

    PubMed

    Olkhanud, Purevdorj B; Damdinsuren, Bazarragchaa; Bodogai, Monica; Gress, Ronald E; Sen, Ranjan; Wejksza, Katarzyna; Malchinkhuu, Enkhzol; Wersto, Robert P; Biragyn, Arya

    2011-05-15

    Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However, it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells, designated tumor-evoked Bregs (tBreg), phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-β-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs, 4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications, as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis. PMID:21444674

  11. Anti-G-CSF treatment induces protective tumor immunity in mouse colon cancer by promoting NK cell, macrophage and T cell responses

    PubMed Central

    Morris, Katherine T.; Castillo, Eliseo F.; Ray, Anita L.; Weston, Lea L.; Nofchissey, Robert A.; Hanson, Joshua A.; Samedi, Von G.; Pinchuk, Irina V.; Hudson, Laurie G.; Beswick, Ellen J.

    2015-01-01

    Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is highly expressed in human and mouse colorectal cancers (CRC). We previously reported that G-CSF stimulated human CRC cell growth and migration, therefore in this study we sought to examine the therapeutic potential of anti-G-CSF treatment for CRC. G-CSF is known to mobilize neutrophils, however its impact on other immune cells has not been well examined. Here, we investigated the effects of therapeutic anti-G-CSF treatment on CRC growth and anti-tumor immune responses. C57BL/6 mice treated with azoxymethane/dextran sodium sulfate (AOM/DSS) to induce neoplasms were administered anti-G-CSF or isotype control antibodies three times a week for three weeks. Animals treated with anti-G-CSF antibodies had a marked decrease in neoplasm number and size compared to the isotype control group. Colon neutrophil and macrophage frequency were unchanged, but the number of macrophages producing IL-10 were decreased while IL-12 producing macrophages were increased. NK cells were substantially increased in colons of anti-G-CSF treated mice, along with IFNγ producing CD4+ and CD8+ T cells. These studies are the first to indicate a crucial role for G-CSF inhibition in promoting protective anti-tumor immunity, and suggest that anti-G-CSF treatment is a potential therapeutic approach for CRC. PMID:26061815

  12. Global T cell dysregulation in non-autoimmune-prone mice promotes rapid development of BAFF-independent, SLE-like autoimmunity1

    PubMed Central

    Stohl, William; Jacob, Noam; Quinn, William J.; Cancro, Michael P.; Gao, Huaxin; Putterman, Chaim; Gao, Xiaoni; Pricop, Luminita; Koss, Michael N.

    2008-01-01

    In otherwise non-autoimmune-prone C57BL/6 (B6) mice rendered genetically deficient in CD152 (CTLA-4), polyclonal hypergammaglobulinemia with increased levels of SLE-associated IgG autoantibodies, glomerular IgG and C3 deposition, and interstitial nephritis all developed by 3-5 weeks of age. Remarkably, superimposing genetic deficiency of BAFF onto CD152 deficiency did not substantially attenuate humoral autoimmunity and immunopathology in these mice, despite the resulting marked reduction in B-lineage cells. Although superimposing a BAFF transgene (resulting in constitutive BAFF overexpression) onto CD152-deficient mice did lead to increases in B-lineage cells and serum levels of certain SLE-associated IgG autoantibodies, renal immunopathology remained largely unaffected. Taken together, these results demonstrate that global T cell dysregulation, even in an otherwise non-autoimmune-prone host, can promote systemic humoral autoimmunity and immunopathology in a BAFF-independent manner. Moreover, supra-physiologic expression of BAFF in the setting of ongoing autoimmunity does not necessarily lead to greater immunopathology. These findings may help explain the limited clinical efficacy appreciated to date of BAFF antagonists in human SLE. PMID:18566449

  13. Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells.

    PubMed Central

    Eisenlohr, L C; Gerhard, W; Hackett, C J

    1987-01-01

    The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants. PMID:2952806

  14. Critical role of the tumor suppressor tuberous sclerosis complex 1 in dendritic cell activation of CD4 T cells by promoting MHC class II expression via IRF4 and CIITA.

    PubMed

    Pan, Hongjie; O'Brien, Thomas F; Wright, Gabriela; Yang, Jialong; Shin, Jinwook; Wright, Kenneth L; Zhong, Xiao-Ping

    2013-07-15

    Dendritic cell (DC) maturation is characterized by upregulation of cell-surface MHC class II (MHC-II) and costimulatory molecules, and production of a variety of cytokines that can shape both innate and adaptive immunity. Paradoxically, transcription of the MHC-II genes, as well as its activator, CIITA, is rapidly silenced during DC maturation. The mechanisms that control CIITA/MHC-II expression and silencing have not been fully understood. We report in this article that the tumor suppressor tuberous sclerosis complex 1 (TSC1) is a critical regulator of DC function for both innate and adaptive immunity. Its deficiency in DCs results in increased mammalian target of rapamycin (mTOR) complex 1 but decreased mTORC2 signaling, altered cytokine production, impaired CIITA/MHC-II expression, and defective Ag presentation to CD4 T cells after TLR4 stimulation. We demonstrate further that IFN regulatory factor 4 can directly bind to CIITA promoters, and decreased IFN regulatory factor 4 expression is partially responsible for decreased CIITA/MHC-II expression in TSC1-deficient DCs. Moreover, we identify that CIITA/MHC-II silencing during DC maturation requires mTOR complex 1 activity. Together, our data reveal unexpected roles of TSC1/mTOR that control multifaceted functions of DCs.

  15. Iris Pigment Epithelium Expressing CD86 (B7-2) Directly Suppresses T Cell Activation In Vitro via Binding to Cytotoxic T Lymphocyte–associated Antigen 4

    PubMed Central

    Sugita, Sunao; Streilein, J. Wayne

    2003-01-01

    A monolayer of pigment epithelium (PE) lines the iris PE (IPE), ciliary body PE, and retina PE of the inner eye, an immune-privileged site. These neural crest-derived epithelial cells participate in ocular immune privilege through poorly defined molecular mechanisms. Murine PE cells cultured from different ocular tissues suppress T cell activation by differing mechanisms. In particular, IPE cells suppress primarily via direct cell to cell contact. By examining surface expression of numerous candidate molecules (tumor necrosis factor receptor [TNFR]1, TNFR2, CD36, CD40, CD47, CD80, CD86, PD-L1, CD95 ligand, and type I interferon receptor), we report that IPE cells uniquely express on their surface the costimulatory molecule CD86. When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation. IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice. We conclude that iris pigment epithelial cells constitutively express cell surface CD86, which enables the cells to contact inhibit T cells via direct interaction with CTLA-4. Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation. PMID:12835481

  16. The Immunomodulator VacA Promotes Immune Tolerance and Persistent Helicobacter pylori Infection through Its Activities on T-Cells and Antigen-Presenting Cells

    PubMed Central

    Djekic, Aleksandra; Müller, Anne

    2016-01-01

    VacA is a pore-forming toxin that has long been known to induce vacuolization in gastric epithelial cells and to be linked to gastric disorders caused by H. pylori infection. Its role as a major colonization and persistence determinant of H. pylori is less well-understood. The purpose of this review is to discuss the various target cell types of VacA and its mechanism of action; specifically, we focus on the evidence showing that VacA targets myeloid cells and T-cells to directly and indirectly prevent H. pylori-specific T-cell responses and immune control of the infection. In particular, the ability of VacA-proficient H. pylori to skew T-cell responses towards regulatory T-cells and the effects of Tregs on H. pylori chronicity are highlighted. The by-stander effects of VacA-driven immunomodulation on extragastric diseases are discussed as well. PMID:27322319

  17. Murine T cell activation is regulated by surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide)

    SciTech Connect

    Warford, Jordan; Doucette, Carolyn D.; Hoskin, David W.; Easton, Alexander S.

    2014-01-10

    Highlights: •Surfen is the first inhibitor of glycosaminoglycan function to be studied in murine T cells. •Surfen reduces T cell proliferation stimulated in vitro and in vivo. •Surfen reduces CD25 expression in T cells activated in vivo but not in vitro. •Surfen increases T cell proliferation when T cell receptor activation is bypassed. •Surfen’s effects are blocked by co-administration of heparin sulfate. -- Abstract: Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) binds to glycosaminoglycans (GAGs) and has been shown to influence their function, and the function of proteoglycans (complexes of GAGs linked to a core protein). T cells synthesize, secrete and express GAGs and proteoglycans which are involved in several aspects of T cell function. However, there are as yet no studies on the effect of GAG-binding agents such as surfen on T cell function. In this study, surfen was found to influence murine T cell activation. Doses between 2.5 and 20 μM produced a graduated reduction in the proliferation of T cells activated with anti-CD3/CD28 antibody-coated T cell expander beads. Surfen (20 mg/kg) was also administered to mice treated with anti-CD3 antibody to activate T cells in vivo. Lymphocytes from surfen-treated mice also showed reduced proliferation and lymph node cell counts were reduced. Surfen reduced labeling with a cell viability marker (7-ADD) but to a much lower extent than its effect on proliferation. Surfen also reduced CD25 (the α-subunit of the interleukin (IL)-2 receptor) expression with no effect on CD69 expression in T cells treated in vivo but not in vitro. When receptor activation was bypassed by treating T cells in vitro with phorbyl myristate acetate (10 ng/ml) and ionomycin (100 ng/ml), surfen treatment either increased proliferation (10 μM) or had no effect (2.5, 5 and 20 μM). In vitro treatment of T cells with surfen had no effect on IL-2 or interferon-γ synthesis and did not alter proliferation of the IL-2 dependent cell

  18. T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells.

    PubMed

    Liu, Yuliang; Xu, Yu; Guo, Shuliang; Chen, Hong

    2016-01-01

    Both TCF-4 and MMP-15 are closely linked to the development of lung cancer, while the regulatory role of TCF-4 in MMP-15 expression is still obscure. Here we found that expression of TCF-4 and MMP-15 was increased in lung cancer cells or tissues versus the normal ones. With gain-or loss-of -function studies, we demonstrated that TCF-4 positively regulated MMP-15 expression in mRNA and protein levels. With reporter gene assay, we found that TCF-4 regulated MMP-15 expression via a potential NF-κB binding element locating at -2833/-2824 in the mouse MMP-15 promoter. With ChIP and immunoblotting assays, we identified that TCF-4 functioned as a co-activator to potentiate the binding between p65 and MMP-15 promoter. Functionally, TCF-4 silence attenuated the migration activity of LLC cells, while additional overexpression of MMP-15 rescued this effect in cell scratch test and transwell migration assay. In xenograft model, TCF-4 silence-improved tumor lesions in lungs and survival time of LLC-tumor bearing mice were abolished by MMP-15 overexpression. In conclusion, we are the first to identify TCF-4 as a co-activator of NF-κB p65 to promote MMP-15 transcription and potentiate the migration activity of the lung cancer cells. Our findings shed light on the therapeutic strategies of this malignancy. PMID:27046058

  19. Interferon-γ Promotes Inflammation and Development of T-Cell Lymphoma in HTLV-1 bZIP Factor Transgenic Mice

    PubMed Central

    Mitagami, Yu; Yasunaga, Jun-ichirou; Kinosada, Haruka; Ohshima, Koichi; Matsuoka, Masao

    2015-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of several inflammatory diseases and a T-cell malignancy, adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ) is the only viral gene that is constitutively expressed in HTLV-1-infected cells, and it has multiple functions on T-cell signaling pathways. HBZ has important roles in HTLV-1-mediated pathogenesis, since HBZ transgenic (HBZ-Tg) mice develop systemic inflammation and T-cell lymphomas, which are similar phenotypes to HTLV-1-associated diseases. We showed previously that in HBZ-Tg mice, HBZ causes unstable Foxp3 expression, leading to an increase in regulatory T cells (Tregs) and the consequent induction of IFN-γ-producing cells, which in turn leads to the development of inflammation in the mice. In this study, we show that the severity of inflammation is correlated with the development of lymphomas in HBZ-Tg mice, suggesting that HBZ-mediated inflammation is closely linked to oncogenesis in CD4+ T cells. In addition, we found that IFN-γ-producing cells enhance HBZ-mediated inflammation, since knocking out IFN-γ significantly reduced the incidence of dermatitis as well as lymphoma. Recent studies show the critical roles of the intestinal microbiota in the development of Tregs in vivo. We found that even germ-free HBZ-Tg mice still had an increased number of Tregs and IFN-γ-producing cells, and developed dermatitis, indicating that an intrinsic activity of HBZ evokes aberrant T-cell differentiation and consequently causes inflammation. These results show that immunomodulation by HBZ is implicated in both inflammation and oncogenesis, and suggest a causal connection between HTLV-1-associated inflammation and ATL. PMID:26296091

  20. Functional analysis of transcription factor binding sites in human promoters

    PubMed Central

    2012-01-01

    Background The binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells. To characterize transcription factor binding site function on a large scale, we predicted and mutagenized 455 binding sites in human promoters. We carried out functional tests on these sites in four different immortalized human cell lines using transient transfections with a luciferase reporter assay, primarily for the transcription factors CTCF, GABP, GATA2, E2F, STAT, and YY1. Results In each cell line, between 36% and 49% of binding sites made a functional contribution to the promoter activity; the overall rate for observing function in any of the cell lines was 70%. Transcription factor binding resulted in transcriptional repression in more than a third of functional sites. When compared with predicted binding sites whose function was not experimentally verified, the functional binding sites had higher conservation and were located closer to transcriptional start sites (TSSs). Among functional sites, repressive sites tended to be located further from TSSs than were activating sites. Our data provide significant insight into the functional characteristics of YY1 binding sites, most notably the detection of distinct activating and repressing classes of YY1 binding sites. Repressing sites were located closer to, and often overlapped with, translational start sites and presented a distinctive variation on the canonical YY1 binding motif. Conclusions The genomic properties that we found to associate with functional TF binding sites on promoters -- conservation, TSS proximity, motifs and their variations -- point the way to improved accuracy in future TFBS predictions. PMID:22951020

  1. T Cells in Fish

    PubMed Central

    Nakanishi, Teruyuki; Shibasaki, Yasuhiro; Matsuura, Yuta

    2015-01-01

    Cartilaginous and bony fish are the most primitive vertebrates with a thymus, and possess T cells equivalent to those in mammals. There are a number of studies in fish demonstrating that the thymus is the essential organ for development of T lymphocytes from early thymocyte progenitors to functionally competent T cells. A high number of T cells in the intestine and gills has been reported in several fish species. Involvement of CD4+ and CD8α+ T cells in allograft rejection and graft-versus-host reaction (GVHR) has been demonstrated using monoclonal antibodies. Conservation of CD4+ helper T cell functions among teleost fishes has been suggested in a number studies employing mixed leukocyte culture (MLC) and hapten/carrier effect. Alloantigen- and virus-specific cytotoxicity has also been demonstrated in ginbuna and rainbow trout. Furthermore, the important role of cell-mediated immunity rather than humoral immunity has been reported in the protection against intracellular bacterial infection. Recently, the direct antibacterial activity of CD8α+, CD4+ T-cells and sIgM+ cells in fish has been reported. In this review, we summarize the recent progress in T cell research focusing on the tissue distribution and function of fish T cells. PMID:26426066

  2. Regulation of Lipid Signaling by Diacylglycerol Kinases during T Cell Development and Function

    PubMed Central

    Krishna, Sruti; Zhong, Xiao-Ping

    2013-01-01

    Diacylglycerol (DAG) and phosphatidic acid (PA) are bioactive lipids synthesized when the T cell receptor binds to a cognate peptide-MHC complex. DAG triggers signaling by recruiting Ras guanyl-releasing protein 1, PKCθ, and other effectors, whereas PA binds to effector molecules that include mechanistic target of rapamycin, Src homology region 2 domain-containing phosphatase 1, and Raf1. While DAG-mediated pathways have been shown to play vital roles in T cell development and function, the importance of PA-mediated signals remains less clear. The diacylglycerol kinase (DGK) family of enzymes phosphorylates DAG to produce PA, serving as a molecular switch that regulates the relative levels of these critical second messengers. Two DGK isoforms, α and ζ, are predominantly expressed in T lineage cells and play an important role in conventional αβ T cell development. In mature T cells, the activity of these DGK isoforms aids in the maintenance of self-tolerance by preventing T cell hyper-activation and promoting T cell anergy. In this review, we discuss the roles of DAG-mediated pathways, PA-effectors, and DGKs in T cell development and function. We also highlight recent work that has uncovered previously unappreciated roles for DGK activity, for instance in invariant NKT cell development, anti-tumor and anti-viral CD8 responses, and the directional secretion of soluble effectors. PMID:23847619

  3. Endothelial microparticles interact with and support the proliferation of T cells.

    PubMed

    Wheway, Julie; Latham, Sharissa L; Combes, Valery; Grau, Georges E R

    2014-10-01

    Endothelial cells closely interact with circulating lymphocytes. Aggression or activation of the endothelium leads to an increased shedding of endothelial cell microparticles (MP). Endothelial MP (EMP) are found in high plasma levels in numerous immunoinflammatory diseases, such as atherosclerosis, sepsis, multiple sclerosis, and cerebral malaria, supporting their role as effectors and markers of vascular dysfunction. Given our recently described role for human brain microvascular endothelial cells (HBEC) in modulating immune responses, we investigated how HBEC-derived MP could interact with and support the proliferation of T cells. Like their mother cells, EMP expressed molecules important for Ag presentation and T cell costimulation, that is, β2-microglobulin, MHC II, CD40, and ICOSL. HBEC were able to take up fluorescently labeled Ags with EMP also containing fluorescent Ags, suggestive of Ag carryover from HBEC to EMP. In cocultures, fluorescently labeled EMP from resting or cytokine-stimulated HBEC formed conjugates with both CD4(+) and CD8(+) subsets, with higher proportions of T cells binding EMP from cytokine-stimulated cells. The increased binding of EMP from cytokinestimulated HBEC to T cells was VCAM-1 and ICAM-1 dependent. Finally, in CFSE T cell proliferation assays using anti-CD3 mAb or T cell mitogens, EMP promoted the proliferation of CD4(+) T cells and that of CD8(+) T cells in the absence of exogenous stimuli and in the T cell mitogenic stimulation. Our findings provide novel evidence that EMP can enhance T cell activation and potentially ensuing Ag presentation, thereby pointing toward a novel role for MP in neuroimmunological complications of infectious diseases.

