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Sample records for promote t-cell binding

  1. Modulating DNA methylation in activated CD8+ T cells inhibits regulatory T cell-induced binding of Foxp3 to the CD8+ T Cell IL-2 promoter.

    PubMed

    Miller, Michelle M; Akaronu, Nnenna; Thompson, Elizabeth M; Hood, Sylvia F; Fogle, Jonathan E

    2015-02-01

    We have previously demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) activated during the course of feline immunodeficiency virus (FIV) infection suppress CD8(+) CTL function in a TGF-β-dependent fashion, inhibiting IFN-γ and IL-2 production and inducing G1 cell-cycle arrest. In this article, we describe the molecular events occurring at the IL-2 promoter leading to suppression of IL-2 production. These experiments demonstrate that Foxp3 induced by lentivirus-activated Tregs in the CD8(+) target cells binds to the IL-2 promoter, actively repressing IL-2 transcription. We further demonstrate that the chronic activation of CD8(+) T cells during FIV infection results in chromatin remodeling at the IL-2 promoter, specifically, demethylation of CpG residues. These DNA modifications occur during active transcription and translation of IL-2; however, these changes render the IL-2 promoter permissive to Foxp3-induced transcriptional repression. These data help explain, in part, the seemingly paradoxical observations that CD8(+) T cells displaying an activation phenotype exhibit altered antiviral function. Further, we demonstrate that blocking demethylation of CpG residues at the IL-2 promoter inhibits Foxp3 binding, suggesting a potential mechanism for rescue and/or reactivation of CD8(+) T cells. Using the FIV model for lentiviral persistence, these studies provide a framework for understanding how immune activation combined with Treg-mediated suppression may affect CD8(+) T cell IL-2 transcription, maturation, and antiviral function.

  2. Th1 cytokines promote T-cell binding to antigen-presenting cells via enhanced hyaluronan production and accumulation at the immune synapse.

    PubMed

    Bollyky, Paul L; Evanko, Stephen P; Wu, Rebecca P; Potter-Perigo, Susan; Long, S Alice; Kinsella, Brian; Reijonen, Helena; Guebtner, Kelly; Teng, Brandon; Chan, Christina K; Braun, Kathy R; Gebe, John A; Nepom, Gerald T; Wight, Thomas N

    2010-05-01

    Hyaluronan (HA) production by dendritic cells (DCs) is known to promote antigen presentation and to augment T-cell activation and proliferation. We hypothesized that pericellular HA can function as intercellular 'glue' directly mediating T cell-DC binding. Using primary human cells, we observed HA-dependent binding between T cells and DCs, which was abrogated upon pre-treatment of the DCs with 4-methylumbelliferone (4-MU), an agent which blocks HA synthesis. Furthermore, T cells regulate HA production by DCs via T cell-derived cytokines in a T helper (Th) subset-specific manner, as demonstrated by the observation that cell-culture supernatants from Th1 but not Th2 clones promote HA production. Similar effects were seen upon the addition of exogenous Th1 cytokines, IL-2, interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). The critical factors which determined the extent of DC-T cell binding in this system were the nature of the pre-treatment the DCs received and their capacity to synthesize HA, as T-cell clones which were pre-treated with monensin, added to block cytokine secretion, bound equivalently irrespective of their Th subset. These data support the existence of a feedforward loop wherein T-cell cytokines influence DC production of HA, which in turn affects the extent of DC-T cell binding. We also document the presence of focal deposits of HA at the immune synapse between T-cells and APC and on dendritic processes thought to be important in antigen presentation. These data point to a pivotal role for HA in DC-T cell interactions at the IS.

  3. Th1 cytokines promote T-cell binding to antigen-presenting cells via enhanced hyaluronan production and accumulation at the immune synapse

    PubMed Central

    Bollyky, Paul L; Evanko, Stephen P; Wu, Rebecca P; Potter-Perigo, Susan; Long, S Alice; Kinsella, Brian; Reijonen, Helena; Guebtner, Kelly; Teng, Brandon; Chan, Christina K; Braun, Kathy R; Gebe, John A; Nepom, Gerald T; Wight, Thomas N

    2010-01-01

    Hyaluronan (HA) production by dendritic cells (DCs) is known to promote antigen presentation and to augment T-cell activation and proliferation. We hypothesized that pericellular HA can function as intercellular ‘glue' directly mediating T cell–DC binding. Using primary human cells, we observed HA-dependent binding between T cells and DCs, which was abrogated upon pre-treatment of the DCs with 4-methylumbelliferone (4-MU), an agent which blocks HA synthesis. Furthermore, T cells regulate HA production by DCs via T cell-derived cytokines in a T helper (Th) subset-specific manner, as demonstrated by the observation that cell-culture supernatants from Th1 but not Th2 clones promote HA production. Similar effects were seen upon the addition of exogenous Th1 cytokines, IL-2, interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α). The critical factors which determined the extent of DC–T cell binding in this system were the nature of the pre-treatment the DCs received and their capacity to synthesize HA, as T-cell clones which were pre-treated with monensin, added to block cytokine secretion, bound equivalently irrespective of their Th subset. These data support the existence of a feedforward loop wherein T-cell cytokines influence DC production of HA, which in turn affects the extent of DC–T cell binding. We also document the presence of focal deposits of HA at the immune synapse between T-cells and APC and on dendritic processes thought to be important in antigen presentation. These data point to a pivotal role for HA in DC–T cell interactions at the IS. PMID:20228832

  4. Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis.

    PubMed

    Nguyen, Dao Xuan; Ehrenstein, Michael R

    2016-06-27

    The interplay between inflammatory and regulatory pathways orchestrates an effective immune response that provides protection from pathogens while limiting injury to host tissue. Tumor necrosis factor (TNF) is a pivotal inflammatory cytokine, but there is conflicting evidence as to whether it boosts or inhibits regulatory T cells (T reg cells). In this study, we show that the therapeutic anti-TNF antibody adalimumab, but not the soluble TNF receptor etanercept, paradoxically promoted the interaction between monocytes and T reg cells isolated from patients with rheumatoid arthritis (RA). Adalimumab bound to monocyte membrane TNF from RA patients and unexpectedly enhanced its expression and its binding to TNF-RII expressed on T reg cells. As a consequence, adalimumab expanded functional Foxp3(+) T reg cells equipped to suppress Th17 cells through an IL-2/STAT5-dependent mechanism. Our data not only highlight the beneficial effect of membrane TNF on T reg cell numbers during chronic inflammation, but in addition reveal how a therapeutic antibody that is thought to act by simply blocking its target can enhance the regulatory properties of this proinflammatory cytokine.

  5. Activated gammadelta T cells promote the activation of uveitogenic T cells and exacerbate EAU development.

    PubMed

    Nian, Hong; Shao, Hui; O'Brien, Rebecca L; Born, Willi K; Kaplan, Henry J; Sun, Deming

    2011-07-29

    To determine how the activation of γδ T cells affects the generation of uveitogenic αβ T cells and the development of experimental autoimmune uveitis (EAU). γδ T cells were isolated from B6 mice immunized with the uveitogenic peptide IRBP(1-20) and αβ T cells from immunized TCR-δ(-/-) mice. Resting γδ T cells were prepared by culture of separated γδ T cells in cytokine-free medium for 3 to 5 days, when they showed downregulation of CD69 expression. Activated γδ T cells were prepared by incubating resting γδ T cells with anti-γδ TCR (GL3) for 2 days. Responder αβ T cells were cocultured with immunizing antigen and antigen-presenting cells. The numbers of antigen-specific T cells expressing IL-17 or IFN-γ were determined by intracellular staining followed by FACS analysis after stimulation, with or without the addition of purified γδ T cells. The cytokines in the culture medium were measured by ELISA. Highly enriched γδ T cells exert widely different effects on autoreactive αβ T cells in EAU, depending on the activation status of the γδ T cells. Whereas nonactivated γδ T cells had little effect on the activation of interphotoreceptor retinoid-binding protein-specific αβ T cells in vitro and in vivo, activated γδ T cells promoted the generation of uveitogenic T cells and exacerbated the development of EAU. The functional ability of γδ T cells is greatly influenced by their activation status. Activated γδ T cells exacerbate EAU through increased activation of uveitogenic T cells.

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

    PubMed Central

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

    2017-01-01

    Adoptive T cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, that 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 MHC 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 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 tumor infiltrating lymphocyte 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 played 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. PMID:23052828

  7. PDZ binding motif of HTLV-1 Tax promotes virus-mediated T-cell proliferation in vitro and persistence in vivo.

    PubMed

    Xie, Li; Yamamoto, Brenda; Haoudi, Abdelali; Semmes, O John; Green, Patrick L

    2006-03-01

    HTLV-1 cellular transformation and disease induction is dependent on expression of the viral Tax oncoprotein. PDZ is a modular protein interaction domain used in organizing signaling complexes in eukaryotic cells through recognition of a specific binding motif in partner proteins. Tax-1, but not Tax-2, contains a PDZ-binding domain motif (PBM) that promotes the interaction with several cellular PDZ proteins. Herein, we investigate the contribution of the Tax-1 PBM in HTLV-induced proliferation and immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. We generated several HTLV-1 and HTLV-2 Tax viral mutants, including HTLV-1deltaPBM, HTLV-2+C22(+PBM), and HTLV-2+ C18(deltaPBM). All Tax mutants maintained the ability to significantly activate the CREB/ATF or NFkappaB signaling pathways. Microtiter proliferation assays revealed that the Tax-1 PBM significantly increases both HTLV-1- and HTLV-2-induced primary T-cell proliferation. In addition, Tax-1 PBM was responsible for the micronuclei induction activity of Tax-1 relative to that of Tax-2. Viral infection and persistence were severely attenuated in rabbits inoculated with HTLV-1deltaPBM. Our results provide the first direct evidence suggesting that PBM-mediated associations between Tax-1 and cellular proteins play a key role in HTLV-induced cell proliferation and genetic instability in vitro and facilitate viral persistence in vivo.

  8. β-Catenin Upregulates the Constitutive and Virus-Induced Transcriptional Capacity of the Interferon Beta Promoter through T-Cell Factor Binding Sites.

    PubMed

    Marcato, Vasco; Luron, Lionel; Laqueuvre, Lucie M; Simon, Dominique; Mansuroglu, Zeyni; Flamand, Marie; Panthier, Jean-Jacques; Souès, Sylvie; Massaad, Charbel; Bonnefoy, Eliette

    2016-01-01

    Rapid upregulation of interferon beta (IFN-β) expression following virus infection is essential to set up an efficient innate antiviral response. Biological roles related to the antiviral and immune response have also been associated with the constitutive production of IFN-β in naive cells. However, the mechanisms capable of modulating constitutive IFN-β expression in the absence of infection remain largely unknown. In this work, we demonstrate that inhibition of the kinase glycogen synthase kinase 3 (GSK-3) leads to the upregulation of the constitutive level of IFN-β expression in noninfected cells, provided that GSK-3 inhibition is correlated with the binding of β-catenin to the IFN-β promoter. Under these conditions, IFN-β expression occurred through the T-cell factor (TCF) binding sites present on the IFN-β promoter independently of interferon regulatory factor 3 (IRF3). Enhancement of the constitutive level of IFN-β per se was able to confer an efficient antiviral state to naive cells and acted in synergy with virus infection to stimulate virus-induced IFN-β expression. Further emphasizing the role of β-catenin in the innate antiviral response, we show here that highly pathogenic Rift Valley fever virus (RVFV) targets the Wnt/β-catenin pathway and the formation of active TCF/β-catenin complexes at the transcriptional and protein level in RVFV-infected cells and mice.

  9. Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma

    PubMed Central

    Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

    2016-01-01

    T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC. PMID:26958940

  10. Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma.

    PubMed

    Hamada, Junichi; Shoda, Katsutoshi; Masuda, Kiyoshi; Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

    2016-03-29

    T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC.

  11. 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.

  12. Human T-cell leukemia virus type 1 Tax requires direct access to DNA for recruitment of CREB binding protein to the viral promoter.

    PubMed

    Lenzmeier, B A; Giebler, H A; Nyborg, J K

    1998-02-01

    Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.

  13. Human T-Cell Leukemia Virus Type 1 Tax Requires Direct Access to DNA for Recruitment of CREB Binding Protein to the Viral Promoter

    PubMed Central

    Lenzmeier, Brian A.; Giebler, Holli A.; Nyborg, Jennifer K.

    1998-01-01

    Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5′-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter. PMID:9447968

  14. Adoptive transfer of regulatory T cells promotes intestinal tumorigenesis and is associated with decreased NK cells and IL-22 binding protein.

    PubMed

    Janakiram, Naveena B; Mohammed, Altaf; Bryant, Taylor; Brewer, Misty; Biddick, Laura; Lightfoot, Stan; Lang, Mark L; Rao, Chinthalapally V

    2015-10-01

    High number of regulatory T cells (Tregs), both circulating and at the tumor site, often indicates a poor prognosis in CRC patient's possibly impairing natural killer (NK) cell function. To determine the role of Tregs in CRC development and their effects on NK cells, we created novel transgenic Rag-Apc mice that lack T cells and develop spontaneous intestinal tumors, and we adoptively transferred Tregs or transiently depleted NK cells during initial stages of tumorigenesis. In 6-weeks old Rag-Apc mice containing microscopic intestinal tumors adoptive transfer of Tregs or transient NK cell depletion dramatically associated with an increase in intestinal tumor multiplicity and tumor size, with significantly decreased survival rates. Importantly, Treg transfer increased small intestinal polyp formation up to 65% (P < 0.0005) and increased colon tumors multiplicities by 84% (P < 0.0001) with a significant decrease in NK cells as compared to control mice. Similarly, in NK depleted mice, colon tumor multiplicities increased up to 40% and small intestinal polyp formation up to 60% (P < 0.0001). Treg transfer or NK cell transient depletion markedly increased interleukin (IL)-22 systemically and the inflammatory signaling molecules P2X7R, and STAT3 in the tumors; and impaired production of the tumor suppressor interferon (IFN)-γ systemically. Notably, IL-22 binding protein (IL-22 BP) was associated with NKs and a significant decrease was seen at the tumor site in mice adoptively transferred with Tregs or depleted of NK cells. Our results suggest that adoptive transfer of Tregs aggressively promote intestinal tumorigenesis by decreasing NK cell number and activity by modulating IL-22 BP.

  15. 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.

  16. CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis.

    PubMed

    Phares, Timothy W; Stohlman, Stephen A; Hwang, Mihyun; Min, Booki; Hinton, David R; Bergmann, Cornelia C

    2012-03-01

    CD4 T cell activation during peripheral infections not only is essential in inducing protective CD8 T cell memory but also promotes CD8 T cell function and survival. However, the contributions of CD4 T cell help to antiviral CD8 T cell immunity during central nervous system (CNS) infection are not well established. Encephalitis induced by the sublethal coronavirus JHMV was used to identify when CD4 T cells regulate CD8 T cell responses following CNS infection. Peripheral expansion of virus-specific CD8 T cells was impaired when CD4 T cells were ablated prior to infection but not at 4 days postinfection. Delayed CD4 T cell depletion abrogated CD4 T cell recruitment to the CNS but only slightly diminished CD8 T cell recruitment. Nevertheless, the absence of CNS CD4 T cells was associated with reduced gamma interferon (IFN-γ) and granzyme B expression by infiltrating CD8 T cells, increased CD8 T cell apoptosis, and impaired control of infectious virus. CD4 T cell depletion subsequent to CD4 T cell CNS migration restored CD8 T cell activity and virus control. Analysis of γc-dependent cytokine expression indicated interleukin-21 (IL-21) as a primary candidate optimizing CD8 T cell activity within the CNS. These results demonstrate that CD4 T cells play critical roles in both enhancing peripheral activation of CD8 T cells and prolonging their antiviral function within the CNS. The data highlight the necessity for temporally and spatially distinct CD4 T cell helper functions in sustaining CD8 T cell activity during CNS infection.

  17. TrkA is a binding partner of NPM-ALK that promotes the survival of ALK(+) T-cell lymphoma.

    PubMed

    Shi, Wenyu; George, Suraj Konnath; George, Bhawana; Curry, Choladda V; Murzabdillaeva, Albina; Alkan, Serhan; Amin, Hesham M

    2017-09-01

    Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK(+) ) T-cell lymphoma is an aggressive neoplasm that is more commonly seen in children and young adults. The pathogenesis of NPM-ALK(+) T-cell lymphoma is not completely understood. Wild-type ALK is a receptor tyrosine kinase that is physiologically expressed in neural tissues during early stages of human development, which suggests that ALK may interact with neurotrophic factors. The aberrant expression of NPM-ALK results from a translocation between the ALK gene on chromosome 2p23 and the NPM gene on chromosome 5q35. The nerve growth factor (NGF) is the first neurotrophic factor attributed to non-neural functions including cancer cell survival, proliferation, and metastasis. These functions are primarily mediated through the tropomyosin receptor kinase A (TrkA). The expression and role of NGF/TrkA in NPM-ALK(+) T-cell lymphoma are not known. In this study, we tested the hypothesis that TrkA signaling is upregulated and sustains the survival of this lymphoma. Our data illustrate that TrkA and NGF are expressed in five NPM-ALK(+) T-cell lymphoma cell lines and TrkA is expressed in 11 of 13 primary lymphoma tumors from patients. In addition, we found evidence to support that NPM-ALK and TrkA functionally interact. A selective TrkA inhibitor induced apoptosis and decreased cell viability, proliferation, and colony formation of NPM-ALK(+) T-cell lymphoma cell lines. These effects were associated with downregulation of cell survival regulatory proteins. Similar results were also observed using specific knockdown of TrkA in NPM-ALK(+) T-cell lymphoma cells by siRNA. Importantly, the inhibition of TrkA signaling was associated with antitumor effects in vivo, because tumor xenografts in mice regressed and the mice exhibited improved survival. In conclusion, TrkA plays an important role in the pathogenesis of NPM-ALK(+) T-cell lymphoma, and therefore, targeting TrkA signaling may represent a novel approach to

  18. T cell intrinsic roles of autophagy in promoting adaptive immunity

    PubMed Central

    Walsh, Craig M.; Bell, Bryan D.

    2010-01-01

    Autophagy, an ancient cellular response where autophagic vacuoles are formed within the cytosol, is induced in response to a variety of cellular insults, including growth factor or nutrient withdrawal, organelle damage and misfolded proteins. Autophagy is rapidly induced in T lymphocytes following antigenic stimulation and blockade of autophagic signaling greatly reduces T cell clonal expansion, suggesting that autophagy is primarily involved in promoting T cell survival. In contrast, a recently identified negative feedback loop involving FADD and caspase-8, limits the level of autophagy in T cells. Failure to activate caspase-8 during T cell mitogenesis leads to hyperactive autophagy and cellular death through a programmed necrotic mechanism. These findings suggest that crosstalk between these cellular processes is essential for T cell activation and homeostasis. PMID:20392618

  19. Methods for quantifying T cell receptor binding affinities and thermodynamics

    PubMed Central

    Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

  20. Baicalin, a natural compound, promotes regulatory T cell differentiation.

    PubMed

    Yang, Ji; Yang, Xue; Li, Ming

    2012-05-16

    CD4(+)CD25(+)Foxp3(+) regulatory T (T(reg)) cells inhibit autoimmunity and protect against tissue injury. The development of these T(reg) cells is controlled by the regulator protein Foxp3, which can be enhanced by the in vitro activation of Foxp3 in the presence of transforming growth factor-beta. However, little is known about alternative methods, such as the use of natural products, for controlling Foxp3-mediated T(reg) cell differentiation. HEK 293 T cells were transfected with Foxp3 expression plasmid, and then treated with different compounds, Foxp3 mRNA expression was determined by real-time RT-PCR. CD4(+)CD25(-)T cells were stimulated with Baicalin, Foxp3 protein expression were analyzed by flow cytometry and confocal microscopy, the regulatory function of T cells stimulated with Baicalin was detected by the carboxyfluorescien succinimidyl ester. We demonstrated that Baicalin, a compound isolated from the Chinese herb Huangqin, induced Foxp3 protein expression in cultured T cells, promoted T(reg) cell differentiation and regulatory activity. Our data also indicated that Baicalin restored Foxp3 expression following its initial interleukin-6-mediated inhibition and induced Foxp3 expression in vitro. These data suggest that Baicalin may promote T(reg) cell differentiation and regulatory activity and may serve as a promising natural immunosuppressive compound for treating autoimmune inflammatory diseases.

  1. Baicalin, a natural compound, promotes regulatory T cell differentiation

    PubMed Central

    2012-01-01

    Background CD4+CD25+Foxp3+ regulatory T (Treg) cells inhibit autoimmunity and protect against tissue injury. The development of these Treg cells is controlled by the regulator protein Foxp3, which can be enhanced by the in vitro activation of Foxp3 in the presence of transforming growth factor-beta. However, little is known about alternative methods, such as the use of natural products, for controlling Foxp3-mediated Treg cell differentiation. Method HEK 293 T cells were transfected with Foxp3 expression plasmid, and then treated with different compounds, Foxp3 mRNA expression was determined by real-time RT-PCR. CD4+CD25-T cells were stimulated with Baicalin, Foxp3 protein expression were analyzed by flow cytometry and confocal microscopy, the regulatory function of T cells stimulated with Baicalin was detected by the carboxyfluorescien succinimidyl ester. Results We demonstrated that Baicalin, a compound isolated from the Chinese herb Huangqin, induced Foxp3 protein expression in cultured T cells, promoted Treg cell differentiation and regulatory activity. Our data also indicated that Baicalin restored Foxp3 expression following its initial interleukin-6-mediated inhibition and induced Foxp3 expression in vitro. Conclusions These data suggest that Baicalin may promote Treg cell differentiation and regulatory activity and may serve as a promising natural immunosuppressive compound for treating autoimmune inflammatory diseases. PMID:22591709

  2. The transcription factor NFAT promotes exhaustion of activated CD8+ T cells

    PubMed Central

    Martinez, Gustavo J.; Pereira, Renata M.; Äijö, Tarmo; Kim, Edward Y.; Marangoni, Francesco; Pipkin, Matthew E.; Togher, Susan; Heissmeyer, Vigo; Zhang, Yi Chen; Crotty, Shane; Lamperti, Edward D.; Ansel, K. Mark; Mempel, Thorsten R.; Lähdesmäki, Harri; Hogan, Patrick G.; Rao, Anjana

    2015-01-01

    SUMMARY During persistent antigen stimulation, CD8+ T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8+ T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8+ T cells, and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8+ T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1. PMID:25680272

  3. CD4+ T cells memorize obesity and promote weight regain.

    PubMed

    Zou, Jianghuan; Lai, Beibei; Zheng, Mingzhu; Chen, Qin; Jiang, Shujun; Song, Anying; Huang, Zan; Shi, Peiliang; Tu, Xin; Wang, Di; Lu, Linrong; Lin, Zhaoyu; Gao, Xiang

    2017-06-19

    Body weight regain often causes failure of obesity therapies while the underlying mechanism remains largely unknown. In this study, we report that immune cells, especially CD4+ T cells, mediate the 'memory' of previous obese status. In a weight gain-loss-regain model, we found that C57BL/6J mice with an obesity history showed a much faster rate of body weight regain. This obesity memory could last for at least 2 months after previously obese mice were kept at the same body weight as non-obese mice. Surprisingly, such obesity memory was abrogated by dexamethasone treatment, whereas immunodeficient Rag1(-/-) and H2A(-/-) mice failed to establish such memory. Rag1(-/-) mice repossessed the obesity memory when immune cells or CD4+ T cells isolated from previously obese mice were transferred. Furthermore, depletion of CD4+ T cells led to obesity memory ablation. Taken together, we conclude that CD4+ T cells mediate obesity memory and promote weight regain.Cellular &Molecular Immunology advance online publication, 19 June 2017; doi:10.1038/cmi.2017.36.

  4. 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

  5. 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.

  6. 6-mercaptopurine promotes energetic failure in proliferating T cells

    PubMed Central

    Fernández-Ramos, Ana A.; Marchetti-Laurent, Catherine; Poindessous, Virginie; Antonio, Samantha; Laurent-Puig, Pierre; Bortoli, Sylvie; Loriot, Marie-Anne; Pallet, Nicolas

    2017-01-01

    The anticancer drug 6-mercaptopurine (6-MP) inhibits de novo purine synthesis and acts as an antiproliferative agent by interfering with protein, DNA and RNA synthesis and promoting apoptosis. Metabolic reprogramming is crucial for tumor progression to foster cancer cells growth and proliferation, and is regulated by mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) as well as the oncogenes Myc and hypoxia inducible factor 1α (HIF-1α). We hypothesized that 6-MP impacts metabolic remodeling through its action on nucleotide synthesis. The aim of our study is to provide a comprehensive characterization of the metabolic changes induced by 6-MP in leukemic T cells. Our results indicate that exposition to 6-MP rapidly reduces intracellular ATP concentration, leading to the activation of AMPK. In turn, mTOR, an AMPK target, was inhibited, and the expression of HIF-1α and Myc was reduced upon 6-MP incubation. As a consequence of these inhibitions, glucose and glutamine fluxes were strongly decreased. Notably, no difference was observed on glucose uptake upon exposition to 6-MP. In conclusion, our findings provide new insights into how 6-MP profoundly impacts cellular energetic metabolism by reducing ATP production and decreasing glycolytic and glutaminolytic fluxes, and how 6-MP modifies human leukemic T cells metabolism with potential antiproliferative effects. PMID:28574837

  7. 6-mercaptopurine promotes energetic failure in proliferating T cells.

    PubMed

    Fernández-Ramos, Ana A; Marchetti-Laurent, Catherine; Poindessous, Virginie; Antonio, Samantha; Laurent-Puig, Pierre; Bortoli, Sylvie; Loriot, Marie-Anne; Pallet, Nicolas

    2017-06-27

    The anticancer drug 6-mercaptopurine (6-MP) inhibits de novo purine synthesis and acts as an antiproliferative agent by interfering with protein, DNA and RNA synthesis and promoting apoptosis. Metabolic reprogramming is crucial for tumor progression to foster cancer cells growth and proliferation, and is regulated by mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) as well as the oncogenes Myc and hypoxia inducible factor 1α (HIF-1α). We hypothesized that 6-MP impacts metabolic remodeling through its action on nucleotide synthesis. The aim of our study is to provide a comprehensive characterization of the metabolic changes induced by 6-MP in leukemic T cells. Our results indicate that exposition to 6-MP rapidly reduces intracellular ATP concentration, leading to the activation of AMPK. In turn, mTOR, an AMPK target, was inhibited, and the expression of HIF-1α and Myc was reduced upon 6-MP incubation. As a consequence of these inhibitions, glucose and glutamine fluxes were strongly decreased. Notably, no difference was observed on glucose uptake upon exposition to 6-MP. In conclusion, our findings provide new insights into how 6-MP profoundly impacts cellular energetic metabolism by reducing ATP production and decreasing glycolytic and glutaminolytic fluxes, and how 6-MP modifies human leukemic T cells metabolism with potential antiproliferative effects.

  8. The α4 Nicotinic Receptor Promotes CD4+ T-Cell Proliferation and a Helper T-Cell Immune Response

    PubMed Central

    Nordman, Jacob C.; Muldoon, Pretal; Clark, Sarah; Damaj, M. Imad

    2014-01-01

    Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell-based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4+CD3+CD4+) and show that this group of cells is responsive to sustained nicotine exposure. In the circulation, spleen, bone marrow, and thymus, we find that nicotine promotes an increase in CD3+CD4+ cells via its activation of the α4 nAChR and regulation of G protein subunit o, G protein regulated–inducer of neurite outgrowth, and CDC42 signaling within T cells. In particular, nicotine is found to promote a helper T cell 2 adaptive immunologic response within T cells that is absent in α4−/− mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T cells, possibly accounting for the effect of smoking on the immune system. PMID:24107512

  9. 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. Copyright © 2015, American Association for the Advancement of Science.

  10. Regulatory T Cell-Enriching Microparticles for Promoting Vascularized Composite Allotransplant Survival

    DTIC Science & Technology

    2016-10-01

    AWARD NUMBER: W81XWH-15-1-0244 TITLE: Regulatory T Cell -Enriching Microparticles for Promoting Vascularized Composite Allotransplant Survival...2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Regulatory T Cell -Enriching Microparticles for Promoting Vascularized Composite Allotransplant Survival...Regulatory T Cell , CTA, Transplant, Biomimetic 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF

  11. 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

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

    PubMed

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

    2012-11-05

    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.

  13. 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.

  14. 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.

  15. 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

  16. Sustained interactions between T cell receptors and antigens promote the differentiation of CD4⁺ memory T cells.

    PubMed

    Kim, Chulwoo; Wilson, Theodore; Fischer, Kael F; Williams, Matthew A

    2013-09-19

    During CD4⁺ T cell activation, T cell receptor (TCR) signals impact T cell fate, including recruitment, expansion, differentiation, trafficking, and survival. To determine the impact of TCR signals on the fate decision of activated CD4⁺ T cells to become end-stage effector or long-lived memory T helper 1 (Th1) cells, we devised a deep-sequencing-based approach that allowed us to track the evolution of TCR repertoires after acute infection. The transition of effector Th1 cells into the memory pool was associated with a significant decrease in repertoire diversity, and the major histocompatibility complex (MHC) class II tetramer off rate, but not tetramer avidity, was a key predictive factor in the representation of individual clonal T cell populations at the memory stage. We conclude that stable and sustained interactions with antigens during the development of Th1 responses to acute infection are a determinative factor in promoting the differentiation of Th1 memory cells.

  17. The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation.

    PubMed

    Attema, Joanne L; Reeves, Raymond; Murray, Vincent; Levichkin, Ilya; Temple, Mark D; Tremethick, David J; Shannon, M Frances

    2002-09-01

    Controlled production of the cytokine IL-2 plays a key role in the mammalian immune system. Expression from the gene is tightly regulated with no detectable expression in resting T cells and a strong induction following T cell activation. The IL-2 proximal promoter (+1 to -300) contains many well-defined transcriptional activation elements that respond to T cell stimulation. To determine the role of chromatin structure in the regulation of interleukin-2 gene transcription, nucleosome assembly across the IL-2 promoter region was examined using in vitro chromatin reconstitution assays. The IL-2 promoter assembles a nucleosome that is both translationally and rotationally positioned, spanning some of the major functional control elements. The binding of transcription factors to these elements, with the exception of the architectural protein HMGA1, was occluded by the presence of the nucleosome. Analysis of the chromatin architecture of the IL-2 gene in Jurkat T cells provided evidence for the presence of a similarly positioned nucleosome in vivo. The region encompassed by this nucleosome becomes remodeled following activation of Jurkat T cells. These observations suggest that the presence of a positioned nucleosome across the IL-2 proximal promoter may play an important role in maintaining an inactive gene in resting T cells and that remodeling of this nucleosome is important for gene activation.

  18. Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion.

    PubMed

    Coffelt, Seth B; Chen, Yung-Yi; Muthana, Munitta; Welford, Abigail F; Tal, Andrea O; Scholz, Alexander; Plate, Karl H; Reiss, Yvonne; Murdoch, Craig; De Palma, Michele; Lewis, Claire E

    2011-04-01

    Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors.

  19. 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.

  20. Mannan-binding lectin, a serum collectin, suppresses T-cell proliferation via direct interaction with cell surface calreticulin and inhibition of proximal T-cell receptor signaling.

    PubMed

    Zhao, Na; Wu, Jie; Xiong, Simin; Zhang, Liyun; Lu, Xiao; Chen, Shangliang; Wu, Qifeng; Wang, Hailan; Liu, Ying; Chen, Zhengliang; Zuo, Daming

    2017-06-01

    Mannan binding lectin (MBL), initially reported to activate the complement pathway, is also known to be involved in the pathogenesis of autoimmune diseases. We report a thus far unknown function of MBL as a suppressor of T-cell activation. MBL markedly inhibited T-cell proliferation induced by anti-CD3 and anti-CD28 antibodies. Moreover, the presence of MBL during T-cell priming interfered with proximal T-cell receptor signaling by decreasing phosphorylation of Lck, ZAP-70, and LAT. MBL bound to T cells through interaction between the collagen-like region of MBL and calreticulin (CRT) expressed on the T-cell surface. The neutralizing antibody against CRT abrogated MBL-mediated suppression of T-cell proliferation, suggesting that MBL down-modulates T-cell proliferation via cell surface CRT. We further demonstrated that the feature of MBL-mediated T-cell suppression is shared by other serum collectins (e.g., C1q and collectin 11). The concentrations of MBL correlated negatively with in vivo T-cell activation status in patients with early-stage silicosis. Furthermore, MBL efficiently inhibited activation and proliferation of autoreactive T cells derived from patients with silicosis, indicating that MBL serves as a negative feedback control of the T-cell responses.-Zhao, N., Wu, J., Xiong, S., Zhang, L., Lu, X., Chen, S., Wu, Q., Wang, H., Liu, Y., Chen, Z., Zuo, D. Mannan-binding lectin, a serum collectin, suppresses T-cell proliferation via direct interaction with cell surface calreticulin and inhibition of proximal T-cell receptor signaling. © FASEB.

  1. Zinc signals promote IL-2-dependent proliferation of T cells.

    PubMed

    Kaltenberg, Jennifer; Plum, Laura M; Ober-Blöbaum, Julia L; Hönscheid, Andrea; Rink, Lothar; Haase, Hajo

    2010-05-01

    Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells.

  2. Promoting metastasis: neutrophils and T cells join forces.

    PubMed

    Fridlender, Zvi G; Albelda, Steven M; Granot, Zvi

    2015-07-01

    The role neutrophils play in cancer is a matter of debate as both pro- and anti-tumor functions have been documented. In a recent publication in Nature, Coffelt et al. identify a new mechanism where neutrophils and T cells cooperate to generate metastasis-supporting immune suppression.

  3. Immunopathology of experimental Chagas' disease: binding of T cells to Trypanosoma cruzi-infected heart tissue.

    PubMed Central

    Mortatti, R C; Maia, L C; de Oliveira, A V; Munk, M E

    1990-01-01

    The immunopathology of Chagas' disease was studied in the experimental model of chronic infection in C57BL/10JT or mice. Sublethal infection with Trypanosoma cruzi, Y strain, induced specific antibodies and a delayed hypersensitivity response to parasite antigens. Mice developed chronic chagasic myocarditis but not skeletal muscle myositis. Binding of T cells to infected heart tissue was investigated during short-term cocultivation of lymphocytes with heart cryostat sections. T cells from infected mice and from normal controls bound equally to myocardium and liver sections from both infected and normal mice. A search in depth was attempted with cells heavily tagged with 99mTc. Labeled T cells from chagasic mice bound to both normal and infected myocardium slices. 99mTc-labeled T cells from controls gave the same binding values. Glass-adherent spleen cells behaved identically to T cells. Prior treatment of the tissue with serum from chronically infected mice did not increase the number of binding cells. Peritoneal macrophages tagged with 99mTc-sulfur colloid also bound to infected myocardium slices. The binding of macrophages was not changed by pretreatment of infected tissue with anti-T, cruzi antibodies. In short, this work did not detect any population of T cells or macrophages which could bind specifically to infected heart tissue to initiate an autoreactive process. Images PMID:2228230

  4. T-cell functional regions of the human IL-3 proximal promoter.

    PubMed

    Ryan, G R; Vadas, M A; Shannon, M F

    1994-10-01

    The human interleukin-3 (IL-3) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post-transcriptional level. We have previously shown that treatment of Jurkat T cells with phytohemaglutinin (PHA) and the phorbol ester, PMA, activated transcription initiation from the IL-3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL-3 gene 5' and 3' flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5' flanking region of the IL-3 gene. The promoter region between -173 and -60 contained the strongest activating elements. The transcription factor AP-1 could bind to this positive activator region of the promoter. We also examined the function of the IL-3 CK-1/CK-2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/PHA.

  5. Adult T-cell leukemia cells are characterized by abnormalities of Helios expression that promote T cell growth.

    PubMed

    Asanuma, Satomi; Yamagishi, Makoto; Kawanami, Katsuaki; Nakano, Kazumi; Sato-Otsubo, Aiko; Muto, Satsuki; Sanada, Masashi; Yamochi, Tadanori; Kobayashi, Seiichiro; Utsunomiya, Atae; Iwanaga, Masako; Yamaguchi, Kazunari; Uchimaru, Kaoru; Ogawa, Seishi; Watanabe, Toshiki

    2013-08-01

    Molecular abnormalities involved in the multistep leukemogenesis of adult T-cell leukemia (ATL) remain to be clarified. Based on our integrated database, we focused on the expression patterns and levels of Ikaros family genes, Ikaros, Helios, and Aiolos, in ATL patients and HTLV-1 carriers. The results revealed profound deregulation of Helios expression, a pivotal regulator in the control of T-cell differentiation and activation. The majority of ATL samples (32/37 cases) showed abnormal splicing of Helios expression, and four cases did not express Helios. In addition, novel genomic loss in Helios locus was observed in 17/168 cases. We identified four ATL-specific short Helios isoforms and revealed their dominant-negative function. Ectopic expression of ATL-type Helios isoform as well as knockdown of normal Helios or Ikaros promoted T-cell growth. Global mRNA profiling and pathway analysis showed activation of several signaling pathways important for lymphocyte proliferation and survival. These data provide new insights into the molecular involvement of Helios function in the leukemogenesis and phenotype of ATL cells, indicating that Helios deregulation is one of the novel molecular hallmarks of ATL.

  6. Interleukin 17-Producing γδT Cells Promote Hepatic Regeneration in Mice

    PubMed Central

    Rao, Raghavendra; Graffeo, Christopher S.; Gulati, Rishabh; Jamal, Mohsin; Narayan, Suchithra; Zambirinis, Constantinos; Barilla, Rocky; Deutsch, Michael; Greco, Stephanie; Ochi, Atsuo; Tomkötter, Lena; Blobstein, Reuven; Avanzi, Antonina; Tippens, Daniel M.; Gelbstein, Yisroel; Heerden, Eliza Van; Miller, George

    2014-01-01

    Background & Aims Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. γδT cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of γδT cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T cell receptor δ chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). Methods We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd−/−, or Clec7a−/− mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. γδT cells were purified by fluorescence-activated cell sorting. Results In mice, partial hepatectomy upregulated expression of CCL20 and ligands of Dectin-1, associated with recruitment and activation of γδT cells and their increased production of interleukin (IL)17 family cytokines. Recruited γδT cells induced production of IL6 by antigen-presenting cells and suppressed expression of interferon γ by natural killer T cells, promoting hepatocyte proliferation. Absence of IL17-producing γδT cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that γδT cells are required for inflammatory responses mediated by IL17 and Dectin-1. Conclusions γδT cells regulate hepatic regeneration by producing IL22 and IL17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for γδT cells to promote hepatic regeneration. PMID:24801349

  7. 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

  8. Chronic exposure to trichloroethylene increases DNA methylation of the Ifng promoter in CD4(+) T cells.

    PubMed

    Gilbert, Kathleen M; Blossom, Sarah J; Erickson, Stephen W; Broadfoot, Brannon; West, Kirk; Bai, Shasha; Li, Jingyun; Cooney, Craig A

    2016-10-17

    CD4(+) T cells in female MRL+/+ mice exposed to solvent and water pollutant trichloroethylene (TCE) skew toward effector/memory CD4(+) T cells, and demonstrate seemingly non-monotonic alterations in IFN-γ production. In the current study we examined the mechanism for this immunotoxicity using effector/memory and naïve CD4(+) T cells isolated every 6 weeks during a 40 week exposure to TCE (0.5mg/ml in drinking water). A time-dependent effect of TCE exposure on both Ifng gene expression and IFN-γ protein production was observed in effector/memory CD4(+) T cells, with an increase after 22 weeks of exposure and a decrease after 40 weeks of exposure. No such effect of TCE was observed in naïve CD4(+) T cells. A cumulative increase in DNA methylation in the CpG sites of the promoter of the Ifng gene was observed in effector/memory, but not naïve, CD4(+) T cells over time. Also unique to the Ifng promoter was an increase in methylation variance in effector/memory compared to naïve CD4(+) T cells. Taken together, the CpG sites of the Ifng promoter in effector/memory CD4(+) T cells were especially sensitive to the effects of TCE exposure, which may help explain the regulatory effect of the chemical on this gene.

  9. Human T-cell leukemia virus type-1 antisense-encoded gene, Hbz, promotes T-lymphocyte proliferation.

    PubMed

    Arnold, Joshua; Zimmerman, Bevin; Li, Min; Lairmore, Michael D; Green, Patrick L

    2008-11-01

    Human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ) is dispensable for HTLV-1-mediated cellular transformation in cell culture, but is required for efficient viral infectivity and persistence in rabbits. In most adult T-cell leukemia (ATL) cells, Tax oncoprotein expression is typically low or undetectable, whereas Hbz gene expression is maintained, suggesting that Hbz expression may support infected cell survival and, ultimately, leukemogenesis. Emerging data indicate that HBZ protein can interact with cAMP response element binding protein (CREB) and Jun family members, altering transcription factor binding and transactivation of both viral and cellular promoters. Herein, lentiviral vectors that express Hbz-specific short hairpin (sh)-RNA effectively decreased both Hbz mRNA and HBZ protein expression in transduced HTLV-1-transformed SLB-1 T cells. Hbz knockdown correlated with a significant decrease in T-cell proliferation in culture. Both SLB-1 and SLB-1-Hbz knockdown cells engrafted into inoculated NOD/SCID(gammachain-/-) mice to form solid tumors that also infiltrated multiple tissues. However, tumor formation and organ infiltration were significantly decreased in animals challenged with SLB-1-Hbz knockdown cells. Our data indicate that Hbz expression enhances the proliferative capacity of HTLV-1-infected T cells, playing a critical role in cell survival and ultimately HTLV-1 tumorigenesis in the infected host.

  10. Human leucocyte antigen class I‐redirected anti‐tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells

    PubMed Central

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

    2016-01-01

    Summary CD4+ T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour‐specific CD4+ T cells occur in low frequency, express relatively low‐affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4+ T cells with tumour‐specific HLA class I‐restricted TCRs prior to adoptive transfer. The lack of help from the co‐receptor CD8 glycoprotein in CD4+ cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4+ and CD8+ T cells expressing wild‐type and a range of affinity‐enhanced TCRs specific for the HLA A*0201‐restricted NY‐ESO‐1‐ and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4+ T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4+ T cells than CD8+ T cells. These results indicate that the CD4+ T cell component of current adoptive therapies using TCRs optimized for CD8+ T cells is below par and that there is room for substantial improvement. PMID:27324616

  11. Lung dendritic cells imprint T cell lung homing and promote lung immunity through the chemokine receptor CCR4

    PubMed Central

    Strassner, James P.

    2013-01-01

    T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC–activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC–imprinted T cells protect against influenza more effectively than do gut and skin DC–imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC–activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC–activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4. PMID:23960189

  12. 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

  13. IL-7 receptor blockade following T cell depletion promotes long-term allograft survival

    PubMed Central

    Mai, Hoa-Le; Boeffard, Françoise; Longis, Julie; Danger, Richard; Martinet, Bernard; Haspot, Fabienne; Vanhove, Bernard; Brouard, Sophie; Soulillou, Jean-Paul

    2014-01-01

    T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti–IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4– and anti-CD8–mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation. PMID:24569454

  14. A novel subset of helper T cells promotes immune responses by secreting GM-CSF

    PubMed Central

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

    2013-01-01

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

  15. Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

    PubMed Central

    Estin, Miriam L.; Thompson, Scott B.; Traxinger, Brianna; Fisher, Marlie H.; Friedman, Rachel S.; Jacobelli, Jordan

    2017-01-01

    Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner. PMID:28320969

  16. Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration.

    PubMed

    Estin, Miriam L; Thompson, Scott B; Traxinger, Brianna; Fisher, Marlie H; Friedman, Rachel S; Jacobelli, Jordan

    2017-04-04

    Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.

  17. Identification of a highly immunogenic HLA-A*01-binding T cell epitope of WT1.

    PubMed

    Asemissen, Anne Marie; Keilholz, Ulrich; Tenzer, Stefan; Müller, Margret; Walter, Steffen; Stevanovic, Stefan; Schild, Hansjörg; Letsch, Anne; Thiel, Eckhard; Rammensee, Hans-Georg; Scheibenbogen, Carmen

    2006-12-15

    The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC. One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer. In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.

  18. CD27 Promotes Survival of Activated T Cells and Complements CD28 in Generation and Establishment of the Effector T Cell Pool

    PubMed Central

    Hendriks, Jenny; Xiao, Yanling; Borst, Jannie

    2003-01-01

    CD27, like CD28, acts in concert with the T cell receptor to support T cell expansion. Using CD27−/− mice, we have shown earlier that CD27 determines the magnitude of primary and memory T cell responses to influenza virus. Here, we have examined the relative contributions of CD27 and CD28 to generation of the virus-specific effector T cell pool and its establishment at the site of infection (the lung), using CD27−/−, CD28−/−, and CD27/CD28−/− mice. We find that primary and memory CD8+ T cell responses to influenza virus are dependent on the collective contribution of both receptors. In the primary response, CD27 and CD28 impact to a similar extent on expansion of virus-specific T cells in draining lymph nodes. CD27 is the principle determinant for accumulation of virus-specific T cells in the lung because it can sustain this response in CD28−/− mice. Unlike CD28, CD27 does not affect cell cycle activity, but promotes survival of activated T cells throughout successive rounds of division at the site of priming and may do so at the site of infection as well. CD27 was found to rescue CD28−/− T cells from death at the onset of division, explaining its capacity to support a T cell response in absence of CD28. PMID:14581610

  19. CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection.

    PubMed

    Ross, Ewan A; Coughlan, Ruth E; Flores-Langarica, Adriana; Bobat, Saeeda; Marshall, Jennifer L; Hussain, Khiyam; Charlesworth, James; Abhyankar, Nikita; Hitchcock, Jessica; Gil, Cristina; López-Macías, Constantino; Henderson, Ian R; Khan, Mahmood; Watson, Steve P; MacLennan, Ian C M; Buckley, Christopher D; Cunningham, Adam F

    2011-08-15

    Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.

  20. 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

  1. Growth Factor Midkine Promotes T-Cell Activation through Nuclear Factor of Activated T Cells Signaling and Th1 Cell Differentiation in Lupus Nephritis.

    PubMed

    Masuda, Tomohiro; Maeda, Kayaho; Sato, Waichi; Kosugi, Tomoki; Sato, Yuka; Kojima, Hiroshi; Kato, Noritoshi; Ishimoto, Takuji; Tsuboi, Naotake; Uchimura, Kenji; Yuzawa, Yukio; Maruyama, Shoichi; Kadomatsu, Kenji

    2017-04-01

    Activated T cells play crucial roles in the pathogenesis of autoimmune diseases, including lupus nephritis (LN). The activation of calcineurin/nuclear factor of activated T cells (NFAT) and STAT4 signaling is essential for T cells to perform various effector functions. Here, we identified the growth factor midkine (MK; gene name, Mdk) as a novel regulator in the pathogenesis of 2,6,10,14-tetramethylpentadecane-induced LN via activation of NFAT and IL-12/STAT4 signaling. Wild-type (Mdk(+/+)) mice showed more severe glomerular injury than MK-deficient (Mdk(-/-)) mice, as demonstrated by mesangial hypercellularity and matrix expansion, and glomerular capillary loops with immune-complex deposition. Compared with Mdk(-/-) mice, the frequency of splenic CD69(+) T cells and T helper (Th) 1 cells, but not of regulatory T cells, was augmented in Mdk(+/+) mice in proportion to LN disease activity, and was accompanied by skewed cytokine production. MK expression was also enhanced in activated CD4(+) T cells in vivo and in vitro. MK induced activated CD4(+) T cells expressing CD69 through nuclear activation of NFAT transcription and selectively increased in vitro differentiation of naive CD4(+) T cells into Th1 cells by promoting IL-12/STAT4 signaling. These results suggest that MK serves an indispensable role in the NFAT-regulated activation of CD4(+) T cells and Th1 cell differentiation, eventually leading to the exacerbation of LN.

  2. A Crucial Role of CXCL14 for Promoting Regulatory T Cells Activation in Stroke

    PubMed Central

    Lee, Hsu-Tung; Liu, Shih-Ping; Lin, Chen-Huan; Lee, Sophie Wei; Hsu, Chung Y.; Sytwu, Huey-Kang; Hsieh, Chia-Hung; Shyu, Woei-Cherng

    2017-01-01

    Inflammatory processes have a detrimental role in the pathophysiology of ischemic stroke. However, little is known about the endogenous anti-inflammatory mechanisms in ischemic brain. Here, we identify CXCL14 as a critical mediator of these mechanisms. CXCL14 levels were upregulated in the ischemic brains of humans and rodents. Moreover, hypoxia inducible factor-1α (HIF-1α) drives hypoxia- or cerebral ischemia (CI)-dependent CXCL14 expression via directly binding to the CXCL14 promoter. Depletion of CXCL14 inhibited the accumulation of immature dendritic cells (iDC) or regulatory T cells (Treg) and increased the infarct volume, whereas the supplementation of CXCL14 had the opposite effects. CXCL14 promoted the adhesion, migration, and homing of circulating CD11c+ iDC to the ischemic tissue via the upregulation of the cellular prion protein (PrPC), PECAM-1, and MMPs. The accumulation of Treg in ischemic areas of the brain was mediated through a cooperative effect of CXCL14 and iDC-secreted IL-2-induced Treg differentiation. Interestingly, CXCL14 largely promoted IL-2-induced Treg differentiation. These findings indicate that CXCL14 is a critical immunomodulator involved in the stroke-induced inflammatory reaction. Passive CXCL14 supplementation provides a tractable path for clinical translation in the improvement of stroke-induced neuroinflammation. PMID:28382159

  3. c-Abl-Mediated Tyrosine Phosphorylation of the T-bet DNA-Binding Domain Regulates CD4+ T-Cell Differentiation and Allergic Lung Inflammation ▿

    PubMed Central

    Chen, An; Lee, Sang-Myeong; Gao, Beixue; Shannon, Stephen; Zhu, Zhou; Fang, Deyu

    2011-01-01

    The tyrosine kinase c-Abl is required for full activation of T cells, while its role in T-cell differentiation has not been characterized. We report that c-Abl deficiency skews CD4+ T cells to type 2 helper T cell (Th2) differentiation, and c-Abl−/− mice are more susceptible to allergic lung inflammation. c-Abl interacts with and phosphorylates T-bet, a Th1 lineage transcription factor. c-Abl-mediated phosphorylation enhances the transcriptional activation of T-bet. Interestingly, three tyrosine residues within the T-bet DNA-binding domain are the predominant sites of phosphorylation by c-Abl. Mutation of these tyrosine residues inhibits the promoter DNA-binding activity of T-bet. c-Abl regulates Th cell differentiation in a T-bet-dependent manner because genetic deletion of T-bet in CD4+ T cells abolishes c-Abl-deficiency-mediated enhancement of Th2 differentiation. Reintroduction of T-bet-null CD4+ T cells with wild-type T-bet, but not its tyrosine mutant, rescues gamma interferon (IFN-γ) production and inhibits Th2 cytokine production. Therefore, c-Abl catalyzes tyrosine phosphorylation of the DNA-binding domain of T-bet to regulate CD4+ T cell differentiation. PMID:21690296

  4. 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

  5. IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain.

    PubMed

    Villegas-Mendez, Ana; Greig, Rachel; Shaw, Tovah N; de Souza, J Brian; Gwyer Findlay, Emily; Stumhofer, Jason S; Hafalla, Julius C R; Blount, Daniel G; Hunter, Christopher A; Riley, Eleanor M; Couper, Kevin N

    2012-07-15

    It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.

  6. Antigen-Presenting Human γδ T Cells Promote Intestinal CD4(+) T Cell Expression of IL-22 and Mucosal Release of Calprotectin.

    PubMed

    Tyler, Christopher J; McCarthy, Neil E; Lindsay, James O; Stagg, Andrew J; Moser, Bernhard; Eberl, Matthias

    2017-03-22

    The cytokine IL-22 plays a critical role in mucosal barrier defense, but the mechanisms that promote IL-22 expression in the human intestine remain poorly understood. As human microbe-responsive Vγ9/Vδ2 T cells are abundant in the gut and recognize microbiota-associated metabolites, we assessed their potential to induce IL-22 expression by intestinal CD4(+) T cells. Vγ9/Vδ2 T cells with characteristics of APCs were generated from human blood and intestinal organ cultures, then cocultured with naive and memory CD4(+) T cells obtained from human blood or the colon. The potency of blood and intestinal γδ T-APCs was compared with that of monocytes and dendritic cells, by assessing CD4(+) T cell phenotypes and proliferation as well as cytokine and transcription factor profiles. Vγ9/Vδ2 T cells in human blood, colon, and terminal ileum acquired APC functions upon microbial activation in the presence of microenvironmental signals including IL-15, and were capable of polarizing both blood and colonic CD4(+) T cells toward distinct effector fates. Unlike monocytes or dendritic cells, gut-homing γδ T-APCs employed an IL-6 independent mechanism to stimulate CD4(+) T cell expression of IL-22 without upregulating IL-17. In human intestinal organ cultures, microbial activation of Vγ9/Vδ2 T cells promoted mucosal secretion of IL-22 and ICOSL/TNF-α-dependent release of the IL-22 inducible antimicrobial protein calprotectin without modulating IL-17 expression. In conclusion, human γδ T-APCs stimulate CD4(+) T cell responses distinct from those induced by myeloid APCs to promote local barrier defense via mucosal release of IL-22 and calprotectin. Targeting of γδ T-APC functions may lead to the development of novel gut-directed immunotherapies and vaccines.

  7. Antigen-Presenting Human γδ T Cells Promote Intestinal CD4+ T Cell Expression of IL-22 and Mucosal Release of Calprotectin

    PubMed Central

    Tyler, Christopher J.; McCarthy, Neil E.; Lindsay, James O.; Moser, Bernhard

    2017-01-01

    The cytokine IL-22 plays a critical role in mucosal barrier defense, but the mechanisms that promote IL-22 expression in the human intestine remain poorly understood. As human microbe–responsive Vγ9/Vδ2 T cells are abundant in the gut and recognize microbiota-associated metabolites, we assessed their potential to induce IL-22 expression by intestinal CD4+ T cells. Vγ9/Vδ2 T cells with characteristics of APCs were generated from human blood and intestinal organ cultures, then cocultured with naive and memory CD4+ T cells obtained from human blood or the colon. The potency of blood and intestinal γδ T-APCs was compared with that of monocytes and dendritic cells, by assessing CD4+ T cell phenotypes and proliferation as well as cytokine and transcription factor profiles. Vγ9/Vδ2 T cells in human blood, colon, and terminal ileum acquired APC functions upon microbial activation in the presence of microenvironmental signals including IL-15, and were capable of polarizing both blood and colonic CD4+ T cells toward distinct effector fates. Unlike monocytes or dendritic cells, gut-homing γδ T-APCs employed an IL-6 independent mechanism to stimulate CD4+ T cell expression of IL-22 without upregulating IL-17. In human intestinal organ cultures, microbial activation of Vγ9/Vδ2 T cells promoted mucosal secretion of IL-22 and ICOSL/TNF-α–dependent release of the IL-22 inducible antimicrobial protein calprotectin without modulating IL-17 expression. In conclusion, human γδ T-APCs stimulate CD4+ T cell responses distinct from those induced by myeloid APCs to promote local barrier defense via mucosal release of IL-22 and calprotectin. Targeting of γδ T-APC functions may lead to the development of novel gut-directed immunotherapies and vaccines. PMID:28330898

  8. Interleukin 2 and interleukin 10 function synergistically to promote CD8(+) T cell cytotoxicity, which is suppressed by regulatory T cells in breast cancer.

    PubMed

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

    2017-03-06

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

  9. CTLA4-Ig Induced T-Cell Anergy Promotes Wnt10b Production and Bone Formation

    PubMed Central

    Roser-Page, Susanne; Vikulina, Tatyana; Zayzafoon, Majd; Weitzmann, M. Neale

    2014-01-01

    Objective Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by severe joint erosion and systemic osteoporosis. Chronic T-cell activation is a hallmark of RA and agents that target the CD28 receptor on T-cells, needed for T-cell activation, are being increasingly employed as therapeutic agents in RA and other inflammatory diseases. Lymphocytes play complex roles in the regulation of the skeleton and although activated T-cells and B-cells secrete cytokines that promote skeletal decline, under physiological conditions lymphocytes also play key protective roles in the stabilization of skeletal mass. Consequently, disruption of T-cell costimulation may have unforeseen consequences on physiological bone turnover. In this study we investigate the impact of pharmacological CD28 T-cell costimulation blockade on physiological bone turnover and structure. Methods C57BL6 mice were treated with Cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, a pharmacological CD28 antagonist, or irrelevant control antibody (Ig) and serum biochemical markers of bone turnover quantified by ELISA. Bone mineral density (BMD) and indices of bone structure were further quantified by dual energy X-ray absorptiometry (DEXA) and micro-computed tomography (μCT) respectively and static and dynamic indices of bone formation quantified using bone histomorphometry. Results Pharmacological disruption of CD28 T-cell costimulation in mice, significantly increased bone mass and enhanced indices of bone structure, a consequence of enhanced bone formation, concurrent with enhanced secretion of the bone anabolic factor Wnt10b by T-cells. Conclusion Inhibition of CD28 co-stimulation by CTLA4-Ig promotes T-cell Wnt10b production and bone formation and may represent a novel anabolic strategy for increasing bone mass in osteoporotic conditions. PMID:24757150

  10. Low-dose controlled release of mTOR inhibitors maintains T cell plasticity and promotes central memory T cells.

    PubMed

    Gammon, Joshua M; Gosselin, Emily A; Tostanoski, Lisa H; Chiu, Yu-Chieh; Zeng, Xiangbin; Zeng, Qin; Jewell, Christopher M

    2017-10-10

    An important goal for improving vaccine and immunotherapy technologies is the ability to provide further control over the specific phenotypes of T cells arising from these agents. Along these lines, frequent administration of rapamycin (Rapa), a small molecule inhibitor of the mammalian target of rapamycin (mTOR), exhibits a striking ability to polarize T cells toward central memory phenotypes (TCM), or to suppress immune function, depending on the concentrations and other signals present during administration. TCM exhibit greater plasticity and proliferative capacity than effector memory T cells (TEFF) and, therefore, polarizing vaccine-induced T cells toward TCM is an intriguing strategy to enhance T cell expansion and function against pathogens or tumors. Here we combined biodegradable microparticles encapsulating Rapa (Rapa MPs) with vaccines composed of soluble peptide antigens and molecular adjuvants to test if this approach allows polarization of differentiating T cells toward TCM. We show Rapa MPs modulate DC function, enhancing secretion of inflammatory cytokines at very low doses, and suppressing function at high doses. While Rapa MP treatment reduced - but did not stop - T cell proliferation in both CD4(+) and CD8(+) transgenic T cell co-cultures, the expanding CD8(+) T cells differentiated to higher frequencies of TCM at low doses of MP Rapa MPs. Lastly, we show in mice that local delivery of Rapa MPs to lymph nodes during vaccination either suppresses or enhances T cell function in response to melanoma antigens, depending on the dose of drug in the depots. In particular, at low Rapa MP doses, vaccines increased antigen-specific TCM, resulting in enhanced T cell expansion measured during subsequent booster injections over at least 100days. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. 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

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

    PubMed Central

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

    2014-01-01

    Adoptive T cell therapy has 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. PMID:21935923

  13. Inactivation of RUNX3/p46 Promotes Cutaneous T-Cell Lymphoma.

    PubMed

    Haider, Ahmed; Steininger, Anne; Ullmann, Reinhard; Hummel, Michael; Dimitrova, Lora; Beyer, Marc; Vandersee, Staffan; Lenze, Dido; Sterry, Wolfram; Assaf, Chalid; Möbs, Markus

    2016-11-01

    The key role of RUNX3 in physiological T-cell differentiation has been extensively documented. However, information on its relevance for the development of human T-cell lymphomas or leukemias is scarce. Here, we show that alterations of RUNX3 by either heterozygous deletion or methylation of its distal promoter can be observed in the tumor cells of 15 of 21 (71%) patients suffering from Sézary syndrome, an aggressive variant of cutaneous T-cell lymphoma. As a consequence, mRNA levels of RUNX3/p46, the isoform controlled by the distal promoter, are significantly lower in Sézary syndrome tumor cells. Re-expression of RUNX3/p46 reduces cell viability and promotes apoptosis in a RUNX3/p46(low) cell line of cutaneous T-cell lymphoma. Based on this, we present evidence that RUNX3 can act as a tumor suppressor in a human T-cell malignancy and suggest that this effect is predominantly mediated through transcripts from its distal promoter, in particular RUNX3/p46.

  14. 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

  15. Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation.

    PubMed Central

    Rooney, J W; Sun, Y L; Glimcher, L H; Hoey, T

    1995-01-01

    The transcription factors NFAT and AP-1 have been shown to be essential for inducible interleukin-2 (IL-2) expression in activated T cells. NFAT has been previously reported to bind to two sites in the IL-2 promoter: in association with AP-1 at the distal antigen response element at -280 and at -135. On the basis of DNase I footprinting with recombinant NFAT and AP-1 proteins, gel shift assays, and transfection experiments, we have identified three additional NFAT sites in the IL-2 promoter. Strikingly, all five NFAT sites are essential for the full induction of promoter activity in response to T-cell receptor stimulation. Four of the five NFAT sites are part of composite elements able to bind AP-1 in association with NFAT. These sites display a diverse range of cooperativity and interdependency on NFAT and AP-1 proteins for binding. One of the NFAT sites directly overlaps the CD28-responsive element. We present evidence that CD28 inducibility is conferred by the AP-1 component in NFAT-AP-1 composite elements. These findings provide further insight into the mechanisms involved in the regulation of the IL-2 promoter. PMID:7565783

  16. Interleukin-17A Promotes CD8+ T Cell Cytotoxicity To Facilitate West Nile Virus Clearance

    PubMed Central

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

    2016-01-01

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

  17. Interleukin-17A Promotes CD8+ T Cell Cytotoxicity To Facilitate West Nile Virus Clearance.

    PubMed

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

    2017-01-01

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

  18. 17q21 asthma-risk variants switch CTCF binding and regulate IL-2 production by T cells

    PubMed Central

    Schmiedel, Benjamin Joachim; Seumois, Grégory; Samaniego-Castruita, Daniela; Cayford, Justin; Schulten, Veronique; Chavez, Lukas; Ay, Ferhat; Sette, Alessandro; Peters, Bjoern; Vijayanand, Pandurangan

    2016-01-01

    Asthma and autoimmune disease susceptibility has been strongly linked to genetic variants in the 17q21 haploblock that alter the expression of ORMDL3; however, the molecular mechanisms by which these variants perturb gene expression and the cell types in which this effect is most prominent are unclear. We found several 17q21 variants overlapped enhancers present mainly in primary immune cell types. CD4+ T cells showed the greatest increase (threefold) in ORMDL3 expression in individuals carrying the asthma-risk alleles, where ORMDL3 negatively regulated interleukin-2 production. The asthma-risk variants rs4065275 and rs12936231 switched CTCF-binding sites in the 17q21 locus, and 4C-Seq assays showed that several distal cis-regulatory elements upstream of the disrupted ZPBP2 CTCF-binding site interacted with the ORMDL3 promoter region in CD4+ T cells exclusively from subjects carrying asthma-risk alleles. Overall, our results suggested that T cells are one of the most prominent cell types affected by 17q21 variants. PMID:27848966

  19. Chemotactic Migration of T Cells towards Dendritic Cells Promotes the Detection of Rare Antigens

    PubMed Central

    Vroomans, Renske M. A.; Marée, Athanasius F. M.; de Boer, Rob J.; Beltman, Joost B.

    2012-01-01

    In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs) has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs) carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data. PMID:23166480

  20. Chemotactic migration of T cells towards dendritic cells promotes the detection of rare antigens.

    PubMed

    Vroomans, Renske M A; Marée, Athanasius F M; de Boer, Rob J; Beltman, Joost B

    2012-01-01

    In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs) has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs) carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data.

  1. HTLV-1 Viral Factor HBZ Induces CCR4 to Promote T-cell Migration and Proliferation.

    PubMed

    Sugata, Kenji; Yasunaga, Jun-Ichirou; Kinosada, Haruka; Mitobe, Yuichi; Furuta, Rie; Mahgoub, Mohamed; Onishi, Chiho; Nakashima, Kazutaka; Ohshima, Koichi; Matsuoka, Masao

    2016-09-01

    Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and other inflammatory diseases in infected individuals. However, a complete understanding of how HTLV-1 transforms T cells is lacking. Expression of the chemokine receptor CCR4 on ATL cells and HTLV-1-infected cells suggested the hypothesis that CCR4 may mediate features of ATL and inflammatory diseases caused by HTLV-1. In this study, we show that the constitutively expressed HTLV-1 bZIP factor (HBZ) encoded by HTLV-1 is responsible for inducing CCR4 and its ability to promote T-cell proliferation and migration. Ectopic expression of HBZ was sufficient to stimulate expression of CCR4 in human and mouse T cells. Conversely, HBZ silencing in ATL cell lines was sufficient to inhibit CCR4 expression. Mechanistic investigations showed that HBZ induced GATA3 expression in CD4(+) T cells, thereby activating transcription from the CCR4 promoter. In an established air pouch model of ATL, we observed that CD4(+) T cells of HBZ transgenic mice (HBZ-Tg mice) migrated preferentially to the pouch, as compared with those in nontransgenic mice. Migration of CD4(+) T cells in HBZ-Tg mice was inhibited by treatment with a CCR4 antagonist. Proliferating (Ki67(+)) CD4(+) T cells were found to express high levels of CCR4 and CD103. Further, CD4(+) T-cell proliferation in HBZ-Tg mice was enhanced by coordinate treatment with the CCR4 ligands CCL17 and 22 and with the CD103 ligand E-cadherin. Consistent with this finding, we found that ATL cells in clinical skin lesions were frequently positive for CCR4, CD103, and Ki67. Taken together, our results show how HBZ activates CCR4 expression on T cells to augment their migration and proliferation, two phenomena linked to HTLV-1 pathogenesis. Cancer Res; 76(17); 5068-79. ©2016 AACR. ©2016 American Association for Cancer Research.

  2. TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer

    PubMed Central

    Sakuishi, Kaori; Ngiow, Shin Foong; Sullivan, Jenna M.; Teng, Michele W. L.; Kuchroo, Vijay K.; Smyth, Mark J.; Anderson, Ana C.

    2013-01-01

    T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8+ T cells arising in chronic diseases such as cancer. In addition to exhausted CD8+ T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3+ Tregs express TIM3. TIM3+ Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3+ Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3+ Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8+ T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8+ T cells. Collectively, our data indicate that TIM3 marks highly suppressive tissue-resident Tregs that play an important role in shaping the antitumor immune response in situ, increasing the value of TIM3-targeting therapeutic strategies against cancer. PMID:23734331

  3. 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.

  4. Aging promotes acquisition of naive-like CD8+ memory T cell traits and enhanced functionalities

    PubMed Central

    Davenport, Bennett; Nguyen, Tom; Victorino, Francisco; Haist, Kelsey; Jhun, Kevin; Karimpour-Fard, Anis; Hunter, Lawrence; Kedl, Ross; Clambey, Eric T.

    2016-01-01

    Protective T cell memory is an acquired trait that is contingent upon the preservation of its constituents and therefore vulnerable to the potentially deleterious effects of organismal aging. Here, however, we have found that long-term T cell memory in a natural murine host-pathogen system can substantially improve over time. Comprehensive molecular, phenotypic, and functional profiling of aging antiviral CD8+ memory T cells (CD8+ TM) revealed a pervasive remodeling process that promotes the gradual acquisition of distinct molecular signatures, of increasingly homogeneous phenotypes, and of diversified functionalities that combine to confer a CD8+ TM–autonomous capacity for enhanced recall responses and immune protection. Notably, the process of CD8+ TM aging is characterized by a progressive harmonization of memory and naive T cell traits, is broadly amenable to experimental acceleration or retardation, and serves as a constitutional component for the “rebound model” of memory T cell maturation. By casting CD8+ TM populations within the temporal framework of their slowly evolving properties, this model establishes a simple ontogenetic perspective on the principal organization of CD8+ T cell memory that may directly inform the development of improved diagnostic, prophylactic, and therapeutic modalities. PMID:27617858

  5. Aging promotes acquisition of naive-like CD8+ memory T cell traits and enhanced functionalities.

    PubMed

    Eberlein, Jens; Davenport, Bennett; Nguyen, Tom; Victorino, Francisco; Haist, Kelsey; Jhun, Kevin; Karimpour-Fard, Anis; Hunter, Lawrence; Kedl, Ross; Clambey, Eric T; Homann, Dirk

    2016-10-03

    Protective T cell memory is an acquired trait that is contingent upon the preservation of its constituents and therefore vulnerable to the potentially deleterious effects of organismal aging. Here, however, we have found that long-term T cell memory in a natural murine host-pathogen system can substantially improve over time. Comprehensive molecular, phenotypic, and functional profiling of aging antiviral CD8+ memory T cells (CD8+ TM) revealed a pervasive remodeling process that promotes the gradual acquisition of distinct molecular signatures, of increasingly homogeneous phenotypes, and of diversified functionalities that combine to confer a CD8+ TM-autonomous capacity for enhanced recall responses and immune protection. Notably, the process of CD8+ TM aging is characterized by a progressive harmonization of memory and naive T cell traits, is broadly amenable to experimental acceleration or retardation, and serves as a constitutional component for the "rebound model" of memory T cell maturation. By casting CD8+ TM populations within the temporal framework of their slowly evolving properties, this model establishes a simple ontogenetic perspective on the principal organization of CD8+ T cell memory that may directly inform the development of improved diagnostic, prophylactic, and therapeutic modalities.

  6. IL-17A produced by γδ T cells promotes tumor growth in hepatocellular carcinoma.

    PubMed

    Ma, Shoubao; Cheng, Qiao; Cai, Yifeng; Gong, Huanle; Wu, Yan; Yu, Xiao; Shi, Liyun; Wu, Depei; Dong, Chen; Liu, Haiyan

    2014-04-01

    Interleukin (IL)-17A is expressed in the tumor microenvironment where it appears to contribute to tumor development, but its precise role in tumor immunity remains controversial. Here, we report mouse genetic evidence that IL-17A is critical for tumor growth. IL-17A-deficient mice exhibited reduced tumor growth, whereas systemic administration of recombinant mouse IL-17A promoted the growth of hepatocellular carcinoma. The tumor-promoting effect of IL-17A was mediated through suppression of antitumor responses, especially CD8(+) T-cell responses. Furthermore, we found that IL-17A was produced mainly by Vγ4 γδ T cells, insofar as depleting Vγ4 γδ T cells reduced tumor growth, whereas adoptive transfer of Vγ4 γδ T cells promoted tumor growth. Mechanistic investigations showed that IL-17A induced CXCL5 production by tumor cells to enhance the infiltration of myeloid-derived suppressor cells (MDSC) to tumor sites in a CXCL5/CXCR2-dependent manner. IL-17A also promoted the suppressive activity of MDSC to reinforce suppression of tumoral immunity. Moreover, we found that MDSC could induce IL-17A-producing γδ T cells via production of IL-1β and IL-23. Conversely, IL-17A could also enhance production of IL-1β and IL-23 in MDSC as a positive feedback. Together, our results revealed a novel mechanism involving cross-talk among γδ T cells, MDSCs, and tumor cells through IL-17A production. These findings offer new insights into how IL-17A influences tumor immunity, with potential implications for the development of tumor immunotherapy.

  7. 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

  8. 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

  9. Upregulation of DR3 expression in CD4+ T cells promotes secretion of IL-17 in experimental autoimmune uveitis

    PubMed Central

    2011-01-01

    Purpose This study investigated the role of death receptor 3 (DR3) in experimental autoimmune uveitis (EAU). Methods EAU was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein (IRBP) 161–180 emulsified with complete Freund’s adjuvant and evaluated with clinical and histopathologic observation. Total protein of draining lymph nodes (DLNs) was extracted from the control, EAU, or recovery phase mice. CD4+ T cells were separated from lymphocytes with magnetic-assisted cell sorting. At the same time, some of the CD4+ T cells were cultured with or without recombinant TL1A (rTL1A, the DR3 ligand) for three days, and the supernatants were collected for the interleukin-17 (IL-17) test. DR3 mRNA and protein levels in CD4+ T cells and the endogenous concentration of TL1A in mice DLNs were assessed with real-time PCR or western blotting. Levels of IL-17 in the supernatants were determined with enzyme-linked immunosorbent assay. Results Histopathological and clinical data revealed severe intraocular inflammation in the immunized mice. The inflammation reached its peak on day 14 in EAU and had resolved in the recovery phase (weeks 4–5 or more after IRBP immunization). CD4+ T cells obtained from EAU (day 7 or 14) had higher levels of DR3 mRNA and protein expression compared with the control group treated with complete Freund’s adjuvant alone and the recovery group. However, the DR3 mRNA and protein levels on day 21 in EAU were similar to those observed in the control and recovery groups. The endogenous levels of TL1A were upregulated in EAU, and decreased in the recovery phase mice. Adding rTL1A increased the production of IL-17 by CD4+ T cells isolated from mice DLNs. Moreover, the increased IL-17 levels in the culture supernatant of CD4+ T cells from EAU were much higher than those from the control and recovery phase mice. However, the effects on promoting IL-17 production in TL1A-stimulated CD4+ T cells were similar

  10. A Transcriptionally Permissive Epigenetic Landscape at the Vasoactive Intestinal Peptide Receptor-1 Promoter Suggests an Euchromatin Nuclear Position in Murine CD4 T Cells

    PubMed Central

    Benton, K.D.; Hermann, R.J.; Vomhof-DeKrey, E.E.; Haring, J.S.; Van der Steen, T.; Smith, J; Dovat, S; Dorsam, G.P.

    2009-01-01

    Summary T cells express receptors for neuropeptides that mediate immunological activities. Vasoactive intestinal peptide receptor – 1 (VPAC1), the prototypical group II G protein coupled receptor, binds two neuropeptides with high affinity, called vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide. During T cell signaling, VPAC1 mRNA expression levels are significantly downregulated through a Src kinase dependent mechanism, thus altering the sensitivity for these neuropeptides during an immune reaction. Presently, it is unknown whether the mechanism that regulates VPAC1 during T cell signaling involves epigenetic changes. Therefore, we hypothesized that the epigenetic landscape consisting of diacetylation at H3K9/14 and trimethylation at H3K4, two transcriptionally permissive histone modifications, would parallel VPAC1 expression showing high enrichment in untreated T cells, but lower enrichment in α-CD3 treated T cells. To this end, quantitative chromatin immunoprecipitation (ChIP) analysis of H3K9/14ac and H3K4me3 was conducted using purified CD4+ T cells, with CD45R+ B cells as a negative control. Our data revealed that these histone modifications at the VPAC1 promoter did indeed parallel its mRNA levels between T and B lymphocytes, but did not decrease during T cell signaling. Collectively, these data strongly imply a euchromatin nuclear position for the VPAC1 locus irrespective of the activation status of T cells. PMID:19729043

  11. Ezrin/Radixin/Moesin proteins and flotillins cooperate to promote uropod formation in T cells.

    PubMed

    Martinelli, Sibylla; Chen, Emily J H; Clarke, Fiona; Lyck, Ruth; Affentranger, Sarah; Burkhardt, Janis K; Niggli, Verena

    2013-01-01

    T cell uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell uropod. Expression of wild type (WT) ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant-negative deletion mutant of ezrin, 1-310 ezrin-EGFP, markedly reduced uropod formation induced by the chemokine SDF-1, T cell front-tail polarity, and capping of PSGL-1 and flotillins. Transfection of T cells with WT or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1-310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and uropod formation, indicating that ERM proteins and flotillins cooperate in uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the uropod of migrating T cells.

  12. Effect of direct albumin binding to sphingosylphosphorylcholine in Jurkat T cells.

    PubMed

    Han, Mijin; Kim, Yu-Lee; Sacket, Santosh J; Kim, Kyeok; Kim, Hyo-Lim; Jo, Ji-Yeong; Ha, Nam-Chul; Im, Dong-Soon

    2007-11-01

    We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.

  13. CD8+ T-cell response promotes evolution of hepatitis C virus nonstructural proteins.

    PubMed

    Ruhl, Marianne; Knuschke, Torben; Schewior, Kevin; Glavinic, Lejla; Neumann-Haefelin, Christoph; Chang, Dae-In; Klein, Marina; Heinemann, Falko M; Tenckhoff, Hannelore; Wiese, Manfred; Horn, Peter A; Viazov, Sergei; Spengler, Ulrich; Roggendorf, Michael; Scherbaum, Norbert; Nattermann, Jacob; Hoffmann, Daniel; Timm, Jörg

    2011-06-01

    Hepatitis C virus (HCV) acquires mutations that allow it to escape the CD8+ T-cell response, although the extent to which this process contributes to viral evolution at the population level is not clear. We studied viral adaptation using data from a large outbreak of HCV genotype 1b infection that occurred among women immunized with contaminated immunoglobulin from 1977 to 1978. The HCV nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients, and mutations were mapped according to their location inside or outside previously described CD8+ T-cell epitopes. A statistical approach was developed to identify sites/regions under reproducible selection pressure associated with HLA class I. The frequency of nonsynonymous mutations was significantly higher inside previously described CD8+ T-cell epitopes than outside-particularly in NS3/4A and NS5B. We identified new regions that are under selection pressure, indicating that not all CD8+ T-cell epitopes have been identified; 6 new epitopes that interact with CD8+ T cells were identified and confirmed in vitro. In some CD8+ T-cell epitopes mutations were reproducibly identified in patients that shared the relevant HLA allele, indicating immune pressure at the population level. There was statistical support for selection of mutations in 18 individual epitopes. Interestingly, 14 of these were restricted by HLA-B allele. HLA class I-associated selection pressure on the nonstructural proteins and here predominantly on NS3/4A and NS5B promotes evolution of HCV. HLA-B alleles have a dominant effect in this selection process. Adaptation of HCV to the CD8+ T-cell response at the population level creates challenges for vaccine design. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  14. Hyperlipidemia Alters Regulatory T Cell Function and Promotes Resistance to Tolerance Induction Through Costimulatory Molecule Blockade

    PubMed Central

    Bagley, J.; Yuan, J.; Chandrakar, A.; Iacomini, J.