  4. Galectin-1 inhibits the viability, proliferation, and Th1 cytokine production of nonmalignant T cells in patients with leukemic cutaneous T-cell lymphoma

    PubMed Central

    Cedeno-Laurent, Filiberto; Watanabe, Rei; Teague, Jessica E.; Kupper, Thomas S.; Clark, Rachael A.

    2012-01-01

    Tumor-derived galectin-1 (Gal-1), a β-galactoside–binding S-type lectin, has been shown to encourage T-cell death and promote T cell–mediated tumor immune escape. In this report, we show that patients with leukemic cutaneous T-cell lymphomas, known to have limited complexity of their T-cell repertoires, have a predominant T helper type-2 (Th2) cytokine profile and significantly elevated plasma levels of Gal-1 compared with healthy controls. Circulating clonal malignant T cells were a major source of Gal-1. The conditioned supernatant of cultured malignant T cells induced a β-galactoside–dependent inhibition of normal T-cell proliferation and a Th2 skewing of cytokine production. These data implicate Gal-1 in development of the Th2 phenotype in patients with advanced-stage cutaneous T-cell lymphoma and highlight the Gal-1–Gal-1 ligand axis as a potential therapeutic target for enhancing antitumor immune responses. PMID:22383798

  5. A BTLA-mediated bait and switch strategy permits Listeria expansion in CD8α(+) DCs to promote long-term T cell responses.

    PubMed

    Yang, Xuanming; Zhang, Xunmin; Sun, Yonglian; Tu, Tony; Fu, May Lynne; Miller, Mendy; Fu, Yang-Xin

    2014-07-01

    Listeria monocytogenes infected CD8α(+) DCs in the spleen are essential for CD8(+) T cell generation. CD8α(+) DCs are also necessary for Listeria expansion and dissemination within the host. The mechanisms that regulate CD8α(+) DCs to allow Listeria expansion are unclear. We find that activating the B and T lymphocyte attenuator (BTLA), a coinhibitory receptor for T cells, suppresses, while blocking BTLA enhances, both the primary and memory CD8 T cell responses against Listeria. Btla(-/-) mice have lower effector and memory CD8(+) T cells while paradoxically also being more resistant to Listeria. Although bacterial entry into Btla(-/-) CD8α(+) DCs is unaffected, Listeria fails to expand within these cells. BTLA signaling limits Fas/FasL-mediated suppression of Listeria expansion within CD8α(+) DCs to more effectively alert adaptive immune cells. This study uncovers a BTLA-mediated strategy used by the host that permits Listeria proliferation to enable increasing T cell responses for long-term protection.

  6. T cell recognition of beryllium.

    PubMed

    Dai, Shaodong; Falta, Michael T; Bowerman, Natalie A; McKee, Amy S; Fontenot, Andrew P

    2013-12-01

    Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by a hypersensitivity to beryllium and characterized by the accumulation of beryllium-specific CD4(+) T cells in the lung. Genetic susceptibility to beryllium-induced disease is strongly associated with HLA-DP alleles possessing a glutamic acid at the 69th position of the β-chain (βGlu69). The structure of HLA-DP2, the most prevalent βGlu69-containing molecule, revealed a unique solvent-exposed acidic pocket that includes βGlu69 and represents the putative beryllium-binding site. The delineation of mimotopes and endogenous self-peptides that complete the αβTCR ligand for beryllium-specific CD4(+) T cells suggests a unique role of these peptides in metal ion coordination and the generation of altered self-peptides, blurring the distinction between hypersensitivity and autoimmunity.

  7. Inflammatory regulatory T cells in the microenvironments of ulcerative colitis and colon carcinoma.

    PubMed

    Kryczek, Ilona; Wang, Lin; Wu, Ke; Li, Wei; Zhao, Ende; Cui, Tracy; Wei, Shuang; Liu, Yan; Wang, Yin; Vatan, Linda; Szeliga, Wojciech; Greenson, Joel K; Roliński, Jacek; Zgodzinski, Witold; Huang, Emina; Tao, Kaixiong; Wang, Guobin; Zou, Weiping

    2016-08-01

    Foxp3(+)CD4(+) regulatory T (Treg) cells are thought to express negligible levels of effector cytokines, and inhibit immune responses and inflammation. Here, we have identified a population of IL-8(+)Foxp3(+)CD4(+) T cells in human peripheral blood, which is selectively increased in the microenvironments of ulcerative colitis and colon carcinoma. Phenotypically, this population is minimally overlapping with IL-17(+)Foxp3(+)CD4(+) T cells, and is different from IL-8(-)Foxp3(+)CD4(+) T cells in the same microenvironment. 40-60% of IL-8(+)Foxp3(+)CD4(+) T cells exhibit naive phenotype and express CD127, whereas IL-8(-)Foxp3(+)CD4(+) cells are basically memory T cells and express minimal CD127. The levels of CXCR5 expression are higher in IL-8(+)Foxp3(+) cells than in IL-8(-)Foxp3(+) cells. IL-2 and TGFβ induce IL-8(+)Foxp3(+) T cells. Exogenous Foxp3 expression promotes IL-8(+)Foxp3(+) T cells and inhibits effector cytokine IFNγ and IL-2 expression. Furthermore, Foxp3 binds to IL-8 proximal promoter and increases its activity. Functionally, IL-8(+)Foxp3(+) T cells inhibit T cell proliferation and effector cytokine production, but stimulate inflammatory cytokine production in the colon tissues, and promote neutrophil trafficking through IL-8. Thus, IL-8(+)Foxp3(+) cells may be an "inflammatory" Treg subset, and possess inflammatory and immunosuppressive dual biological activities. Given their dual roles and localization, these cells may be in a unique position to support tumor initiation and development in human chronic inflammatory environment. PMID:27622054

  8. IL-1R signaling in dendritic cells replaces pattern-recognition receptors in promoting CD8⁺ T cell responses to influenza A virus.

    PubMed

    Pang, Iris K; Ichinohe, Takeshi; Iwasaki, Akiko

    2013-03-01

    Immune responses to vaccines require direct recognition of pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs) on dendritic cells (DCs). Unlike vaccination, infection by a live pathogen often impairs DC function and inflicts additional damage on the host. Here we found that after infection with live influenza A virus, signaling through the interleukin 1 receptor (IL-1R) was required for productive priming of CD8(+) T cells, but signaling through the PRRs TLR7 and RIG-I was not. DCs activated by IL-1 in trans were both required and sufficient for the generation of virus-specific CD8(+) T cell immunity. Our data demonstrate a critical role for a bystander cytokine in the priming of CD8(+) T cells during infection with a live virus.

  9. T-Cell Lymphoma

    MedlinePlus

    ... are extremely rare. T-cell lymphomas can be aggressive (fast-growing) or indolent (slow-growing). Lymphomas are ... also be involved. This group of PTCLs is aggressive and requires combination chemotherapy upon diagnosis. For more ...

  10. Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter

    SciTech Connect

    Dahabieh, Matthew S.; Ooms, Marcel; Malcolm, Tom; Simon, Viviana; Sadowski, Ivan

    2011-09-15

    Transcription from the HIV-1 long terminal repeat (LTR) is mediated by numerous host transcription factors. In this study we characterized an E-box motif (RBE1) within the core promoter that was previously implicated in both transcriptional activation and repression. We show that RBE1 is a binding site for the RBF-2 transcription factor complex (USF1, USF2, and TFII-I), previously shown to bind an upstream viral element, RBE3. The RBE1 and RBE3 elements formed complexes of identical mobility and protein constituents in gel shift assays, both with Jurkat T-cell nuclear extracts and recombinant USF/TFII-I. Furthermore, both elements are regulators of HIV-1 expression; mutations in LTR-luciferase reporters and in HIV-1 molecular clones resulted in decreased transcription, virion production, and proviral expression in infected cells. Collectively, our data indicate that RBE1 is a bona fide RBF-2 binding site and that the RBE1 and RBE3 elements are necessary for mediating proper transcription from the HIV-1 LTR.

  11. Kinetic discrimination in T-cell activation.

    PubMed Central

    Rabinowitz, J D; Beeson, C; Lyons, D S; Davis, M M; McConnell, H M

    1996-01-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model. PMID:8643643

  12. Carbohydrates and T cells: a sweet twosome.

    PubMed

    Avci, Fikri Y; Li, Xiangming; Tsuji, Moriya; Kasper, Dennis L

    2013-04-01

    Carbohydrates as T cell-activating antigens have been generating significant interest. For many years, carbohydrates were thought of as T-independent antigens, however, more recent research had demonstrated that mono- or oligosaccharides glycosidically linked to peptides can be recognized by T cells. T cell recognition of these glycopeptides depends on the structure of both peptide and glycan portions of the antigen. Subsequently, it was discovered that natural killer T cells recognized glycolipids when presented by the antigen presenting molecule CD1d. A transformative insight into glycan-recognition by T cells occurred when zwitterionic polysaccharides were discovered to bind to and be presented by MHCII to CD4+ T cells. Based on this latter observation, the role that carbohydrate epitopes generated from glycoconjugate vaccines had in activating helper T cells was explored and it was found that these epitopes are presented to specific carbohydrate recognizing T cells through a unique mechanism. Here we review the key interactions between carbohydrate antigens and the adaptive immune system at the molecular, cellular and systems levels exploring the significant biological implications in health and disease.

  13. Kinetic Discrimination in T-Cell Activation

    NASA Astrophysics Data System (ADS)

    Rabinowitz, Joshua D.; Beeson, Craig; Lyons, Daniel S.; Davis, Mark M.; McConnell, Harden M.

    1996-02-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.

  14. CLL-cells induce IDOhi CD14+HLA-DRlo myeloid-derived suppressor cells that inhibit T-cell responses and promote TRegs.

    PubMed

    Jitschin, Regina; Braun, Martina; Büttner, Maike; Dettmer-Wilde, Katja; Bricks, Juliane; Berger, Jana; Eckart, Michael J; Krause, Stefan W; Oefner, Peter J; Le Blanc, Katarina; Mackensen, Andreas; Mougiakakos, Dimitrios

    2014-07-31

    Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population that shares certain characteristics including an aberrant myeloid phenotype and the ability to suppress T cells. MDSCs have been predominantly studied in malignant diseases and findings suggest involvement in tumor-associated immune suppression. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults. Immune defects occur already at early disease stages and impact the clinical course. We assessed presence, frequency, association to other immune parameters, and functional properties of circulating CD14(+) cells lacking HLA-DR expression (HLA-DR(lo)) in patients with untreated CLL. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14(+)HLA-DR(lo) cells was significantly increased in CLL patients. Furthermore, MDSCs suppressed in vitro T-cell activation and induced suppressive regulatory T cells (TRegs). The MDSC-mediated modulation of T cells could be attributed to their increased indoleamine 2,3-dioxygenase (IDO) activity. CLL cells induced IDO(hi) MDSCs from healthy donor monocytes suggesting bidirectional crosstalk between CLL-cells, MDSCs, and TRegs. Overall, we identified a MDSC population that expands in CLL. The exact mechanisms responsible for such accumulation remain to be elucidated and it will be of interest to test whether antagonizing suppressive functions of CLL MDSCs could represent a mean for enhancing immune responses.

  15. Immunomodulatory Effects of 1,25-Dihydroxyvitamin D3 on Dendritic Cells Promote Induction of T Cell Hyporesponsiveness to Myelin-Derived Antigens

    PubMed Central

    Willekens, Barbara; Cras, Patrick; Goossens, Herman; Martínez-Cáceres, Eva; Berneman, Zwi N.

    2016-01-01

    While emerging evidence indicates that dendritic cells (DC) play a central role in the pathogenesis of multiple sclerosis (MS), their modulation with immunoregulatory agents provides prospect as disease-modifying therapy. Our observations reveal that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment of monocyte-derived DC results in a semimature phenotype and anti-inflammatory cytokine profile as compared to conventional DC, in both healthy controls and MS patients. Importantly, 1,25(OH)2D3-treated DC induce T cell hyporesponsiveness, as demonstrated in an allogeneic mixed leukocyte reaction. Next, following a freeze-thaw cycle, 1,25(OH)2D3-treated immature DC could be recovered with a 78% yield and 75% viability. Cryopreservation did not affect the expression of membrane markers by 1,25(OH)2D3-treated DC nor their capacity to induce T cell hyporesponsiveness. In addition, the T cell hyporesponsiveness induced by 1,25(OH)2D3-treated DC is antigen-specific and robust since T cells retain their capacity to respond to an unrelated antigen and do not reactivate upon rechallenge with fully mature conventional DC, respectively. These observations underline the clinical potential of tolerogenic DC (tolDC) to correct the immunological imbalance in MS. Furthermore, the feasibility to cryopreserve highly potent tolDC will, ultimately, contribute to the large-scale production and the widely applicable use of tolDC. PMID:27703987

  16. Activation of Flucloxacillin-Specific CD8+ T-Cells With the Potential to Promote Hepatocyte Cytotoxicity in a Mouse Model.

    PubMed

    Nattrass, Ryan; Faulkner, Lee; Vocanson, Marc; Antoine, Daniel J; Kipar, Anja; Kenna, Gerry; Nicolas, Jean-Francois; Park, B Kevin; Naisbitt, Dean J

    2015-07-01

    There are currently no animal models of drug-induced liver injury (DILI) where the adaptive immune system has been shown to damage the liver. Thus, it is difficult to explore the mechanistic basis of the tissue injury. The aim of this study was to use C57BL/6 CD4+-deficient mice with a mutation in the αβ gene encoding for Major histocompatibilty complex (MHC) class II molecules to (1) develop a mouse model of flucloxacillin sensitization, (2) explore whether drug-specific CD8+ kill primary hepatocytes, and (3) analyze perturbations in liver integrity following oral exposure to flucloxacillin. CD8+ T-cells from lymph nodes of flucloxacillin-sensitized mice were stimulated to proliferate, secrete interferon (IFN-γ) and granzyme B, and induce hepatocyte apoptosis in a concentration-dependent manner following ex vivo stimulation. The T-cell response was antigen-specific; T-cells were not activated with other β-lactam antibiotics. Furthermore, T-cell responses only occurred in the presence of flucloxacillin-pulsed antigen presenting cells. In separate experiments, flucloxacillin-specific T-cells were induced to migrate to the mesenteric lymph nodes using retinoic acid, prior to administration of oral flucloxacillin, and analysis of plasma biomarkers of liver injury. Oral exposure to flucloxacillin resulted in mild elevations in alanine aminotransferase, liver, and gall bladder leukocyte infiltration and a marked swelling of the gall bladder. Thus, CD4+-deficient mice represent a promising model to study the role of the adaptive immune system in DILI. PMID:25877613

  17. Visualizing T Cell Migration in situ

    PubMed Central

    Benechet, Alexandre P.; Menon, Manisha; Khanna, Kamal M.

    2014-01-01

    Mounting a protective immune response is critically dependent on the orchestrated movement of cells within lymphoid tissues. The structure of secondary lymphoid organs regulates immune responses by promoting optimal cell–cell and cell–extracellular matrix interactions. Naïve T cells are initially activated by antigen presenting cells in secondary lymphoid organs. Following priming, effector T cells migrate to the site of infection to exert their functions. Majority of the effector cells die while a small population of antigen-specific T cells persists as memory cells in distinct anatomical locations. The persistence and location of memory cells in lymphoid and non-lymphoid tissues is critical to protect the host from re-infection. The localization of memory T cells is carefully regulated by several factors including the highly organized secondary lymphoid structure, the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands. This balance between the anatomy and the ordered expression of cell surface and soluble proteins regulates the subtle choreography of T cell migration. In recent years, our understanding of cellular dynamics of T cells has been advanced by the development of new imaging techniques allowing in situ visualization of T cell responses. Here, we review the past and more recent studies that have utilized sophisticated imaging technologies to investigate the migration dynamics of naïve, effector, and memory T cells. PMID:25120547

  18. Identification of novel Notch target genes in T cell leukaemia

    PubMed Central

    Chadwick, Nicholas; Zeef, Leo; Portillo, Virginia; Fennessy, Carl; Warrander, Fiona; Hoyle, Sarah; Buckle, Anne-Marie

    2009-01-01

    Background Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. Results RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1ΔE or N3ΔE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1). Conclusion The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease. PMID:19508709

  19. Phosphorylation of a Tyrosine Residue on Zap70 by Lck and Its Subsequent Binding via an SH2 Domain May Be a Key Gatekeeper of T Cell Receptor Signaling In Vivo.