    2016-01-01

    Recent work from our laboratory has shown that hyperlipidemia promotes accelerated rejection of vascularized cardiac allografts in mice by inducing anti-donor Th17 reactivity and production of IL-17. Here, we show that hyperlipidemia also affects FoxP3+ regulatory T cells (Tregs). Hyperlipidemia promotes the development of Tregs that express low levels of CD25. Hyperlipidemia also promotes a decrease in central Tregs and an increase in effector Tregs that appears to account for the increase in the frequency of CD25low Tregs. Alterations in Treg subsets also appear to lead to alterations in Treg function. The ability of FoxP3+, CD25high, CD4+ Tregs from hyperlipidemic mice to inhibit proliferation of effector T cells stimulated with anti-CD3 and CD28 was reduced when compared with Tregs from control mice. Regulatory T cells isolated from hyperlipidemic recipients exhibit increased activation of Akt, and a reduction in Bim levels that permits the expansion of FoxP3+CD25lowCD4+ T cells. Hyperlipidemic mice were also resistant to tolerance induction using costimulatory molecule blockade consisting of anti-CD154 and CTLA4Ig, a strategy that requires Tregs. Together, our data suggest that hyperlipidemia profoundly affects Treg subsets and function as well as the ability to induce tolerance. PMID:26079467

  15. Promotion and prevention of autoimmune disease by CD8+ T cells.

    PubMed

    Gravano, David M; Hoyer, Katrina K

    2013-09-01

    Until recently, little was known about the importance of CD8+ T effectors in promoting and preventing autoimmune disease development. CD8+ T cells can oppose or promote autoimmune disease through activities as suppressor cells and as cytotoxic effectors. Studies in several distinct autoimmune models and data from patient samples are beginning to establish the importance of CD8+ T cells in these diseases and to define the mechanisms by which these cells influence autoimmunity. CD8+ effectors can promote disease via dysregulated secretion of inflammatory cytokines, skewed differentiation profiles and inappropriate apoptosis induction of target cells, and work to block disease by eliminating self-reactive cells and self-antigen sources, or as regulatory T cells. Defining the often major contribution of CD8+ T cells to autoimmune disease and identifying the mechanisms by which they alter the pathogenesis of disease is a rapidly expanding area of study and will add valuable information to our understanding of the kinetics, pathology and biology of autoimmune disease.

  16. Hippocampal T cell infiltration promotes neuroinflammation and cognitive decline in a mouse model of tauopathy

    PubMed Central

    Laurent, Cyril; Dorothée, Guillaume; Hunot, Stéphane; Martin, Elodie; Monnet, Yann; Duchamp, Marie; Dong, Yuan; Légeron, François-Pierre; Leboucher, Antoine; Burnouf, Sylvie; Faivre, Emilie; Carvalho, Kévin; Caillierez, Raphaëlle; Zommer, Nadège; Demeyer, Dominique; Jouy, Nathalie; Sazdovitch, Veronique; Schraen-Maschke, Susanna; Delarasse, Cécile; Buée, Luc

    2017-01-01

    Abstract Alzheimer’s disease is characterized by the combined presence of amyloid plaques and tau pathology, the latter being correlated with the progression of clinical symptoms. Neuroinflammatory changes are thought to be major contributors to Alzheimer’s disease pathophysiology, even if their precise role still remains largely debated. Notably, to what extent immune responses contribute to cognitive impairments promoted by tau pathology remains poorly understood. To address this question, we took advantage of the THY-Tau22 mouse model that progressively develops hippocampal tau pathology paralleling cognitive deficits and reappraised the interrelationship between tau pathology and brain immune responses. In addition to conventional astroglial and microglial responses, we identified a CD8-positive T cell infiltration in the hippocampus of tau transgenic mice associated with an early chemokine response, notably involving CCL3. Interestingly, CD8-positive lymphocyte infiltration was also observed in the cortex of patients exhibiting frontemporal dementia with P301L tau mutation. To gain insights into the functional involvement of T cell infiltration in the pathophysiological development of tauopathy in THY-Tau22 mice, we chronically depleted T cells using anti-CD3 antibody. Such anti-CD3 treatment prevented hippocampal T cell infiltration in tau transgenic animals and reverted spatial memory deficits, in absence of tau pathology modulation. Altogether, these data support an instrumental role of hippocampal T cell infiltration in tau-driven pathophysiology and cognitive impairments in Alzheimer’s disease and other tauopathies. PMID:27818384

  17. Hippocampal T cell infiltration promotes neuroinflammation and cognitive decline in a mouse model of tauopathy.

    PubMed

    Laurent, Cyril; Dorothée, Guillaume; Hunot, Stéphane; Martin, Elodie; Monnet, Yann; Duchamp, Marie; Dong, Yuan; Légeron, François-Pierre; Leboucher, Antoine; Burnouf, Sylvie; Faivre, Emilie; Carvalho, Kévin; Caillierez, Raphaëlle; Zommer, Nadège; Demeyer, Dominique; Jouy, Nathalie; Sazdovitch, Veronique; Schraen-Maschke, Susanna; Delarasse, Cécile; Buée, Luc; Blum, David

    2017-01-01

    Alzheimer's disease is characterized by the combined presence of amyloid plaques and tau pathology, the latter being correlated with the progression of clinical symptoms. Neuroinflammatory changes are thought to be major contributors to Alzheimer's disease pathophysiology, even if their precise role still remains largely debated. Notably, to what extent immune responses contribute to cognitive impairments promoted by tau pathology remains poorly understood. To address this question, we took advantage of the THY-Tau22 mouse model that progressively develops hippocampal tau pathology paralleling cognitive deficits and reappraised the interrelationship between tau pathology and brain immune responses. In addition to conventional astroglial and microglial responses, we identified a CD8-positive T cell infiltration in the hippocampus of tau transgenic mice associated with an early chemokine response, notably involving CCL3. Interestingly, CD8-positive lymphocyte infiltration was also observed in the cortex of patients exhibiting frontemporal dementia with P301L tau mutation. To gain insights into the functional involvement of T cell infiltration in the pathophysiological development of tauopathy in THY-Tau22 mice, we chronically depleted T cells using anti-CD3 antibody. Such anti-CD3 treatment prevented hippocampal T cell infiltration in tau transgenic animals and reverted spatial memory deficits, in absence of tau pathology modulation. Altogether, these data support an instrumental role of hippocampal T cell infiltration in tau-driven pathophysiology and cognitive impairments in Alzheimer's disease and other tauopathies. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain.

  18. Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    PubMed Central

    Long, S. Alice; Rieck, Mary; Tatum, Megan; Bollyky, Paul L.; Wu, Rebecca P.; Muller, Isabelle; Ho, Jhon-Chun; Shilling, Heather G.; Buckner, Jane H.

    2011-01-01

    Low antigen dose promotes induction and persistence of Treg in mice, yet few studies have addressed the role of antigen dose in the induction of adaptive CD4+FOXP3+ Treg in humans. To this end, we examined the level of FOXP3 expression in human CD4+CD25− T cells upon activation with autologous antigen presenting cells and varying doses of peptide. Antigen specific T cells expressing FOXP3 were identified by flow cytometry using MHC Class II tetramer (Tmr). We found an inverse relationship between antigen dose and the frequency of FOXP3+ cells for both foreign and self antigen specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr+FOXP3+ T cells was not due to expansion of natural Treg, but instead, we found that induction, proliferation and persistence of FOXP3+ cells was similar in high and low dose cultures whereas proliferation of FOXP3− T cells was favored in high antigen dose cultures. The frequency of FOXP3+ cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3+ cells were maintained with IL-2, but not upon re-stimulation with antigen. Together, these data suggest that low antigen dose favors the transient generation of human antigen specific adaptive Treg over the proliferation of antigen specific FOXP3- effector T cells. These adaptive Treg could function to reduce ongoing inflammatory responses and promote low dose tolerance in humans, especially when antigen exposure and tolerance is transient. PMID:21865550

  19. 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

  20. Natural Killer Cells Promote Early CD8 T Cell Responses against Cytomegalovirus

    PubMed Central

    Robbins, Scott H; Bessou, Gilles; Cornillon, Amélie; Zucchini, Nicolas; Rupp, Brigitte; Ruzsics, Zsolt; Sacher, Torsten; Tomasello, Elena; Vivier, Eric; Koszinowski, Ulrich H; Dalod, Marc

    2007-01-01

    Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-α/β production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-α administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the

  1. Single MHC mutation eliminates enthalpy associated with T cell receptor binding.

    PubMed

    Miller, Peter J; Pazy, Yael; Conti, Brian; Riddle, David; Appella, Ettore; Collins, Edward J

    2007-10-19

    The keystone of the adaptive immune response is T cell receptor (TCR) recognition of peptide presented by major histocompatibility complex (pMHC) molecules. The crystal structure of AHIII TCR bound to MHC, HLA-A2, showed a large interface with an atypical binding orientation. MHC mutations in the interface of the proteins were tested for changes in TCR recognition. From the range of responses observed, three representative HLA-A2 mutants, T163A, W167A, and K66A, were selected for further study. Binding constants and co-crystal structures of the AHIII TCR and the three mutants were determined. K66 in HLA-A2 makes contacts with both peptide and TCR, and has been identified as a critical residue for recognition by numerous TCR. The K66A mutation resulted in the lowest AHIII T cell response and the lowest binding affinity, which suggests that the T cell response may correlate with affinity. Importantly, the K66A mutation does not affect the conformation of the peptide. The change in affinity appears to be due to a loss in hydrogen bonds in the interface as a result of a conformational change in the TCR complementarity-determining region 3 (CDR3) loop. Isothermal titration calorimetry confirmed the loss of hydrogen bonding by a large loss in enthalpy. Our findings are inconsistent with the notion that the CDR1 and CDR2 loops of the TCR are responsible for MHC restriction, while the CDR3 loops interact solely with the peptide. Instead, we present here an MHC mutation that does not change the conformation of the peptide, yet results in an altered conformation of a CDR3.

  2. A novel intronic cAMP response element modulator (CREM) promoter is regulated by activator protein-1 (AP-1) and accounts for altered activation-induced CREM expression in T cells from patients with systemic lupus erythematosus.

    PubMed

    Rauen, Thomas; Benedyk, Konrad; Juang, Yuang-Taung; Kerkhoff, Claus; Kyttaris, Vasileios C; Roth, Johannes; Tsokos, George C; Tenbrock, Klaus

    2011-09-16

    The transcriptional repressor cAMP response element modulator (CREM) α has important roles in normal T cell physiology and contributes to aberrant T cell function in patients with systemic lupus erythematosus (SLE). Recently, we characterized a specificity protein-1-dependent promoter located upstream of the CREM gene that accounts for increased basal CREM expression in SLE T cells and reflects disease activity. Here, we identify a novel intronic CREM promoter (denoted P2) in front of the second exon of the CREM gene that harbors putative binding sites for TATA-binding proteins and the transcriptional activator AP-1. DNA binding studies, chromatin immunoprecipitation, and reporter assays confirmed the functional relevance of these sites, and T cell activation through CD3/CD28 stimulation or phorbol 12-myristate 13-acetate/ionomycin treatment enhances P2 promoter activity. Although the basal CREM levels are increased in T cells from SLE patients compared with healthy controls, there are remarkable differences in the regulation of CREM expression in response to T cell activation. Whereas T cells from healthy individuals display increased CREM expression after T cell activation, most likely through AP-1-dependent up-regulation of the P2 promoter, SLE T cells fail to further increase their basal CREM levels upon T cell activation due to a decreased content of the AP-1 family member c-Fos. Because CREM trans-represses c-fos transcription in SLE T cells, we propose an autoregulatory feedback mechanism between CREM and AP-1. Our findings extend the understanding of CREM gene regulation in the context of T cell activation and disclose another difference in the transcriptional machinery in SLE T cells.

  3. T cells produce an antigen-binding factor with in vivo activity analogous to IgE antibody

    PubMed Central

    1983-01-01

    T cell-dependent activation of resident tissue mast cells is required for the elicitation of delayed-type hypersensitivity skin reactions in mice. A T cell-derived antigen-binding factor that transfers the ability to elicit an immediate hypersensitivity-like skin reaction is described and compared with a hybridoma IgE antibody. Both the T cell factor and IgE mediate reactions with increased vascular permeability and both are mast cell dependent, as they are inactive in two different types of mast cell deficient mice (W/Wv and Sl/Sld). The T cell factor was distinguished from IgE by affinity chromatography using specific anti-IgE and anti-factor antibodies and by a shorter duration of passive sensitization. The T cell factor is a suitable candidate for participation in the mechanism by which T cells activate mast cells in delayed-type hypersensitivity. PMID:6187880

  4. T-cell activation by soluble MHC oligomers can be described by a two-parameter binding model.

    PubMed Central

    Stone, J D; Cochran, J R; Stern, L J

    2001-01-01

    T-cell activation is essential for initiation and control of immune system function. T cells are activated by interaction of cell-surface antigen receptors with major histocompatibility complex (MHC) proteins on the surface of other cells. Studies using soluble oligomers of MHC-peptide complexes and other types of receptor cross-linking agents have supported an activation mechanism that involves T cell receptor clustering. Receptor clustering induced by incubation of T cells with MHC-peptide oligomers leads to the induction of T-cell activation processes, including downregulation of engaged receptors and upregulation of the cell-surface proteins CD69 and CD25. Dose-response curves for these T-cell activation markers are bell-shaped, with different maxima and midpoints, depending on the valency of the soluble oligomer used. In this study, we have analyzed the activation behavior using a mathematical model that describes the binding of multivalent ligands to cell-surface receptors. We show that a simple equilibrium binding model accurately describes the activation data for CD4(+) T cells treated with MHC-peptide oligomers of varying valency. The model can be used to predict activation and binding behavior for T cells and MHC oligomers with different properties. PMID:11606269

  5. Erythropoietin Receptor-Mediated Molecular Crosstalk Promotes T Cell Immunoregulation and Transplant Survival.

    PubMed

    Purroy, Carolina; Fairchild, Robert L; Tanaka, Toshiaki; Baldwin, William M; Manrique, Joaquin; Madsen, Joren C; Colvin, Robert B; Alessandrini, Alessandro; Blazar, Bruce R; Fribourg, Miguel; Donadei, Chiara; Maggiore, Umberto; Heeger, Peter S; Cravedi, Paolo

    2017-08-01

    Although spontaneous kidney transplant acceptance/tolerance occurs in mice and occasionally in humans, mechanisms remain unclear. Herein we test the hypothesis that EPO, a hormone predominantly produced by the adult kidney, has immunomodulating properties that are required for spontaneous kidney graft acceptance. In vitro, in a manner dependent on the EPO receptor and CD131 on antigen-presenting cells, EPO induced the secretion of active TGFβ by antigen-presenting cells, which in turn converted naïve CD4(+) T cells into functional Foxp3(+) regulatory T cells (Treg). In murine transplant models, pharmacologic downregulation of kidney-derived EPO prevented spontaneous Treg generation. In a controlled, prospective cohort clinical study, EPO administration at doses used to correct anemia augmented the frequency of peripheral CD4(+)CD25(+)CD127(lo) T cells in humans with CKD. Furthermore, EPO directly inhibited conventional T cell proliferation in vitro via tyrosine phosphatase SHP-1-dependent uncoupling of IL-2Rβ signaling. Conversely, EPO-initiated signals facilitated Treg proliferation by augmenting IL-2Rγ signaling and maintaining constitutively quenched IL-2Rβ signaling. In additional murine transplant models, recombinant EPO administration prolonged heart allograft survival, whereas pharmacologic downregulation of kidney-derived EPO reduced the expression of TGFβ mRNA and abrogated kidney allograft acceptance. Together, our findings delineate the protolerogenic properties of EPO in inhibiting conventional T cells while simultaneously promoting Treg induction, and suggest that manipulating the EPO/EPO receptor signaling axis could be exploited to prevent and/or treat T cell-mediated pathologies, including transplant rejection. Copyright © 2017 by the American Society of Nephrology.

  6. 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.

  7. Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy.

    PubMed

    Urak, Ryan; Walter, Miriam; Lim, Laura; Wong, ChingLam W; Budde, Lihua E; Thomas, Sandra; Forman, Stephen J; Wang, Xiuli

    2017-01-01

    Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model. Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments. We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once

  8. Caspase Inhibition Blocks Cell Death and Enhances Mitophagy but Fails to Promote T-Cell Lymphoma

    PubMed Central

    Wang, Sih-han; Martin, Sean M.; Harris, Peter S.; Knudson, C. Michael

    2011-01-01

    Caspase-9 is a component of the apoptosome that mediates cell death following release of cytochrome c from mitochondria. Inhibition of Caspase-9 with a dominant negative construct (Casp9DN) blocks apoptosome function, promotes viability and has been implicated in carcinogenesis. Inhibition of the apoptosome in vitro impairs mitochondrial function and promotes mitophagy. To examine whether inhibition of the apoptosome would enhance mitophagy and promote oncogenesis in vivo, transgenic mice were generated that express Casp9DN in the T cell lineage. The effects of Casp9DN on thymocyte viability, mitophagy and thymic tumor formation were examined. In primary thymocytes, Casp9DN delayed dexamethasone (Dex)-induced cell death, altered mitochondrial structure, and decreased oxidant production. Transmission electron microscopy (TEM) revealed that inhibition of the apoptosome resulted in structurally abnormal mitochondria that in some cases were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitochondria being engulfed by autophagosomes (mitophagy), confocal microscopy showed colocalization of LC3-GFP and mitochondria. However, Casp9DN did not significantly accelerate T-cell lymphoma alone, or in combination with Lck-Bax38/1, or with Beclin 1+/− mice, two tumor-prone strains in which altered mitochondrial function has been implicated in promoting tumor development. In addition, heterozygous disruption of Beclin 1 had no effect on T-cell lymphoma formation in Lck-Bax38/1 mice. Further studies showed that Beclin 1 levels had no effect on Casp9DN-induced loss of mitochondrial function. These results demonstrate that neither inhibition of apoptosome function nor Beclin 1 haploinsufficiency accelerate T-cell lymphoma development in mice. PMID:21611191

  9. 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.

  10. HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation.

    PubMed

    Percher, Florent; Curis, Céline; Pérès, Eléonore; Artesi, Maria; Rosewick, Nicolas; Jeannin, Patricia; Gessain, Antoine; Gout, Olivier; Mahieux, Renaud; Ceccaldi, Pierre-Emmanuel; Van den Broeke, Anne; Duc Dodon, Madeleine; Afonso, Philippe V

    2017-06-22

    The human T-lymphotropic virus type 1 (HTLV-1) is efficiently transmitted through cellular contacts. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied in vitro, those facilitating the encounter between infected and target cells remain unknown. In this study, we demonstrate that HTLV-1-infected CD4 T cells secrete a potent chemoattractant, leukotriene B4 (LTB4). LTB4 secretion is dependent on Tax-induced transactivation of the pla2g4c gene, which encodes the cytosolic phospholipase A2 gamma. Inhibition of LTB4 secretion or LTB4 receptor knockdown on target cells reduces T-cell recruitment, cellular contact formation and virus propagation in vitro. Finally, blocking the synthesis of LTB4 in a humanized mouse model of HTLV-1 infection significantly reduces proviral load. This results from a decrease in the number of infected clones while their expansion is not impaired. This study shows the critical role of LTB4 secretion in HTLV-1 transmission both in vitro and in vivo.

  11. Suppressor Mutations within the Core Binding Factor (CBF/AML1) Binding Site of a T-Cell Lymphomagenic Retrovirus

    PubMed Central

    Martiney, Marita J.; Levy, Laura S.; Lenz, Jack

    1999-01-01

    The transcriptional enhancer of the lymphomagenic mouse retrovirus SL3 contains a binding site for the transcription factor core binding factor (CBF; also called AML1, PEBP2, and SEF1). The SL3 CBF binding site is called the core. It differs from the core of the weakly lymphomagenic mouse retrovirus Akv by one nucleotide (the sequences are TGTGGTTAA and TGTGGTCAA, respectively). A mutant virus called SAA that was identical to SL3 except that its core was mutated to the Akv sequence was only moderately attenuated for lymphomagenicity. In most SAA-infected mice, tumor proviruses contained either reversions of the original mutation or one of two novel core sequences. In 20% of the SAA-infected mice, tumor proviruses retained the original SAA/Akv core mutation but acquired one of two additional mutations (underlined), TGCGGTCAA or TGTGGTCTA, that generated core elements called So and T*, respectively. We tested whether the novel base changes in the So and T* cores were suppressor mutations. SL3 mutants that contained So or T* cores in place of the wild-type sequence were generated. These viruses induced T-cell lymphomas in mice more quickly than SAA. Therefore, the mutations in the So and T* cores are indeed second-site suppressor mutations. The suppressor mutations increased CBF binding in vitro and transcriptional activity of the viral long terminal repeats (LTRs) in T lymphocytes to levels comparable to those of SL3. Thus, CBF binding was increased by any of three different nucleotide changes within the sequence of the SAA core. Increased CBF binding resulted in increased LTR transcriptional activity in T cells and in increased viral lymphomagenicity. PMID:9971797

  12. Soluble Factors Secreted by T Cells Promote β-Cell Proliferation

    PubMed Central

    Dirice, Ercument; Kahraman, Sevim; Jiang, Wenyu; El Ouaamari, Abdelfattah; De Jesus, Dario F.; Teo, Adrian K.K.; Hu, Jiang; Kawamori, Dan; Gaglia, Jason L.; Mathis, Diane; Kulkarni, Rohit N.

    2014-01-01

    Type 1 diabetes is characterized by infiltration of pancreatic islets with immune cells, leading to insulin deficiency. Although infiltrating immune cells are traditionally considered to negatively impact β-cells by promoting their death, their contribution to proliferation is not fully understood. Here we report that islets exhibiting insulitis also manifested proliferation of β-cells that positively correlated with the extent of lymphocyte infiltration. Adoptive transfer of diabetogenic CD4+ and CD8+ T cells, but not B cells, selectively promoted β-cell proliferation in vivo independent from the effects of blood glucose or circulating insulin or by modulating apoptosis. Complementary to our in vivo approach, coculture of diabetogenic CD4+ and CD8+ T cells with NOD.RAG1−/− islets in an in vitro transwell system led to a dose-dependent secretion of candidate cytokines/chemokines (interleukin-2 [IL-2], IL-6, IL-10, MIP-1α, and RANTES) that together enhanced β-cell proliferation. These data suggest that soluble factors secreted from T cells are potential therapeutic candidates to enhance β-cell proliferation in efforts to prevent and/or delay the onset of type 1 diabetes. PMID:24089508

  13. The NFAT-1 DNA binding complex in activated T cells contains Fra-1 and JunB.

    PubMed Central

    Boise, L H; Petryniak, B; Mao, X; June, C H; Wang, C Y; Lindsten, T; Bravo, R; Kovary, K; Leiden, J M; Thompson, C B

    1993-01-01

    Activation of T cells induces transcription of the interleukin-2 (IL-2) gene. IL-2 expression is regulated through the binding of transcription factors to multiple sites within the IL-2 enhancer. One such cis-acting element within the IL-2 enhancer is the NFAT-1 (nuclear factor of activated T cells) binding site. NFAT-1 binding activity is absent in resting cells but is induced upon T-cell activation. The induction of NFAT-1 binding activity can be inhibited by cyclosporin A, potentially accounting for the ability of cyclosporin A to inhibit IL-2 production by T cells. We have previously reported that the NFAT-1 binding complex is composed of at least two proteins and that the 5' portion of the NFAT-1 sequence acts as a binding site for one or more proteins from the Ets family of transcription factors. We now report that the 3' portion of the NFAT-1 sequence contains a variant AP-1 binding site. NFAT-1 binding can be specifically inhibited by oligonucleotides containing a consensus AP-1 site. Moreover, mutation of the AP-1 site at the 3' end of the NFAT-1 sequence inhibits both NFAT-1 binding and the ability of the NFAT-1 binding site to activate expression from a reporter plasmid upon T-cell activation. Since AP-1 sites bind dimeric protein complexes composed of individual members of the Fos and Jun families of transcription factors, we used antibodies specific for individual Fos and Jun family members to determine whether they are present in the NFAT-1 binding complex. These experiments demonstrated that the NFAT-1 binding complex contains JunB and Fra-1 proteins. Northern (RNA) blot analyses demonstrate that both fra-1 and junB mRNAs are induced upon T-cell activation, although fra-1 mRNA is present even in quiescent T cells. Of interest, junB is not expressed in quiescent T cells, and it is induced with kinetics that are similar to those for the induction of IL-2 mRNA expression. Taken together, these results suggested that the JunB-Fra-1 heterodimer is the

  14. Nocardia rubra cell-wall skeleton promotes CD4(+) T cell activation and drives Th1 immune response.

    PubMed

    Wang, Guangchuan; Wu, Jie; Miao, Miao; Dou, Heng; Nan, Ning; Shi, Mingsheng; Yu, Guang; Shan, Fengping

    2017-03-15

    Several lines of evidences have shown that Nocardia rubra cell wall skeleton (Nr-CWS) has immunoregulatory and anti-tumor activities. However, there is no information about the effect of Nr-CWS on CD4(+) T cells. The aim of this study was to explore the effect of Nr-CWS on the phenotype and function of CD4(+) T cells. Our results of in vitro experiments showed that Nr-CWS could significantly up-regulate the expression of CD69 and CD25 on CD4(+) T cells, promote the proliferation of CD4(+) T cells, increase the production of IFN-γ, TNF-α and IL-2 in the supernatants, but has no significant effect on the apoptosis and death of CD4(+) T cells. Results of in vivo experiments showed that Nr-CWS could promote the proliferation of CD4(+) T cells, and increase the production of IL-2, IFN-γ and TNF-α (Th1 type cytokines). These data suggest that Nr-CWS can enhance the activation of CD4(+) T cells, promote the proliferation of CD4(+) T cells and the differentiation of CD4(+) T cells to Th1 cells.

  15. The Cytoplasmic Tail of the T Cell Receptor CD3 ε Subunit Contains a Phospholipid-Binding Motif that Regulates T Cell Functions1

    PubMed Central

    DeFord-Watts, Laura M.; Tassin, Tara C.; Becker, Amy M.; Medeiros, Jennifer J.; Albanesi, Joseph P.; Love, Paul E.; Wülfing, Christoph; van Oers, Nicolai S. C.

    2010-01-01

    The CD3 ε subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 ε, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 ε to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and PI(4,5)P2. Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 ε localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 ε lipid-binding domain in T cell biology. PMID:19542373

  16. Core binding factors are necessary for natural killer cell development and cooperate with Notch signaling during T-cell specification

    PubMed Central

    Guo, Yalin; Maillard, Ivan; Chakraborti, Sankhamala; Rothenberg, Ellen V.

    2008-01-01

    CBFβ is the non-DNA binding subunit of the core binding factors (CBFs). Mice with reduced CBFβ levels display profound, early defects in T-cell but not B-cell development. Here we show that CBFβ is also required at very early stages of natural killer (NK)–cell development. We also demonstrate that T-cell development aborts during specification, as the expression of Gata3 and Tcf7, which encode key regulators of T lineage specification, is substantially reduced, as are functional thymic progenitors. Constitutively active Notch or IL-7 signaling cannot restore T-cell expansion or differentiation of CBFβ insufficient cells, nor can overexpression of Runx1 or CBFβ overcome a lack of Notch signaling. Therefore, the ability of the prethymic cell to respond appropriately to Notch is dependent on CBFβ, and both signals converge to activate the T-cell developmental program. PMID:18390836

  17. IL-6 promotes CD4(+) T-cell and B-cell activation during Plasmodium infection.

    PubMed

    Sebina, I; Fogg, L G; James, K R; Soon, M S F; Akter, J; Thomas, B S; Hill, G R; Engwerda, C R; Haque, A

    2017-10-01

    Humoral immunity develops in the spleen during blood-stage Plasmodium infection. This elicits parasite-specific IgM and IgG, which control parasites and protect against malaria. Studies in mice have elucidated cells and molecules driving humoral immunity to Plasmodium, including CD4(+) T cells, B cells, interleukin (IL)-21 and ICOS. IL-6, a cytokine readily detected in Plasmodium-infected mice and humans, is recognized in other systems as a driver of humoral immunity. Here, we examined the effect of infection-induced IL-6 on humoral immunity to Plasmodium. Using P. chabaudi chabaudi AS (PcAS) infection of wild-type and IL-6(-/-) mice, we found that IL-6 helped to control parasites during primary infection. IL-6 promoted early production of parasite-specific IgM but not IgG. Notably, splenic CD138(+) plasmablast development was more dependent on IL-6 than germinal centre (GC) B-cell differentiation. IL-6 also promoted ICOS expression by CD4(+) T cells, as well as their localization close to splenic B cells, but was not required for early Tfh-cell development. Finally, IL-6 promoted parasite control, IgM and IgG production, GC B-cell development and ICOS expression by Tfh cells in a second model, Py17XNL infection. IL-6 promotes CD4(+) T-cell activation and B-cell responses during blood-stage Plasmodium infection, which encourages parasite-specific antibody production. © 2017 John Wiley & Sons Ltd.

  18. 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

  19. The Adaptor Molecule MyD88 Directly Promotes CD8 T cell Responses to Vaccinia Virus

    PubMed Central

    Zhao, Yuan; Trez, Carl De; Flynn, Rachel; Ware, Carl F.; Croft, Michael; Salek-Ardakani, Shahram

    2009-01-01

    Vaccinia Virus (VACV) elicits a robust CD8 T cell response that plays an important role in host resistance. To date, there is little information on the molecules that are essential to generate large pools of VACV-specific effector CD8 T cells. Here, we show that the adaptor molecule MyD88 is critical for the magnitude of primary CD8 T cell responses to both dominant and subdominant VACV epitopes. MyD88−/− mice exhibit profound reduction in CD8 T cell expansion and anti-viral cytokine production. Surprisingly, the defect was not due to impaired antigen-presenting cell function, as MyD88−/− DC matured normally and were able to promote strong CD8 T cell priming following VACV infection. Rather, adoptive transfer experiments demonstrated that intrinsic MyD88-dependent pathways in CD8 T cells were critical. MyD88-deficient CD8 T cells failed to accumulate in wild-type hosts, and poor expansion of MyD88-deficient VACV-specific CD8 T cells resulted after virus infection. In contrast, no defect was evident in the absence of TRIF, TLR2, TLR4, TLR9, and IL-1R. Together, our results highlight an important role for MyD88 in initial anti-viral CD8 T cell responses and suggest that targeting this pathway may be useful in promoting and sustaining anti-VACV immunity. PMID:19414781

  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.

  1. In vivo induction of regulatory T cells promotes allergen tolerance and suppresses allergic contact dermatitis.

    PubMed

    Balmert, Stephen C; Donahue, Cara; Vu, John R; Erdos, Geza; Falo, Louis D; Little, Steven R

    2017-09-10

    Allergic contact dermatitis (ACD) is a common T-cell mediated inflammatory skin condition, characterized by an intensely pruritic rash at the site of contact with allergens like poison ivy or nickel. Current clinical treatments use topical corticosteroids, which broadly and transiently suppress inflammation and symptoms of ACD, but fail to address the underlying immune dysfunction. Here, we present an alternative therapeutic approach that teaches the immune system to tolerate contact allergens by expanding populations of naturally suppressive allergen-specific regulatory T cells (Tregs). Specifically, biodegradable poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) microparticles were engineered to release TGF-β1, Rapamycin, and IL-2, to locally sustain a microenvironment that promotes Treg differentiation. By expanding allergen-specific Tregs and reducing pro-inflammatory effector T cells, these microparticles inhibited destructive hypersensitivity responses to subsequent allergen exposure in an allergen-specific manner, effectively preventing or reversing ACD in previously sensitized mice. Ultimately, this approach to in vivo Treg induction could also enable novel therapies for transplant rejection and autoimmune diseases. Copyright © 2017. Published by Elsevier B.V.

  2. Mutagenesis of beryllium-specific T cell receptors suggests an unusual binding topology for antigen recognition1

    PubMed Central

    Bowerman, Natalie A.; Falta, Michael T.; Mack, Douglas G.; Kappler, John W.; Fontenot, Andrew P.

    2011-01-01

    Unconventional antigens, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of beryllium (Be)-specific T cell receptors (TCR) derived from the lung of a chronic beryllium disease patient. These TCRs are HLA-DP2-restricted and express nearly identical TCR Vβ5.1 chains coupled with different TCR α-chains. Site-specific mutagenesis of all amino acids comprising the complementarity determining regions of the TCRA and TCRB genes showed a dominant role for Vβ5.1 residues in Be recognition, with little contribution from the TCR α-chain. Solvent-exposed residues along the α-helices of the HLA-DP2 α- and β-chains were also mutated to alanine. Two β-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition with no contribution from the HLA-DP2 α-chain. These findings suggest that Be-specific T cells recognize antigen using an unconventional binding topology, with the majority of interactions contributed by TCR Vβ5.1 residues and the HLA-DP2 β1-chain. Thus, unusual docking topologies are not exclusively used by autoreactive T cells, but also for the recognition of unconventional metal antigens, such as Be. PMID:21873524

  3. 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

    2015-09-15

    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.

  4. JEG-3 Trophoblast Cells Producing Human Chorionic Gonadotropin Promote Conversion of Human CD4+FOXP3- T Cells into CD4+FOXP3+ Regulatory T Cells and Foster T Cell Suppressive Activity.

    PubMed

    Poloski, Eileen; Oettel, Anika; Ehrentraut, Stefanie; Luley, Lydia; Costa, Serban Dan; Zenclussen, Ana Claudia; Schumacher, Anne

    2016-03-09

    The pregnancy hormone human Chorionic Gonadotropin (hCG) reportedly modulates innate and adaptive immune responses and contributes thereby to fetal survival. More precisely, hCG has been shown to support human Treg cell homing into the fetal-maternal interface and enhance number and function of Treg cells in murine pregnancy. Here, we aimed to study whether hCG and hCG-producing human trophoblast cell lines induce Treg cells from CD4(+)FOXP3(-) T cells and promote T cell suppressive activity. CD4(+)FOXP3(-) T cells were isolated from peripheral blood of normal pregnant women and cultured in the presence of hCG-producing (JEG-3, HTR-8) and non-producing (SWAN-71) cell lines. To confirm the participation of hCG in Treg cell conversion, the experiments were performed in the presence of anti-hCG and additional experiments were run with recombinant or urine-purified hCG. After culture the number of CD4(+)FOXP3(+) Treg cells as well as the suppressive capacity of total T cells was assessed. hCG-producing JEG-3 cells as well as recombinant and urine-purified hCG induced CD4(+)FOXP3(+) Treg cells from CD4(+)FOXP3(-) T cells. Blockage of hCG impaired Treg cell induction. Moreover, hCG-producing JEG-3 cells increased suppressive activity of CD4(+)FOXP3(-) T cells through an antigen-independent pathway. Our results propose another mechanism through which hCG modulates the female immune system during pregnancy in favor of the fetus.

  5. Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides.

    PubMed

    Wang, Mingjun; Tang, Sheila T; Stryhn, Anette; Justesen, Sune; Larsen, Mette V; Dziegiel, Morten H; Lewinsohn, David M; Buus, Søren; Lund, Ole; Claesson, Mogens H

    2011-04-01

    Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.

  6. Treg-cell depletion promotes chemokine production and accumulation of CXCR3(+) conventional T cells in intestinal tumors.

    PubMed

    Akeus, Paulina; Langenes, Veronica; Kristensen, Jonas; von Mentzer, Astrid; Sparwasser, Tim; Raghavan, Sukanya; Quiding-Järbrink, Marianne

    2015-06-01

    Colorectal cancer (CRC) is one of the most prevalent tumor types worldwide and tumor-infiltrating T cells are crucial for anti-tumor immunity. We previously demonstrated that Treg cells from CRC patients inhibit transendothelial migration of conventional T cells. However, it remains unclear if local Treg cells affect lymphocyte migration into colonic tumors. By breeding APC(Min/+) mice with depletion of regulatory T cells mice, expressing the diphtheria toxin receptor under the control of the FoxP3 promoter, we were able to selectively deplete Treg cells in tumor-bearing mice, and investigate the impact of these cells on the infiltration of conventional T cells into intestinal tumors. Short-term Treg-cell depletion led to a substantial increase in the frequencies of T cells in the tumors, attributed by both increased infiltration and proliferation of T cells in the Treg-cell-depleted tumors. We also demonstrate a selective increase of the chemokines CXCL9 and CXCL10 in Treg-cell-depleted tumors, which were accompanied by accumulation of CXCR3(+) T cells, and increased IFN-γ mRNA expression. In conclusion, Treg-cell depletion increases the accumulation of conventional T cells in intestinal tumors, and targeting Treg cells could be a possible anti-tumor immunotherapy, which not only affects T-cell effector functions, but also their recruitment to tumors.

  7. 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

  8. 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

  9. Non-POU Domain-Containing Octamer-Binding Protein Negatively Regulates HIV-1 Infection in CD4(+) T Cells.

    PubMed

    St Gelais, Corine; Roger, Jonathan; Wu, Li

    2015-08-01

    HIV-1 interacts with numerous cellular proteins during viral replication. Identifying such host proteins and characterizing their roles in HIV-1 infection can deepen our understanding of the dynamic interplay between host and pathogen. We previously identified non-POU domain-containing octamer-binding protein (NonO or p54nrb) as one of host factors associated with catalytically active preintegration complexes (PIC) of HIV-1 in infected CD4(+) T cells. NonO is involved in nuclear processes including transcriptional regulation and RNA splicing. Although NonO has been identified as an HIV-1 interactant in several recent studies, its role in HIV-1 replication has not been characterized. We investigated the effect of NonO on the HIV-1 life cycle in CD4(+) T cell lines and primary CD4(+) T cells using single-cycle and replication-competent HIV-1 infection assays. We observed that short hairpin RNA (shRNA)-mediated stable NonO knockdown in a CD4(+) Jurkat T cell line and primary CD4(+) T cells did not affect cell viability or proliferation, but enhanced HIV-1 infection. The enhancement of HIV-1 infection in Jurkat T cells correlated with increased viral reverse transcription and gene expression. Knockdown of NonO expression in Jurkat T cells modestly enhanced HIV-1 gag mRNA expression and Gag protein synthesis, suggesting that viral gene expression and RNA regulation are the predominantly affected events causing enhanced HIV-1 replication in NonO knockdown (KD) cells. Furthermore, overexpression of NonO in Jurkat T cells reduced HIV-1 single-cycle infection by 41% compared to control cells. Our data suggest that NonO negatively regulates HIV-1 infection in CD4(+) T cells, albeit it has modest effects on early and late stages of the viral life cycle, highlighting the importance of host proteins associated with HIV-1 PIC in regulating viral replication.

  10. 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.

  11. Mitochondrial biogenesis and proteome remodeling promotes one carbon metabolism for T cell activation

    PubMed Central

    Ron-Harel, Noga; Santos, Daniel; Ghergurovich, Jonathan M.; Sage, Peter T.; Reddy, Anita; Lovitch, Scott B.; Dephoure, Noah; Satterstrom, F. Kyle; Sheffer, Michal; Spinelli, Jessica B.; Gygi, Steven; Rabinowitz, Joshua D.; Sharpe, Arlene H.; Haigis, Marcia C.

    2017-01-01

    Summary Naïve T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naïve CD4+ T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture, and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one carbon metabolism that is critical for T cell activation and survival. PMID:27411012

  12. Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance

    PubMed Central

    Soroosh, Pejman; Doherty, Taylor A.; Duan, Wei; Mehta, Amit Kumar; Choi, Heonsik; Adams, Yan Fei; Mikulski, Zbigniew; Khorram, Naseem; Rosenthal, Peter; Broide, David H.

    2013-01-01

    Airway tolerance is the usual outcome of inhalation of harmless antigens. Although T cell deletion and anergy are likely components of tolerogenic mechanisms in the lung, increasing evidence indicates that antigen-specific regulatory T cells (inducible Treg cells [iTreg cells]) that express Foxp3 are also critical. Several lung antigen-presenting cells have been suggested to contribute to tolerance, including alveolar macrophages (MØs), classical dendritic cells (DCs), and plasmacytoid DCs, but whether these possess the attributes required to directly promote the development of Foxp3+ iTreg cells is unclear. Here, we show that lung-resident tissue MØs coexpress TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and that their sampling of harmless airborne antigen and presentation to antigen-specific CD4 T cells resulted in the generation of Foxp3+ Treg cells. Treg cell induction in this model depended on both TGF-β and retinoic acid. Transfer of the antigen-pulsed tissue MØs into the airways correspondingly prevented the development of asthmatic lung inflammation upon subsequent challenge with antigen. Moreover, exposure of lung tissue MØs to allergens suppressed their ability to generate iTreg cells coincident with blocking airway tolerance. Suppression of Treg cell generation required proteases and TLR-mediated signals. Therefore, lung-resident tissue MØs have regulatory functions, and strategies to target these cells might hold promise for prevention or treatment of allergic asthma. PMID:23547101

  13. 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.

  14. 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

    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

  15. 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

  16. Progesterone promotes maternal-fetal tolerance by reducing human maternal T-cell polyfunctionality and inducing a specific cytokine profile.

    PubMed

    Lissauer, David; Eldershaw, Suzy A; Inman, Charlotte F; Coomarasamy, Aravinthan; Moss, Paul A H; Kilby, Mark D

    2015-10-01

    Progesterone is a steroid hormone essential for the maintenance of human pregnancy, and its actions are thought to include promoting maternal immune tolerance of the semiallogenic fetus. We report that exposure of maternal T cells to progesterone at physiological doses induced a unique skewing of the cytokine production profile of CD4(+) and CD8(+) T cells, with reductions not only in potentially deleterious IFN-γ and TNF-α production but also in IL-10 and IL-5. Conversely, production of IL-4 was increased. Maternal T cells also became less polyfunctional, focussing cytokine production toward profiles including IL-4. This was accompanied by reduced T-cell proliferation. Using fetal and viral antigen-specific CD8(+) T-cell clones, we confirmed that this as a direct, nonantigen-specific effect. Yet human T cells lacked conventional nuclear progesterone receptors, implicating a membrane progesterone receptor. CD4(+) and CD8(+) T cells responded to progesterone in a dose-dependent manner, with subtle effects at concentrations comparable to those in maternal blood, but profound effects at concentrations similar to those at the maternal-fetal interface. This characterization of how progesterone modulates T-cell function is important in understanding the normal biology of pregnancy and informing the rational use of progesterone therapy in pregnancies at risk of fetal loss.

  17. Activation of Notch1 promotes development of human CD8(+) single positive T cells in humanized mice.

    PubMed

    Haji, Yoichi; Suzuki, Makiko; Moriya, Kunihiko; So, Takanori; Hozumi, Katsuto; Mizuma, Masamichi; Unno, Michiaki; Ishii, Naoto

    2014-05-02

    Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ(null) (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8(+) single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8(+) SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.

  18. Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma.

    PubMed

    Navarro, Severine; Pickering, Darren A; Ferreira, Ivana B; Jones, Linda; Ryan, Stephanie; Troy, Sally; Leech, Andrew; Hotez, Peter J; Zhan, Bin; Laha, Thewarach; Prentice, Roger; Sparwasser, Tim; Croese, John; Engwerda, Christian R; Upham, John W; Julia, Valerie; Giacomin, Paul R; Loukas, Alex

    2016-10-26

    In the developed world, declining prevalence of some parasitic infections correlates with increased incidence of allergic and autoimmune disorders. Moreover, experimental human infection with some parasitic worms confers protection against inflammatory diseases in phase 2 clinical trials. Parasitic worms manipulate the immune system by secreting immunoregulatory molecules that offer promise as a novel therapeutic modality for inflammatory diseases. We identify a protein secreted by hookworms, anti-inflammatory protein-2 (AIP-2), that suppressed airway inflammation in a mouse model of asthma, reduced expression of costimulatory markers on human dendritic cells (DCs), and suppressed proliferation ex vivo of T cells from human subjects with house dust mite allergy. In mice, AIP-2 was primarily captured by mesenteric CD103(+) DCs and suppression of airway inflammation was dependent on both DCs and Foxp3(+) regulatory T cells (Tregs) that originated in the mesenteric lymph nodes (MLNs) and accumulated in distant mucosal sites. Transplantation of MLNs from AIP-2-treated mice into naïve hosts revealed a lymphoid tissue conditioning that promoted Treg induction and long-term maintenance. Our findings indicate that recombinant AIP-2 could serve as a novel curative therapeutic for allergic asthma and potentially other inflammatory diseases. Copyright © 2016, American Association for the Advancement of Science.

  19. A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia.

    PubMed

    Herranz, Daniel; Ambesi-Impiombato, Alberto; Palomero, Teresa; Schnell, Stephanie A; Belver, Laura; Wendorff, Agnieszka A; Xu, Luyao; Castillo-Martin, Mireia; Llobet-Navás, David; Cordon-Cardo, Carlos; Clappier, Emmanuelle; Soulier, Jean; Ferrando, Adolfo A

    2014-10-01

    Efforts to identify and annotate cancer driver genetic lesions have been focused primarily on the analysis of protein-coding genes; however, most genetic abnormalities found in human cancer are located in intergenic regions. Here we identify a new long range-acting MYC enhancer controlled by NOTCH1 that is targeted by recurrent chromosomal duplications in human T cell acute lymphoblastic leukemia (T-ALL). This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site, interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays. Moreover, analysis of N-Me knockout mice demonstrates a selective and essential role of this regulatory element during thymocyte development and in NOTCH1-induced T-ALL. Together these results identify N-Me as a long-range oncogenic enhancer implicated directly in the pathogenesis of human leukemia and highlight the importance of the NOTCH1-MYC regulatory axis in T cell transformation and as a therapeutic target in T-ALL.

  20. MicroRNA-26a Promotes Regulatory T cells and Suppresses Autoimmune Diabetes in Mice.

    PubMed

    Ma, Hui; Zhang, Shoutao; Shi, Doufei; Mao, Yanhua; Cui, Jianguo

    2016-02-01

    Type-1 diabetes (TID) is an autoimmune disease in which the body's own immune cells attack islet β cells, the cells in the pancreas that produce and release the hormone insulin. Mir-26a has been reported to play functions in cellular differentiation, cell growth, cell apoptosis, and metastasis. However, the role of microRNA-26a (Mir-26a) in autoimmune TID has never been investigated. In our current study, we found that pre-Mir-26a (LV-26a)-treated mice had significantly longer normoglycemic time and lower frequency of autoreactive IFN-γ-producing CD4(+) cells compared with an empty lentiviral vector (LV-Con)-treated non-obese diabetic (NOD) mice. Mir-26a suppresses autoreactive T cells and expands Tregs in vivo and in vitro. Furthermore, in our adoptive transfer study, the groups receiving whole splenocytes and CD25-depleted splenocytes from LV-Con-treated diabetic NOD mice develop diabetes at 3 to 4 weeks of age. In comparison, mice injected with undepleted splenocytes obtained from LV-26a-treated reversal NOD mice develop diabetes after 6-8 weeks. And depletion of CD25(+) cells in the splenocytes of reversed mice abrogates the delay in diabetes onset. In conclusion, Mir-26a suppresses autoimmune diabetes in NOD mice in part through promoted regulatory T cells (Tregs) expression.

  1. c-Myc-Induced Survivin Is Essential for Promoting the Notch-Dependent T Cell Differentiation from Hematopoietic Stem Cells

    PubMed Central

    Haque, Rizwanul; Song, Jianyong; Haque, Mohammad; Lei, Fengyang; Sandhu, Praneet; Ni, Bing; Zheng, Songguo; Fang, Deyu; Yang, Jin-Ming; Song, Jianxun

    2017-01-01

    Notch is indispensable for T cell lineage commitment, and is needed for thymocyte differentiation at early phases. During early stages of T cell development, active Notch prevents other lineage potentials including B cell lineage and myeloid cell (e.g., dendritic cell) lineage. Nevertheless, the precise intracellular signaling pathways by which Notch promotes T cell differentiation remain unclear. Here we report that the transcription factor c-Myc is a key mediator of the Notch signaling–regulated T cell differentiation. In a well-established in vitro differentiation model of T lymphocytes from hematopoietic stem cells, we showed that Notch1 and 4 directly promoted c-Myc expression; dominant-negative (DN) c-Myc inhibited early T cell differentiation. Moreover, the c-Myc expression activated by Notch signaling increased the expression of survivin, an inhibitor of apoptosis (IAP) protein. We further demonstrated that over-expression of c-Myc increased the abundance of survivin and the T cell differentiation thereof, whereas dn c-Myc reduced survivin levels and concomitantly retarded the differentiation. The c-Myc–dependent survivin induction is functionally germane, because Notch-dependent T cell differentiation was canceled by the depletion of survivin. These results identify both c-Myc and survivin as important mediators of the Notch signaling–regulated differentiation of T lymphocytes from hematopoietic stem cells. PMID:28272325

  2. 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

  3. 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.

  4. Retinoic Acid Can Exacerbate T Cell Intrinsic TLR2 Activation to Promote Tolerance

    PubMed Central

    Nguyen, Vivien; Pearson, Kandyce; Kim, Jee-Hyun; Kamdar, Karishma; DePaolo, R. William

    2015-01-01

    The contribution of vitamin A to immune health has been well established. However, recent evidence indicates that its active metabolite, retinoic acid (RA), has the ability to promote both tolerogenic and inflammatory responses. While the outcome of RA-mediated immunity is dependent upon the immunological status of the tissue, the contribution of specific innate signals influencing this response have yet to be delineated. Here, we found that treatment with RA can dampen inflammation during intestinal injury. Importantly, we report a novel and unexpected requirement for TLR2 in RA-mediated suppression. Our data demonstrate that RA treatment enhances TLR2-dependent IL-10 production from T cells and this, in turn, potentiates T regulatory cell (TREG) generation without the need for activation of antigen presenting cells. These data also suggest that combinatorial therapy using RA and TLR2 ligands may be advantageous in the design of therapies to treat autoimmune or inflammatory disease. PMID:25826367

  5. Prolonged antigen presentation by immune complex-binding dendritic cells programs the proliferative capacity of memory CD8 T cells.

    PubMed

    León, Beatriz; Ballesteros-Tato, André; Randall, Troy D; Lund, Frances E

    2014-07-28

    The commitment of naive CD8 T cells to effector or memory cell fates can occur after a single day of antigenic stimulation even though virus-derived antigens (Ags) are still presented by DCs long after acute infection is resolved. However, the effects of extended Ag presentation on CD8 T cells are undefined and the mechanisms that regulate prolonged Ag presentation are unknown. We showed that the sustained presentation of two different epitopes from influenza virus by DCs prevented the premature contraction of the primary virus-specific CD8 T cell response. Although prolonged Ag presentation did not alter the number of memory CD8 T cells that developed, it was essential for programming the capacity of these cells to proliferate, produce cytokines, and protect the host after secondary challenge. Importantly, prolonged Ag presentation by DCs was dependent on virus-specific, isotype-switched antibodies (Abs) that facilitated the capture and cross-presentation of viral Ags by FcγR-expressing DCs. Collectively, our results demonstrate that B cells and Abs can regulate the quality and functionality of a subset of antiviral CD8 T cell memory responses and do so by promoting sustained Ag presentation by DCs during the contraction phase of the primary T cell response.

  6. 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

  7. Hepatic CD206-positive macrophages express amphiregulin to promote the immunosuppressive activity of regulatory T cells in HBV infection.

    PubMed

    Dai, Kai; Huang, Ling; Sun, Xiaomei; Yang, Lihua; Gong, Zuojiong

    2015-12-01

    Hepatitis B virus is a major cause of chronic liver inflammation worldwide. Innate and adaptive immune responses work together to restrain or eliminate hepatitis B virus in the liver. Compromised or failed adaptive immune response results in persistent virus replication and spread. How to promote antiviral immunity is a research focus for hepatitis B virus prevention and therapy. In this study, we investigated the role of macrophages in the regulation of antiviral immunity. We found that F4/80(+)CD206(+)CD80(lo/+) macrophages were a particular hepatic macrophage subset that expressed amphiregulin in our mouse hepatitis B virus infection model. CD206(+) macrophage-derived amphiregulin promoted the immunosuppressive activity of intrahepatic regulatory T cells, demonstrated by higher expression of CTLA-4, ICOS, and CD39, as well as stronger inhibition of antiviral function of CD8(+) T cells. Amphiregulin-neutralizing antibody diminished the effect of CD206(+) macrophages on regulatory T cells. In addition, we found that CD206(+) macrophage-derived amphiregulin activated mammalian target of rapamycin signaling in regulatory T cells, and this mammalian target of rapamycin activation was essential for promotion of regulatory T cell activity by CD206(+) macrophages. Adoptive transfer of CD206(+) macrophages into hepatitis B virus-infected mice increased cytoplasmic hepatitis B virus DNA in hepatocytes and also increased serum hepatitis B surface antigen. The antiviral activity of CD8(+) T cells was decreased after macrophage transfer. Therefore, our research indicated that amphiregulin produced by CD206(+) macrophages plays an important role in modulating regulatory T cell function and subsequently restrains the antiviral activity of CD8(+) T cells. Our study offers new insights into the immunomodulation in hepatitis B virus infection.

  8. 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

  9. Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

    PubMed Central

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

    2015-01-01

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

  10. Endothelial Cell Stimulation Overcomes Restriction and Promotes Productive and Latent HIV-1 Infection of Resting CD4+ T Cells

    PubMed Central

    Baker, Jacob J.; Scott, Geoffrey L.; Davis, Yelena P.; Ho, Yen-Yi; Siliciano, Robert F.

    2013-01-01

    Highly active antiretroviral therapy (HAART) is able to suppress human immunodeficiency virus type 1 (HIV-1) to undetectable levels in the majority of patients, but eradication has not been achieved because latent viral reservoirs persist, particularly in resting CD4+ T lymphocytes. It is generally understood that HIV-1 does not efficiently infect resting CD4+ T cells, and latent infection in those cells may arise when infected CD4+ T lymphoblasts return to resting state. In this study, we found that stimulation by endothelial cells can render resting CD4+ T cells permissible for direct HIV infection, including both productive and latent infection. These stimulated T cells remain largely phenotypically unactivated and show a lower death rate than activated T cells, which promotes the survival of infected cells. The stimulation by endothelial cells does not involve interleukin 7 (IL-7), IL-15, CCL19, or CCL21. Endothelial cells line the lymphatic vessels in the lymphoid tissues and have frequent interactions with T cells in vivo. Our study proposes a new mechanism for infection of resting CD4+ T cells in vivo and a new mechanism for latent infection in resting CD4+ T cells. PMID:23824795

  11. Natural killer cell activation enhances immune pathology and promotes chronic infection by limiting CD8+ T-cell immunity.

    PubMed

    Lang, Philipp A; Lang, Karl S; Xu, Haifeng C; Grusdat, Melanie; Parish, Ian A; Recher, Mike; Elford, Alisha R; Dhanji, Salim; Shaabani, Namir; Tran, Charles W; Dissanayake, Dilan; Rahbar, Ramtin; Ghazarian, Magar; Brüstle, Anne; Fine, Jason; Chen, Peter; Weaver, Casey T; Klose, Christoph; Diefenbach, Andreas; Häussinger, Dieter; Carlyle, James R; Kaech, Susan M; Mak, Tak W; Ohashi, Pamela S

    2012-01-24

    Infections with HIV, hepatitis B virus, and hepatitis C virus can turn into chronic infections, which currently affect more than 500 million patients worldwide. It is generally thought that virus-mediated T-cell exhaustion limits T-cell function, thus promoting chronic disease. Here we demonstrate that natural killer (NK) cells have a negative impact on the development of T-cell immunity by using the murine lymphocytic choriomeningitis virus. NK cell-deficient (Nfil3(-/-), E4BP4(-/-)) mice exhibited a higher virus-specific T-cell response. In addition, NK cell depletion caused enhanced T-cell immunity in WT mice, which led to rapid virus control and prevented chronic infection in lymphocytic choriomeningitis virus clone 13- and reduced viral load in DOCILE-infected animals. Further experiments showed that NKG2D triggered regulatory NK cell functions, which were mediated by perforin, and limited T-cell responses. Therefore, we identified an important role of regulatory NK cells in limiting T-cell immunity during virus infection.

  12. Soluble CD83 Inhibits T Cell Activation by Binding to the TLR4/MD-2 Complex on CD14+ Monocytes

    PubMed Central

    Horvatinovich, Joe M.; Grogan, Elizabeth W.; Norris, Marcus; Steinkasserer, Alexander; Lemos, Henrique; Mellor, Andrew L.; Tcherepanova, Irina Y.; Nicolette, Charles A.

    2017-01-01

    The transmembrane protein CD83, expressed on APCs, B cells, and T cells, can be expressed as a soluble form generated by alternative splice variants and/or by shedding. Soluble CD83 (sCD83) was shown to be involved in negatively regulating the immune response. sCD83 inhibits T cell proliferation in vitro, supports allograft survival in vivo, prevents corneal transplant rejection, and attenuates the progression and severity of autoimmune diseases and experimental colitis. Although sCD83 binds to human PBMCs, the specific molecules that bind sCD83 have not been identified. In this article, we identify myeloid differentiation factor-2 (MD-2), the coreceptor within the TLR4/MD-2 receptor complex, as the high-affinity sCD83 binding partner. TLR4/MD-2 mediates proinflammatory signal delivery following recognition of bacterial LPSs. However, altering TLR4 signaling can attenuate the proinflammatory cascade, leading to LPS tolerance. Our data show that binding of sCD83 to MD-2 alters this signaling cascade by rapidly degrading IL-1R–associated kinase-1, leading to induction of the anti-inflammatory mediators IDO, IL-10, and PGE2 in a COX-2–dependent manner. sCD83 inhibited T cell proliferation, blocked IL-2 secretion, and rendered T cells unresponsive to further downstream differentiation signals mediated by IL-2. Therefore, we propose the tolerogenic mechanism of action of sCD83 to be dependent on initial interaction with APCs, altering early cytokine signal pathways and leading to T cell unresponsiveness. PMID:28193829

  13. The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity

    PubMed Central

    Kidani, Yoko; Elsaesser, Heidi; Hock, M Benjamin; Vergnes, Laurent; Williams, Kevin J; Argus, Joseph P; Marbois, Beth N; Komisopoulou, Evangelia; Wilson, Elizabeth B; Osborne, Timothy F; Graeber, Thomas G; Reue, Karen; Brooks, David G; Bensinger, Steven J

    2013-01-01

    Newly activated CD8+ T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals mediating metabolic reprogramming remain poorly defined. Herein, we demonstrate an essential role for sterol regulatory element binding proteins (SREBPs) in the acquisition of effector cell metabolism. Without SREBP signaling, CD8+ T cells are unable to blast, resulting in markedly attenuated clonal expansion during viral infection. Mechanistic studies indicate that SREBPs are essential to meet the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs are dispensable for homeostatic proliferation, indicating a context-specific requirement for SREBPs in effector responses. These studies provide insights into the molecular signals underlying metabolic reprogramming of CD8+ T cells during the transition from quiescence to activation. PMID:23563690

  14. Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia.

    PubMed

    Rahman, Sunniyat; Magnussen, Michael; León, Theresa E; Farah, Nadine; Li, Zhaodong; Abraham, Brian J; Alapi, Krisztina Z; Mitchell, Rachel J; Naughton, Tom; Fielding, Adele K; Pizzey, Arnold; Bustraan, Sophia; Allen, Christopher; Popa, Teodora; Pike-Overzet, Karin; Garcia-Perez, Laura; Gale, Rosemary E; Linch, David C; Staal, Frank J T; Young, Richard A; Look, A Thomas; Mansour, Marc R

    2017-03-07

    Somatic mutations within non-coding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines, in addition to 3.7% (6/160) of pediatric and 5.5% (9/163) of adult T-ALL patient samples. The majority of indels harbour putative de novo MYB, ETS1 or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provide important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy induced T-ALL, suggesting that such events occur at preferential sites in the non-coding genome.

  15. Changes in chromatin accessibility across the GM-CSF promoter upon T cell activation are dependent on nuclear factor kappaB proteins.

    PubMed

    Holloway, Adele F; Rao, Sudha; Chen, Xinxin; Shannon, M Frances

    2003-02-17

    Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a key cytokine in myelopoiesis and aberrant expression is associated with chronic inflammatory disease and myeloid leukemias. This aberrant expression is often associated with constitutive nuclear factor (NF)-kappaB activation. To investigate the relationship between NF-kappaB and GM-CSF transcription in a chromatin context, we analyzed the chromatin structure of the GM-CSF gene in T cells and the role of NF-kappaB proteins in chromatin remodeling. We show here that chromatin remodeling occurs across a region of the GM-CSF gene between -174 and +24 upon T cell activation, suggesting that remodeling is limited to a single nucleosome encompassing the proximal promoter. Nuclear NF-kappaB levels appear to play a critical role in this process. In addition, using an immobilized template assay we found that the ATPase component of the SWI/SNF chromatin remodeling complex, brg1, is recruited to the GM-CSF proximal promoter in an NF-kappaB-dependent manner in vitro. These results suggest that chromatin remodeling across the GM-CSF promoter in T cells is a result of recruitment of SWI/SNF type remodeling complexes by NF-kappaB proteins binding to the CD28 response region of the promoter.

  16. 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.

  17. Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis.

    PubMed

    Wehr, Alexander; Baeck, Christer; Heymann, Felix; Niemietz, Patricia Maria; Hammerich, Linda; Martin, Christian; Zimmermann, Henning W; Pack, Oliver; Gassler, Nikolaus; Hittatiya, Kanishka; Ludwig, Andreas; Luedde, Tom; Trautwein, Christian; Tacke, Frank

    2013-05-15

    Chronic liver injury characteristically results in hepatic inflammation, which represents a prerequisite for organ fibrosis. Although NKT cells are abundantly present in liver and involved in hepatic inflammation, molecular mechanisms of their recruitment in liver fibrosis remained elusive. We hypothesized that chemokine receptor CXCR6 and its ligand CXCL16 control NKT cell migration and functionality in liver fibrosis. In patients with chronic liver diseases (n = 58), CXCR6 and CXCL16 expression was intrahepatically upregulated compared with controls. In murine liver, Cxcl16 was strongly expressed by endothelium and macrophages, whereas lymphocyte populations (NKT, NK, CD4 T, CD8 T cells) expressed CXCR6. Intravital two-photon microscopy imaging of Cxcr6(+/gfp) and Cxcr6(gfp/gfp) mice and chemotaxis studies in vitro revealed that CXCR6 specifically controls hepatic NKT cell accumulation during the early response upon experimental liver damage. Hepatic invariant NKT cells expressed distinct proinflammatory cytokines including IFN-γ and IL-4 upon injury. CXCR6-deficient mice were protected from liver fibrosis progression in two independent experimental models. Macrophage infiltration and protein levels of inflammatory cytokines IFN-γ, TNF-α, and IL-4 were also reduced in fibrotic livers of Cxcr6(-/-) mice, corroborating that hepatic NKT cells provide essential cytokine signals perpetuating hepatic inflammation and fibrogenesis. Adoptive transfer of NKT cells, but not CD4 T cells, isolated from wild type livers restored hepatic fibrosis in Cxcr6(-/-) mice upon experimental steatohepatitis. Our results demonstrate that hepatic NKT cells accumulate CXCR6-dependent early upon injury, thereby accentuating the inflammatory response in the liver and promoting hepatic fibrogenesis. Interfering with CXCR6/CXCL16 might therefore bear therapeutic potential in liver fibrosis.

  18. Notch1 promotes survival of E2A-deficient T cell lymphomas through pre-T cell receptor-dependent and -independent mechanisms.

    PubMed

    Reschly, Erica J; Spaulding, Christina; Vilimas, Tomas; Graham, W Vallen; Brumbaugh, Rachel L; Aifantis, Iannis; Pear, Warren S; Kee, Barbara L

    2006-05-15

    Loss of E2A transcription factor activity or activation of the intracellular form of Notch1 (ICN) leads to the development of leukemia or lymphoma in humans or mice, respectively. Current models propose that ICN functions by suppressing E2A through a pre-T cell receptor (TCR)-dependent mechanism. Here we show that lymphomas arising in E2A(-/-) mice require the activation of Notch1 for their survival and have accumulated mutations in, or near, the Notch1 PEST domain, resulting in increased stability and signaling. In contrast, lymphomas arising in p53(-/-) mice show the activation of Notch1, but no mutations were identified in ICN. The requirement for Notch1 signaling in E2A(-/-) lymphomas cannot be overcome by ectopic expression of pTalpha; however, pTalpha is required for optimal survival and expansion of these cells. Our findings indicate that the activation of Notch1 is an important "second hit" for the transformation of E2A(-/-) T cell lymphomas and that Notch1 promotes survival through pre-TCR-dependent and -independent mechanisms.

  19. Topical treatment of all-trans retinoic acid inhibits murine melanoma partly by promoting CD8(+) T-cell immunity.

    PubMed

    Yin, Wei; Song, Yan; Liu, Qing; Wu, Yunyun; He, Rui

    2017-10-01

    All-trans retinoic acid (atRA), the main biologically active metabolite of vitamin A, has been implicated in immunoregulation and anti-cancer. A recent finding that vitamin A could decrease the risk of melanoma in humans indicates the beneficial role of atRA in melanoma. However, it remains unknown whether topical application of atRA could inhibit melanoma growth by influencing tumour immunity. We demonstrate topical application of tretinoin ointment (atRA as the active ingredient) effectively inhibited B16F10 melanoma growth. This is accompanied by markedly enhanced CD8(+) T-cell responses, as evidenced by significantly increased proportions of effector CD8(+) T cells expressing granzyme B, tumour necrosis factor-α, or interferon-γ, and Ki67(+) proliferating CD8(+) T cells in atRA-treated tumours compared with vaseline controls. Furthermore, topical atRA treatment promoted the differentiation of effector CD8(+) T cells in draining lymph nodes (DLN) of tumour-bearing mice. Interestingly, atRA did not affect tumoral CD4(+) T-cell response, and even inhibited the differentiation of interferon-γ-expressing T helper type 1 cells in DLN. Importantly, we demonstrated that the tumour-inhibitory effect of atRA was partly dependent on CD8(+) T cells, as CD8(+) T-cell depletion restored tumour volumes in atRA-treated mice, which, however, was still significantly smaller than those in vaseline-treated mice. Finally, we demonstrated that atRA up-regulated MHCI expression in B16F10 cells, and DLN cells from tumour-bearing mice had a significantly higher killing rate when culturing with atRA-treated B16F10 cells. Hence, our study demonstrates that topical atRA treatment effectively inhibits melanoma growth partly by promoting the differentiation and the cytotoxic function of effector CD8(+) T cells. © 2017 John Wiley & Sons Ltd.

  20. HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4+ T Cell Responses

    PubMed Central

    Wang, Mingjun; Larsen, Mette V.; Nielsen, Morten; Harndahl, Mikkel; Justesen, Sune; Dziegiel, Morten H.; Buus, Søren; Tang, Sheila T.; Lund, Ole; Claesson, Mogens H.

    2010-01-01

    Background Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. Methodology/Principal Findings In the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNγ ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4+ or CD8+ T cells prior to the ELISPOT culture revealed that effectors are either CD4+ (the majority of reactivities) or CD8+ T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4+ T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. Conclusions/Significance HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4+ T cell responses restricted by HLA-II molecules. PMID:20479886

  1. Promoter DNA Methylation Pattern Identifies Prognostic Subgroups in Childhood T-Cell Acute Lymphoblastic Leukemia

    PubMed Central

    Borssén, Magnus; Palmqvist, Lars; Karrman, Kristina; Abrahamsson, Jonas; Behrendtz, Mikael; Heldrup, Jesper; Forestier, Erik; Roos, Göran; Degerman, Sofie

    2013-01-01

    Background Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in T-ALL is hitherto undecided. Design and Methods Genome wide promoter DNA methylation analysis was performed in pediatric T-ALL samples (n = 43) using arrays covering >27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary T-ALL group not tested for methylation (n = 32). Results Based on CpG island methylator phenotype (CIMP), T-ALL samples were subgrouped as CIMP+ (high methylation) and CIMP− (low methylation). CIMP− T-ALL patients had significantly worse overall and event free survival (p = 0.02 and p = 0.001, respectively) compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. Conclusions We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric T-ALL. PMID:23762353

  2. Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells.

    PubMed

    Mendoza, Alejandra; Fang, Victoria; Chen, Cynthia; Serasinghe, Madhavika; Verma, Akanksha; Muller, James; Chaluvadi, V Sai; Dustin, Michael L; Hla, Timothy; Elemento, Olivier; Chipuk, Jerry E; Schwab, Susan R

    2017-06-01

    Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P1 receptor (S1P1R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P1R on T cells, and that the requirement for S1P1R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.

  3. 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).

  4. 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.

  5. Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations.

    PubMed

    Peters, Jorieke H; Tjabringa, Geuranne S; Fasse, Esther; de Oliveira, Vivian L; Schalkwijk, Joost; Koenen, Hans J P M; Joosten, Irma

    2013-01-01

    Both keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response. We investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production. A newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry. Our data show that upon co-culture with CD4(pos) or CD8(pos) T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4(pos) and CD8(pos) T-cell populations toward an IL-17(pos) CCR6(pos) RORγt(pos) phenotype in a cell-cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17(pos) cells. We provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases. Copyright © 2012. Published by Elsevier Ireland Ltd.

  6. 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

  7. DC-SIGN promotes Japanese encephalitis virus transmission from dendritic cells to T cells via virological synapses.

    PubMed

    Wang, Ping; Li, Mei; Lu, Wei; Zhang, Di; Hu, Qinxue; Liu, Yalan

    2017-08-31

    Skin-resident dendritic cells (DCs) likely encounter incoming viruses in the first place, and their migration to lymph nodes following virus capture may promote viral replication. However, the molecular mechanisms underlying these processes remain unclear. In the present study, we found that compared to cell-free viruses, DC-bound viruses showed enhanced capture of JEV by T cells. Additionally, JEV infection was increased by co-culturing DCs and T cells. Blocking the C-type lectin receptor DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) with neutralizing antibodies or antagonists blocked JEV transmission to T cells. Live-cell imaging revealed that DCs captured and transferred JEV viral particles to T cells via virological synapses formed at DC-T cell junctions. These findings indicate that DC-SIGN plays an important role in JEV transmission from DCs to T cells and provide insight into how JEV exploits the migratory and antigen-presenting capabilities of DCs to gain access to lymph nodes for dissemination and persistence in the host.

  8. Chemokine receptor CXCR3 deficiency exacerbates murine autoimmune cholangitis by promoting pathogenic CD8(+) T cell activation.

    PubMed

    Ma, Hong-Di; Ma, Wen-Tao; Liu, Qing-Zhi; Zhao, Zhi-Bin; Liu, Mu-Zi-Ying; Tsuneyama, Koichi; Gao, Jin-Ming; Ridgway, William M; Ansari, Aftab A; Gershwin, M Eric; Fei, Yun-Yun; Lian, Zhe-Xiong

    2017-03-01

    CXC Chemokine Receptor 3 (CXCR3) is functionally pleiotropic and not only plays an important role in chemotaxis, but also participates in T cell differentiation and may play a critical role in inducing and maintaining immune tolerance. These observations are particularly critical for autoimmune cholangitis in which CXCR3 positive T cells are found around the portal areas of both humans and mouse models of primary biliary cholangitis (PBC). Herein, we investigated the role of CXCR3 in the pathogenesis of autoimmune cholangitis. We have taken advantage of a unique CXCR3 knockout dnTGFβRII mouse to focus on the role of CXCR3, both by direct observation of its influence on the natural course of disease, as well as through adoptive transfer studies into Rag-/- mice. We report herein that not only do CXCR3 deficient mice develop an exacerbation of autoimmune cholangitis associated with an expanded effector memory T cell number, but also selective adoptive transfer of CXCR3 deficient CD8(+) T cells induces autoimmune cholangitis. In addition, gene microarray analysis of CXCR3 deficient CD8(+) T cells reveal an intense pro-inflammatory profile. Our data suggests that the altered gene profiles induced by CXCR3 deficiency promotes autoimmune cholangitis through pathogenic CD8(+) T cells. These data have significance for human PBC and other autoimmune liver diseases in which therapeutic intervention might be directed to chemokines and/or their receptors.

  9. 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.

  10. 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

  11. 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

    PubMed Central

    Stübig, Thomas; Luetkens, Tim; Hildebrandt, York; Atanackovic, Djordje; Binder, Thomas M. C.; Fehse, Boris; Kröger, Nicolaus

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza. PMID:24757283

  12. Galectin-8 binds specific beta1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells.

    PubMed

    Cárcamo, Claudia; Pardo, Evelyn; Oyanadel, Claudia; Bravo-Zehnder, Marcela; Bull, Paulina; Cáceres, Mónica; Martínez, Jorge; Massardo, Loreto; Jacobelli, Sergio; González, Alfonso; Soza, Andrea

    2006-02-15

    Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a beta-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound alpha1beta1, alpha3beta1 and alpha5beta1 but not alpha2beta1 and alpha4beta1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that alpha5beta1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to alpha1 and alpha5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific beta1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.

  13. Intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate the serum antibody responses induced by dietary antigen.

    PubMed

    Tsuda, Masato; Hosono, Akira; Yanagibashi, Tsutomu; Kihara-Fujioka, Miran; Hachimura, Satoshi; Itoh, Kikuji; Hirayama, Kazuhiro; Takahashi, Kyoko; Kaminogawa, Shuichi

    2010-08-16

    Colonization of the gut by commensal bacteria modulates the induction of oral tolerance and allergy. However, how these intestinal bacteria modulate antigen-specific T cell responses induced by oral antigens remains unclear. In order to investigate this, we used germ-free (GF) ovalbumin (OVA)-specific T cell receptor transgenic (OVA23-3) mice. Conventional (CV) or GF mice were administered an OVA-containing diet. Cytokine production by CD4(+) cells from spleen (SP), mesenteric lymph nodes (MLN) and Peyer's patches (PP) was evaluated by ELISA, as was the peripheral antibody titer. T cell phenotype was assessed by flow cytometry. CD4(+) cells from the SP and MLN of CV and GF mice fed an OVA diet for 3 weeks produced significantly less IL-2 than the corresponding cells from mice receiving a control diet, suggesting that oral tolerance could be induced at the T cell level in the systemic and intestinal immune systems of both bacterial condition of mice. However, we also observed that the T cell hyporesponsiveness induced by dietary antigen was delayed in the systemic immune tissues and was weaker in the intestinal immune tissues of the GF mice. Intestinal MLN and PP CD4(+) T cells from these animals also produced lower levels of IL-10, had less activated/memory type CD45RB(low) cells, and expressed lower levels of CTLA-4 but not Foxp3 compared to their CV counterparts. Furthermore, GF mice produced higher serum levels of OVA-specific antibodies than CV animals. CD40L expression by SP CD4(+) cells from GF mice fed OVA was higher than that of CV mice. These results suggest that intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate serum antibody responses induced by dietary antigens through modulation of the intestinal and systemic T cell phenotype. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Transcriptional regulation of parathyroid hormone-related protein promoter P3 by ETS-1 in adult T-cell leukemia/lymphoma.

    PubMed

    Richard, V; Nadella, M V P; Green, P L; Lairmore, M D; Feuer, G; Foley, J G; Rosol, T J

    2005-07-01

    Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy seen in the majority of adult T-cell leukemia/lymphoma (ATLL) patients with human T-cell lymphotropic virus type-1 (HTLV-1) infection. HTLV-1 Tax has been shown to complex with ETS-1 and SP1 to transactivate the PTHrP P3 promoter. Previously, we established a SCID/bg mouse model of human ATL with RV-ATL cells and showed that PTHrP expression was independent of Tax. In this study, we report an inverse correlation of PTHrP with tax/rex mRNA in multiple HTLV-1-positive cell lines and RV-ATL cells. Stimulation of Jurkat T cells with PMA/ionomycin upregulated the PTHrP P3 promoter by a previously characterized Ets binding site and also induced protein/DNA complex formation identical to that observed in RV-ATL cells. Further, we provide evidence that cotransfection with Ets-1 and constitutively active Mek-1 in HTLV-1-negative transformed T cells with stimulation by PMA/ionomycin not only resulted in a robust induction of PTHrP P3 but also formed a complex with ETS-1/P3 EBS similar to that in ATLL cells. Our data demonstrate that transcriptional regulation of PTHrP in ATLL cells can be controlled by T-cell receptor signaling and the ETS and MAPK ERK pathway in a Tax-independent manner.

  15. Toll like Receptor 2 engagement on CD4(+) T cells promotes TH9 differentiation and function.

    PubMed

    Karim, Ahmad Faisal; Reba, Scott M; Li, Qing; Boom, W Henry; Rojas, Roxana E

    2017-09-01

    We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4(+) T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4(+) T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4(+) T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4(+) T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4(+) T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4(+) T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. 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

  17. 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

  18. RelB-Dependent Stromal Cells Promote T-Cell Leukemogenesis

    PubMed Central

    dos Santos, Nuno R.; Williame, Maryvonne; Gachet, Stéphanie; Cormier, Françoise; Janin, Anne; Weih, Debra; Weih, Falk; Ghysdael, Jacques

    2008-01-01

    Background The Rel/NF-κB transcription factors are often activated in solid or hematological malignancies. In most cases, NF-κB activation is found in malignant cells and results from activation of the canonical NF-κB pathway, leading to RelA and/or c-Rel activation. Recently, NF-κB activity in inflammatory cells infiltrating solid tumors has been shown to contribute to solid tumor initiation and progression. Noncanonical NF-κB activation, which leads to RelB activation, has also been reported in breast carcinoma, prostate cancer, and lymphoid leukemia. Methodology/Principal Findings Here we report a novel role for RelB in stromal cells that promote T-cell leukemogenesis. RelB deficiency delayed leukemia onset in the TEL-JAK2 transgenic mouse model of human T acute lymphoblastic leukemia. Bone marrow chimeric mouse experiments showed that RelB is not required in the hematopoietic compartment. In contrast, RelB plays a role in radio-resistant stromal cells to accelerate leukemia onset and increase disease severity. Conclusions/Significance The present results are the first to uncover a role for RelB in the crosstalk between non-hematopoietic stromal cells and leukemic cells. Thus, besides its previously reported role intrinsic to specific cancer cells, the noncanonical NF-κB pathway may also play a pro-oncogenic role in cancer microenvironmental cells. PMID:18596915

  19. 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.

  20. The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development

    PubMed Central

    Alvarez, John D.; Yasui, Dag H.; Niida, Hiroyuki; Joh, Tadashi; Loh, Dennis Y.; Kohwi-Shigematsu, Terumi

    2000-01-01

    SATB1 is expressed primarily in thymocytes and can act as a transcriptional repressor. SATB1 binds in vivo to the matrix attachment regions (MARs) of DNA, which are implicated in the loop domain organization of chromatin. The role of MAR-binding proteins in specific cell lineages is unknown. We generated SATB1-null mice to determine how SATB1 functions in the T-cell lineage. SATB1-null mice are small in size, have disproportionately small thymi and spleens, and die at 3 weeks of age. At the cellular level, multiple defects in T-cell development were observed. Immature CD3−CD4−CD8− triple negative (TN) thymocytes were greatly reduced in number, and thymocyte development was blocked mainly at the DP stage. The few peripheral CD4+ single positive (SP) cells underwent apoptosis and failed to proliferate in response to activating stimuli. At the molecular level, among 589 genes examined, at least 2% of genes including a proto-oncogene, cytokine receptor genes, and apoptosis-related genes were derepressed at inappropriate stages of T-cell development in SATB1-null mice. For example, IL-2Rα and IL-7Rα genes were ectopically transcribed in CD4+CD8+ double positive (DP) thymocytes. SATB1 appears to orchestrate the temporal and spatial expression of genes during T-cell development, thereby ensuring the proper development of this lineage. Our data provide the first evidence that MAR-binding proteins can act as global regulators of cell function in specific cell lineages. PMID:10716941

  1. T Cells

    MedlinePlus

    ... Cells Share this page Facebook Twitter Email T Cells Definition of MS Myelin Immune-Mediated Disease T ... other immune cells. Three broad categories of T cells Helper T cells augment the immune response by ...