    PubMed

    Thill, Peter A; Weiss, Arthur; Chakraborty, Arup K

    2016-09-15

    The initiation of signaling in T lymphocytes in response to the binding of the T cell receptor (TCR) to cognate ligands is a key step in the emergence of adaptive immune responses. Conventional models posit that TCR signaling is initiated by the phosphorylation of receptor-associated immune receptor activation motifs (ITAMs). The cytoplasmic tyrosine kinase Zap70 binds to phosphorylated ITAMs, is subsequently activated, and then propagates downstream signaling. While evidence for such models is provided by experiments with cell lines, in vivo, Zap70 is bound to phosphorylated ITAMs in resting T cells. However, Zap70 is activated only upon TCR binding to cognate ligand. We report the results of computational studies of a new model for the initiation of TCR signaling that incorporates these in vivo observations. Importantly, the new model is shown to allow better and faster TCR discrimination between self-ligands and foreign ligands. The new model is consistent with many past experimental observations, and experiments that could further test the model are proposed. PMID:27354065

  20. CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells

    PubMed Central

    Herrmann, Andreas; Priceman, Saul J.; Kujawski, Maciej; Xin, Hong; Cherryholmes, Gregory A.; Zhang, Wang; Zhang, Chunyan; Lahtz, Christoph; Kowolik, Claudia; Forman, Steve J.; Kortylewski, Marcin; Yu, Hua

    2014-01-01

    Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte–associated antigen 4 (CTLA4apt) allows gene silencing in exhausted CD8+ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8+ T cells in the tumor milieu; therefore, CTLA4apt fused to a STAT3-targeting siRNA (CTLA4apt–STAT3 siRNA) resulted in internalization into tumor-associated CD8+ T cells and silencing of STAT3, which activated tumor antigen–specific T cells in murine models. Both local and systemic administration of CTLA4apt–STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4apt–STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4apt–STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4apt-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis. PMID:24892807

  1. Krüppel-like factor 2 represses transcription of the telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) in human T cells.

    PubMed

    Hara, Toshifumi; Mizuguchi, Mariko; Fujii, Masahiro; Nakamura, Masataka

    2015-04-01

    In normal human T cells, telomerase activity is strictly regulated. T cells are thought to express telomerase to avoid replicative senescence, unlike most normal somatic cells with definite replicative lifespan. T cells in blood and tissues are usually in a state of quiescence without expression of the limiting catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). In contrast to activation, repression of hTERT transcription has not been studied well. Our previous studies have found an hTERT promoter element with repressive function. Here we identified KLF2, which represses hTERT transcription by binding to the putative promoter element. KLF2 and hTERT exhibited reciprocal mRNA expression patterns in primary human T cells. In activated T cells, KLF2 binding to the hTERT promoter was eliminated, relieving the repression of hTERT transcription found in resting T cells. Our results suggest that KLF2 is involved in strict repression of hTERT expression through binding to the promoter in primary human T cells.

  2. Cannabinoid receptor-2 (CB2) agonist ameliorates colitis in IL-10{sup −/−} mice by attenuating the activation of T cells and promoting their apoptosis

    SciTech Connect

    Singh, Udai P.; Singh, Narendra P.; Singh, Balwan; Price, Robert L.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2012-01-15

    Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Recent studies have shown that the cannabinoid system may play a critical role in mediating protection against intestinal inflammation. However, the effect of cannabinoid receptor induction after chronic colitis progression has not been investigated. Here, we investigate the effect of cannabinoid receptor-2 (CB2) agonist, JWH-133, after chronic colitis in IL-10{sup −/−} mice. JWH-133 effectively attenuated the overall clinical score, and reversed colitis-associated pathogenesis and decrease in body weight in IL-10{sup −/−} mice. After JWH-133 treatment, the percentage of CD4{sup +} T cells, neutrophils, mast cells, natural killer (NK1.1) cells, and activated T cells declined in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN) of mice with chronic colitis. JWH-133 was also effective in ameliorating dextran sodium sulfate (DSS)-induced colitis. In this model, JWH-133 reduced the number and percentage of macrophages and IFN-γ expressing cells that were induced during colitis progression. Treatment with aminoalkylindole 6-iodo-pravadoline (AM630), a CB2 receptor antagonist, reversed the colitis protection provided by JWH-133 treatment. Also, activated T cells were found to undergo apoptosis following JWH-133 treatment both in-vivo and in-vitro. These findings suggest that JWH-133 mediates its effect through CB2 receptors, and ameliorates chronic colitis by inducing apoptosis in activated T cells, reducing the numbers of activated T cells, and suppressing induction of mast cells, NK cells, and neutrophils at sites of inflammation in the LP. These results support the idea that the CB2 receptor agonists may serve as a therapeutic modality against IBD. -- Highlights: ► JWH-133, a cannnabinoid receptor-2 agonist ameliorates experimental colitis. ► JWH-133 suppressed inflammation and

  3. Amino-Terminal Extended Peptide Single-Chain Trimers are Potent Synthetic Agonists for Memory Human CD8+ T cells

    PubMed Central

    Carreno, Beatriz M.; Becker-Hapak, Michelle; Chan, Megan; Lie, Wen-Rong; Wang, Xiaoli; Hansen, Ted H.; Linette, Gerald P.

    2012-01-01

    Upon antigen exposure, most memory T cells undergo re-stimulation induced cell death. Here we describe a novel synthetic agonist, an amino-terminal extended decamer peptide expressed as a single chain trimer, the AT-SCT, that preferentially promotes the growth of memory human CD8+ T cells with minimal re-stimulation-induced cell death. Using the CMV pp65 and melanoma gp100 antigens, we observe the in vitro numerical expansion of a clonally diverse poly-functional population of antigen-specific CD8+ T cells from normal individuals and vaccinated melanoma patients, respectively. Memory CD8+ T cells stimulated with AT-SCT presented on MHC class I/II null cells show reduced cytokine production, slower kinetics of TCR down-regulation and decreased cell death when compared to native nonamer SCT-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT- and SCT-activated T cells. Probing of SCT and AT-SCT peptide-MHC (p-MHC) complexes using fluorochrome-conjugated TCR multimers suggest that nonamer and decamer-linked peptides may be anchored differently to HLA-A2 peptide binding groove. Our findings demonstrate that modified p-MHC structures such as AT-SCT can be engineered as T cell agonists to promote the growth and expansion of memory human CD8+ T cells. PMID:22573808

  4. Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes.

    PubMed

    Karamloo, Fariba; Schmid-Grendelmeier, Peter; Kussebi, Fatimah; Akdis, Mübeccel; Salagianni, Maria; von Beust, Barbara R; Reimers, Andrea; Zumkehr, Judith; Soldatova, Lyudmilla; Housley-Markovic, Zora; Müller, Ulrich; Kündig, Thomas; Kemeny, David M; Spangfort, Michael D; Blaser, Kurt; Akdis, Cezmi A

    2005-11-01

    Novel approaches for the prevention of allergy are required, because of the inevitably increasing prevalence of allergic diseases during the last 30 years. Here, a recombinant chimeric protein, which comprises the whole amino acid sequences of three bee venom major allergens has been engineered and used in prevention of bee venom sensitization in mice. Phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 3) fragments with overlapping amino acids were assembled in a different order in the Api m (1/2/3) chimeric protein, which preserved entire T cell epitopes, whereas B cell epitopes of all three allergens were abrogated. Accordingly, IgE cross-linking leading to mast cell and basophil mediator release was profoundly reduced in humans. Supporting these findings, the Api m (1/2/3) induced 100 to 1000 times less type-1 skin test reactivity in allergic patients. Treatment of mice with Api m (1/2/3) led to a significant reduction of specific IgE development towards native allergen, representing a protective vaccine effect in vivo. These results demonstrate a novel prototype of a preventive allergy vaccine, which preserves the entire T cell epitope repertoire, but bypasses induction of IgE against native allergen, and side effects related to mast cell/basophil IgE FcepsilonRI cross-linking in sensitized individuals. PMID:16206231

  5. Engineered T cell therapies.

    PubMed

    Field, Anne-Christine; Qasim, Waseem

    2015-11-04

    Alongside advancements in gene therapy for inherited immune disorders, the need for effective alternative therapeutic options for other conditions has resulted in an expansion in the field of research for T cell gene therapy. T cells are easily obtained and can be induced to divide robustly ex vivo, a characteristic that allows them to be highly permissible to viral vector-mediated introduction of transgenes. Pioneering clinical trials targeting cancers and infectious diseases have provided safety and feasibility data and important information about persistence of engineered cells in vivo. Here, we review clinical experiences with γ-retroviral and lentiviral vectors and consider the potential of integrating transposon-based vectors as well as specific genome editing with designer nucleases in engineered T cell therapies.

  6. IL-15 Trans-Signaling with the Superagonist RLI Promotes Effector/Memory CD8+ T Cell Responses and Enhances Antitumor Activity of PD-1 Antagonists.

    PubMed

    Desbois, Mélanie; Le Vu, Pauline; Coutzac, Clélia; Marcheteau, Elie; Béal, Coralie; Terme, Magali; Gey, Alain; Morisseau, Sébastien; Teppaz, Géraldine; Boselli, Lisa; Jacques, Yannick; Béchard, David; Tartour, Eric; Cassard, Lydie; Chaput, Nathalie

    2016-07-01

    Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity.

  7. IL-15 Trans-Signaling with the Superagonist RLI Promotes Effector/Memory CD8+ T Cell Responses and Enhances Antitumor Activity of PD-1 Antagonists.

    PubMed

    Desbois, Mélanie; Le Vu, Pauline; Coutzac, Clélia; Marcheteau, Elie; Béal, Coralie; Terme, Magali; Gey, Alain; Morisseau, Sébastien; Teppaz, Géraldine; Boselli, Lisa; Jacques, Yannick; Béchard, David; Tartour, Eric; Cassard, Lydie; Chaput, Nathalie

    2016-07-01

    Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity. PMID:27217584

  8. Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

    PubMed Central

    Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D.; Chorro, Laurent; Carlin, Leo M.; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S.

    2013-01-01

    Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs. PMID:23386724

  9. A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate- and Co-Receptor- Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation

    PubMed Central

    Connell, Bridgette Janine; Chang, Sui-Yuan; Prakash, Ekambaranellore; Yousfi, Rahima; Mohan, Viswaraman; Posch, Wilfried; Wilflingseder, Doris; Moog, Christiane; Kodama, Eiichi N.; Clayette, Pascal; Lortat-Jacob, Hugues

    2016-01-01

    Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1–7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS) as well as to an antibody (mAb 17b) directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1. PMID:27788205

  10. Transcriptional regulator Id2 mediates CD8+ T cell immunity.

    PubMed

    Cannarile, Michael A; Lind, Nicholas A; Rivera, Richard; Sheridan, Alison D; Camfield, Kristin A; Wu, Bei Bei; Cheung, Kitty P; Ding, Zhaoqing; Goldrath, Ananda W

    2006-12-01

    Transcriptional programs that initiate and sustain the proliferation, differentiation and survival of CD8(+) T cells during immune responses are not completely understood. Here we show that inhibitor of DNA binding 2 (Id2), an antagonist of E protein transcription factors, was upregulated in CD8(+) T cells during infection and that expression of Id2 was maintained in memory CD8(+) T cells. Although Id2-deficient naive CD8(+) T cells recognized antigen and proliferated normally early after infection, effector CD8(+) T cells did not accumulate because the cells were highly susceptible to apoptosis. Id2-deficient CD8(+) T cells responding to infection had changes in the expression of genes that influence survival and had altered memory formation. Our data emphasize the importance of Id2 in regulating gene expression by CD8(+) T cells and the magnitude of effector responses, suggesting a mechanism involving Id protein- and E protein-mediated survival and differentiation of mature T cells.

  11. Goblet Cell Derived RELM-β Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation

    PubMed Central

    Bergstrom, Kirk S. B.; Morampudi, Vijay; Chan, Justin M.; Bhinder, Ganive; Lau, Jennifer; Yang, Hyungjun; Ma, Caixia; Huang, Tina; Ryz, Natasha; Sham, Ho Pan; Zarepour, Maryam; Zaph, Colby; Artis, David; Nair, Meera; Vallance, Bruce A.

    2015-01-01

    Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-β is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-β’s role in host defense is unclear. Thus, wildtype and RELM-β gene deficient mice (Retnlb-/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb-/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb-/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-β did not directly increase IEC proliferation, rather RELM-β acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb-/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-β to Retnlb-/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-β and goblet cells play an

  12. The Divergent CD8+ T Cell Adjuvant Properties of LT-IIb and LT-IIc, Two Type II Heat-Labile Enterotoxins, Are Conferred by Their Ganglioside-Binding B Subunits

    PubMed Central

    Hu, John C.; Greene, Christopher J.; King-Lyons, Natalie D.; Connell, Terry D.

    2015-01-01

    Poor immune responses elicited by vaccine antigens can be enhanced by the use of appropriate adjuvants. Type II heat-labile enterotoxins (HLT) produced by Escherichia coli are extremely potent adjuvants that augment both humoral and cellular immunity to co-administered antigens. Recent findings demonstrate that LT-IIb and LT-IIc, two type II HLT adjuvants, exhibit potent, yet distinguishable CD8+ T cell adjuvant properties. While LT-IIc elicits a robust and rapid response at one week after administration, LT-IIb engenders a more gradual and slower expansion of antigen-specific CD8+ T cells that correlates with improved immunity. The variations in immune effects elicited by the HLT adjuvants have been generally attributed to their highly divergent B subunits that mediate binding to various gangliosides on cell surfaces. Yet, HLT adjuvants with point mutations in the B subunit that significantly alter ganglioside binding retain similar adjuvant functions. Therefore, the contribution of the B subunits to adjuvanticity remains unclear. To investigate the influence of the B subunits on the enhancement of immune responses by LT-IIb and LT-IIc, chimeric HLT were engineered in which the B subunits of the two adjuvants were exchanged. Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits. Specifically, the differences in the CD8+ T cell kinetics and protective responses elicited by LT-IIb and LT-IIc did indeed followed their respective B subunits. However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits. Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants. PMID:26565800

  13. Enzymatically quiescent heparanase augments T cell interactions with VCAM-1 and extracellular matrix components under versatile dynamic contexts.

    PubMed

    Sotnikov, Ilya; Hershkoviz, Rami; Grabovsky, Valentin; Ilan, Neta; Cahalon, Liora; Vlodavsky, Israel; Alon, Ronen; Lider, Ofer

    2004-05-01

    During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1 alpha (SDF-1 alpha; CXCL12), in an alpha(4)beta(1)-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in beta(1) integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1 alpha-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1 alpha-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites. PMID:15100255

  14. The human T-cell leukemia/lymphotropic virus type 1 p12I proteins bind the interleukin-2 receptor beta and gammac chains and affects their expression on the cell surface.

    PubMed Central

    Mulloy, J C; Crownley, R W; Fullen, J; Leonard, W J; Franchini, G

    1996-01-01

    p12I is a small hydrophobic protein encoded by the human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) that interacts with the 16-kDa component of the H+ vacuolar ATPase and cooperates with bovine papillomavirus 1 E5 oncoprotein in cell transformation. Just as an important step in E5 action appears to be its binding to the platelet-derived growth factor receptor, it was found that p12I binds specifically to both the beta and gamma(c) chains of the interleukin-2 receptor (IL-2R). The IL-2R beta and gamma(c) chains associated with p12I are endoglycosidase-H sensitive, suggesting that their interaction occurs in a pre-Golgi compartment. p12I stabilizes the immature forms of the IL-2R beta and gamma(c) chains and decreases their cell surface expression. The interactions of p12I with IL-2R beta and gamma(c) may have important implications in the immunosuppressive effect of HTLV-1 in vivo as well as in the ligand-independent HTLV-1-mediated T-cell proliferation. PMID:8648694

  15. Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-κB binding activity and TNF-α secretion of T cells

    PubMed Central

    Pohlers, Dirk; Schmidt-Weber, Carsten B; Franch, Angels; Kuhlmann, Jürgen; Bräuer, Rolf; Emmrich, Frank; Kinne, Raimund W

    2002-01-01

    The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-α secretion, and more strongly induced NF-κB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies. PMID:12010568

  16. Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.

    PubMed

    Rose, David M; Liu, Shouchun; Woodside, Darren G; Han, Jaewon; Schlaepfer, David D; Ginsberg, Mark H

    2003-06-15

    Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1). PMID:12794117

  17. Roles of the six peptide-binding pockets of the HLA-A2 molecule in allorecognition by human cytotoxic T-cell clones.

    PubMed

    Matsui, M; Hioe, C E; Frelinger, J A

    1993-01-15

    To evaluate the contribution of the major histocompatibility complex class I pockets to the binding of self-peptides recognized by alloreactive cytotoxic T-lymphocyte (CTL) clones, we have constructed an extensive library of HLA-A2 mutants with different amino acid substitutions in each of the six pockets. When these mutants were tested in cytotoxicity assays with a panel of HLA-A2-specific alloreactive CTL clones, each CTL clone showed a unique pattern of reactivity, implying the different contributions of each pocket to binding individual peptides. We noted that the majority of the mutants in pocket B significantly affect recognition by the CTL clones. Unexpectedly, the mutations influencing allorecognition are found in all other pockets as well. Overall, this study demonstrates that each of the six peptide-binding pockets plays an important and distinct role in binding of self-peptides required for recognition of the HLA-A2 molecule by alloreactive CTLs. PMID:7678462

  18. Preventing T cell rejection of pig xenografts.

    PubMed

    Higginbotham, Laura; Ford, Mandy L; Newell, Kenneth A; Adams, Andrew B

    2015-11-01

    Xenotransplantation is a potential solution to the limited supply of donor organs. While early barriers to xenograft acceptance, such as hyperacute rejection, are now largely avoided through genetic engineering, the next frontier in successful xenograft survival will require prevention of T cell-mediated rejection. Most successful immunosuppressive regimens in xenotransplantation utilize T cell depletion with antibody therapy. Additionally, the use of T cell costimulatory blockade - specifically blockade of the CD40-CD154 pathway - shows promise with several reports of long-term xenograft survival. Additional therapies, such as transgenic expression of T cell coinhibitory molecules or transfer of immunomodulatory cells to promote tolerance, may be necessary to achieve reliable long-term xenograft acceptance. Further studies in pre-clinical models are essential in order to optimize these regimens prior to trials in patients.

  19. T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells

    PubMed Central

    Conley, James M.; Gallagher, Michael P.; Berg, Leslie J.

    2016-01-01

    Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation and CD8 T cell function are discussed here. We propose that inducible T cell kinase (ITK) acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen. PMID:26973653

  20. Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

    PubMed

    He, Jing; Tsai, Louis M; Leong, Yew Ann; Hu, Xin; Ma, Cindy S; Chevalier, Nina; Sun, Xiaolin; Vandenberg, Kirsten; Rockman, Steve; Ding, Yan; Zhu, Lei; Wei, Wei; Wang, Changqi; Karnowski, Alexander; Belz, Gabrielle T; Ghali, Joanna R; Cook, Matthew C; Riminton, D Sean; Veillette, André; Schwartzberg, Pamela L; Mackay, Fabienne; Brink, Robert; Tangye, Stuart G; Vinuesa, Carola G; Mackay, Charles R; Li, Zhanguo; Yu, Di

    2013-10-17

    Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory.

  1. HCV-induced miR146a controls SOCS1/STAT3 and cytokine expression in monocytes to promote regulatory T-cell development.

    PubMed

    Ren, J P; Ying, R S; Cheng, Y Q; Wang, L; El Gazzar, M; Li, G Y; Ning, S B; Moorman, J P; Yao, Z Q

    2016-10-01

    Host innate and adaptive immune responses must be tightly regulated by an intricate balance between positive and negative signals to ensure their appropriate onset and termination while fighting pathogens and avoiding autoimmunity; persistent pathogens may usurp these regulatory machineries to dampen host immune responses for their persistence in vivo. Here, we demonstrate that miR146a is up-regulated in monocytes from hepatitis C virus (HCV)-infected individuals compared to control subjects. Interestingly, miR146a expression in monocytes without HCV infection increased, whereas its level in monocytes with HCV infection decreased, following Toll-like receptor (TLR) stimulation. This miR146a induction by HCV infection and differential response to TLR stimulation were recapitulated in vitro in monocytes co-cultured with hepatocytes with or without HCV infection. Importantly, inhibition of miR146a in monocytes from HCV-infected patients led to a decrease in IL-23, IL-10 and TGF-β expressions through the induction of suppressor of cytokine signalling 1 (SOCS1) and the inhibition of signal transducer and activator transcription 3 (STAT3), and this subsequently resulted in a decrease in regulatory T cells (Tregs) accumulated during HCV infection. These results suggest that miR146a may regulate SOCS1/STAT3 and cytokine signalling in monocytes, directing T-cell differentiation and balancing immune clearance and immune injury during chronic viral infection.

  2. Defective CD8 T Cell Memory Following Acute Infection Without CD4 T Cell Help

    NASA Astrophysics Data System (ADS)

    Sun, Joseph C.; Bevan, Michael J.

    2003-04-01

    The CD8+ cytotoxic T cell response to pathogens is thought to be CD4+ helper T cell independent because infectious agents provide their own inflammatory signals. Mice that lack CD4+ T cells mount a primary CD8 response to Listeria monocytogenes equal to that of wild-type mice and rapidly clear the infection. However, protective memory to a challenge is gradually lost in the former animals. Memory CD8+ T cells from normal mice can respond rapidly, but memory CD8+ T cells that are generated without CD4 help are defective in their ability to respond to secondary encounters with antigen. The results highlight a previously undescribed role for CD4 help in promoting protective CD8 memory development.

  3. The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase.

    PubMed Central

    Franchini, G; Mulloy, J C; Koralnik, I J; Lo Monico, A; Sparkowski, J J; Andresson, T; Goldstein, D J; Schlegel, R

    1993-01-01

    The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses. Images PMID:8230493

  4. Designer T cells by T cell receptor replacement.

    PubMed

    Sommermeyer, Daniel; Neudorfer, Julia; Weinhold, Monika; Leisegang, Matthias; Engels, Boris; Noessner, Elfriede; Heemskerk, Mirjam H M; Charo, Jehad; Schendel, Dolores J; Blankenstein, Thomas; Bernhard, Helga; Uckert, Wolfgang

    2006-11-01

    T cell receptor (TCR) gene transfer is a convenient method to produce antigen-specific T cells for adoptive therapy. However, the expression of two TCR in T cells could impair their function or cause unwanted effects by mixed TCR heterodimers. With five different TCR and four different T cells, either mouse or human, we show that some TCR are strong--in terms of cell surface expression--and replace weak TCR on the cell surface, resulting in exchange of antigen specificity. Two strong TCR are co-expressed. A mouse TCR replaces human TCR on human T cells. Even though it is still poorly understood why some TCRalpha/beta combinations are preferentially expressed on T cells, our data suggest that, in the future, designer T cells with exclusive tumor reactivity can be generated by T cell engineering. PMID:17051621

  5. KLF2 deficiency in T cells results in unrestrained cytokine production and bystander chemokine receptor upregulation

    PubMed Central

    Weinreich, Michael A.; Takada, Kensuke; Skon, Cara; Reiner, Steven L.; Jameson, Stephen C.; Hogquist, Kristin A.

    2009-01-01

    SUMMARY The transcription factor KLF2 regulates T cell trafficking by promoting expression of the lipid binding receptor, S1P1, and the selectin, CD62L. Recently, it was proposed that KLF2 also represses the expression of chemokine receptors. We confirm the upregulation of the chemokine receptor CXCR3 on KLF2 deficient T cells. However, we show that this is a cell nonautonomous effect, as revealed by CXCR3 upregulation on WT bystander cells in mixed bone marrow chimeras with KLF2 deficient cells. Furthermore, we show that KLF2 deficient T cells overproduce IL-4, leading to the upregulation of CXCR3 through an IL-4 receptor and eomesodermin dependent pathway. Consistent with the increased IL-4 production, we find high levels of serum IgE in mice with T cell specific KLF2 deficiency. Our findings support a model where KLF2 regulates T cell trafficking by direct regulation of S1P1 and CD62L, and restrains spontaneous cytokine production in naive T cells. PMID:19592277

  6. Neem leaf glycoprotein promotes dual generation of central and effector memory CD8(+) T cells against sarcoma antigen vaccine to induce protective anti-tumor immunity.

    PubMed

    Ghosh, Sarbari; Sarkar, Madhurima; Ghosh, Tithi; Guha, Ipsita; Bhuniya, Avishek; Saha, Akata; Dasgupta, Shayani; Barik, Subhasis; Bose, Anamika; Baral, Rathindranath

    2016-03-01

    We have previously shown that Neem Leaf Glycoprotein (NLGP) mediates sustained tumor protection by activating host immune response. Now we report that adjuvant help from NLGP predominantly generates CD44(+)CD62L(high)CCR7(high) central memory (TCM; in lymph node) and CD44(+)CD62L(low)CCR7(low) effector memory (TEM; in spleen) CD8(+) T cells of Swiss mice after vaccination with sarcoma antigen (SarAg). Generated TCM and TEM participated either to replenish memory cell pool for sustained disease free states or in rapid tumor eradication respectively. TCM generated after SarAg+NLGP vaccination underwent significant proliferation and IL-2 secretion following SarAg re-stimulation. Furthermore, SarAg+NLGP vaccination helps in greater survival of the memory precursor effector cells at the peak of the effector response and their maintenance as mature memory cells, in comparison to single modality treatment. Such response is corroborated with the reduced phosphorylation of FOXO in the cytosol and increased KLF2 in the nucleus associated with enhanced CD62L, CCR7 expression of lymph node-resident CD8(+) T cells. However, spleen-resident CD8(+) T memory cells show superior efficacy for immediate memory-to-effector cell conversion. The data support in all aspects that SarAg+NLGP demonstrate superiority than SarAg vaccination alone that benefits the host by rapid effector functions whenever required, whereas, central-memory cells are thought to replenish the memory cell pool for ultimate sustained disease free survival till 60 days following post-vaccination tumor inoculation.

  7. Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells, but not Neutrophils, Promote Viral Clearance after Therapy with Cyclophosphamide and Dexamethasone.

    PubMed

    Mostafa, Heba H; Vogel, Peter; Srinivasan, Ashok; Russell, Charles J

    2016-09-01

    In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus-specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential. PMID:27589232

  8. Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells, but not Neutrophils, Promote Viral Clearance after Therapy with Cyclophosphamide and Dexamethasone

    PubMed Central

    Mostafa, Heba H.; Vogel, Peter; Srinivasan, Ashok; Russell, Charles J.

    2016-01-01

    In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus—specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential. PMID:27589232

  9. Malaria drives T cells to exhaustion

    PubMed Central

    Wykes, Michelle N.; Horne-Debets, Joshua M.; Leow, Chiuan-Yee; Karunarathne, Deshapriya S.

    2014-01-01

    Malaria is a significant global burden but after >30 years of effort there is no vaccine on the market. While the complex life cycle of the parasite presents several challenges, many years of research have also identified several mechanisms of immune evasion by Plasmodium spp. Recent research on malaria, has investigated the programmed cell death-1 (PD-1) pathway which mediates exhaustion of T cells, characterized by poor effector functions and recall responses and in some cases loss of the cells by apoptosis. Such studies have shown exhaustion of CD4+ T cells and an unappreciated role for CD8+ T cells in promoting sterile immunity against blood stage malaria. This is because PD-1 mediates up to a 95% reduction in numbers and functional capacity of parasite-specific CD8+ T cells, thus masking their role in protection. The role of T cell exhaustion during malaria provides an explanation for the absence of sterile immunity following the clearance of acute disease which will be relevant to future malaria-vaccine design and suggests the need for novel therapeutic solutions. This review will thus examine the role of PD-1-mediated T cell exhaustion in preventing lasting immunity against malaria. PMID:24904561

  10. Aberrant TAL1 activation is mediated by an interchromosomal interaction in human T-cell acute lymphoblastic leukemia.

    PubMed

    Patel, B; Kang, Y; Cui, K; Litt, M; Riberio, M S J; Deng, C; Salz, T; Casada, S; Fu, X; Qiu, Y; Zhao, K; Huang, S

    2014-02-01

    Long-range chromatin interactions control metazoan gene transcription. However, the involvement of intra- and interchromosomal interactions in development and oncogenesis remains unclear. TAL1/SCL is a critical transcription factor required for the development of all hematopoietic lineages; yet, aberrant TAL1 transcription often occurs in T-cell acute lymphoblastic leukemia (T-ALL). Here, we report that oncogenic TAL1 expression is regulated by different intra- and interchromosomal loops in normal hematopoietic and leukemic cells, respectively. These intra- and interchromosomal loops alter the cell-type-specific enhancers that interact with the TAL1 promoter. We show that human SET1 (hSET1)-mediated H3K4 methylations promote a long-range chromatin loop, which brings the +51 enhancer in close proximity to TAL1 promoter 1 in erythroid cells. The CCCTC-binding factor (CTCF) facilitates this long-range enhancer/promoter interaction of the TAL1 locus in erythroid cells while blocking the same enhancer/promoter interaction of the TAL1 locus in human T-cell leukemia. In human T-ALL, a T-cell-specific transcription factor c-Maf-mediated interchromosomal interaction brings the TAL1 promoter into close proximity with a T-cell-specific regulatory element located on chromosome 16, activating aberrant TAL1 oncogene expression. Thus, our study reveals a novel molecular mechanism involving changes in three-dimensional chromatin interactions that activate the TAL1 oncogene in human T-cell leukemia. PMID:23698277

  11. I spy alloreactive T cells.

    PubMed

    Alegre, Maria-Luisa

    2015-01-28

    High-throughput sequencing of the T cell receptor Vβ CDR3 region allowed longitudinal tracking of alloreactive T cells in kidney transplant patients, revealing clonal deletion as a mechanism of transplantation tolerance (Morris et al., this issue). PMID:25632032

  12. The glycocalyx promotes cooperative binding and clustering of adhesion receptors.

    PubMed

    Xu, Guang-Kui; Qian, Jin; Hu, Jinglei

    2016-05-18

    Cell adhesion plays a pivotal role in various biological processes, e.g., immune responses, cancer metastasis, and stem cell differentiation. The adhesion behaviors depend subtly on the binding kinetics of receptors and ligands restricted at the cell-substrate interfaces. Although much effort has been directed toward investigating the kinetics of adhesion molecules, the role of the glycocalyx, anchored on cell surfaces as an exterior layer, is still unclear. In this paper, we propose a theoretical approach to study the collective binding kinetics of a few and a large number of binders in the presence of the glycocalyx, representing the cases of initial and mature adhesions of cells, respectively. The analytical results are validated by finding good agreement with our Monte Carlo simulations. In the force loading case, the on-rate and affinity increase as more bonds form, whereas this cooperative effect is not observed in the displacement loading case. The increased thickness and stiffness of the glycocalyx tend to decrease the affinity for a few bonds, while they have less influence on the affinity for a large number of bonds. Moreover, for a flexible membrane with thermally-excited shape fluctuations, the glycocalyx is exhibited to promote the formation of bond clusters, mainly due to the cooperative binding of binders. This study helps to understand the cooperative kinetics of adhesion receptors under physiologically relevant loading conditions and sheds light on the novel role of the glycocalyx in cell adhesion. PMID:27102288

  13. Auto-reactive T cells revised. Overestimation based on methodology?

    PubMed

    Thorlacius-Ussing, Gorm; Sørensen, Jesper F; Wandall, Hans H; Pedersen, Anders Elm

    2015-05-01

    Autoreactive T cells have been identified in most autoimmune diseases and recently even in healthy individuals. Similar, T cells that recognize either wild-type or tumorspecific tumor antigens have been increasingly reported to develop spontaneously in cancer patients. This insight has become possible mainly due to novel immunoassays which have revolutionized the discovery of rare antigen specific T cells. At present, the major dogma that explains this increasing number of reports of autoreactive T cells is that autoreactive T cells are counteracted by CD4+CD25+ regulatory T (Treg) cells in vivo, in particular in healthy individuals, whereas dysfunction in Tregs or Treg responsiveness may unmask the autoreactive T cell responses in patients with autoimmune diseases. However, studies that identify autoreactive T cells are usually performed by culturing T cells with antigen presenting cells loaded with E. coli produced recombinant protein or unmodified synthetic HLA binding peptides. Our concern is that this approach may ignore the presence of natural genetic variation and post-translational modifications such as e.g. the complex nature of N- and O-linked glycosylation of mammalian proteins. Thus, T cell antigen reactivities identified with unmodified antigens in vitro may in part represent in vitro T cell activation against neo-epitopes and not true in vivo autoreactivity as postulated. This methodological problem may have implications for the interpretation of the frequent reporting of autoreactive T cells in autoimmunity, T cell responses to wild-type tumor antigens in cancer patients and most important for the increasing reports on naïve T cells with specificity against self-antigens in healthy individuals. Here, we discuss and provide examples for the possibility that the experimental methodology applied to document T cell reactivity against unmodified protein or peptide may lead to overinterpretation of the reported frequencies of autoreactive CD4+ and CD8+ T

  14. Fe(2+) binding on amyloid β-peptide promotes aggregation.

    PubMed

    Boopathi, Subramaniam; Kolandaivel, Ponmalai

    2016-09-01

    The metal ions Zn(2+) , Cu(2+) , and Fe(2+) play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06-2X/6-311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ1-42 -Zn(2+) , Aβ1-42 -Cu(2+) , and Aβ1-42 -Fe(2+) systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N-terminal. The present investigation, the Aβ1-42 -Zn(2+) system possess three turn conformations separated by coil structure. Zn(2+) binding caused the loss of the helical structure of N-terminal residues which transformed into the S-shaped conformation. Zn(2+) has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu(2+) binds with peptide, β sheet formation is observed at the N-terminal residues of the peptide. Fe(2+) binding is to promote the formation of Glu22-Lys28 salt-bridge which stabilized the turn conformation in the Phe19-Gly25 residues, subsequently β sheets were observed at His13-Lys18 and Gly29-Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe(2+) binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe(2+) promotes the oligomerization by enhancing the peptide-peptide interaction. Proteins 2016; 84:1257-1274. © 2016 Wiley Periodicals, Inc. PMID:27214008

  15. Fe(2+) binding on amyloid β-peptide promotes aggregation.

    PubMed

    Boopathi, Subramaniam; Kolandaivel, Ponmalai

    2016-09-01

    The metal ions Zn(2+) , Cu(2+) , and Fe(2+) play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06-2X/6-311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ1-42 -Zn(2+) , Aβ1-42 -Cu(2+) , and Aβ1-42 -Fe(2+) systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N-terminal. The present investigation, the Aβ1-42 -Zn(2+) system possess three turn conformations separated by coil structure. Zn(2+) binding caused the loss of the helical structure of N-terminal residues which transformed into the S-shaped conformation. Zn(2+) has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu(2+) binds with peptide, β sheet formation is observed at the N-terminal residues of the peptide. Fe(2+) binding is to promote the formation of Glu22-Lys28 salt-bridge which stabilized the turn conformation in the Phe19-Gly25 residues, subsequently β sheets were observed at His13-Lys18 and Gly29-Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe(2+) binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe(2+) promotes the oligomerization by enhancing the peptide-peptide interaction. Proteins 2016; 84:1257-1274. © 2016 Wiley Periodicals, Inc.