  2. Upregulation of DR3 expression in CD4⁺ T cells promotes secretion of IL-17 in experimental autoimmune uveitis.

    PubMed

    Qin, Tingyu

    2011-01-01

    This study investigated the role of death receptor 3 (DR3) in experimental autoimmune uveitis (EAU). EAU was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein (IRBP) 161-180 emulsified with complete Freund's adjuvant and evaluated with clinical and histopathologic observation. Total protein of draining lymph nodes (DLNs) was extracted from the control, EAU, or recovery phase mice. CD4⁺ T cells were separated from lymphocytes with magnetic-assisted cell sorting. At the same time, some of the CD4⁺ T cells were cultured with or without recombinant TL1A (rTL1A, the DR3 ligand) for three days, and the supernatants were collected for the interleukin-17 (IL-17) test. DR3 mRNA and protein levels in CD4⁺ T cells and the endogenous concentration of TL1A in mice DLNs were assessed with real-time PCR or western blotting. Levels of IL-17 in the supernatants were determined with enzyme-linked immunosorbent assay. Histopathological and clinical data revealed severe intraocular inflammation in the immunized mice. The inflammation reached its peak on day 14 in EAU and had resolved in the recovery phase (weeks 4-5 or more after IRBP immunization). CD4⁺ T cells obtained from EAU (day 7 or 14) had higher levels of DR3 mRNA and protein expression compared with the control group treated with complete Freund's adjuvant alone and the recovery group. However, the DR3 mRNA and protein levels on day 21 in EAU were similar to those observed in the control and recovery groups. The endogenous levels of TL1A were upregulated in EAU, and decreased in the recovery phase mice. Adding rTL1A increased the production of IL-17 by CD4⁺ T cells isolated from mice DLNs. Moreover, the increased IL-17 levels in the culture supernatant of CD4⁺ T cells from EAU were much higher than those from the control and recovery phase mice. However, the effects on promoting IL-17 production in TL1A-stimulated CD4⁺ T cells were similar between the controland

  3. Inhibition of TGF-β1 Signaling Promotes Central Memory T Cell Differentiation

    PubMed Central

    Takai, Shinji; Schlom, Jeffrey; Tucker, Joanne; Tsang, Kwong Y.; Greiner, John W.

    2013-01-01

    The present study affirmed that isolated CD8+ T cells express mRNA and produce TGF-β following cognate peptide recognition. Blockage of endogenous TGF-β with either a TGF-β-blocking antibody or a small molecule inhibitor of TGF-βRI enhances the generation of CD62Lhigh/CD44high central memory CD8+ T cells accompanied with a robust recall response. Interestingly, the augmentation within the central memory T cell pool occurs in lieu of cellular proliferation or activation, but with the expected increase in the ratio of the Eomes/T-bet transcriptional factors. Yet, the signal transduction pathway(s) seems to be non-canonical, independent of SMAD or mTOR signaling. Enhancement of central memory generation by TGF-β blockade is also confirmed in human PBMCs. The findings underscore the role(s) that autocrine TGF-β plays in T cell homeostasis and, in particular, the balance of effector/memory and central/memory T cells. These results may provide a rationale to targeting TGF-β signaling to enhance antigen-specific CD8+ T cell memory against a lethal infection or cancer. PMID:23904158

  4. Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

    PubMed

    von Burg, Nicole; Chappaz, Stéphane; Baerenwaldt, Anne; Horvath, Edit; Bose Dasgupta, Somdeb; Ashok, Devika; Pieters, Jean; Tacchini-Cottier, Fabienne; Rolink, Antonius; Acha-Orbea, Hans; Finke, Daniela

    2014-09-02

    Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

  5. Human mesenchymal stem cells promote survival of T cells in a quiescent state.

    PubMed

    Benvenuto, Federica; Ferrari, Stefania; Gerdoni, Ezio; Gualandi, Francesca; Frassoni, Francesco; Pistoia, Vito; Mancardi, Gianluigi; Uccelli, Antonio

    2007-07-01

    Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response, as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells, we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl-2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall, MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state, providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article.

  6. 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

  7. FK506 BINDING PROTEIN 12 DEFICIENCY IN ENDOTHELIAL AND HEMATOPOIETIC CELLS DECREASES REGULATORY T CELLS AND CAUSES HYPERTENSION

    PubMed Central

    Chiasson, Valorie L.; Talreja, Deepa; Young, Kristina J.; Chatterjee, Piyali; Banes-Berceli, Amy K.; Mitchell, Brett M.

    2011-01-01

    Patients treated with the immunosuppressive drug tacrolimus (FK506), which binds FK506 Binding Protein 12 (FKBP12) then inhibits the calcium-dependent phosphatase calcineurin, exhibit decreased regulatory T cells, endothelial dysfunction, and hypertension; however the mechanisms and whether altered T cell polarization play a role are unknown. Tacrolimus treatment of mice for 1 week dose-dependently decreased CD4+/FoxP3+ (regulatory T cells) and increased CD4+/IL-17+ (T helper 17) cells in the spleen, and caused endothelial dysfunction and hypertension. To determine the mechanisms, we crossed floxed FKBP12 mice with Tie2-Cre mice to generate offspring lacking FKBP12 in endothelial and hematopoietic cells only (FKBP12EC KO). Given FKBP12’s role in inhibiting TGF-β receptor activation, Tie2-Cre-mediated deletion of FKBP12 increased TGF-β receptor activation and SMAD2/3 signaling. FKBP12EC KO mice exhibited increased vascular expression of genes and proteins related to endothelial cell activation and inflammation. Serum levels of the pro-inflammatory cytokines IL-2, IL-6, IFNγ, IL-17a, IL-21, and IL-23 were increased significantly suggesting a Th17 cell-mediated inflammatory state. Flow cytometry studies confirmed this as splenocyte levels of CD4+/IL-17+ cells were increased significantly while CD4+/FoxP3+ cells were decreased in FKBP12EC KO mice. Furthermore, spleens from FKBP12EC KO mice showed increased STAT3 activation, involved in Th17 cell induction, and decreased STAT5 activation, involved in regulatory T cell induction. FKBP12EC KO mice also exhibited endothelial dysfunction and hypertension. These data suggest that tacrolimus, through its activation of TGF-β receptors in endothelial and hematopoietic cells, may cause endothelial dysfunction and hypertension by activating endothelial cells, reducing Tregs, and increasing Th17 cell polarization and inflammation. PMID:21518963

  8. Automatic sequence design of major histocompatibility complex class I binding peptides impairing CD8+ T cell recognition.

    PubMed

    Ogata, Koji; Jaramillo, Alfonso; Cohen, William; Briand, Jean-Paul; Connan, Francine; Choppin, Jeannine; Muller, Sylviane; Wodak, Shoshana J

    2003-01-10

    An automatic protein design procedure was used to compute amino acid sequences of peptides likely to bind the HLA-A2 major histocompatibility complex (MHC) class I allele. The only information used by the procedure are a structural template, a rotamer library, and a well established classical empirical force field. The calculations are performed on six different templates from x-ray structures of HLA-A0201-peptide complexes. Each template consists of the bound peptide backbone and the full atomic coordinates of the MHC protein. Sequences within 2 kcal/mol of the minimum energy sequence are computed for each template, and the sequences from all the templates are combined and ranked by their energies. The five lowest energy peptide sequences and five other low energy sequences re-ranked on the basis of their similarity to peptides known to bind the same MHC allele are chemically synthesized and tested for their ability to bind and form stable complexes with the HLA-A2 molecule. The most efficient binders are also tested for inhibition of the T cell receptor recognition of two known CD8(+) T effectors. Results show that all 10 peptides bind the expected MHC protein. The six strongest binders also form stable HLA-A2-peptide complexes, albeit to varying degrees, and three peptides display significant inhibition of CD8(+) T cell recognition. These results are rationalized in light of our knowledge of the three-dimensional structures of the HLA-A2-peptide and HLA-A2-peptide-T cell receptor complexes.

  9. Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4+ T Cells.

    PubMed

    Li, Chuan; Wang, Hai-Bo; Kuang, Wen-Dong; Ren, Xiao-Xin; Song, Shu-Ting; Zhu, Huan-Zhang; Li, Qiang; Xu, Li-Ran; Guo, Hui-Jun; Wu, Li; Wang, Jian-Hua

    2017-01-01

    HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4(+) T cells. Furthermore, Naf1 knockdown in resting CD4(+) T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy.

  10. Random migration and signal integration promote rapid and robust T cell recruitment.

    PubMed

    Textor, Johannes; Henrickson, Sarah E; Mandl, Judith N; von Andrian, Ulrich H; Westermann, Jürgen; de Boer, Rob J; Beltman, Joost B

    2014-08-01

    To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance.

  11. Random Migration and Signal Integration Promote Rapid and Robust T Cell Recruitment

    PubMed Central

    Textor, Johannes; Henrickson, Sarah E.; Mandl, Judith N.; von Andrian, Ulrich H.; Westermann, Jürgen; de Boer, Rob J.; Beltman, Joost B.

    2014-01-01

    To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance. PMID:25102014

  12. 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

  13. 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.

  14. IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis.

    PubMed

    Coffelt, Seth B; Kersten, Kelly; Doornebal, Chris W; Weiden, Jorieke; Vrijland, Kim; Hau, Cheei-Sing; Verstegen, Niels J M; Ciampricotti, Metamia; Hawinkels, Lukas J A C; Jonkers, Jos; de Visser, Karin E

    2015-06-18

    Metastatic disease remains the primary cause of death for patients with breast cancer. The different steps of the metastatic cascade rely on reciprocal interactions between cancer cells and their microenvironment. Within this local microenvironment and in distant organs, immune cells and their mediators are known to facilitate metastasis formation. However, the precise contribution of tumour-induced systemic inflammation to metastasis and the mechanisms regulating systemic inflammation are poorly understood. Here we show that tumours maximize their chance of metastasizing by evoking a systemic inflammatory cascade in mouse models of spontaneous breast cancer metastasis. We mechanistically demonstrate that interleukin (IL)-1β elicits IL-17 expression from gamma delta (γδ) T cells, resulting in systemic, granulocyte colony-stimulating factor (G-CSF)-dependent expansion and polarization of neutrophils in mice bearing mammary tumours. Tumour-induced neutrophils acquire the ability to suppress cytotoxic T lymphocytes carrying the CD8 antigen, which limit the establishment of metastases. Neutralization of IL-17 or G-CSF and absence of γδ T cells prevents neutrophil accumulation and downregulates the T-cell-suppressive phenotype of neutrophils. Moreover, the absence of γδ T cells or neutrophils profoundly reduces pulmonary and lymph node metastases without influencing primary tumour progression. Our data indicate that targeting this novel cancer-cell-initiated domino effect within the immune system--the γδ T cell/IL-17/neutrophil axis--represents a new strategy to inhibit metastatic disease.

  15. Capicua deficiency induces autoimmunity and promotes follicular helper T cell differentiation via derepression of ETV5

    PubMed Central

    Park, Sungjun; Lee, Seungwon; Lee, Choong-Gu; Park, Guk Yeol; Hong, Hyebeen; Lee, Jeon-Soo; Kim, Young Min; Lee, Sung Bae; Hwang, Daehee; Choi, Youn Soo; Fryer, John D.; Im, Sin-Hyeog; Lee, Seung-Woo; Lee, Yoontae

    2017-01-01

    High-affinity antibody production through the germinal centre (GC) response is a pivotal process in adaptive immunity. Abnormal development of follicular helper T (TFH) cells can induce the GC response to self-antigens, subsequently leading to autoimmunity. Here we show the transcriptional repressor Capicua/CIC maintains peripheral immune tolerance by suppressing aberrant activation of adaptive immunity. CIC deficiency induces excessive development of TFH cells and GC responses in a T-cell-intrinsic manner. ETV5 expression is derepressed in Cic null TFH cells and knockdown of Etv5 suppresses the enhanced TFH cell differentiation in Cic-deficient CD4+ T cells, suggesting that Etv5 is a critical CIC target gene in TFH cell differentiation. Furthermore, we identify Maf as a downstream target of the CIC–ETV5 axis in this process. These data demonstrate that CIC maintains T-cell homeostasis and negatively regulates TFH cell development and autoimmunity. PMID:28855737

  16. The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed β2-Microglobulin through Distinct Binding Sites.

    PubMed

    Merkle, Patrick S; Irving, Melita; Hongjian, Song; Ferber, Mathias; Jørgensen, Thomas J D; Scholten, Kirsten; Luescher, Immanuel; Coukos, George; Zoete, Vincent; Cuendet, Michel A; Michielin, Olivier; Rand, Kasper D

    2017-08-01

    T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR-pMHC complex formation significantly stabilizes distinct CDR loops of the TCR, it does not trigger structural changes in receptor segments remote from the binding interface. Intriguingly, our HDX measurements reveal that the TCR α-constant domain (C- and F-strand) directly interacts with the unbound MHC light chain, β2-microglobulin (β2m). Surface plasmon resonance measurements corroborated a binding event between TCR and β2m with a dissociation constant of 167 ± 20 μM. We propose a model structure for the TCR-β2m complex based on a refined protein-protein docking approach driven by HDX data and information from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of β2m on T-cell cytotoxicity, suggesting an alternate role for β2m binding. Overall, we show that binding of β2m to the TCR occurs in vitro and, as such, not only should be considered in structure-function studies of the TCR-pMHC complex but also could play a hitherto unidentified role in T-cell function in vivo.

  17. 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.

  18. Transforming growth factor-β promotes ‘death by neglect’ in post-activated human T cells

    PubMed Central

    Sillett, H K; Cruickshank, S M; Southgate, J; Trejdosiewicz, L K

    2001-01-01

    Transforming growth factor-β (TGF-β) is central to the wound repair processes that follow local trauma and inflammation. In order to mimic the early events of wound-healing, we studied the effects of TGF-β on mitogen-stimulated peripheral blood cells. TGF-β added at the initiation of mitogenesis did not significantly alter T-cell activation, proliferation, CD45 isoform switching, or activation-induced cell death. By contrast, TGF-β added 72 hr post-activation (or later) enhanced the cumulative increase in apoptotic T cells. TGF-β had no effect on mitogen-induced up-regulation of Fas (CD95) or Fas ligand and did not enhance killing of the Fas-sensitive Jurkat cell line by activated T cells. Furthermore, TGF-β had no direct effect on levels of mRNA for members of the bcl family (bcl-X, bfl-1, bik, bak, bax, bcl-2 and mcl-1). These findings suggest that TGF-β does not directly induce apoptosis via the Fas system or by direct effects on bcl proteins. However, interleukin-2, which can ‘rescue’ lymphocytes from spontaneous apoptosis due to cytokine deprivation, abolished the pro-apoptotic effects of TGF-β on post-activated T cells, thus demonstrating that TGF-β increases the cytokine-dependence of T cells for survival. We propose a novel role for TGF-β in the suppression of inflammation by promoting the elimination of post-activated T cells once the initiating stimulus has been resolved. PMID:11298829

  19. Fas-Fas ligand interactions are essential for the binding to and killing of activated macrophages by gamma delta T cells.

    PubMed

    Dalton, Jane E; Howell, Gareth; Pearson, Jayne; Scott, Phillip; Carding, Simon R

    2004-09-15

    Gammadelta T cells have a direct role in resolving the host immune response to infection by eliminating populations of activated macrophages. Macrophage reactivity resides within the Vgamma1/Vdelta6.3 subset of gammadelta T cells, which have the ability to kill activated macrophages following infection with Listeria monocytogenes (Lm). However, it is not known how gammadelta T cell macrophage cytocidal activity is regulated, or what effector mechanisms gammadelta T cells use to kill activated macrophages. Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta-/- mice were incubated with splenocytes from wild-type and Fas ligand (FasL)-deficient mice (gld), the ability of Vgamma1 T cells to bind macrophages was shown to be dependent upon Fas-FasL interactions. Combinations of anti-TCR and FasL Abs completely abolished binding to and killing of activated macrophages by Vgamma1 T cells. In addition, confocal microscopy showed that Fas and the TCR colocalized on Vgamma1 T cells at points of contact with macrophages. Collectively, these studies identify an accessory or coreceptor-like function for Fas-FasL that is essential for the interaction of Vgamma1 T cells with activated macrophages and their elimination during the resolution stage of pathogen-induced immune responses. Copyright 2004 The American Association of Immunologists, Inc.

  20. 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

  1. Acquired Senescent T-Cell Phenotype Correlates with Clinical Severity in GATA Binding Protein 2-Deficient Patients.

    PubMed

    Ruiz-García, Raquel; Rodríguez-Vigil, Carmen; Marco, Francisco Manuel; Gallego-Bustos, Fernando; Castro-Panete, María José; Diez-Alonso, Laura; Muñoz-Ruiz, Carlos; Ruiz-Contreras, Jesús; Paz-Artal, Estela; González-Granado, Luis Ignacio; Allende, Luis Miguel

    2017-01-01

    GATA binding protein 2 (GATA2) deficiency is a rare disorder of hematopoiesis, lymphatics, and immunity caused by spontaneous or autosomal dominant mutations in the GATA2 gene. Clinical manifestations range from neutropenia, lymphedema, deafness, to severe viral and mycobacterial infections, bone marrow failure, and acute myeloid leukemia. Patients also present with monocytopenia, dendritic cell, B- and natural killer (NK)-cell deficiency. We studied the T-cell and NK-cell compartments of four GATA2-deficient patients to assess if changes in these lymphocyte populations could be correlated with clinical phenotype. Patients with more severe clinical complications demonstrated a senescent T-cell phenotype whereas patients with lower clinical score had undetectable changes relative to controls. In contrast, patients' NK-cells demonstrated an immature/activated phenotype that did not correlate with clinical score, suggesting an intrinsic NK-cell defect. These studies will help us to determine the contribution of T- and NK-cell dysregulation to the clinical phenotype of GATA2 patients, and may help to establish the most accurate therapeutic options for these patients. Asymptomatic patients may be taken into consideration for hematopoietic stem cell transplantation when dysregulation of T-cell and NK-cell compartment is present.

  2. Selective binding of CD4 and CD8 T-cells to antigen presenting cells for enrichment of CMV and HIV specific T-lymphocytes.

    PubMed

    Li Pira, Giuseppina; Ivaldi, Federico; Manca, Fabrizio

    2012-02-28

    Adherent antigen presenting cells (APC) pulsed with protein or peptide antigens were used to capture specific CD4 or CD8 T-cells derived from established T-cell lines or from PBMC of immune subjects based on physiological interaction between TCR and MHC-peptide complex. This method could be applied independently of epitope specificity, HLA restriction alleles, activation markers and secreted cytokines, parameters required by other methods for selection of specific T cells. Non specific T-cells were removed by applying a 1g force that did not affect binding of specific T-lymphocytes. Lymphocyte selection was specific and the average recovery was 36% for CD4 T-cells. CD8 T-cells proved trickier to purify, since solid phase APC were recognized as targets for cytotoxicity. Specificity was comparable to CD4 cells, but the average recovery for CD8 cells was 26%. No residual alloreactivity was detected in expanded T-cells. Frequency and recovery of specific T-cells were comparable to other current technologies, such as generation of T-cell lines and cytokine capture method. Since antigen and IL2 are the only reagents added to the cultures, this physiological procedure can be proposed for selection and expansion of pathogen specific T-cells not only for research purposes, but also for adoptive reconstitution of immunocompromised subjects if performed under GMP conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. 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

  4. Cytomegalovirus-Specific IL-10-Producing CD4+ T Cells Are Governed by Type-I IFN-Induced IL-27 and Promote Virus Persistence

    PubMed Central

    Marsden, Morgan; Stacey, Maria A.; Abdul-Karim, Juneid; Gimeno Brias, Silvia; Costa Bento, Diana; Ghazal, Peter; Weaver, Casey T.; Carlesso, Gianluca; Clare, Simon; Godkin, Andrew; Jones, Gareth W.; Humphreys, Ian R.

    2016-01-01

    CD4+ T cells support host defence against herpesviruses and other viral pathogens. We identified that CD4+ T cells from systemic and mucosal tissues of hosts infected with the β-herpesviridae human cytomegalovirus (HCMV) or murine cytomegalovirus (MCMV) express the regulatory cytokine interleukin (IL)-10. IL-10+CD4+ T cells co-expressed TH1-associated transcription factors and chemokine receptors. Mice lacking T cell-derived IL-10 elicited enhanced antiviral T cell responses and restricted MCMV persistence in salivary glands and secretion in saliva. Thus, IL-10+CD4+ T cells suppress antiviral immune responses against CMV. Expansion of this T-cell population in the periphery was promoted by IL-27 whereas mucosal IL-10+ T cell responses were ICOS-dependent. Infected Il27rα-deficient mice with reduced peripheral IL-10+CD4+ T cell accumulation displayed robust T cell responses and restricted MCMV persistence and shedding. Temporal inhibition experiments revealed that IL-27R signaling during initial infection was required for the suppression of T cell immunity and control of virus shedding during MCMV persistence. IL-27 production was promoted by type-I IFN, suggesting that β-herpesviridae exploit the immune-regulatory properties of this antiviral pathway to establish chronicity. Further, our data reveal that cytokine signaling events during initial infection profoundly influence virus chronicity. PMID:27926930

  5. 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

  6. INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

    PubMed Central

    Andrés-Delgado, Laura; Antón, Olga M.; Bartolini, Francesca; Ruiz-Sáenz, Ana; Correas, Isabel; Gundersen, Gregg G.

    2012-01-01

    T cell antigen receptor–proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor–induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells. PMID:22986496

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

    PubMed

    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

    2015-04-21

    Epigenetic regulation of lineage-specific genes is important for the differentiation and function of T cells. Ten-eleven translocation (Tet) proteins catalyze 5-methylcytosine (5 mC) conversion to 5-hydroxymethylcytosine (5 hmC) to mediate DNA demethylation. However, the roles of Tet proteins in the immune response are unknown. Here, we characterized the genome-wide distribution of 5 hmC in CD4(+) T cells and found that 5 hmC marks putative regulatory elements in signature genes associated with effector cell differentiation. Moreover, Tet2 protein was recruited to 5 hmC-containing regions, dependent on lineage-specific transcription factors. Deletion of Tet2 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.

  8. Tumour-experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands

    PubMed Central

    Domaica, Carolina I; Fuertes, Mercedes B; Rossi, Lucas E; Girart, María V; Ávila, Damián E; Rabinovich, Gabriel A; Zwirner, Norberto W

    2009-01-01

    Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-γ secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co-culture with some melanomas, activated CD4+ T cells promoted degranulation, and NKG2D- and NKp46-dependent IFN-γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4+, CD8+ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. PMID:19498463

  9. 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

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

    PubMed

    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

    2015-11-01

    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.

  11. P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells.

    PubMed

    Figliuolo, Vanessa R; Savio, Luiz Eduardo Baggio; Safya, Hanaa; Nanini, Hayandra; Bernardazzi, Cláudio; Abalo, Alessandra; de Souza, Heitor S P; Kanellopoulos, Jean; Bobé, Pierre; Coutinho, Cláudia M L M; Coutinho-Silva, Robson

    2017-03-07

    P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RB(low). Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.

  12. CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells*

    PubMed Central

    Masuda, Mari; Maruyama, Tomoko; Ohta, Tsutomu; Ito, Akihiko; Hayashi, Tomayoshi; Tsukasaki, Kunihiko; Kamihira, Shimeru; Yamaoka, Shoji; Hoshino, Hiroo; Yoshida, Teruhiko; Watanabe, Toshiki; Stanbridge, Eric J.; Murakami, Yoshinori

    2010-01-01

    CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients. PMID:20215110

  13. 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.

  14. Characterization of a 70-kDa, EBV gp350/220-binding protein on HSB-2 T cells.

    PubMed

    Hedrick, J A; Lao, Z; Lipps, S G; Wang, Y; Todd, S C; Lambris, J D; Tsoukas, C D

    1994-11-15

    EBV binds and infects HSB-2 T cells via a receptor distinct from CD21. To further study this novel EBV receptor, we expressed the first 470 amino acids of the EBV-gp350/220 using the baculovirus expression system. The recombinant gp350/220(1-470) has a m.w. of 95 kDa, reacts with anti-gp350/220 Abs, and binds CD21 in ELISA. Radiolabeled gp350/220(1-470) binds both HSB-2 and Raji cells. The gp350/220(1-470) protein also inhibits EBV binding to both HSB-2 and Raji, detected by flow cytometry. Lysates of HSB-2 cells compete with CD21 for binding to gp350/220(1-470), suggesting that the two receptors bind related epitopes on the recombinant protein. Scatchard analysis reveals that gp350/220(1-470) binds to 34,000 high affinity sites/HSB-2 cell (Kd = 0.92 x 10(-8) M) compared with the 97,000 high affinity sites bound/Raji cell (Kd = 1.78 x 10(-8) M). Utilizing a gp350/220(1-470)-affinity matrix, we identify a 70-kDa (55-kDa nonreduced) protein on the surfaces of 125I-labeled HSB-2 cells. Binding of this protein to the matrix is inhibited by anti-gp350/220 Ab 72A1. In summary, we characterize a novel EBV-binding molecule on HSB-2 cells, compare its reactivity with gp350/220 to that of CD21, and provide evidence of a gp350/220-reactive, 70-kDa protein on the surfaces of HSB-2 cells. In view of previous evidence of HSB-2 infectivity by EBV, we propose that the 70 kDa protein represents the novel EBV receptor.

  15. 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

  16. Changes in T cell subsets identify responders to FcR non-binding anti-CD3 mAb (teplizumab) in patients with Type 1 diabetes

    PubMed Central

    Tooley, James E; Vudattu, Nalini; Choi, Jinmyung; Cotsapas, Chris; Devine, Lesley; Raddassi, Khadir; Ehlers, Mario R; McNamara, James G; Harris, Kristina M; Kanaparthi, Sai; Phippard, Deborah; Herold, Kevan C

    2016-01-01

    The mechanisms whereby immune therapies affect progression of Type 1 diabetes (T1D) are not well understood. Teplizumab, an FcR non-binding anti-CD3 mAb, has shown efficacy in multiple randomized clinical trials. We previously reported an increase in the frequency of circulating CD8+ central memory (CD8CM) T cells in clinical responders, but the generalizability of this finding and the molecular effects of teplizumab on these T cells have not been evaluated. We analyzed data from 2 randomized clinical studies of teplizumab in patients with new and recent onset T1D. At the conclusion of therapy clinical responders showed a significant reduction in circulating CD4+ effector memory (CD4EM) T cells. Afterwards, there was an increase in the frequency and absolute number of CD8CM T cells. In vitro, teplizumab expanded CD8CM T cells by proliferation and conversion of non-CM T cells. Nanostring analysis of gene expression of CD8CM T cells from responders and non-responders vs placebo-treated control subjects identified decreases in expression of genes associated with immune activation and increases in expression of genes associated with T cell differentiation and regulation. We conclude that CD8CM T cells with decreased activation and regulatory gene expression are associated with clinical responses to teplizumab in patients with T1D. PMID:26518356

  17. Schisandrin B inhibits Th1/Th17 differentiation and promotes regulatory T cell expansion in mouse lymphocytes.

    PubMed

    Chen, Zhaoyang; Guo, Min; Song, Guohua; Gao, Jiping; Zhang, Yinhong; Jing, Zhijie; Liu, Tianfu; Dong, Chuan

    2016-06-01

    Schisandrin B (Sch-B), the most abundant active ingredient of the fruit of Schisandra chinensis, has been proposed to have antioxidant, anti-tumor and anti-inflammatory effects. The present study was undertaken to investigate the effect of Sch-B on differentiation of T helper cells (Th). Using mouse splenic lymphocytes stimulated with concanavalin A (Con A) in vitro and ex vivo as inflammation models, we found that Sch-B significantly inhibited secretion of Th1 and Th17 related cytokines, such as IFN-γ and IL-17. In addition, we found that Sch-B suppressed the differentiation of naive CD4+ T cells into Th1 and Th17 cells, while promoted their differentiation into the regulatory T cells (Treg) in vitro. We further found that Sch-B suppressed transcription of Th1-related T-box transcription factor, T-bet, and Th17-related transcription factor, retinoid related orphan receptor gamma t (RORγt), while enhanced transcription of Treg-related transcription factor forkhead box protein 3 (Foxp3) in naive CD4+ T cells under Th cell polarization conditions. Furthermore, the effect of Sch-B on the T cell differentiation was abrogated by heme oxygenase-1 (HO-1) inhibitor zinc protoporphyrin. Taken together, we conclude that Sch-B can modulate differentiation of naïve CD4+ T cells into specific lineages of effector cells, which may have potential benefits for treatment of autoimmune diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

    SciTech Connect

    Carlow, D.A.; Teh, H.S.; Marth, J.

    1995-02-15

    In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4{sup +}8{sup +} thymocytes upon TCR cross-linking. In contrast, expression of Tgtp in peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of {approx}50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation. 51 refs., 6 figs.

  19. IL-21 Promotes Pulmonary Fibrosis through the Induction of Pro-fibrotic CD8+ T Cells

    PubMed Central

    Brodeur, Tia Y.; Robidoux, Tara E.; Weinstein, Jason S.; Craft, Joseph; Swain, Susan L.; Marshak-Rothstein, Ann

    2015-01-01

    Type 2 effector production of IL-13, a demonstrated requirement in models of fibrosis, is routinely ascribed to CD4+ Th2 cells. We now demonstrate a major role for CD8+ T cells in a murine model of sterile lung injury. These pulmonary CD8+ T cells differentiate into IL-13-producing Tc2 and play a major role in a bleomycin-induced model of fibrosis. Differentiation of these Tc2 cells in the lung requires IL-21, and bleomycin treated IL-21- and IL-21R-deficient mice develop inflammation but not fibrosis. Moreover, IL-21R-expressing CD8+ cells are sufficient to reconstitute the fibrotic response in the IL-21R-deficient mice. We further show that the combination of IL-4 and IL-21 skews naïve CD8+ T cells to produce IL-21, which in turn acts in an autocrine manner to support robust IL-13 production. Our data reveal a novel pathway involved in the onset and regulation of pulmonary fibrosis, and identify Tc2 cells as key mediators of fibrogenesis. PMID:26519529

  20. Galectin-8 Ameliorates Murine Autoimmune Ocular Pathology and Promotes a Regulatory T Cell Response

    PubMed Central

    Sampson, James F.; Hasegawa, Eiichi; Mulki, Lama; Suryawanshi, Amol; Jiang, Shuhong; Chen, Wei-Sheng; Rabinovich, Gabriel A.; Connor, Kip M.; Panjwani, Noorjahan

    2015-01-01

    Galectins have emerged as potent immunoregulatory agents that control chronic inflammation through distinct mechanisms. Here, we report that treatment with Galectin-8 (Gal-8), a tandem-repeat member of the galectin family, reduces retinal pathology and prevents photoreceptor cell damage in a murine model of experimental autoimmune uveitis. Gal-8 treatment increased the number of regulatory T cells (Treg) in both the draining lymph node (dLN) and the inflamed retina. Moreover, a greater percentage of Treg cells in the dLN and retina of Gal-8 treated animals expressed the inhibitory coreceptor cytotoxic T lymphocyte antigen (CTLA)-4, the immunosuppressive cytokine IL-10, and the tissue-homing integrin CD103. Treg cells in the retina of Gal-8-treated mice were primarily inducible Treg cells that lack the expression of neuropilin-1. In addition, Gal-8 treatment blunted production of inflammatory cytokines by retinal T helper type (TH) 1 and TH17 cells. The effect of Gal-8 on T cell differentiation and/or function was specific for tissues undergoing an active immune response, as Gal-8 treatment had no effect on T cell populations in the spleen. Given the need for rational therapies for managing human uveitis, Gal-8 emerges as an attractive therapeutic candidate not only for treating retinal autoimmune diseases, but also for other TH1- and TH17-mediated inflammatory disorders. PMID:26126176

  1. Tissue damage-associated "danger signals" influence T-cell responses that promote the progression of preneoplasia to cancer.

    PubMed

    He, Ying; Zha, Jikun; Wang, Yamin; Liu, Wenhua; Yang, Xuanming; Yu, Ping

    2013-01-15

    T-cell responses may be shaped by sterile "danger signals" that are constituted by damage-associated molecular patterns (DAMP). However, whether and what type of adaptive immune responses are triggered in vivo by DAMPs induced by tumor progression are not well characterized. In this study, we report that the production of HMGB1, an established DAMP released by dying cells, was critical for tumor progression in an established mouse model of prostate cancer. HMGB1 was required for the activation and intratumoral accumulation of T cells that expressed cytokine lymphotoxinα(1)β(2) (LT) on their surface. Intriguingly, these tumor-activated T cells recruited macrophages to the lesion and were essential to promote the preneoplasia to invasive carcinoma in an LTβ receptor (LTβR)-dependent manner. Taken together, our findings suggest that the release of HMGB1 as an endogenous danger signal is important for priming an adaptive immune response that promotes malignant progression, with implications for cancer prevention and therapy.

  2. ICOSL expression in human bone marrow-derived mesenchymal stem cells promotes induction of regulatory T cells

    PubMed Central

    Lee, Hyun-Joo; Kim, Si-Na; Jeon, Myung-Shin; Yi, TacGhee; Song, Sun U.

    2017-01-01

    Mesenchymal stem cells (MSCs) can modulate lymphocyte proliferation and function. One of the immunomodulatory functions of MSCs involves CD4+CD25+FoxP3+ regulatory T cells (Tregs), which negatively regulate inflammatory responses. MSC-mediated Treg induction is supposed to be regulated by mechanisms requiring both soluble and cell contact-dependent factors. Although the involvement of soluble factors has been revealed, the contact-dependent mechanisms in MSC-mediated Treg induction remain unclear. We attempted to identify molecule(s) other than secreted factors that are responsible for MSC-mediated Treg induction and to uncover the underlying mechanisms. Under in vitro Treg-inducing conditions, ICOSL expression in MSCs coincided with Treg induction in co-cultures of MSCs with CD4+ T cells. When cultured in a transwell plate, MSCs failed to induce Tregs. Neutralization or knockdown of ICOSL significantly reduced Tregs and their IL-10 release. ICOSL overexpression in MSCs promoted induction of functional Tregs. ICOSL-ICOS signaling promoted Treg differentiation from CD4+ T cells through activation of the phosphoinositide 3-kinase-Akt pathway. MSCs primed with Interleukin-1β significantly induced Tregs through ICOSL upregulation. We demonstrated that the Treg-inducing activity of MSCs is proportionate to their basal ICOSL expression. This study provides evidence that ICOSL expression in human MSCs plays an important role in contact-dependent regulation of MSC-mediated Treg induction. PMID:28290526

  3. Deletion of calcineurin and myocyte enhancer factor 2 (MEF2) binding domain of Cabin1 results in enhanced cytokine gene expression in T cells.

    PubMed

    Esau, C; Boes, M; Youn, H D; Tatterson, L; Liu, J O; Chen, J

    2001-11-19

    Cabin1 binds calcineurin and myocyte enhancer factor 2 (MEF2) through its COOH-terminal region. In cell lines, these interactions were shown to inhibit calcineurin activity after T cell receptor (TCR) signaling and transcriptional activation of Nur77 by MEF2. The role of these interactions under physiological conditions was investigated using a mutant mouse strain that expresses a truncated Cabin1 lacking the COOH-terminal calcineurin and MEF2 binding domains. T and B cell development and thymocyte apoptosis were normal in mutant mice. In response to anti-CD3 stimulation, however, mutant T cells expressed significantly higher levels of interleukin (IL)-2, IL-4, IL-9, IL-13, and interferon gamma than wild-type T cells. The enhanced cytokine gene expression was not associated with change in nuclear factor of activated T cells (NF-AT)c or NF-ATp nuclear translocation but was preceded by the induction of a phosphorylated form of MEF2D in mutant T cells. Consistent with the enhanced cytokine expression, mutant mice had elevated levels of serum immunoglobulin (Ig)G1, IgG2b, and IgE and produced more IgG1 in response to a T cell-dependent antigen. These findings suggest that the calcineurin and MEF2 binding domain of Cabin1 is dispensable for thymocyte development and apoptosis, but is required for proper regulation of T cell cytokine expression probably through modulation of MEF2 activity.

  4. IFN-γ promotes transendothelial migration of CD4(+) T cells across the blood-brain barrier.

    PubMed

    Sonar, Sandip Ashok; Shaikh, Shagufta; Joshi, Nupura; Atre, Ashwini N; Lal, Girdhari

    2017-07-06

    Transendothelial migration (TEM) of Th1 and Th17 cells across the blood-brain barrier (BBB) has a critical role in the development of experimental autoimmune encephalomyelitis (EAE). How cytokines produced by inflammatory Th1 and Th17 cells damage the endothelial BBB and promote transendothelial migration of immune cells into the central nervous system (CNS) during autoimmunity is not understood. We therefore investigated the effect of various cytokines on brain endothelial cells. Among the various cytokines tested, such as Th1 (IFN-γ, IL-1α, IL-1β, TNF-α, IL-12), Th2 (IL-3, IL-4, IL-6 and IL-13), Th17 (IL-17A, IL-17F, IL-21, IL-22, IL-23, GM-CSF) and Treg-specific cytokines (IL-10 and TGF-β), IFN-γ predominantly showed increased expression of ICAM-1, VCAM-1, MAdCAM-1, H2-K(b) and I-A(b) molecules on brain endothelial cells. Furthermore, IFN-γ induced transendothelial migration of CD4(+) T cells from the apical (luminal side) to the basal side (abluminal side) of the endothelial monolayer to chemokine CCL21 in a STAT-1-dependent manner. IFN-γ also favored the transcellular route of TEM of CD4(+) T cells. Multicolor immunofluorescence and confocal microscopic analysis showed that IFN-γ induced relocalization of ICAM-1, PECAM-1, ZO-1 and VE-cadherin in the endothelial cells, which affected the migration of CD4(+) T cells. These findings reveal that the IFN-γ produced during inflammation could contribute towards disrupting the BBB and promoting TEM of CD4(+) T cells. Our findings also indicate that strategies that interfere with the activation of CNS endothelial cells may help in controlling neuroinflammation and autoimmunity.Immunology and Cell Biology advance online publication, 1 August 2017; doi:10.1038/icb.2017.56.

  5. Vaccines combined with immune checkpoint antibodies promote cytotoxic T cell activity and tumor eradication

    PubMed Central

    Ali, Omar A.; Lewin, Sarah A.; Dranoff, Glenn; Mooney, David J.

    2015-01-01

    We demonstrate that a poly(lactide-co-glycolide) (PLG) cancer vaccine can be used in combination with immune checkpoint antibodies, anti–CTLA-4 or anti–PD-1, to enhance cytotoxic T cell (CTL) activity and induce the regression of solid B16 tumors in mice. Combination therapy obviated the need for vaccine boosting and significantly skewed intratumoral reactions toward CTL activity, resulting in the regression of B16 tumors up to 50mm2 in size and 75% survival rates. These data suggests that combining material-based cancer vaccines with checkpoint antibodies has the potential to mediate tumor regression in humans. PMID:26669718

  6. The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells.

    PubMed

    Richard, Arianne C; Tan, Cuiyan; Hawley, Eric T; Gomez-Rodriguez, Julio; Goswami, Ritobrata; Yang, Xiang-Ping; Cruz, Anthony C; Penumetcha, Pallavi; Hayes, Erika T; Pelletier, Martin; Gabay, Odile; Walsh, Matthew; Ferdinand, John R; Keane-Myers, Andrea; Choi, Yongwon; O'Shea, John J; Al-Shamkhani, Aymen; Kaplan, Mark H; Gery, Igal; Siegel, Richard M; Meylan, Françoise

    2015-04-15

    The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9. Copyright © 2015 by The American Association of Immunologists, Inc.