  16. Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity.

    PubMed

    Berezhnoy, Alexey; Castro, Iris; Levay, Agata; Malek, Thomas R; Gilboa, Eli

    2014-01-01

    Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8(+) T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8(+) T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8(+) T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8(+) T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8(+) T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.

  17. Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity

    PubMed Central

    Berezhnoy, Alexey; Castro, Iris; Levay, Agata; Malek, Thomas R.; Gilboa, Eli

    2013-01-01

    Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8+ T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8+ T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8+ T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8+ T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8+ T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge. PMID:24292708

  18. Cutting edge: maresin-1 engages regulatory T cells to limit type 2 innate lymphoid cell activation and promote resolution of lung inflammation.

    PubMed

    Krishnamoorthy, Nandini; Burkett, Patrick R; Dalli, Jesmond; Abdulnour, Raja-Elie E; Colas, Romain; Ramon, Sesquile; Phipps, Richard P; Petasis, Nicos A; Kuchroo, Vijay K; Serhan, Charles N; Levy, Bruce D

    2015-02-01

    Asthma is a chronic inflammatory disease that fails to resolve. Recently, a key role for type 2 innate lymphoid cells (ILC2s) was linked to asthma pathogenesis; however, mechanisms for ILC2 regulation remain to be determined. In this study, metabololipidomics of murine lungs identified temporal changes in endogenous maresin 1 (MaR1) during self-limited allergic inflammation. Exogenous MaR1 reduced lung inflammation and ILC2 expression of IL-5 and IL-13 and increased amphiregulin. MaR1 augmented de novo generation of regulatory T cells (Tregs), which interacted with ILC2s to markedly suppress cytokine production in a TGF-β-dependent manner. Ab-mediated depletion of Tregs interrupted MaR1 control of ILC2 expression of IL-13 in vivo. Together, the findings uncover Tregs as potent regulators of ILC2 activation; MaR1 targets Tregs and ILC2s to restrain allergic lung inflammation, suggesting MaR1 as the basis for a new proresolving therapeutic approach to asthma and other chronic inflammatory diseases.

  19. In silico prediction of the T-cell and IgE-binding epitopes of Per a 6 and Bla g 6 allergens in cockroaches.

    PubMed

    Chen, Hao; Yang, Hai-Wei; Wei, Ji-Fu; Tao, Ai-Lin

    2014-10-01

    Per a 6 and Bla g 6 are cockroach allergens found in Periplaneta americana and Blattella germanica, respectively. The objective of the present study was to predict the B‑ and T‑cell epitopes of the Per a 6 and Bla g 6 allergens. Three immunoinformatics tools, the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server, were used to predict the potential B‑cell epitopes, while Net‑MHCIIpan‑2.0 and NetMHCII‑2.2 were used to predict the T‑cell epitopes of the two allergens. As a result, seven peptides were predicted in the Per a 6 allergen and seven peptides were predicted in the Bla g 6 allergen in the B‑cell epitope predictions. In the T‑cell prediction, the Per a 6 allergen was predicted to have nine strongly binding nonamer core epitope sequences (IC50<50 nm) and 28 weakly binding sequences (50 nm

  20. The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8+ T cells

    PubMed Central

    Kurachi, Makoto; Barnitz, R. Anthony; Yosef, Nir; Odorizzi, Pamela M.; Dilorio, Michael A.; Lemieux, Madeleine E.; Yates, Kathleen; Godec, Jernej; Klatt, Martin G.; Regev, Aviv; Wherry, E. John; Haining, W. Nicholas

    2014-01-01

    The transcription factor BATF is required for interleukin 17 (IL-17)-producing helper T cell (TH17) and follicular helper T cell (TFH) differentiation. Here, we show that BATF also has a fundamental role in regulating effector CD8+ T cell differentiation. BATF-deficient CD8+ T cells show profound defects in effector expansion and undergo proliferative and metabolic catastrophe early after antigen encounter. BATF, together with IRF4 and Jun proteins, binds to and promotes early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors, while paradoxically repressing genes encoding effector molecules (IFN-γ and granzyme B). Thus, BATF amplifies TCR-dependent transcription factor expression and augments inflammatory signal propagation but restrains effector gene expression. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved. PMID:24584090

  1. Interleukin-6 expands homeostatic space for peripheral T cells.

    PubMed

    Hong, Changwan; Nam, Anna S; Keller, Hilary R; Ligons, Davinna L; Park, Joo-Young; Yoon, Hee-Won; Park, Joseph J; Luckey, Megan A; Park, Jung-Hyun

    2013-11-01

    T cell homeostasis and survival is dependent on interleukin-7 (IL-7). Immune activation, however, downregulates IL-7 receptor expression on T cells so that T cell survival during activation must be maintained independently of IL-7. The pro-inflammatory cytokine IL-6 shares common signaling pathways with IL-7 and can promote T cell survival in vitro. But whether IL-6 promotes T cell survival and homeostasis in vivo is not clear. Notably, IL-6 overexpression results in massive plasmacytosis and autoimmunity so that an IL-6 effect on in vivo T cell survival has remained untested. To overcome this limitation, here we generated IL-6 transgenic mice on an immunoglobulin heavy chain (IgH) deficient background which rendered them B cell deficient. Notably, such IgH(KO)IL6(Tg) mice were free of any signs of inflammation or autoimmunity and remained healthy throughout the course of analysis. In these mice, we found that IL-6 overexpression significantly increased peripheral T cell numbers, but importantly without increasing thymopoiesis. Moreover, IL-6 signaled T cells maintained their naïve phenotype and did not express activation/memory markers, suggesting that increased T cell numbers were due to increased T cell survival and not because of expansion of activated T cells. Mechanistically, we found that IL-6 signaling induced expression of pro-survival factors Mcl-1 and Pim-1/-2 but not Bcl-2. Thus, IL-6 is a T cell homeostatic cytokine that expands T cell space and can maintain the naïve T cell pool. PMID:23988623

  2. MHC class II transactivator represses human IL-4 gene transcription by interruption of promoter binding with CBP/p300, STAT6 and NFAT1 via histone hypoacetylation

    PubMed Central

    Zhou, Xiaorong; Jiang, Yang; Lu, Liming; Ding, Qing; Jiao, Zhijun; Zhou, Yun; Xin, Lijun; Chou, Kuang-Yen

    2007-01-01

    In addition to its property of enhancing major histocompatibility complex (MHC) class II expression, the class II transactivator (CIITA) was recently demonstrated to be involved in T helper type 1/type 2 (Th1/Th2) differentiation by regulating interleukin-4 (IL-4) gene transcription. There was however, controversy regarding whether CIITA promotes or suppresses IL-4 expression in the experiments with transgenic mice. To clarify the discrepancy by using simpler experimental systems, human Jurkat T cells that express IL-4 but not interferon-γ, even if stimulated with phorbol 12-myristate 13-acetate plus ionomycin, were used for CIITA transfection. Significant suppression of IL-4 gene expression was demonstrated. Simultaneously, histones H3 and H4 in the IL-4 promoter were hypoacetylated. The suppression could be totally reversed by the histone deacetylatase inhibitor trichostatin A. Furthermore, the IL-4 expression was determined in primarily established human Th1/Th2 cells to which CIITA small interference RNA (siRNA) had been introduced. A substantially increased level of IL-4 was recorded in the CIITA siRNA-transfected Th1 cells, which was in parallel with significantly enhanced acetylation in histone H3 of the IL-4 promoter. Chromatin immunoprecipitation analysis indicated that CIITA abrogated the binding of coactivator CBP/p300 and transcription factors STAT6/NFAT1 to IL-4 promoter in the CIITA-transfected cells. In conclusion, CIITA was active in the repression of transcription activation of human IL-4 gene in both the T-cell line and the primary human CD4 T cells by preventing transcription factors from binding to IL-4 promoter through histone hypoacetylation. Our data confirm a potential significant role of CIITA in controlling Th1/Th2 differentiation via modulation of IL-4 gene activation. PMID:17645498

  3. A stochastic T cell response criterion

    PubMed Central

    Currie, James; Castro, Mario; Lythe, Grant; Palmer, Ed; Molina-París, Carmen

    2012-01-01

    The adaptive immune system relies on different cell types to provide fast and coordinated responses, characterized by recognition of pathogenic challenge, extensive cellular proliferation and differentiation, as well as death. T cells are a subset of the adaptive immune cellular pool that recognize immunogenic peptides expressed on the surface of antigen-presenting cells by means of specialized receptors on their membrane. T cell receptor binding to ligand determines T cell responses at different times and locations during the life of a T cell. Current experimental evidence provides support to the following: (i) sufficiently long receptor–ligand engagements are required to initiate the T cell signalling cascade that results in productive signal transduction and (ii) counting devices are at work in T cells to allow signal accumulation, decoding and translation into biological responses. In the light of these results, we explore, with mathematical models, the timescales associated with T cell responses. We consider two different criteria: a stochastic one (the mean time it takes to have had N receptor–ligand complexes bound for at least a dwell time, τ, each) and one based on equilibrium (the time to reach a threshold number N of receptor–ligand complexes). We have applied mathematical models to previous experiments in the context of thymic negative selection and to recent two-dimensional experiments. Our results indicate that the stochastic criterion provides support to the thymic affinity threshold hypothesis, whereas the equilibrium one does not, and agrees with the ligand hierarchy experimentally established for thymic negative selection. PMID:22745227

  4. Estrogen Therapy Delays Autoimmune Diabetes and Promotes the Protective Efficiency of Natural Killer T-Cell Activation in Female Nonobese Diabetic Mice.

    PubMed

    Gourdy, Pierre; Bourgeois, Elvire A; Levescot, Anaïs; Pham, Linh; Riant, Elodie; Ahui, Marie-Louise; Damotte, Diane; Gombert, Jean-Marc; Bayard, Francis; Ohlsson, Claes; Arnal, Jean-François; Herbelin, André

    2016-01-01

    Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention. PMID:26485613

  5. Intraepithelial macrophage infiltration is related to a high number of regulatory T cells and promotes a progressive course of HPV-induced vulvar neoplasia.

    PubMed

    van Esch, Edith M G; van Poelgeest, Mariette I E; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Jordanova, Ekaterina S; van der Burg, Sjoerd H

    2015-02-15

    Human papilloma virus (HPV)-induced usual-type vulvar intraepithelial neoplasia (uVIN) is infiltrated by myeloid cells but the type and role of these cells is unclear. We used triple immunofluorescent confocal microscopy to locate, identify and quantify myeloid cells based on their staining pattern for CD14, CD33 and CD163 in a cohort of 43 primary and 20 recurrent uVIN lesions, 21 carcinomas and 26 normal vulvar tissues. The progressive course of uVIN is characterized by an increase in both intraepithelial and stromal mature M1 and M2 macrophages. While the M2 macrophages outnumber M1 macrophages in healthy controls and uVIN, they are matched in number by M1 macrophages in cancer. Importantly, uVIN patients with a dense intraepithelial infiltration with mature CD14+ macrophages (irrespective of M1 or M2 type) displayed approximately a six times higher risk to develop a recurrence and a high number of these cells constituted an independent prognostic factor for recurrence. In addition, a dense intraepithelial CD14+ cell infiltration was associated with high numbers of intraepithelial CD4+ Tregs and low numbers of stromal CD8+TIM3+ T cells. Patients with low numbers of intraepithelial CD14+ cells and high numbers of stromal CD8+TIM3+ cells showed the best recurrence-free survival. These data clearly show the importance of the local immune response in HPV-induced vulvar neoplasia and may be of help in predicting the prognosis of patients or their response to immunotherapy.

  6. An Oral Salmonella-Based Vaccine Inhibits Liver Metastases by Promoting Tumor-Specific T-Cell-Mediated Immunity in Celiac and Portal Lymph Nodes: A Preclinical Study.

    PubMed

    Vendrell, Alejandrina; Mongini, Claudia; Gravisaco, María José; Canellada, Andrea; Tesone, Agustina Inés; Goin, Juan Carlos; Waldner, Claudia Inés

    2016-01-01

    Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4(+) and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8(+)IFN-γ(+)) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination. PMID:26973649

  7. Estrogen Therapy Delays Autoimmune Diabetes and Promotes the Protective Efficiency of Natural Killer T-Cell Activation in Female Nonobese Diabetic Mice.

    PubMed

    Gourdy, Pierre; Bourgeois, Elvire A; Levescot, Anaïs; Pham, Linh; Riant, Elodie; Ahui, Marie-Louise; Damotte, Diane; Gombert, Jean-Marc; Bayard, Francis; Ohlsson, Claes; Arnal, Jean-François; Herbelin, André

    2016-01-01

    Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention.

  8. An Oral Salmonella-Based Vaccine Inhibits Liver Metastases by Promoting Tumor-Specific T-Cell-Mediated Immunity in Celiac and Portal Lymph Nodes: A Preclinical Study

    PubMed Central

    Vendrell, Alejandrina; Mongini, Claudia; Gravisaco, María José; Canellada, Andrea; Tesone, Agustina Inés; Goin, Juan Carlos; Waldner, Claudia Inés

    2016-01-01

    Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4+ and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8+IFN-γ+) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination. PMID:26973649

  9. An Oral Salmonella-Based Vaccine Inhibits Liver Metastases by Promoting Tumor-Specific T-Cell-Mediated Immunity in Celiac and Portal Lymph Nodes: A Preclinical Study.

    PubMed

    Vendrell, Alejandrina; Mongini, Claudia; Gravisaco, María José; Canellada, Andrea; Tesone, Agustina Inés; Goin, Juan Carlos; Waldner, Claudia Inés

    2016-01-01

    Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4(+) and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8(+)IFN-γ(+)) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination.

  10. T cell activation regulates CD6 alternative splicing by transcription dynamics and SRSF1.

    PubMed

    da Glória, Vânia G; Martins de Araújo, Mafalda; Mafalda Santos, Ana; Leal, Rafaela; de Almeida, Sérgio F; Carmo, Alexandre M; Moreira, Alexandra

    2014-07-01

    The T cell-surface glycoprotein CD6 is a modulator of cellular responses and has been implicated in several autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. During Ag presentation, CD6 is targeted to the immunological synapse in a ligand binding-dependent manner, in which CD6 domain 3 directly contacts CD166, expressed on the APC. T cell activation results in the induction of CD6Δd3, an alternatively spliced isoform that lacks the ligand-binding domain and thus no longer localizes at the immunological synapse. In this study, we investigated the molecular mechanisms regulating the expression of CD6Δd3 upon human primary T cell activation. Using chromatin immunoprecipitation, we observed an increase in RNA polymerase II occupancy along the CD6 gene and augmented CD6 transcription. We showed that activation leads to transcription-related chromatin modifications, revealed by higher CD6 acetylation levels. Modulation of chromatin conformation using a histone deacetylase inhibitor that increases transcription rate causes an increase of exon 5 skipping. We further showed that the splicing factor SRSF1 binds to a regulatory element in CD6 intron 4, activating exon 5 splicing and promoting exon 5 inclusion. Concomitant with T cell activation-induced exon 5 skipping, we observed a downregulation of SRSF1. Using RNA immunoprecipitation, we showed that in activated T cells, SRSF1 recruitment to the CD6 transcript is impaired by increased chromatin acetylation levels. We propose that upon T cell activation, SRSF1 becomes limiting, and its function in CD6 exon 5 splicing is countered by an increase in CD6 transcription, dependent on chromatin acetylation.

  11. The function of the octamer-binding site in the DRA promoter

    SciTech Connect

    Voliva, C.F.; Jabrane-Ferrat, N.; Peterlin, B.M.

    1996-06-01

    The octamer binding site, which is located immediately upstream of the poorly conserved DRA TATA sequence, is important for high levels of expression of this human major histocompatibility class II gene in B cells. In this study, we demonstrate that the substitution of the DRA TATA sequence with the TATA box from the adenovirus Elb promoter removes the requirement for the octamer binding site for high levels of expression from the DRA promoter. Since only the TATA box from the Elb but not the DRA promoters binds the TATA binding protein, we conclude that the octamer binding site helps to recruit TBP to the DRA promoter. 32 refs., 7 figs.

  12. Inhibition of cell-to-cell transmission of human T-cell lymphotropic virus type 1 in vitro by carbohydrate-binding agents.

    PubMed

    Balestrieri, Emanuela; Ascolani, Arianna; Igarashi, Yasuhiro; Oki, Toshikazu; Mastino, Antonio; Balzarini, Jan; Macchi, Beatrice

    2008-08-01

    Peripheral blood mononuclear cells (PBMCs) from healthy individuals can be infected by human T-lymphotropic virus type 1 (HTLV-1) upon cocultivation of the PBMCs with irradiated HTLV-1-transformed human MT-2 cells. This model system closely mimics HTLV-1 transmission through cell-to-cell contact. Carbohydrate-binding agents (CBAs) such as the alpha(1,3)/alpha(1,6)mannose-specific Hippeastrum hybrid agglutinin and the GlcNAc-specific Urtica dioica agglutinin, and also the small, nonpeptidic alpha(1,2)-mannose-specific antibiotic pradimicin A, were able to efficiently prevent cell-to-cell HTLV-1 transmission at nontoxic concentrations, as evidenced by the lack of appearance of virus-specific mRNA and of the viral protein Tax in the acceptor cells. Consistently, antivirally active doses of CBAs fully prevented HTLV-1-induced stimulation of PBMC growth. The inhibitory effects of CBAs on HTLV-1 transmission were also evident when HTLV-1-infected C5MJ cells were used in place of MT-2 cells as a virus donor cell line. The anti-HTLV-1 properties of the CBAs highlight the importance of the envelope glycans in events underlying HTLV-1 passage from cell to cell and indicate that CBAs should be further investigated for their potential to prevent HTLV-1 infection, including mother-to-child virus transmission by cell-to-cell contact through breast milk feeding. PMID:18505856

  13. Phenotypic models of T cell activation.

    PubMed

    Lever, Melissa; Maini, Philip K; van der Merwe, P Anton; Dushek, Omer

    2014-09-01

    T cell activation is a crucial checkpoint in adaptive immunity, and this activation depends on the binding parameters that govern the interactions between T cell receptors (TCRs) and peptide-MHC complexes (pMHC complexes). Despite extensive experimental studies, the relationship between the TCR-pMHC binding parameters and T cell activation remains controversial. To make sense of conflicting experimental data, a variety of verbal and mathematical models have been proposed. However, it is currently unclear which model or models are consistent or inconsistent with experimental data. A key problem is that a direct comparison between the models has not been carried out, in part because they have been formulated in different frameworks. For this Analysis article, we reformulated published models of T cell activation into phenotypic models, which allowed us to directly compare them. We find that a kinetic proofreading model that is modified to include limited signalling is consistent with the majority of published data. This model makes the intriguing prediction that the stimulation hierarchy of two different pMHC complexes (or two different TCRs that are specific for the same pMHC complex) may reverse at different pMHC concentrations.

  14. The novel C24D synthetic polypeptide inhibits binding of placenta immunosuppressive ferritin to human T cells and elicits anti-breast cancer immunity in vitro and in vivo.

    PubMed

    Solodeev, Inna; Zahalka, Muayad A; Moroz, Chaya

    2014-09-01

    Immune tolerance mechanisms supporting normal human pregnancy are exploited by breast cancer and other malignancies. We cloned from human placenta and breast cancer cells the novel human immunomodulator named placenta immunosuppressive ferritin (PLIF). PLIF is composed of a ferritin heavy chain-like domain and a novel cytokine-like domain, named C48. Both intact PLIF and C48 inhibit T cell proliferation. Blocking PLIF by specific antibodies in a tolerant breast cancer model in nude mice resulted in tumor cell apoptosis and rejection. This prompted us to study active immune preventive strategies targeting PLIF activity. Currently, we report on the design and synthesis of the novel C24D polypeptide, which inhibits the binding of PLIF to T cells and therefore inhibits the immune suppressive effect of PLIF. The effect of C24D on the generation of anti-breast cancer cytotoxic T lymphocytes (CTLs) was studied in vitro in cultures of MCF-7 (HLA-A2(+)) or T47D (HLA-A2(-)) breast cancer cells incubated with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. We found that C24D treatment exclusively induced development of CTLs. On reactivation by their specific target cells, the CTLs secreted interferon-γ and induced target apoptosis. Anti-MCF-7 CTLs were cross-cytotoxic to MDA-MB-231 (HLA-A2(+)) triple-negative breast cancer but not to T47D. Moreover, C24D treatment in vivo inhibited the growth of MCF-7 tumors engrafted in immune-compromised nude mice transfused with naïve allogeneic human PBMCs. Our results demonstrate that C24D treatment breakdown breast cancer induced tolerance enabling the initiation of effective anti-tumor immune response. PMID:25246274

  15. The novel C24D synthetic polypeptide inhibits binding of placenta immunosuppressive ferritin to human T cells and elicits anti-breast cancer immunity in vitro and in vivo.

    PubMed

    Solodeev, Inna; Zahalka, Muayad A; Moroz, Chaya

    2014-09-01

    Immune tolerance mechanisms supporting normal human pregnancy are exploited by breast cancer and other malignancies. We cloned from human placenta and breast cancer cells the novel human immunomodulator named placenta immunosuppressive ferritin (PLIF). PLIF is composed of a ferritin heavy chain-like domain and a novel cytokine-like domain, named C48. Both intact PLIF and C48 inhibit T cell proliferation. Blocking PLIF by specific antibodies in a tolerant breast cancer model in nude mice resulted in tumor cell apoptosis and rejection. This prompted us to study active immune preventive strategies targeting PLIF activity. Currently, we report on the design and synthesis of the novel C24D polypeptide, which inhibits the binding of PLIF to T cells and therefore inhibits the immune suppressive effect of PLIF. The effect of C24D on the generation of anti-breast cancer cytotoxic T lymphocytes (CTLs) was studied in vitro in cultures of MCF-7 (HLA-A2(+)) or T47D (HLA-A2(-)) breast cancer cells incubated with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. We found that C24D treatment exclusively induced development of CTLs. On reactivation by their specific target cells, the CTLs secreted interferon-γ and induced target apoptosis. Anti-MCF-7 CTLs were cross-cytotoxic to MDA-MB-231 (HLA-A2(+)) triple-negative breast cancer but not to T47D. Moreover, C24D treatment in vivo inhibited the growth of MCF-7 tumors engrafted in immune-compromised nude mice transfused with naïve allogeneic human PBMCs. Our results demonstrate that C24D treatment breakdown breast cancer induced tolerance enabling the initiation of effective anti-tumor immune response.

  16. Detection of an autoreactive T-cell population within the polyclonal repertoire that undergoes distinct autoimmune regulator (Aire)-mediated selection.

    PubMed

    Taniguchi, Ruth T; DeVoss, Jason J; Moon, James J; Sidney, John; Sette, Alessandro; Jenkins, Marc K; Anderson, Mark S

    2012-05-15

    The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting the display of tissue-specific antigens in the thymus. To study the influence of Aire on thymic selection in a physiological setting, we used tetramer reagents to detect autoreactive T cells specific for the Aire-dependent tissue-specific antigen interphotoreceptor retinoid-binding protein (IRBP), in the polyclonal repertoire. Two class II tetramer reagents were designed to identify T cells specific for two different peptide epitopes of IRBP. Analyses of the polyclonal T-cell repertoire showed a high frequency of activated T cells specific for both IRBP tetramers in Aire(-/-) mice, but not in Aire(+/+) mice. Surprisingly, although one tetramer-binding T-cell population was efficiently deleted in the thymus in an Aire-dependent manner, the second tetramer-binding population was not deleted and could be detected in both the Aire(-/-) and Aire(+/+) T-cell repertoires. We found that Aire-dependent thymic deletion of IRBP-specific T cells relies on intercellular transfer of IRBP between thymic stroma and bone marrow-derived antigen-presenting cells. Furthermore, our data suggest that Aire-mediated deletion relies not only on thymic expression of IRBP, but also on proper antigen processing and presentation of IRBP by thymic antigen-presenting cells.

  17. Sialylation regulates peripheral tolerance in CD4+ T cells.

    PubMed

    Brennan, Patrick J; Saouaf, Sandra J; Van Dyken, Steve; Marth, Jamey D; Li, Bin; Bhandoola, Avinash; Greene, Mark I

    2006-05-01

    Decreased binding by the 6C10 auto-antibody serves as a unique marker for CD4+ T cell unresponsiveness after the induction of T cell tolerance in Vbeta8.1 TCR transgenic mice. We further define the nature of the epitope recognized by the 6C10 antibody to be a subset of Thy-1 bearing incompletely sialylated N-linked glycans, and furthermore, we demonstrate that tolerant CD4+ T cells have an increased degree of cell-surface sialylation. To test the significance of the altered glycosylation state identified by the 6C10 auto-antibody in the tolerant CD4+ T cell population, surface sialic acid was cleaved enzymatically. Treatment of purified peripheral CD4+ T cells with Vibrio cholerae sialidase (VCS) leads to increased 6C10 binding, significantly enhances proliferation in the tolerant CD4+ population and corrects defects in phosphotyrosine signaling observed in the tolerant CD4+ T cell. Furthermore, in vivo administration of VCS enhances proliferation in both tolerant and naive CD4+ T cell subsets. These studies suggest that sialylation of glycoproteins on the surface of the CD4+ T cell contributes to the regulation of T cell responsiveness in the tolerant state. PMID:16291658

  18. A "hotspot" for autoimmune T cells in type 1 diabetes.

    PubMed

    Stadinski, Brian D; Obst, Reinhard; Huseby, Eric S

    2016-06-01

    The ability of a single T cell antigen receptor (TCR) to cross-react with multiple antigens allows the finite number of T cells within an organism to respond to the compendium of pathogen challenges faced during a lifetime. Effective immune surveillance, however, comes at a price. TCR cross-reactivity can allow molecular mimics to spuriously activate autoimmune T cells; it also underlies T cell rejection of organ transplants and drives graft-versus-host disease. In this issue of the JCI, Cole and colleagues provide insight into how an insulin-reactive T cell cross-reacts with pathogen-derived antigens by focusing on a limited portion of the peptides to provide a hotspot for binding. These findings dovetail with recent studies of alloreactive and autoimmune TCRs and suggest that the biochemical principles that govern conventional protein-protein interactions may allow the specificity and cross-reactivity profiles of T cells to be predicted. PMID:27183386

  19. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy

    PubMed Central

    Dai, Hanren; Wang, Yao; Lu, Xuechun

    2016-01-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  20. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    PubMed

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy.

  1. NAD+ regulates Treg cell fate and promotes allograft survival via a systemic IL-10 production that is CD4+ CD25+ Foxp3+ T cells independent

    PubMed Central

    Elkhal, Abdallah; Rodriguez Cetina Biefer, Hector; Heinbokel, Timm; Uehara, Hirofumi; Quante, Markus; Seyda, Midas; Schuitenmaker, Jeroen M.; Krenzien, Felix; Camacho, Virginia; de la Fuente, Miguel A.; Ghiran, Ionita; Tullius, Stefan G.

    2016-01-01

    CD4+ CD25+ Foxp3+ Tregs have been shown to play a central role in immune homeostasis while preventing from fatal inflammatory responses, while Th17 cells have traditionally been recognized as pro-inflammatory mediators implicated in a myriad of diseases. Studies have shown the potential of Tregs to convert into Th17 cells, and Th17 cells into Tregs. Increasing evidence have pointed out CD25 as a key molecule during this transdifferentiation process, however molecules that allow such development remain unknown. Here, we investigated the impact of NAD+ on the fate of CD4+ CD25+ Foxp3+ Tregs in-depth, dissected their transcriptional signature profile and explored mechanisms underlying their conversion into IL-17A producing cells. Our results demonstrate that NAD+ promotes Treg conversion into Th17 cells in vitro and in vivo via CD25 cell surface marker. Despite the reduced number of Tregs, known to promote homeostasis, and an increased number of pro-inflammatory Th17 cells, NAD+ was able to promote an impressive allograft survival through a robust systemic IL-10 production that was CD4+ CD25+ Foxp3+ independent. Collectively, our study unravels a novel immunoregulatory mechanism of NAD+ that regulates Tregs fate while promoting allograft survival that may have clinical applications in alloimmunity and in a wide spectrum of inflammatory conditions. PMID:26928119

  2. Regulation of Lipid Specific and Vitamin Specific Non-MHC Restricted T Cells by Antigen Presenting Cells and Their Therapeutic Potentials

    PubMed Central

    Salio, Mariolina; Cerundolo, Vincenzo

    2015-01-01

    Since initial reports, more than 25 years ago, that T cells recognize lipids in the context on non-polymorphic CD1 molecules, our understanding of antigen presentation to non-peptide-specific T cell populations has deepened. It is now clear that αβ T cells bearing semi-invariant T cell receptor, as well as subsets of γδ T cells, recognize a variety of self and non-self lipids and contribute to shaping immune responses via cross talk with dendritic cells and B cells. Furthermore, it has been demonstrated that small molecules derived from the microbial riboflavin biosynthetic pathway (vitamin B2) bind monomorphic MR1 molecules and activate mucosal-associated invariant T cells, another population of semi-invariant T cells. Novel insights in the biological relevance of non-peptide-specific T cells have emerged with the development of tetrameric CD1 and MR1 molecules, which has allowed accurate enumeration and functional analysis of CD1- and MR1-restricted T cells in humans and discovery of novel populations of semi-invariant T cells. The phenotype and function of non-peptide-specific T cells will be discussed in the context of the known distribution of CD1 and MR1 molecules by different subsets of antigen-presenting cells at steady state and following infection. Concurrent modulation of CD1 transcription and lipid biosynthetic pathways upon TLR stimulation, coupled with efficient lipid antigen processing, result in the increased cell surface expression of antigenic CD1–lipid complexes. Similarly, MR1 expression is almost undetectable in resting APC and it is upregulated following bacterial infection, likely due to stabilization of MR1 molecules by microbial antigens. The tight regulation of CD1 and MR1 expression at steady state and during infection may represent an important mechanism to limit autoreactivity, while promoting T cell responses to foreign antigens. PMID:26284072

  3. Interleukin-21 Is a Critical Regulator of CD4 and CD8 T Cell Survival during Priming under Interleukin-2 Deprivation Conditions

    PubMed Central

    Khattar, Mithun; Miyahara, Yoshihiro; Schroder, Paul M.; Xie, Aini; Chen, Wenhao; Stepkowski, Stanislaw M.

    2014-01-01

    Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8+ T cells was reduced, whereas CD4+ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8+ T cells in comparison to CD4+ T cells. However, using transwell co-culture assays we have found that CD4+ T cells could rescue the survival of CD8+ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8+ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4+ and CD8+ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses. PMID:24416451

  4. T Cell Metabolic Fitness in Anti-Tumor Immunity

    PubMed Central

    Siska, Peter J.; Rathmell, Jeffrey C.

    2015-01-01

    SUMMARY T cell metabolism plays a central role to support and shape immune responses and may play a key role in anti-tumor immunity. T cell metabolism is normally held under tight regulation in an immune response of glycolysis to promote effector T cell expansion and function. However, tumors may deplete nutrients, generate toxic products, or stimulate conserved negative feedback mechanisms, such as through PD-1, to impair effector T cell nutrient uptake and metabolic fitness. In addition, regulatory T cells are favored in low glucose conditions and may inhibit anti-tumor immune responses. Here we review how the tumor microenvironment modifies metabolic and functional pathways in T cells and how these changes may uncover new targets and challenges for cancer immunotherapy and treatment. PMID:25773310

  5. Kinase-independent function of RIP1, critical for mature T-cell survival and proliferation

    PubMed Central

    Dowling, John P; Cai, Yubo; Bertin, John; Gough, Peter J; Zhang, Jianke

    2016-01-01

    The death receptor, Fas, triggers apoptotic death and is essential for maintaining homeostasis in the peripheral lymphoid organs. RIP1 was originally cloned when searching for Fas-binding proteins and was later shown to associate also with the signaling complex of TNFR1. Although Fas exclusively induces apoptosis, TNFR1 primarily activates the pro-survival/pro-inflammatory NF-κB pathway. Mutations in Fas lead to lymphoproliferative (lpr) diseases, and deletion of TNFR1 results in defective innate immune responses. However, the function of RIP1 in the adult lymphoid system has not been well understood, primarily owing to perinatal lethality in mice lacking the entire RIP1 protein in germ cells. This current study investigated the requirement for RIP1 in the T lineage using viable RIP1 mutant mice containing a conditional and kinase-dead RIP1 allele. Disabling the kinase activity of RIP1 had no obvious impact on the T-cell compartment. However, T-cell-specific deletion of RIP1 led to a severe T-lymphopenic condition, owing to a dramatically reduced mature T-cell pool in the periphery. Interestingly, the immature T-cell compartment in the thymus appeared intact. Further analysis showed that mature RIP1−/− T cells were severely defective in antigen receptor-induced proliferative responses. Moreover, the RIP1−/− T cells displayed greatly increased death and contained elevated caspase activities, an indication of apoptosis. In total, these results revealed a novel, kinase-independent function of RIP1, which is essential for not only promoting TCR-induced proliferative responses but also in blocking apoptosis in mature T cells. PMID:27685623

  6. Inducible T-cell receptor expression in precursor T cells for leukemia control.

    PubMed

    Hoseini, S S; Hapke, M; Herbst, J; Wedekind, D; Baumann, R; Heinz, N; Schiedlmeier, B; Vignali, D A A; van den Brink, M R M; Schambach, A; Blazar, B R; Sauer, M G

    2015-07-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T-cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. As expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8(+) T-cell development was required to obtain a mature T-cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T-cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity.