  7. The TNF-family ligand TL1A and its receptor DR3 promote T-cell mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9 producing T cells1

    PubMed Central

    Richard, Arianne C.; Tan, Cuiyan; Hawley, Eric T.; Gomez-Rodriguez, Julio; Goswami, Ritobrata; Yang, Xiang-ping; Cruz, Anthony C.; Penumetcha, Pallavi; Hayes, Erika T.; Pelletier, Martin; Gabay, Odile; Walsh, Matthew; Ferdinand, John R.; Keane-Myers, Andrea; Choi, Yongwon; O'Shea, John J.; Al-Shamkhani, Aymen; Kaplan, Mark H.; Gery, Igal; Siegel, Richard M.; Meylan, Françoise

    2015-01-01

    The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation, and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3 deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. Here we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9. PMID:25786692

  8. Nude mice produce a T cell-derived antigen-binding factor that mediates the early component of delayed-type hypersensitivity.

    PubMed

    Herzog, W R; Meade, R; Pettinicchi, A; Ptak, W; Askenase, P W

    1989-03-15

    The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is caused by the sequential action of two different T cells. An early-acting, DTH-initiating T cell produces an Ag-specific T cell factor, that is analogous to IgE antibody and initiates DTH by sensitizing the local tissues for release of the vasoactive amine serotonin. In picryl chloride or oxazolone contact sensitivity, this T cell factor is Ag-specific, but MHC unrestricted. We, therefore, hypothesized that DTH-initiating T cells are primitive T cells with Ag receptors that can bind Ag without MHC restriction. In order to characterize the origin of this DTH-initiating T cell and the conditions that are necessary for its development, we contact-sensitized various strains of immunodeficient mice. Surprisingly, we found that the early phase of DTH was present in athymic nude mice. In contrast, the early component of DTH was absent in mice with severe combined immunodeficiency. These mice lack T and B cells, but have NK cells. These findings suggested that the early component of DTH was not caused by NK cells, and was caused by cells belonging to a lineage from a rearranging gene family. The early component of DTH in nude mice was Ag specific, was caused by MHC unrestricted Thy-1+ T cells, and was mediated by Ag-binding, Ag-specific T cell factors. We found that DTH-initiating, T cell-derived, Ag-binding molecules from nude mice and normal CBA/J mice had the same functional properties. The early component of DTH was elicited in two different systems (contact sensitivity and SRBC-specific DTH) in two strains of nude mice (BALB/c athymic nudes and CByB6F1/J-nu) from two different suppliers, but not in BALB/c and athymic nudes from a third supplier. From these findings we concluded that DTH-initiating T cells, which produce IgE-like Ag-specific T cell factors, are present in some strains of athymic nude mice and thus are relatively thymic independent T cells.

  9. CBAP promotes thymocyte negative selection by facilitating T-cell receptor proximal signaling

    PubMed Central

    Ho, K-C; Chiang, Y-J; Lai, A C-Y; Liao, N-S; Chang, Y-J; Yang-Yen, H-F; Yen, J J-Y

    2014-01-01

    T-cell receptor (TCR)-transduced signaling is critical to thymocyte development at the CD4/CD8 double-positive stage, but the molecules involved in this process are not yet fully characterized. We previously demonstrated that GM-CSF/IL-3/IL-5 receptor common β-chain-associated protein (CBAP) modulates ZAP70-mediated T-cell migration and adhesion. On the basis of the high expression of CBAP during thymocyte development, we investigated the function of CBAP in thymocyte development using a CBAP knockout mouse. CBAP-deficient mice showed normal early thymocyte development and positive selection. In contrast, several negative selection models (including TCR transgene, superantigen staphylococcal enterotoxin B, and anti-CD3 antibody treatment) revealed an attenuation of TCR-induced thymocyte deletion in CBAP knockout mice. This phenotype correlated with a reduced accumulation of BIM upon TCR crosslinking in CBAP-deficient thymocytes. Loss of CBAP led to reduced TCR-induced phosphorylation of proteins involved in both proximal and distal signaling events, including ZAP70, LAT, PLCγ1, and JNK1/2. Moreover, TCR-induced association of LAT signalosome components was reduced in CBAP-deficient thymocytes. Our data demonstrate that CBAP is a novel component in the TCR signaling pathway and modulates thymocyte apoptosis during negative selection. PMID:25393474

  10. High Density Lipoproteins Selectively Promote the Survival of Human Regulatory T-cells.

    PubMed

    Rueda, Cesar M; Rodriguez-Perea, Ana Lucia; Moreno-Fernandez, Maria; Jackson, Courtney M; Melchior, John T; Davidson, W Sean; Chougnet, Claire A

    2017-04-04

    High-density lipoproteins (HDL) appear to affect regulatory T cell (Treg) homeostasis, as suggested by the increased Treg counts in HDL-treated mice and by the positive correlation between Treg frequency and HDL-C levels in statin-treated healthy adults. However, the underlying mechanisms remain unclear. Herein, we show that HDL, not LDL, significantly decreased the apoptosis of human Treg in vitro, whereas they did not alter naive or memory CD4+ T-cell survival. Similarly, oleic acid bound to serum albumin increased Treg survival. Treg bound and internalized high amounts of HDL compared to other subsets, which might arise from the higher expression of the scavenger receptor class B-type I by Treg; accordingly, blocking this receptor hindered HDL-mediated Treg survival. Mechanistically, we showed that HDL increased Treg ATP concentration and mitochondrial activity, enhancing basal respiration, maximal respiration and spare respiratory capacity. Blockade of fatty acid oxidation by etoxomir abolished the HDL-mediated enhanced survival and mitochondrial activity. Our findings thus suggest that Treg can specifically internalize HDL from their microenvironment and use them as an energy source. Furthermore, a novel implication of our data is that enhanced Treg survival may contribute to HDL anti-inflammatory properties.

  11. T cell-intrinsic ASC critically promotes TH17-mediated experimental autoimmune encephalomyelitis

    PubMed Central

    Martin, Bradley N.; Wang, Chenhui; Zhang, Cun-jin; Kang, Zizhen; Gulen, Muhammet Fatih; Zepp, Jarod A.; Zhao, Junjie; Bian, Guanglin; Do, Jeong-su; Min, Booki; Pavicic, Paul G.; El-Sanadi, Caroline; Fox, Paul L.; Akitsu, Aoi; Iwakura, Yoichiro; Sarkar, Anasuya; Wewers, Mark D.; Kaiser, William J.; Mocarski, Edward S.; Rothenberg, Marc E.; Hise, Amy G.; Dubyak, George R.; Ransohoff, Richard M.; Li, Xiaoxia

    2017-01-01

    IL-1β is critical for TH17 cell survival, expansion, and effector function in vivo during autoimmune responses, including EAE. However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis is poorly defined. In the present study, we uncovered a novel T cell-intrinsic inflammasome that drives IL-1β production during TH17-mediated EAE pathogenesis. TCR activation induced pro-IL-1β expression, while ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on TH17 but not TH1 cells, and ATP-treated TH17 cells showed enhanced survival compared to ATP-treated TH1 cells, suggesting autocrine action of TH17-derived IL-1β. Together, these data reveal a critical role for IL-1β produced by a TH17 cell-intrinsic ASC-NLRP3-Caspase-8 inflammasome during CNS inflammation. PMID:26998763

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

    PubMed Central

    Lee, Soyoung; Yamada, Yohei; Tonsho, Makoto; Boskovic, Svjetlan; Nadazdin, Ognjenka; Schoenfeld, David; Cappetta, Kate; Atif, Muhammad; Smith, Rex-Neal; Cosimi, A. Benedict; Benichou, Gilles; Kawai, Tatsuo

    2014-01-01

    Renal allograft tolerance has been achieved in MHC-mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow (DBM) transplantation. To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel conditioning regimen, the “delayed protocol” in which 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 CD2highCD8+ 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 CD2high 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 CD2high 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. PMID:24165326

  13. Interleukin 10 promotes immune response by increasing the survival of activated CD8(+) T cells in human papillomavirus 16-infected cervical cancer.

    PubMed

    Li, Li; Ma, Yan; Liu, Shuang; Zhang, Jin; Xu, Xin-Yan

    2016-10-11

    Human papillomavirus (HPV)-specific CD8(+) T cells are present in HPV-infected cervical cancer patients and have demonstrated potent antitumor properties. However, these cells cannot control tumor progression in most patients. To investigate the underlying mechanisms involved in suppressing or promoting CD8(+) T cell functions, we focused on interleukin 10 (IL-10), a pleiotropic cytokine with controversial roles in antitumor immunity. We found that compared to healthy controls, circulating CD8(+) T cells in HPV 16-infected cervical cancer patients expressed significantly higher levels of IL-10. Interestingly, these CD8(+) T cells from cervical cancer patients, but not those from healthy controls, responded to HPV 16 E6/E7 peptide stimulation by increasing IL-10 expression, demonstrating an antigen-specific IL-10 release. Addition of exogenous IL-10 improved the survival, but did not increase the proliferation, of peptide-stimulated CD8(+) T cells. CD8(+) T cells cultured in the presence of IL-10 also resulted in significantly higher interferon gamma (IFN-gamma) and granzyme B concentration, primarily due to improved cell survival. In resected cervical tumors, the frequency of tumor-infiltrating IL-10(+) CD8(+) T cells was positively correlated with the frequency of tumor-infiltrating IFN-gamma(+) and granzyme B(+) CD8(+) T cells. Tumor-associated macrophages were more potent than peripheral blood monocyte-derived macrophages at inducing IL-10 expression in CD8(+) T cells, possibly explaining the elevated IL-10(+) CD8(+) T cell frequency in cervical cancer patients. Together, these results are consistent with an immunostimulatory role of IL-10, which promoted CD8(+) T cell response by increasing the survival of activated CD8(+) T cells.

  14. Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif.

    PubMed

    Xu, Chenqi; Gagnon, Etienne; Call, Matthew E; Schnell, Jason R; Schwieters, Charles D; Carman, Christopher V; Chou, James J; Wucherpfennig, Kai W

    2008-11-14

    Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

  15. CD8+ T cells promote proliferation of benign prostatic hyperplasia epithelial cells under low androgen level via modulation of CCL5/STAT5/CCND1 signaling pathway.

    PubMed

    Yang, Yang; Hu, Shuai; Liu, Jie; Cui, Yun; Fan, Yu; Lv, Tianjing; Liu, Libo; Li, Jun; He, Qun; Han, Wenke; Yu, Wei; Sun, Yin; Jin, Jie

    2017-02-20

    Previous studies by our group have shown that low intra-prostatic dihydrotestosterone (DHT) induced BPH epithelial cells (BECs) to recruit CD8+ T cells. However, the influence of the recruited CD8+ T cells on BECs under a low androgen level is still unknown. Here, we found CD8+ T cells have the capacity to promote proliferation of BECs in low androgen condition. Mechanism dissection revealed that interaction between CD8+ T cells and BECs through secretion of CCL5 might promote the phosphorylation of STAT5 and a higher expression of CCND1 in BECs. Suppressed CCL5/STAT5 signals via CCL5 neutralizing antibody or STAT5 inhibitor Pimozide led to reverse CD8+ T cell-enhanced BECs proliferation. IHC analysis from Finasteride treated patients showed PCNA expression in BECs was highly correlated to the level of CD8+ T cell infiltration and the expression of CCL5. Consequently, our data indicated infiltrating CD8+ T cells could promote the proliferation of BECs in low androgen condition via modulation of CCL5/STAT5/CCND1 signaling. The increased secretion of CCL5 from the CD8+ T cells/BECs interaction might help BECs survive in a low DHT environment. Targeting these signals may provide a new potential therapeutic approach to better treat BPH patients who failed the therapy of 5α-reductase inhibitors.

  16. CD8+ T cells promote proliferation of benign prostatic hyperplasia epithelial cells under low androgen level via modulation of CCL5/STAT5/CCND1 signaling pathway

    PubMed Central

    Yang, Yang; Hu, Shuai; Liu, Jie; Cui, Yun; Fan, Yu; Lv, Tianjing; Liu, Libo; Li, Jun; He, Qun; Han, Wenke; Yu, Wei; Sun, Yin; Jin, Jie

    2017-01-01

    Previous studies by our group have shown that low intra-prostatic dihydrotestosterone (DHT) induced BPH epithelial cells (BECs) to recruit CD8+ T cells. However, the influence of the recruited CD8+ T cells on BECs under a low androgen level is still unknown. Here, we found CD8+ T cells have the capacity to promote proliferation of BECs in low androgen condition. Mechanism dissection revealed that interaction between CD8+ T cells and BECs through secretion of CCL5 might promote the phosphorylation of STAT5 and a higher expression of CCND1 in BECs. Suppressed CCL5/STAT5 signals via CCL5 neutralizing antibody or STAT5 inhibitor Pimozide led to reverse CD8+ T cell-enhanced BECs proliferation. IHC analysis from Finasteride treated patients showed PCNA expression in BECs was highly correlated to the level of CD8+ T cell infiltration and the expression of CCL5. Consequently, our data indicated infiltrating CD8+ T cells could promote the proliferation of BECs in low androgen condition via modulation of CCL5/STAT5/CCND1 signaling. The increased secretion of CCL5 from the CD8+ T cells/BECs interaction might help BECs survive in a low DHT environment. Targeting these signals may provide a new potential therapeutic approach to better treat BPH patients who failed the therapy of 5α-reductase inhibitors. PMID:28216616

  17. 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.

  18. 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.

  19. Sorafenib combined with HER-2 targeted vaccination can promote effective T cell immunity in vivo.

    PubMed

    Sunay, Melek M E; Foote, Jeremy B; Leatherman, James M; Edwards, Justin P; Armstrong, Todd D; Nirschl, Christopher J; Hicks, Jessica; Emens, Leisha A

    2017-05-01

    The tumor microenvironment (TME) is established and maintained through complex interactions between tumor cells and host stromal elements. Therefore, therapies that target multiple cellular components of the tumor may be most effective. Sorafenib, a multi-kinase inhibitor, alters signaling pathways in both tumor cells and host stromal cells. Thus, we explored the potential immune-modulating effects of sorafenib in a murine HER-2-(neu) overexpressing breast tumor model alone and in combination with a HER-2 targeted granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting vaccine (3T3neuGM). In vitro, sorafenib inhibited the growth of HER-2 overexpressing NT2.5 tumor cells, inducing apoptosis. Sorafenib also interfered with ERK MAPK, p38 MAPK, and STAT3 signaling, as well as cyclin D expression, but did not affect HER-2 or AKT signaling. In vivo, single agent sorafenib disrupted the tumor-associated vasculature and induced tumor cell apoptosis, effectively inducing the regression of established NT2.5 tumors in immune competent FVB/N mice. Immune depletion studies demonstrated that both CD4(+) and CD8(+) T cells were required for tumor regression. Sorafenib treatment did not impact the rate of tumor clearance induced by vaccination with 3T3neuGM in tumor-bearing FVB/N mice relative to either sorafenib treatment or vaccination alone. In vivo studies further demonstrated that sorafenib enhanced the accumulation of both CD4(+) and CD8(+) T cells into the TME of vaccinated mice. Together, these findings suggest that GM-CSF-secreting cellular immunotherapy may be integrated with sorafenib without impairing vaccine-based immune responses. Copyright © 2017. Published by Elsevier B.V.

  20. 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

  1. Characterization of the androgen-regulated prostate-specific T cell receptor gamma-chain alternate reading frame protein (TARP) promoter.

    PubMed

    Cheng, Wing-Shing; Giandomenico, Valeria; Pastan, Ira; Essand, Magnus

    2003-08-01

    TARP (T cell receptor gamma-chain alternate reading frame protein) is uniquely expressed in males in prostate epithelial cells and prostate cancer cells. Here we demonstrate that TARP expression is regulated by testosterone at the transcriptional level through specific binding of androgen receptor to an androgen response element in the proximal TARP promoter. We further demonstrate that the promoter specifically initiates reporter gene expression in TARP-positive prostate cancer cell lines. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the prostate-specific antigen (PSA) enhancer. We found that in the prostatic adenocarcinoma cell line LNCaP, the transcriptional activity of the regulatory sequence consisting of a TARP promoter and PSA enhancer is 20 times higher than the activity of a regulatory sequence consisting of the PSA promoter and PSA enhancer. Thus, our studies define a regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in prostate cancer gene therapy.

  2. Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells

    PubMed Central

    Davey, Gayle M.; Schober, Sonya L.; Endrizzi, Bart T.; Dutcher, Angela K.; Jameson, Stephen C.; Hogquist, Kristin A.

    1998-01-01

    During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes. PMID:9815264

  3. DEPTOR is a direct NOTCH1 target that promotes cell proliferation and survival in T-cell leukemia.

    PubMed

    Hu, Y; Su, H; Liu, C; Wang, Z; Huang, L; Wang, Q; Liu, S; Chen, S; Zhou, J; Li, P; Chen, Z; Liu, H; Qing, G

    2017-02-23

    Aberrant activation of NOTCH1 signaling plays a vital role in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Yet the molecular events downstream of NOTCH1 that drive T-cell leukemogenesis remain incompletely understood. Starting from genome-wide gene-expression profiling to seek important NOTCH1 transcriptional targets, we identified DEP-domain containing mTOR-interacting protein (DEPTOR), which was previously shown to be important in multiple myeloma but remains functionally unclear in other hematological malignancies. Mechanistically, we demonstrated NOTCH1 directly bound to and activated the human DEPTOR promoter in T-ALL cells. DEPTOR depletion abolished cellular proliferation, attenuated glycolytic metabolism and enhanced cell death, while ectopically expressed DEPTOR significantly promoted cell growth and glycolysis. We further showed that DEPTOR depletion inhibited while its overexpression enhanced AKT activation in T-ALL cells. Importantly, AKT inhibition completely abrogated DEPTOR-mediated cell growth advantages. Moreover, DEPTOR depletion in a human T-ALL xenograft model significantly delayed T-ALL onset and caused a substantial decrease of AKT activation in leukemic blasts. We thus reveal a novel mechanism involved in NOTCH1-driven leukemogenesis, identifying the transcriptional control of DEPTOR and its regulation of AKT as additional key elements of the leukemogenic program activated by NOTCH1.

  4. MiR-34a promotes DCs development and inhibits their function on T cell activation by targeting WNT1

    PubMed Central

    Chen, Si; Xia, Fei; Sun, Di; Fang, Deyu; Xiong, Sidong; Jin, Liping; Zhang, Jinping

    2017-01-01

    MicroRNAs serve important functions in numerous biological processes. Whether microRNAs also act on dendritic cell (DC) differentiation and function remains unclear. In this study, both conventional DCs (cDCs) and plasmacytoid DCs (pDCs) were increased in miR-34a overexpressing bone marrow chimeric and transgenic (TG) mice. Further experiments showed that miR-34a promoted preDC differentiated into cDCs and pDCs without affecting the proliferation and apoptosis of DCs. Luciferase report assay and Western blot experiments demonstrated that WNT1 is the direct target of miR-34a in DCs. Interestingly, miR-34a overexpressing cDCs also produced a large amount of IL-17a and suppressed T cell activation because of the inhibition of TCF1 expression, thus increasing RORγT expression. Taken together, miR-34a promotes preDC to differentiate into cDCs and pDCs, as well as inhibits the function of cDCs on the activation of CD4+ T cells by producing IL-17a. PMID:28199987

  5. CD226 ligation protects against EAE by promoting IL-10 expression via regulation of CD4+ T cell differentiation

    PubMed Central

    Chen, Kun; Zhang, Chunmei; Song, Chaojun; Fang, Liang; Xu, Zhuwei; Yang, Kun; Jin, Boquan; Wang, Qintao; Chen, Lihua

    2016-01-01

    Treatment targeting CD226 can ameliorate experimental autoimmune encephalomyelitis (EAE), the widely accepted model of MS. However, the mechanisms still need to be elucidated. Here we showed that CD226 blockage by anti-CD226 blocking mAb LeoA1 efficiently promoted IL-10 production in human peripheral blood monocytes (PBMC) or in mixed lymphocyte culture (MLC) system, significantly induced the CD4+IL-10+ T cell differentiation while suppressing the generation of Th1 and Th17. Furthermore, CD226 pAb administration in vivo reduced the onset of EAE in mice by promoting IL-10 production and regulating T cell differentiation. Concomitantly, the onset and severity of EAE were reduced and the serum IL-10 expression levels were increased in CD226 knockout mice than that in control mice when both received EAE induction. These novel findings confirmed that CD226 played a pivotal role in mediating autoimmune diseases such as EAE. Furthermore, to our knowledge, we show for the first time that IL-10 is an important contributor in the inhibitory effects of CD226 ligation on EAE. PMID:26942885

  6. Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells

    PubMed Central

    Pedersen, Natasja Wulff; Chandran, P. Anoop; Qian, Yu; Rebhahn, Jonathan; Petersen, Nadia Viborg; Hoff, Mathilde Dalsgaard; White, Scott; Lee, Alexandra J.; Stanton, Rick; Halgreen, Charlotte; Jakobsen, Kivin; Mosmann, Tim; Gouttefangeas, Cécile; Chan, Cliburn; Scheuermann, Richard H.; Hadrup, Sine Reker

    2017-01-01

    Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore difficult to detect. We used a highly heterogeneous dataset from a recent MHC multimer proficiency panel to assess if MHC multimer-binding CD8+ T cells could be analyzed with computational solutions currently available, and if such analyses would reduce the technical variation across different laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0.01 to 1.5% of lymphocytes within samples from two donors. Experience from this analysis shows that all three programs can be used for the identification of high to intermediate frequency of MHC multimer-binding T cell populations, with results very similar to that of manual gating. For the less frequent populations (<0.1% of live, single lymphocytes), SWIFT outperformed the other tools. As used in this study, none of the algorithms offered a completely automated pipeline for identification of MHC multimer populations, as varying degrees of human interventions were needed to complete the analysis. In this study, we demonstrate the feasibility of using automated analysis pipelines for assessing and identifying even rare populations of antigen-responsive T cells and discuss the main

  7. T-cell memory responses elicited by yellow fever vaccine are targeted to overlapping epitopes containing multiple HLA-I and -II binding motifs.

    PubMed

    de Melo, Andréa Barbosa; Nascimento, Eduardo J M; Braga-Neto, Ulisses; Dhalia, Rafael; Silva, Ana Maria; Oelke, Mathias; Schneck, Jonathan P; Sidney, John; Sette, Alessandro; Montenegro, Silvia M L; Marques, Ernesto T A

    2013-01-01

    The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4(+) and CD8(+) T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, "promiscuous" T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.

  8. CD103 or LFA-1 engagement at the immune synapse between cytotoxic T cells and tumor cells promotes maturation and regulates T-cell effector functions.

    PubMed

    Franciszkiewicz, Katarzyna; Le Floc'h, Audrey; Boutet, Marie; Vergnon, Isabelle; Schmitt, Alain; Mami-Chouaib, Fathia

    2013-01-15

    T-cell adhesion/costimulatory molecules and their cognate receptors on target cells play a major role in T-cell receptor (TCR)-mediated activities. Here, we compared the involvement of CD103 and LFA-1, and their respective ligands, in the maturation of the cytotoxic immune synapse (cIS) and in the activation of CTL effector functions. Our results indicate that cytotoxicity toward cancer cells and, to a lesser extent, cytokine production by specific CTL require, together with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1. Flow-based adhesion assay showed that engagement of CD103 or LFA-1, together with TCR, enhances the strength of the T-cell/target cell interaction. Moreover, electron microscopic analyses showed that integrin-dependent mature cIS (mcIS) displays a cohesive ultrastructure, with tight membrane contacts separated by extensive clefts. In contrast, immature cIS (icIS), which is unable to trigger target cell lysis, is loose, with multiple protrusions in the effector cell membrane. Experiments using confocal microscopy revealed polarized cytokine release and degranulation at the mcIS associated with target cell killing, whereas icIS is characterized by failure of IFN-γ and granzyme B relocalization. Thus, interactive forces between CTL and epithelial tumor cells, mainly regulated by integrin engagement, correlate with maturity and the ultrastructure of the cIS and influence CTL effector functions. These results provide new insights into molecular mechanisms regulating antitumor CTL responses and may lead to the development of more efficient cancer immunotherapy strategies.

  9. 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.

  10. Persistent p55TNFR expression impairs T cell responses during chronic tuberculosis and promotes reactivation.

    PubMed

    Dambuza, Ivy M; Keeton, Roanne; Hsu, Nai-Jen; Allie, Nasiema; Quesniaux, Valérie F J; Ryffel, Bernhard; Jacobs, Muazzam

    2016-12-20

    The pleiotropic activities of TNF are mediated by two structurally related but functionally distinct type I transmembrane receptors, p55TNFR and p75TNFR expressed in most cell types, that can be cleaved and act as TNF scavengers. Here, we investigated the effect of persistent p55TNFR cell surface expression during aerosol inhalation challenge with virulent M. tuberculosis H37Rv. We demonstrated that persistency of p55TNFR in macrophage cultures increased the synthesis of soluble TNF, p75TNFR and NO, however, had no effects on bacteria killing ability. Furthermore, it did not facilitate enhanced protection to primary acute M. tuberculosis infection in p55(∆NS) mice. Without exacerbated lung inflammation, we found a compensatory increase in p75TNFR shedding and decrease in bioactive TNF in BAL of p55(∆NS) mice after M. tuberculosis challenge. Defective expressions of CD44 and INFγ attributed to an impaired T cell response during persistent p55TNFR expression that caused marginal transient susceptibility during chronic infection. Moreover, persistent p55TNFR expression induced early reactivation during latent tuberculosis infection. These data indicate a prominent role of p55TNFR shedding in Th1 mediated protection against chronic and latent tuberculosis infection.

  11. Persistent p55TNFR expression impairs T cell responses during chronic tuberculosis and promotes reactivation

    PubMed Central

    Dambuza, Ivy M.; Keeton, Roanne; Hsu, Nai-Jen; Allie, Nasiema; Quesniaux, Valérie F. J.; Ryffel, Bernhard; Jacobs, Muazzam

    2016-01-01

    The pleiotropic activities of TNF are mediated by two structurally related but functionally distinct type I transmembrane receptors, p55TNFR and p75TNFR expressed in most cell types, that can be cleaved and act as TNF scavengers. Here, we investigated the effect of persistent p55TNFR cell surface expression during aerosol inhalation challenge with virulent M. tuberculosis H37Rv. We demonstrated that persistency of p55TNFR in macrophage cultures increased the synthesis of soluble TNF, p75TNFR and NO, however, had no effects on bacteria killing ability. Furthermore, it did not facilitate enhanced protection to primary acute M. tuberculosis infection in p55∆NS mice. Without exacerbated lung inflammation, we found a compensatory increase in p75TNFR shedding and decrease in bioactive TNF in BAL of p55∆NS mice after M. tuberculosis challenge. Defective expressions of CD44 and INFγ attributed to an impaired T cell response during persistent p55TNFR expression that caused marginal transient susceptibility during chronic infection. Moreover, persistent p55TNFR expression induced early reactivation during latent tuberculosis infection. These data indicate a prominent role of p55TNFR shedding in Th1 mediated protection against chronic and latent tuberculosis infection. PMID:27995986

  12. Relevance of regulatory T cell promotion of donor-specific tolerance in solid organ transplantation

    PubMed Central

    Sagoo, Pervinder; Lombardi, Giovanna; Lechler, Robert I.

    2012-01-01

    Current clinical strategies to control the alloimmune response after transplantation do not fully prevent induction of the immunological processes which lead to acute and chronic immune-mediated graft rejection, and as such the survival of a solid organ allograft is limited. Experimental research on naturally occurring CD4+CD25highFoxP3+ Regulatory T cells (Tregs) has indicated their potential to establish stable long-term graft acceptance, with the promise of providing a more effective therapy for transplant recipients. Current approaches for clinical use are based on the infusion of freshly isolated or ex vivo polyclonally expanded Tregs into graft recipients with an aim to redress the in vivo balance of T effector cells to Tregs. However mounting evidence suggests that regulation of donor-specific immunity may be central to achieving immunological tolerance. Therefore, the next stages in optimizing translation of Tregs to organ transplantation will be through the refinement and development of donor alloantigen-specific Treg therapy. The altering kinetics and intensity of alloantigen presentation pathways and alloimmune priming following transplantation may indeed influence the specificity of the Treg required and the timing or frequency at which it needs to be administered. Here we review and discuss the relevance of antigen-specific regulation of alloreactivity by Tregs in experimental and clinical studies of tolerance and explore the concept of delivering an optimal Treg for the induction and maintenance phases of achieving transplantation tolerance. PMID:22811678

  13. Hypoxia-induced CCL28 promotes recruitment of regulatory T cells and tumor growth in liver cancer

    PubMed Central

    Wang, Li; Zhu, Zhifeng; Lu, Rong; Yao, Zhi

    2016-01-01

    Tumor cells craft microenvironment to overcome growth disadvantages and adjust to escape the immunosurveillance during tumorigenesis and metastasis. The evolving adaption to the changing microenvironment is exemplified by the development of strategies to deal with hypoxia resulted from fast proliferation of the tumor cells. In this study, we found that hypoxia hepatocellular carcinoma (HCC) cells recruited Regulatory T cells (Tregs) and expressed more Chemokine (C-C motif) ligand 28 (CCL28). Deletion of CCL28 inhibited Treg recruitment. Furthermore, overexpression of CCL28 promoted tumor growth and Treg infiltration in vivo. Enhanced angiogenesis and VEGF expression was also observed. Moreover, inhibition of HIF1α reversed hypoxia-induced CCL28 upregulation. Taken together, our results demonstrate that HCC recruits Tregs to promote angiogenesis under hypoxic condition by upregulating CCL28 expression. These findings establish a link between Tregs and hypoxia in HCC growth and may provide a new potential therapeutic target for treating HCC. PMID:27716621

  14. Upregulation of Bcl-2 and Its Promoter Signals in CD4+ T Cells during Neuromyelitis Optica Remission

    PubMed Central

    Yang, Tao; Wang, Su; Yang, Xiao; Zheng, Qi; Wang, Lei; Li, Qian; Wei, Mingyan; Du, Zongpan; Fan, Yongping

    2017-01-01

    The homeostatic balance between production and elimination of CD4+ T cells in peripheral blood plays an important role in patients with neuromyelitis optica (NMO). The objective of the present study was to evaluate the anti-apoptosis genes Bcl-2 and its promoter signal (nuclear factor kappa-light-chain-enhancer of activated B cells, NFκB) in CD4+ T cells. Healthy subjects (HS, n = 25) and patients with multiple sclerosis (MS) (n = 25) and NMO (n = 30) in remission were consecutively enrolled in this prospective study between May and December 2015. CD4+ T cells were isolated using magnetic beads coated with anti-CD4 monoclonal antibodies, and gene expression of Bcl-2, NFκB, phosphatidylinositol-4, 5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt), and MAP kinase kinase kinase 7 (MAP3K7) was measured by real-time reverse transcription-polymerase chain reaction (rt-PCR). Cytokines of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were detected using human cytokine multiplex assay. Bcl-2 and NFκB gene expressions were elevated in NMO patients (1.63 ± 0.25; 2.35 ± 0.25) compared with those of HS (0.90 ± 0.11; 1.42 ± 0.22) and/or MS patients (1.03 ± 0.18; 1.55 ± 0.20) (P < 0.05). MAP3K7, but not Akt, was increased in NMO patients (1.23 ± 0.18; 1.56 ± 0.22) (P < 0.01) and was a significant factor related to elevated NFκB gene expressions (P < 0.001). On the other hand, IL-1β and TNF-α were also detected in the study and the results showed that both were elevated in NMO patients (23.84 ± 1.81; 56.40 ± 2.45) (P < 0.01; P < 0.05, respectively). We propose that MAP3K7 induced by IL-1β and TNF-α but not Akt promotes NFκB expression and, in turn, prolongs Bcl-2–mediated survival of CD4+ T cells in NMO patients. PMID:28174515

  15. Invariant Natural Killer T Cells Promote T Cell Immunity by Modulating the Function of Lung Dendritic Cells during Chlamydia pneumoniae Infection.

    PubMed

    Shekhar, Sudhanshu; Joyee, Antony George; Gao, Xiaoling; Peng, Ying; Wang, Shuhe; Yang, Jie; Yang, Xi

    2015-01-01

    In this study, we examined the effect of invariant natural killer T (iNKT) cells on the function of lung dendritic cells (LDCs) in eliciting protective immunity against Chlamydia pneumoniae (Cpn) lung infection. We employed a combination of approaches including the use of iNKT cell-deficient, Jα18-knockout (KO) mice and LDC adoptive transfer. We found that iNKT cells significantly altered the number, phenotype and cytokine profile of LDCs following infection. Furthermore, coculture of T cells with LDCs from Cpn-infected wild-type (WT) and KO mice induced type-1 and type-2 responses, respectively. More importantly, upon adoptive transfer, LDCs from Cpn-infected WT mice (WT-LDCs) conferred protective immunity, whereas LDCs from KO mice (KO-LDCs) increased the severity of disease after challenge infection. Further cytokine analyses of the lung tissues and lung-draining lymph node cells showed that KO-LDC-recipient mice exhibited a type-2 cytokine production pattern, while WT-LDC recipients exhibited a type-1 cytokine profile. Taken together, our results provide in vivo evidence that iNKT cells play a critical role in modulating LDC function to generate protective T-cell immunity, particularly in a clinically relevant intracellular bacterial infection. © 2014 S. Karger AG, Basel.

  16. 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.

  17. NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization

    PubMed Central

    2010-01-01

    Background Nitrogen dioxide (NO2) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO2 is also produced endogenously in the lung during acute inflammatory responses. NO2 can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c+ antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c+ cells in NO2-promoted allergic sensitization. Methods We systemically depleted CD11c+ cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO2 followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c+ cells from wildtype mice were studied after exposure to NO2 and ovalbumin for cellular phenotype by flow cytometry and in vitro cytokine production. Results Transient depletion of CD11c+ cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c+ cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO2 exposure. By 48 hours, CD11c+MHCII+ DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c+CD11b- and CD11c+CD11b+ pulmonary cells exposed to NO2 in vivo increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647+ CD11c+MHCII+ DCs present in MLN from NO2-exposed mice by 48 hours. Co-cultures of ova-specific CD4+ T cells from naïve mice and CD11c+ pulmonary cells from NO2-exposed mice produced IL-1

  18. NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization.

    PubMed

    Hodgkins, Samantha R; Ather, Jennifer L; Paveglio, Sara A; Allard, Jenna L; LeClair, Laurie A Whittaker; Suratt, Benjamin T; Boyson, Jonathan E; Poynter, Matthew E

    2010-07-26

    Nitrogen dioxide (NO2) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO2 is also produced endogenously in the lung during acute inflammatory responses. NO2 can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c+ antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c+ cells in NO2-promoted allergic sensitization. We systemically depleted CD11c+ cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO2 followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c+ cells from wildtype mice were studied after exposure to NO2 and ovalbumin for cellular phenotype by flow cytometry and in vitro cytokine production. Transient depletion of CD11c+ cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c+ cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO2 exposure. By 48 hours, CD11c+MHCII+ DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c+CD11b- and CD11c+CD11b+ pulmonary cells exposed to NO2 in vivo increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647+ CD11c+MHCII+ DCs present in MLN from NO2-exposed mice by 48 hours. Co-cultures of ova-specific CD4+ T cells from naïve mice and CD11c+ pulmonary cells from NO2-exposed mice produced IL-1, IL-12p70, and IL-6 in vitro

  19. 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).

  20. IP-10-Mediated T Cell Homing Promotes Cerebral Inflammation over Splenic Immunity to Malaria Infection

    PubMed Central

    Nie, Catherine Q.; Bernard, Nicholas J.; Norman, M. Ursula; Amante, Fiona H.; Lundie, Rachel J.; Crabb, Brendan S.; Heath, William R.; Engwerda, Christian R.; Hickey, Michael J.; Schofield, Louis; Hansen, Diana S.

    2009-01-01

    Plasmodium falciparum malaria causes 660 million clinical cases with over 2 million deaths each year. Acquired host immunity limits the clinical impact of malaria infection and provides protection against parasite replication. Experimental evidence indicates that cell-mediated immune responses also result in detrimental inflammation and contribute to severe disease induction. In both humans and mice, the spleen is a crucial organ involved in blood stage malaria clearance, while organ-specific disease appears to be associated with sequestration of parasitized erythrocytes in vascular beds and subsequent recruitment of inflammatory leukocytes. Using a rodent model of cerebral malaria, we have previously found that the majority of T lymphocytes in intravascular infiltrates of cerebral malaria-affected mice express the chemokine receptor CXCR3. Here we investigated the effect of IP-10 blockade in the development of experimental cerebral malaria and the induction of splenic anti-parasite immunity. We found that specific neutralization of IP-10 over the course of infection and genetic deletion of this chemokine in knockout mice reduces cerebral intravascular inflammation and is sufficient to protect P. berghei ANKA-infected mice from fatality. Furthermore, our results demonstrate that lack of IP-10 during infection significantly reduces peripheral parasitemia. The increased resistance to infection observed in the absence of IP-10-mediated cell trafficking was associated with retention and subsequent expansion of parasite-specific T cells in spleens of infected animals, which appears to be advantageous for the control of parasite burden. Thus, our results demonstrate that modulating homing of cellular immune responses to malaria is critical for reaching a balance between protective immunity and immunopathogenesis. PMID:19343215

  1. Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection.

    PubMed

    Wiesner, Darin L; Specht, Charles A; Lee, Chrono K; Smith, Kyle D; Mukaremera, Liliane; Lee, S Thera; Lee, Chun G; Elias, Jack A; Nielsen, Judith N; Boulware, David R; Bohjanen, Paul R; Jenkins, Marc K; Levitz, Stuart M; Nielsen, Kirsten

    2015-03-01

    Pulmonary mycoses are often associated with type-2 helper T (Th2) cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection.

  2. 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.

  3. Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry

    PubMed Central

    Bi, Shuguang; Hong, Patrick W.; Lee, Benhur; Baum, Linda G.

    2011-01-01

    Interaction of cell surface glycoproteins with endogenous lectins on the cell surface regulates formation and maintenance of plasma membrane domains, clusters signaling complexes, and controls the residency time of glycoproteins on the plasma membrane. Galectin-9 is a soluble, secreted lectin that binds to glycoprotein receptors to form galectin–glycoprotein lattices on the cell surface. Whereas galectin-9 binding to specific glycoprotein receptors induces death of CD4 Th1 cells, CD4 Th2 cells are resistant to galectin-9 death due to alternative glycosylation. On Th2 cells, galectin-9 binds cell surface protein disulfide isomerase (PDI), increasing retention of PDI on the cell surface and altering the redox status at the plasma membrane. Cell surface PDI regulates integrin function on platelets and also enhances susceptibility of T cells to infection with HIV. We find that galectin-9 binding to PDI on Th2 cells results in increased cell migration through extracellular matrix via β3 integrins, identifying a unique mechanism to regulate T-cell migration. In addition, galectin-9 binding to PDI on T cells potentiates infection with HIV. We identify a mechanism for regulating cell surface redox status via a galectin–glycoprotein lattice, to regulate distinct T-cell functions. PMID:21670307

  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

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

    2005-12-27

    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.

  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. CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis.