  7. Inducible T-cell receptor expression in precursor T cells for leukemia control.

    PubMed

    Hoseini, S S; Hapke, M; Herbst, J; Wedekind, D; Baumann, R; Heinz, N; Schiedlmeier, B; Vignali, D A A; van den Brink, M R M; Schambach, A; Blazar, B R; Sauer, M G

    2015-07-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T-cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. As expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8(+) T-cell development was required to obtain a mature T-cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T-cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

  8. Immediate Dysfunction of Vaccine-Elicited CD8+ T Cells Primed in the Absence of CD4+ T Cells

    PubMed Central

    Provine, Nicholas M.; Larocca, Rafael A.; Aid, Malika; Penaloza-MacMaster, Pablo; Badamchi-Zadeh, Alexander; Borducchi, Erica N.; Yates, Kathleen B.; Abbink, Peter; Kirilova, Marinela; Ng’ang’a, David; Bramson, Jonathan; Haining, W. Nicholas

    2016-01-01

    CD4+ T cell help is critical for optimal CD8+ T cell memory differentiation and maintenance in many experimental systems. In addition, many reports have identified reduced primary CD8+ T cell responses in the absence of CD4+ T cell help, which often coincides with reduced Ag or pathogen clearance. In this study, we demonstrate that absence of CD4+ T cells at the time of adenovirus vector immunization of mice led to immediate impairments in early CD8+ T cell functionality and differentiation. Unhelped CD8+ T cells exhibited a reduced effector phenotype, decreased ex vivo cytotoxicity, and decreased capacity to produce cytokines. This dysfunctional state was imprinted within 3 d of immunization. Unhelped CD8+ T cells expressed elevated levels of inhibitory receptors and exhibited transcriptomic exhaustion and anergy profiles by gene set enrichment analysis. Dysfunctional, impaired effector differentiation also occurred following immunization of CD4+ T cell–deficient mice with a poxvirus vector. This study demonstrates that following priming with viral vectors, CD4+ T cell help is required to promote both the expansion and acquisition of effector functions by CD8+ T cells, which is accomplished by preventing immediate dysfunction. PMID:27448585

  9. Molecular Insights for Optimizing T Cell Receptor Specificity Against Cancer

    PubMed Central

    Hebeisen, Michael; Oberle, Susanne G.; Presotto, Danilo; Speiser, Daniel E.; Zehn, Dietmar; Rufer, Nathalie

    2013-01-01

    Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses. PMID:23801991

  10. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    NASA Astrophysics Data System (ADS)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  11. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits.

    PubMed

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-07-13

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  12. An Interleukin-33-Mast Cell-Interleukin-2 Axis Suppresses Papain-Induced Allergic Inflammation by Promoting Regulatory T Cell Numbers

    PubMed Central

    Morita, Hideaki; Arae, Ken; Unno, Hirotoshi; Miyauchi, Kousuke; Toyama, Sumika; Nambu, Aya; Oboki, Keisuke; Ohno, Tatsukuni; Motomura, Kenichiro; Matsuda, Akira; Yamaguchi, Sachiko; Narushima, Seiko; Kajiwara, Naoki; Iikura, Motoyasu; Suto, Hajime; McKenzie, Andrew N.J.; Takahashi, Takao; Karasuyama, Hajime; Okumura, Ko; Azuma, Miyuki; Moro, Kazuyo; Akdis, Cezmi A.; Galli, Stephen J.; Koyasu, Shigeo; Kubo, Masato; Sudo, Katsuko; Saito, Hirohisa; Matsumoto, Kenji; Nakae, Susumu

    2015-01-01

    SUMMARY House dust mite-derived proteases contribute to allergic disorders in part by disrupting epithelial barrier function. Interleukin-33 (IL-33), produced by lung cells after exposure to protease allergens, can induce innate-type airway eosinophilia by activating natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2), to secrete Th2 type-cytokines. Because IL-33 also can induce mast cells (MCs) to secrete Th2 type-cytokines, MCs are thought to cooperate with NH cells in enhancing protease or IL-33-mediated innate-type airway eosinophilia. However, we found that MC-deficient KitW-sh/W-sh mice exhibited exacerbated protease-induced lung inflammation associated with reduced numbers of regulatory T (Treg) cells. Moreover, IL-2 produced by IL-33-stimulated MCs promoted expansion of numbers of Treg cells, thereby suppressing development of papain- or IL-33-induced airway eosinophilia. We have thus identified a unique anti-inflammatory pathway that can limit induction of innate-type allergic airway inflammation mediated by NH cells. PMID:26200013

  13. An Interleukin-33-Mast Cell-Interleukin-2 Axis Suppresses Papain-Induced Allergic Inflammation by Promoting Regulatory T Cell Numbers.

    PubMed

    Morita, Hideaki; Arae, Ken; Unno, Hirotoshi; Miyauchi, Kousuke; Toyama, Sumika; Nambu, Aya; Oboki, Keisuke; Ohno, Tatsukuni; Motomura, Kenichiro; Matsuda, Akira; Yamaguchi, Sachiko; Narushima, Seiko; Kajiwara, Naoki; Iikura, Motoyasu; Suto, Hajime; McKenzie, Andrew N J; Takahashi, Takao; Karasuyama, Hajime; Okumura, Ko; Azuma, Miyuki; Moro, Kazuyo; Akdis, Cezmi A; Galli, Stephen J; Koyasu, Shigeo; Kubo, Masato; Sudo, Katsuko; Saito, Hirohisa; Matsumoto, Kenji; Nakae, Susumu

    2015-07-21

    House dust mite-derived proteases contribute to allergic disorders in part by disrupting epithelial barrier function. Interleukin-33 (IL-33), produced by lung cells after exposure to protease allergens, can induce innate-type airway eosinophilia by activating natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2), to secrete Th2 type-cytokines. Because IL-33 also can induce mast cells (MCs) to secrete Th2 type-cytokines, MCs are thought to cooperate with NH cells in enhancing protease or IL-33-mediated innate-type airway eosinophilia. However, we found that MC-deficient Kit(W-sh/W-sh) mice exhibited exacerbated protease-induced lung inflammation associated with reduced numbers of regulatory T (Treg) cells. Moreover, IL-2 produced by IL-33-stimulated MCs promoted expansion of numbers of Treg cells, thereby suppressing development of papain- or IL-33-induced airway eosinophilia. We have thus identified a unique anti-inflammatory pathway that can limit induction of innate-type allergic airway inflammation mediated by NH cells.

  14. An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells.

    PubMed

    Massoud, Amir Hossein; Charbonnier, Louis-Marie; Lopez, David; Pellegrini, Matteo; Phipatanakul, Wanda; Chatila, Talal A

    2016-09-01

    Mechanisms by which regulatory T (Treg) cells fail to control inflammation in asthma remain poorly understood. We show that a severe asthma-associated polymorphism in the gene encoding the interleukin (IL)-4 receptor alpha chain (Il4ra(R576)) promotes conversion of induced Treg (iTreg) cells toward a T helper 17 (TH17) cell fate. This skewing is mediated by the recruitment by IL-4Rα(R576) of the growth-factor-receptor-bound protein 2 (GRB2) adaptor protein, which drives IL-17 expression by activating a pathway that involves extracellular-signal-regulated kinase, IL-6 and the transcription factor STAT3. Treg cell-specific deletion of genes that regulate TH17 cell differentiation, including Il6ra and RAR-related orphan receptor gamma (Rorc), but not of Il4 or Il13, prevented exacerbated airway inflammation in mice expressing Il4ra(R576) (hereafter referred to as Il4ra(R576) mice). Furthermore, treatment of Il4ra(R576) mice with a neutralizing IL-6-specific antibody prevented iTreg cell reprogramming into TH17-like cells and protected against severe airway inflammation. These findings identify a previously unknown mechanism for the development of mixed TH2-TH17 cell inflammation in genetically prone individuals and point to interventions that stabilize iTreg cells as potentially effective therapeutic strategies. PMID:27479084

  15. Increased 5-hydroxymethylcytosine in CD4(+) T cells in systemic lupus erythematosus.

    PubMed

    Zhao, Ming; Wang, Jing; Liao, Wei; Li, Duo; Li, Mengying; Wu, Haijing; Zhang, Yiqun; Gershwin, M Eric; Lu, Qianjin

    2016-05-01

    One of the major disappointments in autoimmunity has been the relative lack of informative data when genomewide associations (GWAS) have been applied to patients with systemic lupus erythematosus (SLE). Indeed, there is increasing evidence that SLE is characterized by widespread epigenetic changes. 5-Hydroxymethylcytosine (5-hmC) is a newly discovered modified form of cytosine suspected to be an important epigenetic modification in embryonic development, cell differentiation and cancer. DNA methylation dynamics have already been implicated in the pathogenesis of SLE, while little is known about hydroxymethylation in this process. Here, we show an increased 5-hmC level in genomic DNA in CD4(+) T cells of patients with SLE compared with healthy controls, accompanied by the up-regulated expression of the Ten-eleven translocation TET2 and TET3, which can enzymatically convert 5-methylcytosine (5-mC) to 5-hmC. Moreover, we present the differential patterns of DNA hydroxymethylation in genome-wide promoter regions in SLE CD4(+) T cells compared with healthy controls. We identified 2748 genes with increased 5-hmC levels in promoter regions in SLE CD4(+) T cells, which were enriched in critical pathways, including neurotrophin signaling, WNT signaling, MAPK signaling, calcium signaling and the mTOR signaling pathway. Through a combined analysis of differential DNA hydroxymethylation profile and gene expression profile in SLE CD4(+) T cells, we found 131 genes with the increased 5-hmC in promoter regions and up-regulated expression in SLE CD4(+) T cells compared with healthy controls, including selected immune-related genes, i.e. SOCS1, NR2F6 and IL15RA, which were also confirmed by ChIP-qPCR. Furthermore, we demonstrate that CTCF, as a transcription factor, can mediate DNA hydroxymethylation and contribute to overexpression of SOCS1 in CD4(+) T cells through binding to the promoter region of SOCS1. Taken together, our study reveals a critical differential 5-hmC in the

  16. Consensus nomenclature for CD8+ T cell phenotypes in cancer

    PubMed Central

    Apetoh, Lionel; Smyth, Mark J.; Drake, Charles G.; Abastado, Jean-Pierre; Apte, Ron N.; Ayyoub, Maha; Blay, Jean-Yves; Bonneville, Marc; Butterfield, Lisa H.; Caignard, Anne; Castelli, Chiara; Cavallo, Federica; Celis, Esteban; Chen, Lieping; Colombo, Mario P.; Comin-Anduix, Begoña; Coukos, Georges; Dhodapkar, Madhav V.; Dranoff, Glenn; Frazer, Ian H.; Fridman, Wolf-Hervé; Gabrilovich, Dmitry I.; Gilboa, Eli; Gnjatic, Sacha; Jäger, Dirk; Kalinski, Pawel; Kaufman, Howard L.; Kiessling, Rolf; Kirkwood, John; Knuth, Alexander; Liblau, Roland; Lotze, Michael T.; Lugli, Enrico; Marincola, Francesco; Melero, Ignacio; Melief, Cornelis J.; Mempel, Thorsten R.; Mittendorf, Elizabeth A.; Odun, Kunle; Overwijk, Willem W.; Palucka, Anna Karolina; Parmiani, Giorgio; Ribas, Antoni; Romero, Pedro; Schreiber, Robert D.; Schuler, Gerold; Srivastava, Pramod K.; Tartour, Eric; Valmori, Danila; van der Burg, Sjoerd H.; van der Bruggen, Pierre; van den Eynde, Benoît J.; Wang, Ena; Zou, Weiping; Whiteside, Theresa L.; Speiser, Daniel E.; Pardoll, Drew M.; Restifo, Nicholas P.; Anderson, Ana C.

    2015-01-01

    Whereas preclinical investigations and clinical studies have established that CD8+ T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8+ T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8+ T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8+ T cell immunity, leading to the emergence of dysfunctional CD8+ T cells. The existence of different types of CD8+ T cells in cancer calls for a more precise definition of the CD8+ T cell immune phenotypes in cancer and the abandonment of the generic terms “pro-tumor” and “antitumor.” Based on recent studies investigating the functions of CD8+ T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8+ T cells in cancer. PMID:26137416

  17. Egr2-dependent gene expression profiling and ChIP-Seq reveal novel biologic targets in T cell anergy

    PubMed Central

    Zheng, Yan; Zha, Yuanyuan; Spaapen, Robbert M.; Mathew, Rebecca; Barr, Kenneth; Bendelac, Albert; Gajewski, Thomas F.

    2013-01-01

    T cell anergy is one of the mechanisms contributing to peripheral tolerance, particularly in the context of progressively growing tumors and in tolerogenic treatments promoting allograft acceptance. We recently reported that early growth response gene 2 (Egr2) is a critical transcription factor for the induction of anergy in vitro and in vivo, which was identified based on its ability to regulate the expression of inhibitory signaling molecules diacylglycerol kinase (DGK)-α and -ζ. We reasoned that other transcriptional targets of Egr2 might encode additional factors important for T cell anergy and immune regulation. Thus, we conducted two sets of genome-wide screens: gene expression profiling of wild type versus Egr2-deleted T cells treated under anergizing conditions, and a ChIP-Seq analysis to identify genes that bind Egr2 in anergic cells. Merging of these data sets revealed 49 targets that are directly regulated by Egr2. Among these are inhibitory signaling molecules previously reported to contribute to T cell anergy, but unexpectedly, also cell surface molecules and secreted factors, including lymphocyte-activation gene 3 (Lag3), Class-I-MHC-restricted T cell associated molecule (Crtam), Semaphorin 7A (Sema7A), and chemokine CCL1. These observations suggest that anergic T cells might not simply be functionally inert, and may have additional functional properties oriented towards other cellular components of the immune system. PMID:23548837

  18. T-Cell Receptor-Transduced T Cells: Clinical Experience.

    PubMed

    Robbins, Paul F

    2015-01-01

    The large number of T-cell epitopes that have been found to be processed and presented on human tumors, now numbering in the hundreds, provides a rich source of targets for therapeutic interventions aimed at inducing durable tumor regression. Vaccination strategies aimed at inducing responses to these antigens have been largely ineffective, and it has been challenging to generate large numbers of T cells with the functional capacity to mediate durable tumor regressions in adoptive immunotherapy strategies in patients who have common epithelial malignancies. The ability to generate T-cell receptors that recognize shared as well as unique antigens expressed in a wide variety of common tumor types that include lung, breast, ovarian, gastrointestinal, urothelial, and genitourinary cancers provides an opportunity to develop widely applicable therapies based on the adoptive transfer of autologous T cells transduced with those receptors.

  19. Expression of the Mouse Pre-T Cell Receptor α Gene Is Controlled by an Upstream Region Containing a Transcriptional Enhancer

    PubMed Central

    Reizis, Boris; Leder, Philip

    1999-01-01

    The pre-T cell receptor α (pTα) protein is a critical component of the pre-T cell receptor complex in early thymocytes. The expression of the pTα gene is one of the earliest markers of the T cell lineage and occurs exclusively in pre-T cells. To investigate the molecular basis of thymocyte-specific gene expression, we searched for the genomic elements regulating transcription of the mouse pTα gene. We now report that expression of the pTα gene is primarily controlled by an upstream genomic region, which can drive thymocyte-specific expression of a marker gene in transgenic mice. Within this region, we have identified two specific DNase-hypersensitive sites corresponding to a proximal promoter and an upstream transcriptional enhancer. The pTα enhancer appears to function preferentially in pre-T cell lines and binds multiple nuclear factors, including YY1. The enhancer also contains two G-rich stretches homologous to a critical region of the thymocyte-specific lck proximal promoter. Here we show that these sites bind a common nuclear factor and identify it as the zinc finger protein ZBP-89. Our data establish a novel experimental model for thymocyte-specific gene expression and suggest an important role for ZBP-89 in T cell development. PMID:10330446

  20. CD8 controls T cell cross-reactivity.

    PubMed

    Wooldridge, Linda; Laugel, Bruno; Ekeruche, Julia; Clement, Mathew; van den Berg, Hugo A; Price, David A; Sewell, Andrew K

    2010-10-15

    Estimates of human αβ TCR diversity suggest that there are <10(8) different Ag receptors in the naive T cell pool, a number that is dwarfed by the potential number of different antigenic peptide-MHC (pMHC) molecules that could be encountered. Consequently, an extremely high degree of cross-reactivity is essential for effective T cell immunity. Ag recognition by T cells is unique in that it involves a coreceptor that binds at a site distinct from the TCR to facilitate productive engagement of the pMHC. In this study, we show that the CD8 coreceptor controls T cell cross-reactivity for pMHCI Ags, thereby ensuring that the peripheral T cell repertoire is optimally poised to negotiate the competing demands of responsiveness in the face of danger and quiescence in the presence of self.