    PubMed

    Joshi, Anjali; Punke, Erin B; Sedano, Melina; Beauchamp, Bethany; Patel, Rima; Hossenlopp, Cassady; Alozie, Ogechika K; Gupta, Jayanta; Mukherjee, Debabrata; Garg, Himanshu

    2017-03-22

    CCR5 is the major co-receptor for HIV and polymorphisms in the CCR5 gene as well as promoter region that alter cell surface expression have been associated with disease progression. We determined the relationship between CCR5 promoter polymorphisms and CD4 decline and other immunopathological features like immune activation and CD4+ T cell apoptosis in HIV patients. CCR5 promoter haplotype HHC was significantly associated with higher CD4 counts in patients. The relative promoter activity (RPA) of each haplotype was determined in vitro and combined promoter activity based on both alleles (CRPA) was assigned to each patients. Interestingly, CCR5 CRPA correlated inversely with CD4 counts and CD4:CD8 ratio specifically in viremic patients. In normal individuals, the CRPA correlated with the number of CCR5+ CD4+ T cells in the peripheral blood suggesting an effect on CCR5 expression. In a subset of high viremic patients harboring R5 tropic HIV, there was a strong correlation between CCR5 CRPA and both CD4 counts and CD4 T cell apoptosis. Our study demonstrates that, CCR5 promoter polymorphisms correlate with CD4 T cell loss possibly by regulating CD4 T cell apoptosis in HIV patients. Furthermore, assigning CRPAs to each patient is a new method of translating genotype to phenotype.

  8. Regulation of T cell Receptor Activation by Dynamic Membrane Binding of the CD3ε Cytoplasmic Tyrosine-Based Motif

    PubMed Central

    Xu, Chenqi; Gagnon, Etienne; Call, Matthew E.; Schnell, Jason R.; Schwieters, Charles D.; Carman, Christopher V.; Chou, James J.; Wucherpfennig, Kai W.

    2008-01-01

    Summary Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live cell imaging studies showed a close interaction of the CD3ε cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer (FRET) between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3ε residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance (NMR) structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3ε ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation. PMID:19013279

  9. Environmental contaminant trichloroethylene promotes autoimmune disease and inhibits T-cell apoptosis in MRL(+/+) mice.

    PubMed

    Gilbert, Kathleen M; Pumford, Neil R; Blossom, Sarah J

    2006-12-01

    The ability of environmental contaminant trichloroethylene to alter immune function and promote autoimmunity was tested in female MRL(+/+) mice. MRL(+/+) mice exposed to occupationally relevant doses of trichloroethylene in their drinking water for 32 weeks developed autoantibodies and pathological evidence of autoimmune hepatitis. The ability of trichloroethylene (TCE) to promote autoimmunity was associated with the expansion of activated (CD44(hi) CD62L(lo)) CD4(+) T-lymphocytes that produced increased levels of the pro-inflammatory cytokine interferon (IFN)-gamma. Activated T-lymphocytes can accumulate if activation-induced apoptosis is suppressed. Consequently, the effect of TCE on apoptosis in CD4(+) T-lymphocytes was investigated. These experiments were conducted with TCE and one of the major oxidative metabolites of trichloroethylene, namely trichloroacetaldehyde hydrate (TCAH). CD4(+) T-lymphocytes isolated from MRL(+/+) mice exposed to TCE or TCAH in their drinking water for 4 weeks were resistant to activation-induced apoptosis in vitro. The TCE-or TCAH-induced decrease in activation-induced apoptosis was associated with decreased expression of FasL, one of the cell surface molecules that mediate apoptosis. These results suggest that exposure to the common water contaminant TCE or its metabolite TCAH inhibits activation-induced apoptosis in CD4+ T-lymphocytes, thereby promoting autoimmune disease by suppressing the process that would otherwise delete activated self-reactive T-lymphocytes.

  10. Genetic variants of the IL22 promoter associate to onset of psoriasis before puberty and increased IL-22 production in T cells.

    PubMed

    Nikamo, Pernilla; Cheuk, Stanley; Lysell, Josefin; Enerbäck, Charlotta; Bergh, Kerstin; Xu Landén, Ning; Eidsmo, Liv; Ståhle, Mona

    2014-06-01

    Most psoriasis susceptibility genes were identified in cohorts of mixed clinical phenotypes and the exploration of genes in clinical subtypes is scarce. IL-22 has an established role in host defense and in psoriasis skin pathology, reflecting the delicate balance between control of infection and immunopathology. In a case-control study, we compared the genetic association to IL22 in psoriasis onset in patients between 0-9 (n=207), 10-20 (n=394), and 21-40 (n=468) years with healthy controls (n=1,529). Logistic regression analysis revealed association to regulatory elements in the IL22 promoter confined to onset of psoriasis before puberty (odds ratio=1.45, P<0.0007). The associated variants contain putative binding sites for AhR, a potent inducer of IL-22 expression. In a luciferase assay, transcriptional activity of a high-risk gene variant resulted in 80% higher promoter activity (P=0.012) compared with a low-risk variant. Ex vivo stimulated T cells from peripheral blood were analyzed with flow cytometry. Children with psoriasis carrying a high-risk variant produced 1.7 times more IL-22 compared with low-risk variants (P=0.042). Our combined genetic and functional data support the notion that a genetic IL22 variant that promotes epithelial barrier defense is preferentially enriched in and may precipitate the onset of psoriasis at an early age.

  11. IL-15 promotes regulatory T cell function and protects against diabetes development in NK-depleted NOD mice.

    PubMed

    Xia, Jinxing; Liu, Wentao; Hu, Biliang; Tian, Zhigang; Yang, Yongguang

    2010-02-01

    IL-15, an anti-apoptotic cytokine, has been reported to promote the survival and function of NK cells and T cells, including regulatory T cells (Tregs). Here we examined the effect of repeated injections of IL-15 on the development of diabetes in NOD mice. Injection of recombinant murine IL-15, once a day for 2 weeks, neither inhibited nor accelerated diabetes development in untreated NOD mice. However, treatment with IL-15 significantly reduced the incidence and delayed the onset of diabetes in NOD mice that were depleted of NK cells, while NK cell depletion alone had no protection against the disease development. The protective effect in IL-15-treated, NK cell-depleted NOD mice was associated with an increase in immunosuppressive activity of CD4(+)CD25(+) Tregs. IL-15 also enhanced Foxp3 expression in CD4(+)CD25(+) cells in an in vitro culture system, and such an effect of IL-15 was abrogated by IL-15-activated NK cells. Inhibition of IL-15-induced Foxp3 expression by IL-15-activated NK cells likely resulted from their IFN-gamma production, as recombinant IFN-gamma, or the culture supernatant of IL-15-activated wild-type mouse NK cells but not of IL-15-activated IFN-gamma-deficient NK cells, mediated a similar inhibition. IFN-gamma also diminished the stimulatory effect of IL-15 on Treg function in vitro. These results indicate that IL-15 has the potential to promote Treg function and protect against diabetes development in NOD mice, but such an activity can be eliminated by simultaneous activation of NK cells in IL-15-treated mice.

  12. 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

  13. In vitro differentiation from naive to mature E-selectin binding CD4 T cells: acquisition of skin-homing properties occurs independently of cutaneous lymphocyte antigen expression.

    PubMed

    Takahashi, Ryo; Mizukawa, Yoshiko; Yamazaki, Yoshimi; Hayakawa, Kazuhito; Hayakawa, Jun; Kudo, Akihiko; Shiohara, Tetsuo

    2003-12-01

    We previously showed that skin-homing CD4 T cells in peripheral blood can be subdivided into three populations on the basis of the expression pattern of the cutaneous lymphocyte Ag (CLA) and fucosyltransferase VII (FucT-VII): FucT-VII(+)CLA(-), FucT-VII(+)CLA(+), and FucT-VII(-)CLA(+). In view of the known late appearance of CLA during T cell differentiation, T cells programmed to attain skin-homing properties may start to generate E-selectin-binding epitopes at early stages of differentiation before induction of CLA expression. To this end, the in vitro differentiation from naive to CLA(+) memory T cells was followed after activation with anti-CD3 mAb. Here we demonstrate that naive skin-homing CD4 T cell precursors undergo a linear differentiation process from the FucT-VII(+)CLA(-) phenotype to the FucT-VII(+)CLA(+) phenotype and eventually to the FucT-VII(-)CLA(+) phenotype. The appearance of the FucT-VII(+)CLA(-) subset coincided with or could be immediately followed by the generation of E-selectin binding epitopes, and even after E-selectin-binding epitopes were no longer detectable, CLA remained expressed for prolonged periods of time, suggesting that induction of functional E-selectin ligands depends primarily on the expression of FucT-VII, but not CLA. Immunofluorescence and confocal microscopy studies of these T cells confirm that most E-selectin ligands were found independently of CLA expression.

  14. Binding of host-cell factors to DNA sequences in the long terminal repeat of human T-cell leukemia virus type I: implications for viral gene expression

    SciTech Connect

    Nyborg, J.K.; Dynan, W.S.; Chen, I.S.Y.; Wachsman, W.

    1988-03-01

    Efficient expression of human T-cell leukemia virus type I (HTLV-I) genes requires both host and viral proteins and is dependent on DNA sequences in the proviral long terminal repeats (LTRs). The authors have used DNase I-protection assays (footprinting) to construct a map of protein-DNA interactions over a 250-nucleotide region of the LTR upstream of the start site for viral RNA synthesis. They find that a host factor (host expression factor 1, or HEF-1) binds to the imperfect 21-nucleotide repeats that have previously been implicated in HTLV-I gene expression. HEF-1 binding activity is present in preparations from both lymphoid and nonlymphoid cells lines. However, the boundaries of the protected regions and the presence of a flanking DNase-hypersensitive site vary with cell type. Several regions of binding are detected in addition to the HEF-1 sites, including a complex group of sites 40-90 nucleotides upstream of the RNA start site. A comparison of HTLV-I transformed T lymphocytes that do and do not express the viral trans-activating protein p40/sup xI/ shows that none of the observed features of the DNase I footprint pattern correlate directly with the presence of this protein in the extract. These results suggest (i) that the primary recognition of promoter elements in the HTLV-I LTR involves specific interactions with host-cell proteins and (ii) that p40/sup xI/ influences the activity of one or more of these proteins, rather than interacting directly with the DNA.

  15. MicroRNA-939 down-regulates CD2-associated protein by targeting promoter in HEK-293T cells.

    PubMed

    Huang, Yu-Ping; Qiu, Ling-Zhi; Zhou, Guo-Ping

    2016-01-01

    CD2-associated protein (CD2AP) serves as a slit diaphragm (SD) protein and plays essential roles in maintaining podocyte integrity and reducing proteinuria. MicroRNAs (miRNAs) are novel regulators of gene expression. Podocyte-specific loss of miRNAs would lead to significant proteinuria. Here, we report new evidence in which miRNAs may function to suppress CD2AP expression through a transcriptional way. By scanning human CD2AP promoter in silico for sequences complementary to known miRNAs, we chose miR-939, miR-148b*, miR-191*, miR-638 as four candidates and transfected them into HEK-293T cells. Dual-luciferase reporter assay identified that only miR-939 significantly reduced the relative luciferase activity of CD2AP promoter region. Further analysis confirmed that the mRNA and protein expressions of CD2AP were also down-regulated by miR-939. In conclusion, we have identified that miR-939 targets CD2AP promoter sequences and suppresses its gene expression. These findings suggest that miRNAs may mediate podocyte injury via reducing the expression of the SD proteins, such as CD2AP.

  16. Thymic B cells promote thymus-derived regulatory T cell development and proliferation.

    PubMed

    Lu, Fang-Ting; Yang, Wei; Wang, Yin-Hu; Ma, Hong-Di; Tang, Wei; Yang, Jing-Bo; Li, Liang; Ansari, Aftab A; Lian, Zhe-Xiong

    2015-07-01

    Thymic CD4(+) FoxP3(+) regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cell-cell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3(+) Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for co-stimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFβ. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells.

  17. A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells

    PubMed Central

    Shepard, Blythe D.; Natarajan, Niranjana; Protzko, Ryan J.; Acres, Omar W.; Pluznick, Jennifer L.

    2013-01-01

    Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies. PMID:23840901

  18. One base pair change abolishes the T cell-restricted activity of a kB-like proto-enhancer element from the interleukin 2 promoter.

    PubMed Central

    Briegel, K; Hentsch, B; Pfeuffer, I; Serfling, E

    1991-01-01

    The inducible, T cell-specific enhancers of murine and human Interleukin 2 (Il-2) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif. When cloned in multiple copies this so-called TCEd (distal T cell element) acts as an inducible proto-enhancer element in E14 T lymphoma cells, but not in HeLa cells. In extracts of induced, Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA. The binding of the most prominent factor, named TCF-1 (T cell factor 1), is correlated with the proto-enhancer activity of TCEd. TCF-1 consists of two polypeptides of about 50 kD and 105 kD; the former seems to be related to the 50 kD polypeptide of NF-kB. Purified NF-kB is also able to bind to the TCEd, but TCF-1 binds stronger than NF-kB to TCEd DNA. The conversion of the TCEd to a 'perfect' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and, as a functional consequence, to the activity of the 'converted' TCEd motifs in HeLa cells. Thus, the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells. These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene. Images PMID:1945879

  19. Dnmt3a haploinsufficiency cooperates with oncogenic Kras to promote an early-onset T-cell acute lymphoblastic leukemia

    PubMed Central

    Chang, Yuan-I; Kong, Guangyao; Ranheim, Erik A; Tu, Po-Shu; Yu, Yi-Shan; Zhang, Jing

    2017-01-01

    Mutations in DNA methyltransferase 3A (DNMT3A) are prevalent in various myeloid and lymphoid malignancies. The most common DNMT3A R882 mutations inhibit methyltransferase activity of the remaining wild-type DNMT3A proteins at a heterozygous state due to their dominant-negative activity. Reports and COSMIC database analysis reveal significantly different frequencies of R882 mutations in myeloid versus T-cell malignancies, inspiring us to investigate whether downregulation of DNMT3A regulates malignancies of different lineages in a dose-dependent manner. In a competitive transplant setting, the survival of recipients with KrasG12D/+; Dnmt3a+/- bone marrow (BM) cells was significantly shortened than that of recipients with KrasG12D/+ cells. Moreover, all of the recipients with KrasG12D/+; Dnmt3a+/- cells developed a lethal T-cell acute lymphoblastic leukemia (T-ALL) without significant myeloproliferative neoplasm (MPN) phenotypes, while ~20% of recipients with KrasG12D/+ cells developed MPN with or without T-ALL. This is in sharp contrast to the recipients with KrasG12D/+; Dnmt3a-/- cells, in which ~60% developed a lethal myeloid malignancy (MPN or acute myeloid leukemia [AML]). Our data suggest that in the context of oncogenic Kras, loss of Dnmt3a promotes myeloid malignancies, while Dnmt3a haploinsufficiency induces T-ALL. This dose-dependent phenotype is highly consistent with the prevalence of DNMT3A R882 mutations in AML versus T-ALL in human. PMID:28386358

  20. 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.

  1. 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.

  2. 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.

  3. CD4 T Cells Promote Rather than Control Tuberculosis in the Absence of PD-1–Mediated Inhibition

    PubMed Central

    Barber, Daniel L.; Mayer-Barber, Katrin D.; Feng, Carl G.; Sharpe, Arlene H.; Sher, Alan

    2014-01-01

    Although CD4 T cells are required for host resistance to Mycobacterium tuberculosis, they may also contribute to pathology. In this study, we examine the role of the inhibitory receptor PD-1 and its ligand PD-L1 during M. tuberculosis infection. After aerosol exposure, PD-1 knockout (KO) mice develop high numbers of M. tuberculosis-specific CD4 T cells but display markedly increased susceptibility to infection. Importantly, we show that CD4 T cells themselves drive the increased bacterial loads and pathology seen in infected PD-1 KO mice, and PD-1 deficiency in CD4 T cells is sufficient to trigger early mortality. PD-L1 KO mice also display enhanced albeit less severe susceptibility, indicating that T cells are regulated by multiple PD ligands during M. tuberculosis infection. M. tuberculosis-specific CD8 T cell responses were normal in PD-1 KO mice, and CD8 T cells only had a minor contribution to the exacerbated disease in the M. tuberculosis-infected PD-1 KO and PD-L1 KO mice. Thus, in the absence of the PD-1 pathway, M. tuberculosis benefits from CD4 T cell responses, and host resistance requires inhibition by PD-1 to prevent T cell-driven exacerbation of the infection. PMID:21172867

  4. Co-expression of TIM-3 and CEACAM1 promotes T cell exhaustion in colorectal cancer patients.

    PubMed

    Zhang, Yang; Cai, Pengcheng; Li, Lei; Shi, Liang; Chang, Panpan; Liang, Tao; Yang, Qianqian; Liu, Yang; Wang, Lin; Hu, Lihua

    2017-02-01

    T-cell immunoglobulin domain and mucin domain-3(TIM-3) is an activation induced inhibitory molecule involved in immune tolerance and is recently reported to induce T cell exhaustion which is mediated by carcinoembryonic antigen cell adhesion molecule 1(CEACAM1), another well-known molecule expressed on activated T cells and involved in T cell inhibition. To investigate the expression of TIM-3 and CEACAM1 on circulating CD8(+) T cells and tumor infiltrating lymphocytes (TILs), 65 diagnosed colorectal cancer (CRC) patients and 38 healthy controls were enrolled in this study and the results showed that TIM-3 and CEACAM1 were both highly expressed on circulating CD8(+) T cells in CRC patients and elevated on TILs compared with paraneoplastic T cells. Furthermore, TIM-3(+)CEACAM1(+) CD8(+) T cells represented the most dysfunctional population with the least IFN-γ production. In addition, the expressions of TIM-3 and CEACAM1 were correlated with advanced stage and could be independent risk factors for CRC. We for the first time to our knowledge suggested that co-expression of TIM-3 and CEACAM1 can mediate T cell exhaustion and may be potential biomarkers for CRC prediction, highlighting the possibility of being immunotherapy targets.

  5. Dermal γδ T Cells Do Not Freely Re-Circulate Out of Skin and Produce IL-17 to Promote Neutrophil Infiltration during Primary Contact Hypersensitivity

    PubMed Central

    Jiang, Xiaodong; Park, Chang Ook; Geddes Sweeney, Jenna; Yoo, Min Jae; Gaide, Olivier; Kupper, Thomas Seth

    2017-01-01

    The role of mouse dermal γδ T cells in inflammatory skin disorders and host defense has been studied extensively. It is known that dendritic epidermal T cells (DETC) have a monomorphic γδ T cell receptor (TCR) and reside in murine epidermis from birth. We asked if dermal γδ cells freely re-circulated out of skin, or behaved more like dermal resident memory T cells (TRM) in mice. We found that, unlike epidermal γδ T cells (DETC), dermal γδ cells are not homogeneous with regard to TCR, express the tissue resident T cell markers CD69 and CD103, bear skin homing receptors, and produce IL-17 and IL-22. We created GFP+: GFP− parabiotic mice and found that dermal γδ T cells re-circulate very slowly—more rapidly than authentic αβ TCR TRM, but more slowly than the recently described dermal αβ TCR T migratory memory cells (TMM). Mice lacking the TCR δ gene (δ-/-) had a significant reduction of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). We created mice deficient in dermal γδ T cells but not DETC, and these mice also showed a markedly reduced CHS response after DNFB challenge. The infiltration of effector T cells during CHS was not reduced in dermal γδ T cell-deficient mice; however, infiltration of Gr-1+CD11b+ neutrophils, as well as ear swelling, was reduced significantly. We next depleted Gr-1+ neutrophils in vivo, and demonstrated that neutrophils are required for ear swelling, the accepted metric for a CHS response. Depletion of IL-17-producing dermal Vγ4+ cells and neutralization of IL-17 in vivo, respectively, also led to a significantly reduced CHS response and diminished neutrophil infiltration. Our findings here suggest that dermal γδ T cells have an intermediate phenotype of T cell residence, and play an important role in primary CHS through producing IL-17 to promote neutrophil infiltration. PMID:28081153

  6. In vivo regulation of interleukin-2 receptor alpha gene transcription by the coordinated binding of constitutive and inducible factors in human primary T cells.

    PubMed Central

    Algarté, M; Lécine, P; Costello, R; Plet, A; Olive, D; Imbert, J

    1995-01-01

    IL-2R alpha transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. To analyse the mechanisms used to activate IL-2R alpha transcription as well as those used to block it in non-expressing cells, we determined the protein-DNA interactions at the IL-2R alpha locus in three different cell types using the DMS/LMPCR genomic footprinting method. CD25/IL-2R alpha can be efficiently induced in primary human T cells since approximately 100% express this gene when receiving an appropriate combination of mitogenic stimuli. To understand why IL-2R alpha is not expressed in other haematopoietic cell types, we analysed BJAB B lymphoma cells which do not express the IL-2R alpha gene and contain constitutively active nuclear NF-kappa B. Primary fibroblasts from embryo and adult skin were selected to examine the mechanisms that may be used to keep the IL-2R alpha gene inactive in non-haematopoietic cells. The three main results are: (i) the stable in vivo occupancy of IL-2R alpha kappa B element in resting T cells, most probably by constitutive NF-kappa B p50 homodimer that could impair SRF binding to the flanking SRE/CArG box; (ii) its inducible occupancy by NF-kappa B p50-p65 associated with the binding of an SRE/CArG box DNA-binding factor upon mitogenic stimulation; and (iii) a correlation between the precommitment of T cells to activation and the presence of stable preassembled protein-DNA complexes in contrast with the bare IL-2R alpha locus in non-T cells. Images PMID:7588634

  7. T cells in pregnancy.

    PubMed

    Piccinni, Marie-Pierre

    2005-01-01

    Maternal tolerance of the fetal allograft could be the result of the integration of numerous mechanisms promoted by different cells present in the decidua. Decidual macrophages and dendritic cells, which are found in close association with T lymphocytes are the most potent activators of T lymphocyte responses and could play a sentinel function for the immune system, initiating antigen-specific T cell responses to fetal antigens. T cell cytokines produced in response to fetal molecules could have a role in the maintenance or in the failure of pregnancy. The levels of LIF, IL-4, IL-10 and M-CSF produced by decidual T cells of women suffering from unexplained spontaneous abortion are lower than those of normal pregnant women indicating that these cytokines may contribute to the maintenance of pregnancy. T cells from the cumulus oophorus surrounding the preimplantation embryo produce LIF and IL-4. These findings suggest that cytokines produced by maternal T cells create a suitable microenvironment for preimplantation embryo development and maintenance of pregnancy. T cell cytokine profile could be modulated by the hormones present in the microenvironment of T cells: high doses of progesterone present at fetomaternal interface and in the cumulus induce the production of IL-4 and LIF, whereas relaxin induces IFN-gamma production.

  8. 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.

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

    PubMed

    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

    Radiation-induced pulmonary fibrosis results from thoracic radiation therapy and severely limits radiation therapy approaches. CD4(+)CD25(+)FoxP3(+) 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. 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. 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. 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. Copyright © 2015. Published by Elsevier Inc.

  10. Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

    PubMed Central

    2010-01-01

    Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is

  11. B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile

    PubMed Central

    DeFuria, Jason; Belkina, Anna C.; Jagannathan-Bogdan, Madhumita; Snyder-Cappione, Jennifer; Carr, Jordan David; Nersesova, Yanina R.; Markham, Douglas; Strissel, Katherine J.; Watkins, Amanda A.; Zhu, Min; Allen, Jessica; Bouchard, Jacqueline; Toraldo, Gianluca; Jasuja, Ravi; Obin, Martin S.; McDonnell, Marie E.; Apovian, Caroline; Denis, Gerald V.; Nikolajczyk, Barbara S.

    2013-01-01

    Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D. PMID:23479618

  12. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells

    PubMed Central

    Hurton, Lenka V.; Singh, Harjeet; Najjar, Amer M.; Switzer, Kirsten C.; Mi, Tiejuan; Maiti, Sourindra; Olivares, Simon; Rabinovich, Brian; Huls, Helen; Forget, Marie-Andrée; Datar, Vrushali; Kebriaei, Partow; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

    2016-01-01

    Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials. PMID:27849617

  13. Constitutive signaling from an engineered IL-7 receptor promotes durable tumor elimination by tumor redirected T-cells.

    PubMed

    Shum, Thomas; Omer, Bilal; Tashiro, Haruko; Kruse, Robert L; Wagner, Dimitrios L; Parikh, Kathan; Yi, Zhongzhen; Sauer, Tim; Liu, Daofeng; Parihar, Robin; Castillo, Paul; Liu, Hao; Brenner, Malcolm K; Metelitsa, Leonid S; Gottschalk, Stephen; Rooney, Cliona M

    2017-08-22

    Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but are associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL-7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Co-expressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and anti-tumor activity during repeated exposure to tumor cells, without T cell dysfunction or autonomous T cell growth. Furthermore, C7R co-expressing CAR-T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer. Copyright ©2017, American Association for Cancer Research.

  14. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

    PubMed

    Hurton, Lenka V; Singh, Harjeet; Najjar, Amer M; Switzer, Kirsten C; Mi, Tiejuan; Maiti, Sourindra; Olivares, Simon; Rabinovich, Brian; Huls, Helen; Forget, Marie-Andrée; Datar, Vrushali; Kebriaei, Partow; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2016-11-29

    Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR(+) T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19(+) leukemia. Long-lived T cells were CD45RO(neg)CCR7(+)CD95(+), phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR(+) T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

  15. A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells

    PubMed Central

    Jaalouk, Diana E; Lejeune, Laurence; Couture, Clément; Galipeau, Jacques

    2006-01-01

    Background T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2) promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes. Results First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT) element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg). Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP) fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 μM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA). This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A. Conclusion These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be potentially exploited in

  16. G-CSF treatment promotes apoptosis of autoreactive T cells to restrict the inflammatory cascade and accelerate recovery in experimental allergic encephalomyelitis.

    PubMed

    Peng, Wei

    2017-03-01

    G-CSF is a hematopoietic growth factor that regulates the proliferation, differentiation and survival of myeloid lineage cells, which has protective effects in autoimmune neuroinflammatory diseases such as EAE. Here we use EAE model treated by G-CSF to address the hypothesis that G-CSF inhibits the proliferative response of splenic T cells via the enhancement of apoptosis, and this priming effect of G-CSF depends on the cell cycle. Our results show that G-CSF administration reduced EAE frequency and severity of attacks. The inflammatory cells and demyelination areas were decreased in the CNS of G-CSF-treated mice. G-CSF treatment altered cytokine profiles in vivo to inhibit the productions of IFN-γ, IL-1β, IL-2, TNF-α, IL-17 and NO, while the secretions of IL-4 and IL-10 were increased. Splenic T cells from G-CSF-treated mice showed significantly lower proliferative response to specific antigen MOG35-55 stimulation. G-CSF enhanced the percentage of a CD4(+)CD25(+) T cell subset in spleen T cells. Moreover, G-CSF promoted the G0/G1 to S phase transition of MOG35-55 autoreactive T cells inducing apoptosis and elevating Bax gene expression of apoptosis marker. These findings indicate that G-CSF treatment induces the apoptosis of MOG35-55 autoreactive T cells, which decreases the production of pro-inflammatory cytokines and NO, suppresses the proliferation of autoreactive T cells and elevates a CD4(+)CD25(+) T cell subset to inhibit inflammatory infiltration and demyelination within CNS of EAE. The conclusions of G-CSF treatment in EAE mice suggest that G-CSF is clinically applicable and may be considered for future use in therapeutic measures for multiple sclerosis treatment.

  17. Infiltrating T cells promote prostate cancer metastasis via modulation of FGF11→miRNA-541→androgen receptor (AR)→MMP9 signaling

    PubMed Central

    Hu, Shuai; Li, Lei; Yeh, Shuyuan; Cui, Yun; Li, Xin; Chang, Hong-Chiang; Jin, Jie; Chang, Chawnshang

    2014-01-01

    Early clinical studies suggested infiltrating T cells might be associated with poor outcomes in prostate cancer (PCa) patients. The detailed mechanisms how T cells contribute to PCa progression, however, remained unclear. Here, we found PCa cells have a better capacity to recruit more CD4(+) T cells than the surrounding normal prostate cells via secreting more chemokines-CXCL9. The consequences of more recruited CD4(+) T cells to PCa might then lead to enhance PCa cell invasion. Mechanism dissection revealed that infiltrating CD4(+) T cells might function through the modulation of FGF11→miRNA-541 signals to suppress PCa androgen receptor (AR) signals. The suppressed AR signals might then alter the MMP9 signals to promote the PCa cell invasion. Importantly, suppressed AR signals via AR-siRNA or anti-androgen Enzalutamidein PCa cells also enhanced the recruitment of T cells and the consequences of this positive feed back regulation could then enhance the PCa cell invasion. Targeting these newly identified signals viaFGF11-siRNA, miRNA-541 inhibitor or MMP9 inhibitor all led to partially reverse the enhanced PCa cell invasion. Results from in vivo mouse models also confirmed the in vitro cell lines in co-culture studies. Together, these results concluded that infiltrating CD4(+) T cells could promote PCa metastasis via modulation of FGF11→miRNA-541→AR→MMP9 signaling. Targeting these newly identified signals may provide us a new potential therapeutic approach to better battle PCa metastasis. PMID:25135278

  18. 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.

  19. Immunotherapy of murine sarcomas with auto-anti-idiotypic monoclonal antibodies which bind to tumor-specific T cells.

    PubMed

    Nelson, K A; George, E; Swenson, C; Forstrom, J W; Hellström, K E

    1987-09-15

    Hybridomas producing monoclonal antibodies (mAb) were obtained from BALB/c mice immunized against either of two transplanted, chemically induced syngeneic sarcomas, MCA-1490 or MCA-1511. Two mAb, 4.72 and 5.96, were obtained, one from each immunization. They were found to have apparent anti-idiotypic specificity in that they, when injected s.c., primed naive BALB/c mice for delayed-type hypersensitivity that was specific for the immunizing tumor and required homology at genes linked to the Igh-1 allotype locus. Neither mAb bound tumor antigen. When mice with established transplants of MCA-1490 or MCA-1511 were treated by repeated i.p. injections of the appropriate anti-idiotypic mAb (4.72 and 5.96, respectively), a significant reduction in tumor growth was observed in those mice that had received the appropriate mAb. The idiotope defined by mAb 4.72 was expressed by T cells in mice responding to MCA-1490. mAb 4.72 bound to T cell suppressor factors that were specific for MCA-1490 and were derived from T cell hybridomas or sera of mice bearing MCA-1490. mAb 4.72 also bound to cells from lymph nodes draining the area of a growing MCA-1490 tumor. It was used, in combination with cell sorting, to establish a T cell line, which mediated delayed-type hypersensitivity to MCA-1490 and inhibited the outgrowth of MCA-1490 in BALB/c mice. Thus, mAb specific for idiotopes on T cells responding to syngeneic tumor antigen had both direct immunotherapeutic activity and could be used to establish cultures of tumor-reactive T cells.

  20. TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells.

    PubMed

    Yarosh, Christopher A; Tapescu, Iulia; Thompson, Matthew G; Qiu, Jinsong; Mallory, Michael J; Fu, Xiang-Dong; Lynch, Kristen W

    2015-10-15

    PSF (a.k.a. SFPQ) is a ubiquitously expressed, essential nuclear protein with important roles in DNA damage repair and RNA biogenesis. In stimulated T cells, PSF binds to and suppresses the inclusion of CD45 exon 4 in the final mRNA; however, in resting cells, TRAP150 binds PSF and prevents access to the CD45 RNA, though the mechanism for this inhibition has remained unclear. Here, we show that TRAP150 binds a region encompassing the RNA recognition motifs (RRMs) of PSF using a previously uncharacterized, 70 residue region we have termed the PSF-interacting domain (PID). TRAP150's PID directly inhibits the interaction of PSF RRMs with RNA, which is mediated through RRM2. However, interaction of PSF with TRAP150 does not appear to inhibit the dimerization of PSF with other Drosophila Behavior, Human Splicing (DBHS) proteins, which is also dependent on RRM2. Finally, we use RASL-Seq to identify ∼40 T cell splicing events sensitive to PSF knockdown, and show that for the majority of these, PSF's effect is antagonized by TRAP150. Together these data suggest a model in which TRAP150 interacts with dimeric PSF to block access of RNA to RRM2, thereby regulating the activity of PSF toward a broad set of splicing events in T cells.

  1. Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment

    PubMed Central

    Yuan, Jinyun; Wang, Jianbin; Crain, Karen; Fearns, Colleen; Kim, Kenneth A; Hua, Kevin L; Gregory, Philip D; Holmes, Michael C; Torbett, Bruce E

    2012-01-01

    HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4+ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4+ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4+ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4+ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4+ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4+ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4+ T cells during HIV-1 infection. PMID:22273578

  2. Low Intraprostatic DHT Promotes the Infiltration of CD8+ T Cells in BPH Tissues via Modulation of CCL5 Secretion

    PubMed Central

    Fan, Yu; Liu, Jie; Xiao, Fei; Li, Xin; Yu, Wei; Cui, Yun; Sun, Mengkui; Lv, Tianjing; Jin, Jie

    2014-01-01

    Clinical studies suggested thatandrogen might be associated with infiltrating T cells in prostate of benign prostatic hyperplasia (BPH) patients, but detail of T-cell subset and mechanism still remained unclear. The present study tested the hypothesis that intraprostatic 5α-dihydrotestosterone (DHT) exerts effects on T cells recruitment by BPH epithelial cells. Prostate tissues from 64 cases of BPH patients after transurethral resection of prostate (TURP) were divided into 2 groups: (1) no medication history; (2) administration of 5α-reductase type II inhibitor-finasteride 5 mg daily for at least 6 months before surgery. Group 2 presented significantly higher CD8+ T cells infiltration than group 1, but no changes in CD4+ T cells (immunohistochemistry and flow cytometry). In vitro study more CD8+ T cell migrated to the prostate tissue lysates from group 2 and BPH-1 cells in low DHT condition. Transcription of chemokine (C-C motif) Ligand 5 (CCL5) mRNA in BPH-1 cells and chemokine (C-C motif) receptor 5 (CCR5) mRNA in CD8+ T cells were upregulated in low DHT condition (q-PCR). CCL5 expression was also identified to be higher in group 2 prostate tissues by IHC. This study suggested that intraprostatic DHT may participate in regulating inflammatory response which was induced by human prostatic epithelial cell, via modulating CCL5 secretion. PMID:24808637

  3. A Mutational Analysis of the Binding of Staphylococcal Enterotoxins B and C3 to the T Cell Receptor β Chain and Major Histocompatibility Complex Class II

    PubMed Central

    Leder, Lukas; Llera, Andrea; Lavoie, Pascal M.; Lebedeva, Marina I.; Li, Hongmin; Sékaly, Rafick-Pierre; Bohach, Gregory A.; Gahr, Pamala J.; Schlievert, Patrick M.; Karjalainen, Klaus; Mariuzza, Roy A.

    1998-01-01

    The three-dimensional structure of the complex between a T cell receptor (TCR) β chain (mouse Vβ8.2Jβ2.1Cβ1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 Å resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the β–SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR β chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR β chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR–SAG and SAG–MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the β–SEC3 complex (“hot spot” residues) are strictly conserved among enterotoxins reactive with mouse Vβ8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vβ-binding specificities. PMID:9500785

  4. 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

  5. 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

  6. The Vα14 invariant natural killer T cell TCR forces microbial glycolipids and CD1d into a conserved binding mode

    PubMed Central

    Li, Yali; Girardi, Enrico; Wang, Jing; Yu, Esther Dawen; Painter, Gavin F.; Kronenberg, Mitchell

    2010-01-01

    Invariant natural killer T cells (iNKT cells) rapidly produce effector cytokines. In this study, we report the first crystal structures of the iNKT cell T cell receptor (TCR) bound to two natural, microbial glycolipids presented by CD1d. Binding of the TCR induced CDR3-α–dependent structural changes in the F′ roof of CD1d; these changes resemble those occurring in the absence of TCR engagement when the highly potent synthetic antigen α-galactosylceramide (α-GalCer) binds CD1d. Furthermore, in the Borrelia burgdorferi α–galactosyl diacylglycerol–CD1d complex, TCR binding caused a marked repositioning of the galactose sugar into an orientation that closely resembles α-GalCer. The TCR-dependent reorientation of the sugar, together with the induced CD1d fit, may explain the weaker potency of the microbial antigens compared with α-GalCer. We propose that the TCR of iNKT cells binds with a conserved footprint onto CD1d, regardless of the bound glycolipid antigen, and that for microbial antigens this unique binding mode requires TCR-initiated conformational changes. PMID:20921281

  7. IL-7 promotes Glut1 trafficking and glucose uptake via STAT5-mediated activation of Akt to support T-cell survival.

    PubMed

    Wofford, Jessica A; Wieman, Heather L; Jacobs, Sarah R; Zhao, Yuxing; Rathmell, Jeffrey C

    2008-02-15

    Lymphocyte homeostasis requires coordination of metabolic processes with cellular energetic and biosynthetic demands but mechanisms that regulate T-cell metabolism are uncertain. We show that interleukin-7 (IL-7) is a key regulator of glucose uptake in T lymphocytes. To determine how IL-7 affects glucose uptake, we analyzed IL-7 signaling mechanisms and regulation of the glucose transporter, Glut1. The IL-7 receptor (IL-7R) stimulated glucose uptake and cell-surface localization of Glut1 in a manner that required IL-7R Y449, which promoted rapid signal transducer and activator of transcription 5 (STAT5) activation and a delayed yet sustained activation of Akt. Each pathway was necessary for IL-7 to promote glucose uptake, as Akt1(-/-) T cells or PI3-kinase inhibition and RNAi of STAT5 led to defective glucose uptake in response to IL-7. STAT5 and Akt acted in a linear pathway, with STAT5-mediated transcription leading to Akt activation, which was necessary for STAT5 and IL-7 to promote glucose uptake and prevent cell death. Importantly, IL-7 required glucose uptake to promote cell survival. These data demonstrate that IL-7 promotes glucose uptake via a novel signaling mechanism in which STAT5 transcriptional activity promotes Akt activation to regulate Glut1 trafficking and glucose uptake that is critical for IL-7 to prevent T-cell death and maintain homeostasis.

  8. Spreading the load: antigen transfer between migratory and lymph node-resident dendritic cells promotes T cell priming.

    PubMed

    Mueller, Scott N

    2017-08-28

    Dendritic cells (DC) are specialized in the processing and presentation of antigen for the activation of lymphocytes. Multiple subsets of DCs exist with distinct functions and roles in the initiation of immune responses. DCs found within tissues acquire antigens or become infected by pathogens and migrate to local draining lymph nodes (LN) where they can directly stimulate T cells. These migratory DCs can also transfer antigens to LN-resident DCs and may indirectly enhance T cell priming. In this issue of the European Journal of Immunology, Gurevich et al. [Eur. J. Immunol. 2017. 47: XXXX-XXXX] elegantly demonstrate the influence of the transfer of antigen from migratory DCs to resident DCs on the dynamics of CD8 T cell priming in mice. Using both in vitro imaging to visualise antigen dissemination and intravital 2-photon microscopy to track T cell clustering with migratory and resident DCs, antigen-donor DC were found to efficiently distribute antigen to recipient DC. This process, which involved LFA-1, enhanced the recruitment of CD8(+) T cells into the response and rescued priming when DCs were impaired in presentation capacity. Together, these findings shed light on the dynamics of the transfer of antigens between DCs in vivo for the efficient priming of cytotoxic T cell responses. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  9. IL-7 inhibits tumor growth by promoting T cell-mediated antitumor immunity in Meth A model.