  1. Basonuclin Regulates a Subset of Ribosomal RNA Genes in HaCaT Cells

    PubMed Central

    Zhang, Shengliang; Wang, Junwen; Tseng, Hung

    2007-01-01

    Basonuclin (Bnc1), a cell-type-specific ribosomal RNA (rRNA) gene regulator, is expressed mainly in keratinocytes of stratified epithelium and gametogenic cells of testis and ovary. Previously, basonuclin was shown in vitro to interact with rRNA gene (rDNA) promoter at three highly conserved sites. Basonuclin's high affinity binding site overlaps with the binding site of a dedicated and ubiquitous Pol I transcription regulator, UBF, suggesting that their binding might interfere with each other if they bind to the same promoter. Knocking-down basonuclin in mouse oocytes eliminated approximately one quarter of RNA polymerase I (Pol I) transcription foci, without affecting the BrU incorporation of the remaining ones, suggesting that basonuclin might regulate a subset of rDNA. Here we show, via chromatin immunoprecipitation (ChIP), that basonuclin is associated with rDNA promoters in HaCaT cells, a spontaneously established human keratinocyte line. Immunoprecipitation data suggest that basonuclin is in a complex that also contains the subunits of Pol I (RPA194, RPA116), but not UBF. Knocking-down basonuclin in HaCaT cells partially impairs the association of RPA194 to rDNA promoter, but not that of UBF. Basonuclin-deficiency also reduces the amount of 47S pre-rRNA, but this effect can be seen only after cell-proliferation related rRNA synthesis has subsided at a higher cell density. DNA sequence of basonuclin-bound rDNA promoters shows single nucleotide polymorphisms (SNPs) that differ from those associated with UBF-bound promoters, suggesting that basonuclin and UBF interact with different subsets of promoters. In conclusion, our results demonstrate basonuclin's functional association with rDNA promoters and its interaction with Pol I in vivo. Our data also suggest that basonuclin-Pol I complex transcribes a subset of rDNA. PMID:17878937

  2. T-cell help dependence of memory CD8+ T-cell expansion upon vaccinia virus challenge relies on CD40 signaling.

    PubMed

    Bedenikovic, Gregor; Crouse, Josh; Oxenius, Annette

    2014-01-01

    Due to their capacity to differentiate into long-lived memory cells, CD8(+) T cells are able to resolve subsequent infections faster than during the primary response. Among other factors, CD4(+) T cells play a crucial role during primary and secondary CD8(+) T-cell responses. However, the timing and mechanisms by which they influence CD8(+) T cells may differ in primary and secondary responses. Here, we demonstrate that during both primary and secondary vaccinia virus infection, CD4(+) T cells are necessary to promote CD8(+) T-cell responses. While CD4(+) T cells contributed to memory CD8(+) T-cell development, they were even more important during memory recall responses during challenge, as absence of CD4(+) T cells during challenge resulted in markedly decreased proliferation and increased apoptosis. T-cell help during primary and secondary responses was mediated via CD40 signaling, with DCs being an integral part of that pathway. As opposed to primary CD8(+) T-cell responses where only a combination of agonistic CD40 signaling and provision of IL-2 could substitute for T-cell help, agonistic CD40 triggering alone was sufficient to rescue memory CD8(+) T-cell responses in absence of T-cell help in the context of vaccinia virus infection.

  3. Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene.

    PubMed Central

    Ho, I C; Vorhees, P; Marin, N; Oakley, B K; Tsai, S F; Orkin, S H; Leiden, J M

    1991-01-01

    In addition to its role in the recognition of foreign antigens, the T cell receptor (TCR) alpha gene serves as a model system for studies of developmentally-regulated, lineage-specific gene expression in T cells. TCR alpha gene expression is restricted to cells of the TCR alpha/beta+ lineage, and is controlled by a T cell-specific transcriptional enhancer located 4.5 kb 3' to the C alpha gene segment. The TCR alpha enhancer contains four nuclear protein binding sites called T alpha 1-T alpha 4. In this report we describe the identification and characterization of a novel human cDNA, hGATA-3 that binds to the T alpha 3 element of the human TCR alpha enhancer. hGATA-3 contains a zinc finger domain that is highly related to the DNA-binding domain of the erythroid-specific transcription factor, GATA-1, and binds to a region of T alpha 3 that contains a consensus GATA binding site (AGATAG). Northern blot analyses of hematopoietic cell lines demonstrate that hGATA-3 is expressed exclusively in T cells. Overexpression of hGATA-3 in HeLa cells or human B cells specifically activated transcription from a co-transfected reporter plasmid containing two copies of the T alpha 3 binding site located upstream of the minimal SV40 promoter. Taken together these results demonstrate that hGATA-3 is a novel lineage-specific hematopoietic transcription factor that appears to play an important role in regulating the T cell-specific expression of the TCR alpha gene. Images PMID:1827068

  4. B and T cell screen

    MedlinePlus

    Direct immunofluorescence; E-rosetting; T and B lymphocyte assays; B and T lymphocyte assays ... to distinguish between T and B cells. The E-rosetting test identifies T cells and direct immunofluorescence ...

  5. 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

  6. Glycolysis determines dichotomous regulation of T cell subsets in hypoxia

    PubMed Central

    Xu, Yang; Zhang, Ming; Savoldo, Barbara; Metelitsa, Leonid S.; Rodgers, John; Yustein, Jason T.; Neilson, Joel R.

    2016-01-01

    Hypoxia occurs in many pathological conditions, including chronic inflammation and tumors, and is considered to be an inhibitor of T cell function. However, robust T cell responses occur at many hypoxic inflammatory sites, suggesting that functions of some subsets are stimulated under low oxygen conditions. Here, we investigated how hypoxic conditions influence human T cell functions and found that, in contrast to naive and central memory T cells (TN and TCM), hypoxia enhances the proliferation, viability, and cytotoxic action of effector memory T cells (TEM). Enhanced TEM expansion in hypoxia corresponded to high hypoxia-inducible factor 1α (HIF1α) expression and glycolytic activity compared with that observed in TN and TCM. We determined that the glycolytic enzyme GAPDH negatively regulates HIF1A expression by binding to adenylate-uridylate–rich elements in the 3′-UTR region of HIF1A mRNA in glycolytically inactive TN and TCM. Conversely, active glycolysis with decreased GAPDH availability in TEM resulted in elevated HIF1α expression. Furthermore, GAPDH overexpression reduced HIF1α expression and impaired proliferation and survival of T cells in hypoxia, indicating that high glycolytic metabolism drives increases in HIF1α to enhance TEM function during hypoxia. This work demonstrates that glycolytic metabolism regulates the translation of HIF1A to determine T cell responses to hypoxia and implicates GAPDH as a potential mechanism for controlling T cell function in peripheral tissue. PMID:27294526

  7. Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

    PubMed

    Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

    2015-07-01

    Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection. PMID:25834094

  8. Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

    PubMed

    Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

    2015-07-01

    Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

  9. Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter.

    PubMed

    Petri, V; Hsieh, M; Brenowitz, M

    1995-08-01

    A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I "footprint" titration techniques. These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes. The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined. Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 degrees C. An unusually negative value of the apparent standard heat capacity change, delta Cp degrees = -3.5 +/- 0.5 kcal/mol.K, was determined from these data. The dependence of the TBP-E4 promoter interaction on [KCl] indicates that 3.6 +/- 0.3 K+ ions are displaced upon complex formation. Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7. Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I "footprinting" techniques. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 degrees C (ka = 5.2 +/- 0.5) x 10(5) M-1 s-1) confirms the results obtained by Hawley and co-workers [Hoopes, B.C., LeBlanc, J.F., & Hawley, D.K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them through TBP concentrations of 636 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells.

    PubMed

    Zimmerman, Mary A; Singh, Nagendra; Martin, Pamela M; Thangaraju, Muthusamy; Ganapathy, Vadivel; Waller, Jennifer L; Shi, Huidong; Robertson, Keith D; Munn, David H; Liu, Kebin

    2012-06-15

    Butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit protective effects toward inflammatory diseases such as ulcerative colitis (UC) and inflammation-mediated colorectal cancer. Recent studies have shown that chronic IFN-γ signaling plays an essential role in inflammation-mediated colorectal cancer development in vivo, whereas genome-wide association studies have linked human UC risk loci to IFNG, the gene that encodes IFN-γ. However, the molecular mechanisms underlying the butyrate-IFN-γ-colonic inflammation axis are not well defined. Here we showed that colonic mucosa from patients with UC exhibit increased signal transducer and activator of transcription 1 (STAT1) activation, and this STAT1 hyperactivation is correlated with increased T cell infiltration. Butyrate treatment-induced apoptosis of wild-type T cells but not Fas-deficient (Fas(lpr)) or FasL-deficient (Fas(gld)) T cells, revealing a potential role of Fas-mediated apoptosis of T cells as a mechanism of butyrate function. Histone deacetylase 1 (HDAC1) was found to bind to the Fas promoter in T cells, and butyrate inhibits HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation in T cells. Knocking down gpr109a or slc5a8, the genes that encode for receptor and transporter of butyrate, respectively, resulted in altered expression of genes related to multiple inflammatory signaling pathways, including inducible nitric oxide synthase (iNOS), in mouse colonic epithelial cells in vivo. Butyrate effectively inhibited IFN-γ-induced STAT1 activation, resulting in inhibition of iNOS upregulation in human colon epithelial and carcinoma cells in vitro. Our data thus suggest that butyrate delivers a double-hit: induction of T cell apoptosis to eliminate the source of inflammation and suppression of IFN-γ-mediated inflammation in colonic epithelial cells, to suppress colonic inflammation.

  11. Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells

    PubMed Central

    Zimmerman, Mary A.; Singh, Nagendra; Martin, Pamela M.; Thangaraju, Muthusamy; Ganapathy, Vadivel; Waller, Jennifer L.; Shi, Huidong; Robertson, Keith D.; Munn, David H.

    2012-01-01

    Butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit protective effects toward inflammatory diseases such as ulcerative colitis (UC) and inflammation-mediated colorectal cancer. Recent studies have shown that chronic IFN-γ signaling plays an essential role in inflammation-mediated colorectal cancer development in vivo, whereas genome-wide association studies have linked human UC risk loci to IFNG, the gene that encodes IFN-γ. However, the molecular mechanisms underlying the butyrate-IFN-γ-colonic inflammation axis are not well defined. Here we showed that colonic mucosa from patients with UC exhibit increased signal transducer and activator of transcription 1 (STAT1) activation, and this STAT1 hyperactivation is correlated with increased T cell infiltration. Butyrate treatment-induced apoptosis of wild-type T cells but not Fas-deficient (Faslpr) or FasL-deficient (Fasgld) T cells, revealing a potential role of Fas-mediated apoptosis of T cells as a mechanism of butyrate function. Histone deacetylase 1 (HDAC1) was found to bind to the Fas promoter in T cells, and butyrate inhibits HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation in T cells. Knocking down gpr109a or slc5a8, the genes that encode for receptor and transporter of butyrate, respectively, resulted in altered expression of genes related to multiple inflammatory signaling pathways, including inducible nitric oxide synthase (iNOS), in mouse colonic epithelial cells in vivo. Butyrate effectively inhibited IFN-γ-induced STAT1 activation, resulting in inhibition of iNOS upregulation in human colon epithelial and carcinoma cells in vitro. Our data thus suggest that butyrate delivers a double-hit: induction of T cell apoptosis to eliminate the source of inflammation and suppression of IFN-γ-mediated inflammation in colonic epithelial cells, to suppress colonic inflammation. PMID:22517765

  12. Monoclonal regulatory T cells provide insights into T cell suppression

    PubMed Central

    Gubser, Céline; Schmaler, Mathias; Rossi, Simona W.; Palmer, Ed

    2016-01-01

    Regulatory T cells (Tregs) have a crucial role in maintaining lymphocyte homeostasis. However an understanding of how Tregs function at a cellular and molecular level has not yet been fully elucidated. Here, we make use of a T cell receptor (TCR) transgenic, Rag−/− mouse expressing a Forkhead-Box-Protein P3 (Foxp3) transgene. This mouse provides a source of monoclonal CD4+ Foxp3+ T cells with a defined specificity. Here we show that monoclonal B3K506 Tregs are functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is a parameter, which correlates with enhanced suppression. PMID:27210828

  13. Genome-wide DNA methylation analysis identifies hypomethylated genes regulated by FOXP3 in human regulatory T cells.

    PubMed

    Zhang, Yuxia; Maksimovic, Jovana; Naselli, Gaetano; Qian, Junyan; Chopin, Michael; Blewitt, Marnie E; Oshlack, Alicia; Harrison, Leonard C

    2013-10-17

    Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4(+) T cells (Naive). We detected 2315 differentially methylated cytosine-guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

  14. Upstream regulatory regions required to stabilize binding to the TATA sequence in an adenovirus early promoter.

    PubMed

    Garcia, J; Wu, F; Gaynor, R

    1987-10-26

    Of the five early adenovirus promoters, the early region 3 (E3) promoter is one of the most strongly induced by the E1A protein. To identify cellular proteins involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, DNase I footprinting using partially purified Hela cell extracts was performed. Four regions of the E3 promoter serve as binding domains for cellular proteins. These regions are found between -156 to -179 (site IV), -83 to -103 (site III), -47 to -67 (site II), and -16 to -37 (site I), relative to the start of transcription. Examination of the DNA sequences in each binding domain suggests that site III likely serves as a binding site for activator protein 1 (AP-1), site II for the cyclic AMP regulatory element binding protein (CREB), and site I for a TATA binding factor. The factors binding to either site II or III were sufficient to stabilize binding to the TATA sequence (site I). Mutagenesis studies indicated that both sites II and III, in addition to site I, are needed for complete basal and E1A-induced transcription. These results suggest that multiple cellular factors are involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, and that either of two upstream regions are capable of stabilizing factor binding to the TATA sequence.

  15. Establishment and characterization of a cell based artificial antigen-presenting cell for expansion and activation of CD8+ T cells ex vivo.

    PubMed

    Gong, Weijuan; Ji, Mingchun; Cao, Zhengfeng; Wang, Liheng; Qian, Yayun; Hu, Maozhi; Qian, Li; Pan, Xingyuan

    2008-02-01

    Artificial antigen-presenting cells are expected to stimulate the expansion and acquisition of optimal therapeutic features of T cells before infusion. Here CD32 that binds to a crystallizable fragment of IgG monoclonal antibody was genetically expressed on human K562 leukemia cells to provide a ligand for T-cell receptor. CD86 and 4-1BBL, which are ligands of co-stimulating receptors of CD28 and 4-1BB, respectively, were also expressed on K562 cells. Then we accomplished the artificial antigen-presenting cells by coupling K32/CD86/4-1BBL cell with OKT3 monoclonal antibody against CD3, named K32/CD86/4-1BBL/OKT3 cells. These artificial modified cells had the abilities of inducing CD8+ T cell activation, promoting CD8+ T cell proliferation, division, and long-term growth, inhibiting CD8+ T cell apoptosis, and enhancing CD8+ T cell secretion of IFN-gamma and perforin. Furthermore, antigen-specific cytotoxic T lymphocytes could be retained in the culture stimulated with K32/CD86/4-1BBL/OKT3 cells at least within 28 days. This approach was robust, simple, reproducible and economical for expansion and activation of CD8+ T cells and may have important therapeutic implications for adoptive immunotherapy. PMID:18318994

  16. Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function

    PubMed Central

    Voo, Kui S.; Bover, Laura; Harline, Megan L.; Vien, Long T.; Facchinetti, Valeria; Arima, Kazuhiko; Kwak, Larry W.; Liu, Yong J.

    2013-01-01

    Current cancer vaccines induce tumor-specific T cell responses without sustained tumor regression because immunosuppressive elements within the tumor induce exhaustion of effector T cells and infiltration of immune-suppressive regulatory T cells (Tregs). Therefore, much effort has been made to generate agonistic Abs targeting members of the TNFR superfamily, such as OX40, 4- 1BB, and GITR, expressed on effector T cells and Tregs, to reinvigorate T cell effector function and block Treg-suppressive function. In this article, we describe the development of a panel of anti-human OX40 agonistic mouse mAbs that could promote effector CD4+ and CD8+ T cell proliferation, inhibit the induction of CD4+ IL-10 -producing type 1 regulatory T cells, inhibit the expansion of ICOS+IL-10+ Tregs, inhibit TGF-b–induced FOXP3 expression on naive CD4+ T cells, and block natural Treg–suppressive function. We humanized two anti–human OX40 mAb clones, and they retained the potency of their parental clones. These Abs should provide broad opportunities for potential combination therapy to treat a wide realm of cancers and preventative vaccines against infectious diseases. PMID:24014877

  17. Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function.

    PubMed

    Voo, Kui S; Bover, Laura; Harline, Megan L; Vien, Long T; Facchinetti, Valeria; Arima, Kazuhiko; Kwak, Larry W; Liu, Yong J

    2013-10-01

    Current cancer vaccines induce tumor-specific T cell responses without sustained tumor regression because immunosuppressive elements within the tumor induce exhaustion of effector T cells and infiltration of immune-suppressive regulatory T cells (Tregs). Therefore, much effort has been made to generate agonistic Abs targeting members of the TNFR superfamily, such as OX40, 4-1BB, and GITR, expressed on effector T cells and Tregs, to reinvigorate T cell effector function and block Treg-suppressive function. In this article, we describe the development of a panel of anti-human OX40 agonistic mouse mAbs that could promote effector CD4(+) and CD8(+) T cell proliferation, inhibit the induction of CD4(+) IL-10 -producing type 1 regulatory T cells, inhibit the expansion of ICOS(+)IL-10(+) Tregs, inhibit TGF-β-induced FOXP3 expression on naive CD4(+) T cells, and block natural Treg-suppressive function. We humanized two anti-human OX40 mAb clones, and they retained the potency of their parental clones. These Abs should provide broad opportunities for potential combination therapy to treat a wide realm of cancers and preventative vaccines against infectious diseases. PMID:24014877

  18. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  19. Transient Surface CCR5 Expression by Naive CD8+ T Cells within Inflamed Lymph Nodes Is Dependent on High Endothelial Venule Interaction and Augments Th Cell-Dependent Memory Response.

    PubMed

    Askew, David; Su, Charles A; Barkauskas, Deborah S; Dorand, R Dixon; Myers, Jay; Liou, Rachel; Nthale, Joseph; Huang, Alex Y

    2016-05-01

    In inflamed lymph nod