    PubMed

    Tang, Jian-Cai; Shen, Guo-Bo; Wang, Shi-Min; Wan, Yong-Sheng; Wei, Yu-Quan

    2014-01-01

    Immune suppression is well documented during tumor progression, which includes loss of effect of T cells and expansion of T regulatory (Treg) cells. IL-7 plays a key role in the proliferation, survival and homeostasis of T cells and displays a potent antitumor activity in vivo. In the present study, we investigated the antitumor effect of IL-7 in Meth A model. IL-7 inhibited tumor growth and prolonged the survival of tumor-bearing mice with corresponding increases in the frequency of CD4 and CD8 T cells, Th1 (CD4(+)IFN-γ(+)), Tc1 (CD8(+)IFN-γ(+)) and T cells cytolytic activity against Meth A cells. Neutralization of CD4 or CD8 T cells reversed the antitumor benefit of IL-7. Furthermore, IL-7 decreased regulatory T Foxp3 as well as cells suppressive activity with a reciprocal increase in SMAD7. In addition, we observed an increase of the serum concentrations of IL-6 and IFN-γ, and a significant decrease of TGF-β and IL-10 after IL-7 treatment. Taken together, these results indicate that IL-7 augments T cell-mediated antitumor immunity and improves the effect of antitumor in Meth A model.

  10. ALPHAVIRUS REPLICON PARTICLES ACTING AS ADJUVANTS PROMOTE CD8+ T CELL RESPONSES TO CO-DELIVERED ANTIGEN

    PubMed Central

    Thompson, Joseph M.; Whitmore, Alan C.; Staats, Herman F.; Johnston, Robert E.

    2013-01-01

    Alphavirus replicon particles induce strong antibody and CD8+ T cell responses to expressed antigens in numerous experimental systems. We have recently demonstrated that Venezuelan equine encephalitis virus replicon particles (VRP) possess adjuvant activity for systemic and mucosal antibody responses. In this report, we demonstrate that VRP induced an increased and balanced serum IgG subtype response to co-delivered antigen, with simultaneous induction of antigen-specific IgG1 and IgG2a antibodies, and increased both systemic and mucosal antigen-specific CD8+ T cell responses, as measured by an IFN-γ ELISPOT assay. Additionally, VRP further increased antigen-specific T cell immunity in an additive fashion following co-delivery with the TLR ligand, CpG DNA. VRP infection led to recruitment of CD8+ T cells into the mucosal compartment, possibly utilizing the mucosal homing receptor, as this integrin was upregulated on CD8+ T cells in the draining lymph node of VRP-infected animals, where VRP-infected dendritic cells reside. This newly recognized ability of VRP to mediate increased T cell response towards co-delivered antigen provides the potential to both define the molecular basis of alphavirus-induced immunity, and improve alphavirus-based vaccines. PMID:18582997

  11. Alphavirus replicon particles acting as adjuvants promote CD8+ T cell responses to co-delivered antigen.

    PubMed

    Thompson, Joseph M; Whitmore, Alan C; Staats, Herman F; Johnston, Robert E

    2008-08-05

    Alphavirus replicon particles induce strong antibody and CD8+ T cell responses to expressed antigens in numerous experimental systems. We have recently demonstrated that Venezuelan equine encephalitis virus replicon particles (VRP) possess adjuvant activity for systemic and mucosal antibody responses. In this report, we demonstrate that VRP induced an increased and balanced serum IgG subtype response to co-delivered antigen, with simultaneous induction of antigen-specific IgG1 and IgG2a antibodies, and increased both systemic and mucosal antigen-specific CD8+ T cell responses, as measured by an IFN-gamma ELISPOT assay. Additionally, VRP further increased antigen-specific T cell immunity in an additive fashion following co-delivery with the TLR ligand, CpG DNA. VRP infection led to recruitment of CD8+ T cells into the mucosal compartment, possibly utilizing the mucosal homing receptor, as this integrin was upregulated on CD8+ T cells in the draining lymph node of VRP-infected animals, where VRP-infected dendritic cells reside. This newly recognized ability of VRP to mediate increased T cell response towards co-delivered antigen provides the potential to both define the molecular basis of alphavirus-induced immunity, and improve alphavirus-based vaccines.

  12. ICOS-ligand expression on plasmacytoid dendritic cells supports breast cancer progression by promoting the accumulation of immunosuppressive CD4+ T cells.

    PubMed

    Faget, Julien; Bendriss-Vermare, Nathalie; Gobert, Michael; Durand, Isabelle; Olive, Daniel; Biota, Cathy; Bachelot, Thomas; Treilleux, Isabelle; Goddard-Leon, Sophie; Lavergne, Emilie; Chabaud, Sylvie; Blay, Jean Yves; Caux, Christophe; Ménétrier-Caux, Christine

    2012-12-01

    Human breast tumors are infiltrated by memory CD4(+) T cells along with increased numbers of regulatory T cells (Treg) and plasmacytoid dendritic cells (pDC) that facilitate immune escape and correlate with poor prognosis. Here, we report that inducible costimulatory molecule (ICOS), a T cell costimulatory molecule of the CTLA4/PD1/CD28 family, is expressed mostly by tumor-associated Treg in primary breast tumors. A large proportion of these ICOS(+) Treg were Ki67(+) and this evident proliferative expansion was found to rely on interactions with tumor-associated pDC. Indeed, tumor-associated Treg highly expanded in presence of pDC but failed to proliferate under CD3/CD28 signal. In vitro experiments revealed that the addition of a neutralizing anti-ICOS antibody blocked pDC-induced Treg expansion and interleukin-10 secretion by memory CD4(+) T cells, establishing a pivotal role for ICOS in this process. Supporting these findings, the presence of ICOS(+) cells in clinical specimens of breast cancer correlated with a poor prognosis. Together, our results highlight an important relationship between Treg and pDC in breast tumors, and show that ICOS/ICOS-L interaction is a central event in immunosuppression of tumor-associated memory CD4(+) T cells. These findings strongly rationalize antibody-mediated ICOS blockade as a powerful clinical strategy to correct immune escape and promote therapeutic responses in breast cancer.

  13. The human fetal placenta promotes tolerance against the semiallogeneic fetus by inducing regulatory T cells and homeostatic M2 macrophages.

    PubMed

    Svensson-Arvelund, Judit; Mehta, Ratnesh B; Lindau, Robert; Mirrasekhian, Elahe; Rodriguez-Martinez, Heriberto; Berg, Göran; Lash, Gendie E; Jenmalm, Maria C; Ernerudh, Jan

    2015-02-15

    A successful pregnancy requires that the maternal immune system is instructed to a state of tolerance to avoid rejection of the semiallogeneic fetal-placental unit. Although increasing evidence supports that decidual (uterine) macrophages and regulatory T cells (Tregs) are key regulators of fetal tolerance, it is not known how these tolerogenic leukocytes are induced. In this article, we show that the human fetal placenta itself, mainly through trophoblast cells, is able to induce homeostatic M2 macrophages and Tregs. Placental-derived M-CSF and IL-10 induced macrophages that shared the CD14(+)CD163(+)CD206(+)CD209(+) phenotype of decidual macrophages and produced IL-10 and CCL18 but not IL-12 or IL-23. Placental tissue also induced the expansion of CD25(high)CD127(low)Foxp3(+) Tregs in parallel with increased IL-10 production, whereas production of IFN-γ (Th1), IL-13 (Th2), and IL-17 (Th17) was not induced. Tregs expressed the suppressive markers CTLA-4 and CD39, were functionally suppressive, and were induced, in part, by IL-10, TGF-β, and TRAIL. Placental-derived factors also limited excessive Th cell activation, as shown by decreased HLA-DR expression and reduced secretion of Th1-, Th2-, and Th17-associated cytokines. Thus, our data indicate that the fetal placenta has a central role in promoting the homeostatic environment necessary for successful pregnancy. These findings have implications for immune-mediated pregnancy complications, as well as for our general understanding of tissue-induced tolerance.

  14. CD39/ENTPD1 expression by CD4+Foxp3+ regulatory T cells promotes hepatic metastatic tumor growth in mice

    PubMed Central

    SUN, XIAOFENG; WU, YAN; GAO, WENDA; ENJYOJI, KEIICHI; CSIZMADIA, EVA; MÜLLER, CHRISTA E.; MURAKAMI, TAKASHI; ROBSON, SIMON C.

    2010-01-01

    Background & Aims Adenosine mediates immune suppression and is generated by the ectonucleotidases CD39 (ENTPD1) and CD73 that are expressed on vascular endothelial cells and regulatory T cells (Treg). Although tumor-infiltrating immune cells include Foxp3+ Treg, it is not clear whether local adenosine generation by Treg promotes tumor growth in a CD39-dependent manner. In this study, we have examined the impact of CD39 expression by Treg on effector immune cell responses to hepatic metastases in vivo. Methods and Results A model of hepatic metastatic cancer was developed with portal vein infusion of luciferase-expressing melanoma B16/F10 cells and MCA38 colon cancer cells in wild type and mutant mice null for Cd39. Chimeric mice were generated by bone marrow transplantation (BMT) using Cd39 null or wild type (wt) C57BL6 donors and irradiated recipient mice. We demonstrate that hepatic growth of melanoma metastatic tumors was strongly inhibited in mice with Cd39 null vasculature or in wild type mice with circulating Cd39 null bone marrow-derived cells. We show functional CD39 expression on CD4+Foxp3+ Treg suppressed anti-tumor immunity mediated by NK cells in vitro and in vivo. Lastly, inhibition of CD39 activity by POM-1 (polyoxometalate-1), a pharmacological inhibitor of NTPDase activity, significantly inhibited tumor growth (P < .001). Conclusions CD39 expression on Treg inhibits NK activity and is permissive for metastatic growth. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy for secondary hepatic malignancies. PMID:20546740

  15. Ovarian cancer stem cells promote tumour immune privilege and invasion via CCL5 and regulatory T cells.

    PubMed

    You, Yanan; Li, Yiying; Li, Ming; Mi, Lei; Wu, Mengyao; Qu, Ying; Yuan, Yan; Chen, Tong; Jiang, Hua

    2017-09-04

    Emerging evidence indicates a link between the increased proportion of regulatory T cells (Tregs) and reduced survival in patients who have been diagnosed with cancer. Cancer stem cells (CSCs) have been indicated to play a vital role in tumour initiation, drug resistance and recurrence. However, the relationship between Tregs and CSCs remains largely unknown. Here, we sorted out ovarian cancer stem-like side population (SP) cells and CD133(+) cells to investigate the influence of ovarian CSCs on Tregs. Among the various immune-related molecules that we assessed, C-C motif chemokine ligand 5 (CCL5) was the most elevated in ovarian CSCs relative to that in the non-CSCs. The expression of its receptor, C-C motif chemokine receptor 5 (CCR5), was also increased on the surface of Tregs in ovarian cancer patients. This receptor-ligand expression profile indicated that ovarian CSCs recruit Tregs via CCL5-CCR5 interactions. We further assessed the expression of interleukin-10 (IL-10) in Tregs cultured with different cancer cells. Tregs cultured in conditioned medium (CM) from ovarian CD133(+) cells expressed a higher level of IL-10 than Tregs cultured in CM from CD133(-) cells, indicating that Tregs exert pronounced immune-inhibitory functions in CSC-rich environments. Furthermore, co-culture with ovarian cancer cell lines induced the expression of matrix metalloproteinase-9 (MMP9) in Tregs, which in turn enhanced the degradation of the extracellular matrix and enabled the invasion of tumour cells, thereby facilitating tumour metastasis. For the first time here, our findings describe the relationship between ovarian CSCs and Tregs, and demonstrated that these two cell populations cooperate to promote tumour immune tolerance and enhance tumour progression. This article is protected by copyright. All rights reserved. © 2017 British Society for Immunology.

  16. RNA binding of T-cell intracellular antigen-1 (TIA-1) C-terminal RNA recognition motif is modified by pH conditions.

    PubMed

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

    2013-09-06

    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.

  17. 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

  18. P-selectin glycoprotein ligand-1 in T cells.

    PubMed

    Abadier, Michael; Ley, Klaus

    2017-05-01

    We review P-selectin glycoprotein ligand-1 (PSGL-1) as a selectin and chemokine-binding adhesion molecule. PSGL-1 is widely studied in neutrophils. Here, we focus on T cells, because PSGL-1 was recently described as a major immunomodulatory molecule during viral infection. PSGL-1 also plays a crucial role in T-cell homeostasis by binding to lymphoid chemokines, and can induce tolerance by enhancing the functions of regulatory T cells. PSGL-1 was originally described as a leukocyte ligand for P-selectin, but it is actually a ligand for all selectins (P-, L- and E-selectin), binds chemokines, activates integrins and profoundly affects T-cell biology. It has been shown recently that PSGL-1 can modulate T cells during viral infection by acting as a negative regulator for T-cell functions. Absence of PSGL-1 promotes effector CD4 and CD8 T-cell differentiation and prevents T-cell exhaustion. Consistent with this, tumor growth was significantly reduced in PSGL-1-deficient mice because of an enhanced number of effector T cells together with reduced levels of inhibitory receptors that induce T-cell exhaustion. PSGL-1 is the best-studied selectin ligand and has become a posterchild of versatility in leukocyte adhesion, inflammation and immunology. The direct involvement of PSGL-1 in T-cell biology suggests that it might be a drug target. Indeed, PSGL-1 has been tested in some clinical trials and recently, PSGL-1 blockers were proposed as a potential cotherapy in cancer immunotherapy.

  19. Identification of a functional NF-kappa B binding site in the murine T cell receptor beta 2 locus

    PubMed Central

    1989-01-01

    We have identified a sequence in the TCR beta 2 locus that is homologous to the kappa B site in the Ig kappa light chain enhancer. This element, TCR beta-B, is located in the vicinity of previously identified T cell-specific DNase1 hypersensitive sites. Transfection analysis shows that a 60-bp fragment encompassing this site is preferentially active in T cells stimulated with phorbol esters or the HTLV-1 tax gene product compared with a B cell line that constitutively expresses NF-kappa B. Our results provide the first evidence for transcriptional regulatory sequences residing within the J beta 2-C beta 2 intron and suggest the possible involvement of these sequences in modulation of TCR beta gene expression upon cellular activation. PMID:2530301

  20. The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27.

    PubMed

    Apetoh, Lionel; Quintana, Francisco J; Pot, Caroline; Joller, Nicole; Xiao, Sheng; Kumar, Deepak; Burns, Evan J; Sherr, David H; Weiner, Howard L; Kuchroo, Vijay K

    2010-09-01

    Type 1 regulatory T cells (Tr1 cells ) that produce interleukin 10 (IL-10) are instrumental in the prevention of tissue inflammation, autoimmunity and graft-versus-host disease. The transcription factor c-Maf is essential for the induction of IL-10 by Tr1 cells, but the molecular mechanisms that lead to the development of these cells remain unclear. Here we show that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR), which was induced by IL-27, acted in synergy with c-Maf to promote the development of Tr1 cells. After T cell activation under Tr1-skewing conditions, the AhR bound to c-Maf and promoted transactivation of the Il10 and Il21 promoters, which resulted in the generation of Tr1 cells and the amelioration of experimental autoimmune encephalomyelitis. Manipulating AhR signaling could therefore be beneficial in the resolution of excessive inflammatory responses.

  1. The NF-kappa B binding site is necessary for efficient replication of simian immunodeficiency virus of macaques in primary macrophages but not in T cells in vitro.

    PubMed Central

    Bellas, R E; Hopkins, N; Li, Y

    1993-01-01

    We demonstrate here that the nuclear factor-kappa B (NF-kappa B) binding site in the simian immunodeficiency virus (SIVmac) long terminal repeat is essential for efficient virus replication in primary alveolar macrophages but dispensable for efficient replication in primary T cells. Mutation of the NF-kappa B site does not seriously impair replication of a T-cell-tropic SIVmac239 or a macrophagetropic SIVmacEm* in peripheral blood lymphocytes or established CD4+ cell lines; however, mutation of the NF-kappa B site prevents efficient SIVmacEm* replication in primary alveolar macrophages. These data suggest that efficient replication in primary macrophages requires both envelope and long terminal repeat determinants. Images PMID:8474179

  2. 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. Copyright © 2016 by The American Association of Immunologists, Inc.

  3. Structure and Function of a Fungal Adhesin that Binds Heparin and Mimics Thrombospondin-1 by Blocking T Cell Activation and Effector Function

    PubMed Central

    Brandhorst, T. Tristan; Roy, René; Wüthrich, Marcel; Nanjappa, Som; Filutowicz, Hanna; Galles, Kevin; Tonelli, Marco; McCaslin, Darrell R.; Satyshur, Kenneth; Klein, Bruce

    2013-01-01

    Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1. PMID:23853587

  4. The HMGB1–CXCL12 Complex Promotes Inflammatory Cell Infiltration in Uveitogenic T Cell-Induced Chronic Experimental Autoimmune Uveitis

    PubMed Central

    Yun, Juan; Jiang, Guomin; Wang, Yunsong; Xiao, Tong; Zhao, Yuan; Sun, Deming; Kaplan, Henry J.; Shao, Hui

    2017-01-01

    It is largely unknown how invading autoreactive T cells initiate the pathogenic process inside the diseased organ in organ-specific autoimmune diseases. In experimental autoimmune uveitis (EAU) induced by uveitogenic, interphotoreceptor retinoid-binding protein (IRBP)-specific T cells (tEAU) in mice, we have previously reported that high mobility group box 1 (HMGB1) released as a consequence of the direct interaction between IRBP-specific T cells and retinal parenchymal cells is an early and critical mediator in induction of intraocular inflammation. Our present study explored the roles of HMGB1 in intraocular inflammation, focusing on its role in recruiting inflammatory cells into the eye. Our results showed that supernatants from retinal explants either stimulated with HMGB1 or cocultured with IRBP-specific T cells attracted leukocytes. Notably, HMGB1 antagonists blocked supernatant-induced chemoattraction when present from the start of coculture, but not when added to the culture supernatants after coculture, indicating that molecules released by HMGB1-treated retinal cells are chemoattractive. Moreover, CXCL12 levels in the coculture supernatants were dependent on HMGB1, since they were increased in the cocultures and reduced when HMGB1 antagonists were added at the beginning of the coculture. When either anti-CXCL12 Ab was added to the supernatants after coculture or the responding lymphocytes were pretreated with Ab against CXCL12 specific receptor, CXCR4, chemoattraction by the coculture supernatants was decreased. Finally, induction of tEAU was significantly inhibited by a CXCR4 antagonist, AMD3100, at the time of autoreactive T cell transfer. Our study demonstrates that, at a very early stage of intraocular inflammation initiated by uveitogenic autoreactive T cells, synergism between HMGB1 and CXCL12 is crucial for the infiltration of inflammatory cells. PMID:28261206

  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. B cells promote induction of experimental autoimmune encephalomyelitis by facilitating reactivation of T cells in the CNS

    PubMed Central

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

    2014-01-01

    The efficacy of rituximab treatment in multiple sclerosis has renewed interest in the role of B cells in CNS autoimmunity. Here we show that B cells are the predominant MHC class II+ subset in the naïve CNS in mice, and they constitutively express pro-inflammatory cytokines. Incidence of experimental autoimmune encephalomyelitis (EAE) 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 to Th17 cells in μMT mice, causing an increased Th17:Th1 ratio in the CNS at EAE 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. PMID:24367024

  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. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis.

    PubMed

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C

    2008-07-01

    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  9. 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

  10. T Cell Receptor-Engineered T Cells to Treat Solid Tumors: T Cell Processing Toward Optimal T Cell Fitness

    PubMed Central

    van Steenbergen-Langeveld, Sabine; van Brakel, Mandy; Groot-van Ruijven, Corrien M.; van Elzakker, Pascal M.M.L.; van Krimpen, Brigitte; Sleijfer, Stefan; Debets, Reno

    2014-01-01

    Abstract Therapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T cells specific for carbonic anhydrase IX (CAIX), we observed toxicities that (most likely) indicated in vivo function of CAR T cells as well as low T cell persistence and clinical response rates. The latter observations were confirmed by later clinical trials in other solid tumor types and other gene-modified T cells. To improve the efficacy of T cell therapy, we have redefined in vitro conditions to generate T cells with young phenotype, a key correlate with clinical outcome. For their impact on gene-modified T cell phenotype and function, we have tested various anti-CD3/CD28 mAb-based T cell activation and expansion conditions as well as several cytokines prior to and/or after gene transfer using two different receptors: CAIX CAR and MAGE-C2(ALK)/HLA-A2 TCR. In a total set of 16 healthy donors, we observed that T cell activation with soluble anti-CD3/CD28 mAbs in the presence of both IL15 and IL21 prior to TCR gene transfer resulted in enhanced proportions of gene-modified T cells with a preferred in vitro phenotype and better function. T cells generated according to these processing methods demonstrated enhanced binding of pMHC, and an enhanced proportion of CD8+, CD27+, CD62L+, CD45RA+T cells. These new conditions will be translated into a GMP protocol in preparation of a clinical adoptive therapy trial to treat patients with MAGE-C2-positive tumors. PMID:25423330

  11. 3,3′-Diindolylmethane Ameliorates Experimental Autoimmune Encephalomyelitis by Promoting Cell Cycle Arrest and Apoptosis in Activated T Cells through MicroRNA Signaling Pathways

    PubMed Central

    Rouse, Michael; Rao, Roshni; Nagarkatti, Mitzi

    2014-01-01

    3,3′-Diindolylmethane (DIM) is a naturally derived indole found in cruciferous vegetables that has great potential as a novel and effective therapeutic agent. In the current study, we investigated the effects of DIM post-treatment on the regulation of activated T cells during the development of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. We demonstrated that the administration of DIM 10 days after EAE induction was effective at ameliorating disease parameters, including inflammation and central nervous system cellular infiltration. MicroRNA (miRNA) microarray analysis revealed an altered miRNA profile in brain infiltrating CD4+ T cells following DIM post-treatment of EAE mice. Additionally, bioinformatics analysis suggested the involvement of DIM-induced miRNAs in pathways and processes that halt cell cycle progression and promote apoptosis. Additional studies confirmed that DIM impacted these cellular processes in activated T cells. Further evidence indicated that DIM treatment significantly upregulated several miRNAs (miR-200c, miR-146a, miR-16, miR-93, and miR-22) in brain CD4+ T cells during EAE while suppressing their associated target genes. Similarly, we found that overexpression of miR-16 in primary CD4+ T cells led to significant downregulation of both mRNA and protein levels of cyclin E1 and B-cell lymphoma-2, which play important roles in regulating cell cycle progression and apoptosis. Collectively, these studies demonstrate that DIM post-treatment leads to the amelioration of EAE development by suppressing T-cell responses through the induction of select miRNAs that control cell cycle progression and mediate apoptosis. PMID:24898268

  12. Binding of a soluble alpha beta T-cell receptor to superantigen/major histocompatibility complex ligands.

    PubMed Central

    Kappler, J; White, J; Kozono, H; Clements, J; Marrack, P

    1994-01-01

    The genes for the alpha and beta chains of a murine T-cell receptor were truncated just prior to the portions encoding the transmembrane regions and introduced into baculovirus by recombination. Insect cells infected with the virus secreted a soluble form of the receptor that could be purified to homogeneity. This soluble receptor reacted with a set of six monoclonal antibodies originally raised to different epitopes on the natural transmembrane-region-containing receptor and bound with appropriate specificity to a cell surface complex of the human major histocompatibility complex class II molecule DR1 with the bacterial superantigen staphylococcal enterotoxin B. Images PMID:8078904

  13. 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.

  14. 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.

  15. 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.

  16. HIV-1 Rev–binding protein accelerates cellular uptake of iron to drive Notch-induced T cell leukemogenesis in mice

    PubMed Central

    Khwaja, Shariq S.; Liu, Hudan; Tong, Caili; Jin, Fang; Pear, Warren S.; van Deursen, Jan; Bram, Richard J.

    2010-01-01

    Somatic activating mutations in Notch1 contribute to the pathogenesis of T cell acute lymphoblastic lymphoma (T-ALL), but how activated Notch1 signaling exerts this oncogenic effect is not completely understood. Here we identify HIV-1 Rev–binding protein (Hrb), a component of the clathrin-mediated endocytosis machinery, as a critical mediator of Notch-induced T-ALL development in mice. Hrb was found to be a direct transcriptional target of Notch1, and Hrb loss reduced the incidence or delayed the onset of T-ALL in mouse models in which activated Notch1 signaling either contributes to or drives leukemogenesis. Consistent with this observation, Hrb supported survival and proliferation of hematopoietic and T cell precursor cells in vitro. We demonstrated that Hrb accelerated the uptake of transferrin, which was required for upregulation of the T cell protooncogene p21. Indeed, iron-deficient mice developed Notch1-induced T-ALL substantially more slowly than control mice, further supporting a critical role for iron uptake during leukemogenesis. Taken together, these results reveal that Hrb is a critical Notch target gene that mediates lymphoblast transformation and disease progression via its ability to satisfy the enhanced demands of transformed lymphoblasts for iron. Further, our data suggest that Hrb may be targeted to improve current treatment or design novel therapies for human T-ALL patients. PMID:20516639

  17. HIV-1 Rev-binding protein accelerates cellular uptake of iron to drive Notch-induced T cell leukemogenesis in mice.

    PubMed

    Khwaja, Shariq S; Liu, Hudan; Tong, Caili; Jin, Fang; Pear, Warren S; van Deursen, Jan; Bram, Richard J

    2010-07-01

    Somatic activating mutations in Notch1 contribute to the pathogenesis of T cell acute lymphoblastic lymphoma (T-ALL), but how activated Notch1 signaling exerts this oncogenic effect is not completely understood. Here we identify HIV-1 Rev-binding protein (Hrb), a component of the clathrin-mediated endocytosis machinery, as a critical mediator of Notch-induced T-ALL development in mice. Hrb was found to be a direct transcriptional target of Notch1, and Hrb loss reduced the incidence or delayed the onset of T-ALL in mouse models in which activated Notch1 signaling either contributes to or drives leukemogenesis. Consistent with this observation, Hrb supported survival and proliferation of hematopoietic and T cell precursor cells in vitro. We demonstrated that Hrb accelerated the uptake of transferrin, which was required for upregulation of the T cell protooncogene p21. Indeed, iron-deficient mice developed Notch1-induced T-ALL substantially more slowly than control mice, further supporting a critical role for iron uptake during leukemogenesis. Taken together, these results reveal that Hrb is a critical Notch target gene that mediates lymphoblast transformation and disease progression via its ability to satisfy the enhanced demands of transformed lymphoblasts for iron. Further, our data suggest that Hrb may be targeted to improve current treatment or design novel therapies for human T-ALL patients.

  18. The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vγ9Vδ2 T cell activation by dendritic cells

    PubMed Central

    Castella, Barbara; Kopecka, Joanna; Sciancalepore, Patrizia; Mandili, Giorgia; Foglietta, Myriam; Mitro, Nico; Caruso, Donatella; Novelli, Francesco; Riganti, Chiara; Massaia, Massimo

    2017-01-01

    Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation. PMID:28580927

  19. IL-21 promotes lupus-like disease in chronic graft-versus-host disease through both CD4 T cell- and B cell-intrinsic mechanisms.

    PubMed

    Nguyen, Vinh; Luzina, Irina; Rus, Horea; Tegla, Cosmin; Chen, Ching; Rus, Violeta

    2012-07-15

    T cell-driven B cell hyperactivity plays an essential role in driving autoimmune disease development in systemic lupus erythematosus. IL-21 is a member of the type I cytokine family with pleiotropic activities. It regulates B cell differentiation and function, promotes T follicular helper (T(FH)) cell and Th17 cell differentiation, and downregulates the induction of T regulatory cells. Although IL-21 has been implicated in systemic lupus erythematosus, the relative importance of IL-21R signaling in CD4(+) T cells versus B cells is not clear. To address this question, we took advantage of two induced models of lupus-like chronic graft-versus-host disease by using wild-type or IL-21R(-/-) mice as donors in the parent-into-F1 model and as hosts in the Bm12→B6 model. We show that IL-21R expression on donor CD4(+) T cells is essential for sustaining T(FH) cell number and subsequent help for B cells, resulting in autoantibody production and more severe lupus-like renal disease, but it does not alter the balance of Th17 cells and regulatory T cells. In contrast, IL-21R signaling on B cells is critical for the induction and maintenance of germinal centers, plasma cell differentiation, autoantibody production, and the development of renal disease. These results demonstrate that IL-21 promotes autoimmunity in chronic graft-versus-host disease through both CD4(+) T cell- and B cell-intrinsic mechanisms and suggest that IL-21 blockade may attenuate B cell hyperactivity, as well as the aberrant T(FH) cell pathway that contributes to lupus pathogenesis.

  20. Trogocytosis of MHC-I/Peptide Complexes Derived from Tumors and Infected Cells Enhances Dendritic Cell Cross-Priming and Promotes Adaptive T Cell Responses

    PubMed Central

    Zhang, Qian-Jin; Li, Xiao-Lin; Wang, David; Huang, Xiao-Cong; Mathis, J. Michael; Duan, Wei-Ming; Knight, David; Shi, Runhua; Glass, Jonathan; Zhang, Dong-Qing; Eisenbach, Lea; Jefferies, Wilfred A.

    2008-01-01

    The transporter associated with antigen processing (TAP) and the major histocompatibility complex class I (MHC-I), two important components of the MHC-I antigen presentation pathway, are often deficient in tumor cells. The restoration of their expression has been shown to restore the antigenicity and immunogenicity of tumor cells. However, it is unclear whether TAP and MHC-I expression in tumor cells can affect the induction phase of the T cell response. To address this issue, we expressed viral antigens in tumors that are either deficient or proficient in TAP and MHC-I expression. The relative efficiency of direct immunization or immunization through cross-presentation in promoting adaptive T cell responses was compared. The results demonstrated that stimulation of animals with TAP and MHC-I proficient tumor cells generated antigen specific T cells with greater killing activities than those of TAP and MHC-I deficient tumor cells. This discrepancy was traced to differences in the ability of dendritic cells (DCs) to access and sample different antigen reservoirs in TAP and MHC-I proficient versus deficient cells and thereby stimulate adaptive immune responses through the process of cross-presentation. In addition, our data suggest that the increased activity of T cells is caused by the enhanced DC uptake and utilization of MHC-I/peptide complexes from the proficient cells as an additional source of processed antigen. Furthermore, we demonstrate that immune-escape and metastasis are promoted in the absence of this DC ‘arming’ mechanism. Physiologically, this novel form of DC antigen sampling resembles trogocytosis, and acts to enhance T cell priming and increase the efficacy of adaptive immune responses against tumors and infectious pathogens. PMID:18769733

  1. 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

  2. Immunological and structural homology between human T-cell leukemia virus type I envelope glycoprotein and a region of human interleukin-2 implicated in binding the. beta. receptor

    SciTech Connect

    Kohtz, D.S.; Kohtz, J.D.; Puszkin, S. ); Altman, A. )

    1988-02-01

    The N-terminal segment of human interleukin-2 (hIL-2) appears to mediate binding of the {beta} hIL-2 receptor. An affinity-purified antibody prepared against this peptide segment (p81) is shown here to cross-react with a homologous region of the human T-cell leukemia virus type I (HTLV-I) envelope glycoprotein, raising the interesting possibility that the envelope glycoprotein of HTLV-I can interact with the {beta} hIL-2 receptor.

  3. Conditional PDK1 ablation promotes epidermal and T cell-mediated dysfunctions leading to inflammatory skin disease

    PubMed Central

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

    2015-01-01

    Phosphoinositide dependent kinase-1 (PDK1) is a key signaling molecule downstream of the phosphatidylinositol 3-kinase (PI-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 Th2 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 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 which prevents skin immune infiltration and inflammation. PMID:26099023

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. 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.

  6. The weak, fine-tuned binding of ubiquitous transcription factors to the Il-2 enhancer contributes to its T cell-restricted activity.

    PubMed Central

    Hentsch, B; Mouzaki, A; Pfeuffer, I; Rungger, D; Serfling, E

    1992-01-01

    The T lymphocyte-specific enhancers of the murine and human Interleukin 2 (Il-2) genes harbour several binding sites for ubiquitous transcription factors. All these sites for the binding of AP-1, NF-kB or Oct-1 are non-canonical sites, i.e. they differ in one or a few base pairs from consensus sequences for the optimal binding of these factors. Although the factors bind weakly to these sites, the latter are functionally important because their mutation to non-binding sites results in a decrease of inducible activity of the Il-2 enhancer. Conversion of three sites to canonical binding sites of Octamer factors, AP-1 and NF-kB results in a drastic increase in enhancer activity and the induction of the Il-2 enhancer in non-T cells, such as B cell lines, murine L cells and human HeLa cells. The introduction of two or three canonical sites into the enhancer leads to a further increase of its activity. Il-2 enhancer induction is also observed in B cells when the concentration of AP-1 and Oct factors increases as a result of cotransfections with FosB and Octamer expression plasmids. When Il-2 enhancer constructs carrying canonical factor binding sites were injected into Xenopus oocytes the strong binding of ubiquitous factors substantially overcomes the silencing effect of negatively acting factors present in resting primary T lymphocytes. These results suggest a fine-tuned interplay between ubiquitous and lymphoid-specific factors binding to and transactivating the Il-2 enhancer and show that the binding affinity of ubiquitous factors to the enhancer contributes to its cell-type specific activity. Moreover, we believe that a dramatic increase of transcriptional activity brought about by single point mutations at strategic important factor binding sites may also have relevance to the activation of nuclear oncogenes. Images PMID:1614851

  7. Human Blood CD1c(+) Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4(+) T Cells.

    PubMed

    Leal Rojas, Ingrid M; Mok, Wai-Hong; Pearson, Frances E; Minoda, Yoshihito; Kenna, Tony J; Barnard, Ross T; Radford, Kristen J

    2017-01-01

    Dendritic cells (DC) initiate the differentiation of CD4(+) helper T cells into effector cells including Th1 and Th17 responses that play an important role in inflammation and autoimmune disease pathogenesis. In mice, Th1 and Th17 responses are regulated by different conventional (c) DC subsets, with cDC1 being the main producers of IL-12p70 and inducers of Th1 responses, while cDC2 produce IL-23 to promote Th17 responses. The role that human DC subsets play in memory CD4(+) T cell activation is not known. This study investigated production of Th1 promoting cytokine IL-12p70, and Th17 promoting cytokines, IL-1β, IL-6, and IL-23, by human blood monocytes, CD1c(+) DC, CD141(+) DC, and plasmacytoid DC and examined their ability to induce Th1 and Th17 responses in memory CD4(+) T cells. Human CD1c(+) DC produced IL-12p70, IL-1β, IL-6, and IL-23 in response to R848 combined with LPS or poly I:C. CD141(+) DC were also capable of producing IL-12p70 and IL-23 but were not as proficient as CD1c(+) DC. Activated CD1c(+) DC were endowed with the capacity to promote both Th1 and Th17 effector function in memory CD4(+) T cells, characterized by high production of interferon-γ, IL-17A, IL-17F, IL-21, and IL-22. These findings support a role for CD1c(+) DC in autoimmune inflammation where Th1/Th17 responses play an important role in disease pathogenesis.

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

    PubMed Central

    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 CD44hi, CD62Llo, 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. PMID:25395302

  9. Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8⁺ T Cell Epitopes.

    PubMed

    Becker, Pablo D; Nörder, Miriam; Weissmann, Sebastian; Ljapoci, Ronny; Erfle, Volker; Drexler, Ingo; Guzmán, Carlos A

    2014-07-22

    Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

  10. IL-23-independent induction of IL-17 from γδT cells and innate lymphoid cells promotes experimental intraocular neovascularization.

    PubMed

    Hasegawa, Eiichi; Sonoda, Koh-Hei; Shichita, Takashi; Morita, Rimpei; Sekiya, Takashi; Kimura, Akihiro; Oshima, Yuji; Takeda, Atsunobu; Yoshimura, Takeru; Yoshida, Shigeo; Ishibashi, Tatsuro; Yoshimura, Akihiko

    2013-02-15

    Choroidal neovascularization (CNV) is a characteristic of age-related macular degeneration. Genome-wide association studies have provided evidence that the immune system is involved in the pathogenesis of age-related macular degeneration; however, the role of inflammatory cytokines in CNV has not been established. In this study, we demonstrated that IL-17 had a strong potential for promoting neovascularization in a vascular endothelial growth factor-independent manner in laser-induced experimental CNV in mice. Infiltrated γδT cells and Thy-1(+) innate lymphoid cells, but not Th17 cells, were the main sources of IL-17 in injured eyes. IL-23 was dispensable for IL-17 induction in the eye. Instead, we found that IL-1β and high-mobility group box 1 strongly promoted IL-17 expression by γδT cells. Suppression of IL-1β and high-mobility group box 1, as well as depletion of γδT cells, reduced IL-17 levels and ameliorated experimental CNV. Our findings suggest the existence of a novel inflammatory cytokine network that promotes neovascularization in the eye.

  11. Identification and characterization of a critical CP2-binding element in the human interleukin-4 promoter.

    PubMed

    Casolaro, V; Keane-Myers, A M; Swendeman, S L; Steindler, C; Zhong, F; Sheffery, M; Georas, S N; Ono, S J

    2000-11-24

    Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

  12. Endothelial CD47 promotes Vascular Endothelial-cadherin tyrosine phosphorylation and participates in T-cell recruitment at sites of inflammation in vivo

    PubMed Central

    Azcutia, Veronica; Stefanidakis, Michael; Tsuboi, Naotake; Mayadas, Tanya; Croce, Kevin J.; Fukuda, Daiju; Aikawa, Masanori; Newton, Gail; Luscinskas, Francis W.

    2012-01-01

    At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte Signal Regulatory Proteinγ (SIRPγ) regulate human T-cell TEM. The role of endothelial CD47 in T-cell TEM in vivo, however, has not been explored. Here, CD47−/− mice showed reduced recruitment of blood T-cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-α induced inflammation. Reconstitution of CD47−/− mice with wild type bone marrow (BM) cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47−/− endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in BM chimera mice. In an in vitro human system, CD47 on both HUVEC and T-cells were required for TEM. Although previous studies showed CD47-dependent signaling required Gαi coupled pathways, this was not the case for endothelial CD47 because pertussis toxin (PTX), which inactivates Gαi, had no inhibitory effect, whereas Gαi was required by the T-cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Antibody-induced crosslinking of CD47 revealed robust actin cytoskeleton reorganization and Src and Pyk-2 kinase dependent tyrosine phosphorylation of the VE-cadherin cytoplasmic tail. This signaling was PTX insensitive suggesting that endothelial CD47 signaling is independent of Gαi. These findings suggest that engagement of endothelial CD47 by its ligands triggers “outside-in” signals in endothelium that facilitate leukocyte TEM. PMID:22815286

  13. Characterization of the Canine MHC Class I DLA-88*50101 Peptide Binding Motif as a Prerequisite for Canine T Cell Immunotherapy

    PubMed Central

    Barth, Sharon M.; Schreitmüller, Christian M.; Proehl, Franziska; Oehl, Kathrin; Lumpp, Leonie M.; Kowalewski, Daniel J.; Di Marco, Moreno; Sturm, Theo; Backert, Linus; Schuster, Heiko; Stevanović, Stefan; Rammensee, Hans-Georg; Planz, Oliver

    2016-01-01

    There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA) class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients. PMID:27893789

  14. Different Thermodynamic Binding Mechanisms and Peptide Fine Specificities Associated with a Panel of Structurally Similar High-Affinity T Cell Receptors

    SciTech Connect

    Jones, L.; Colf, L; Bankovich, A; Stone, J; Gao, Y; Chan, C; Huang, R; Garcia, K; Kranz, D

    2008-01-01

    To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L{sup d} as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1{Beta}, 2{alpha}, 3{alpha}, or 3{Beta}. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L{sup d}. Four of the five TCRs examined bound to QL9-L{sup d} in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1{Beta} mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L{sup d} and a single amino acid peptide variant of QL9, called QL9-Y5-Ld. While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1{Beta} exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L{sup d} was solved and compared to structures of the 2C TCR/QL9-L{sup d} complex and three high-affinity TCR/QL9-L{sup d} complexes. Our findings show that the QL9-L{sup d} complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.

  15. Polypyrimidine tract-binding protein is critical for the turnover and subcellular distribution of CD40 ligand mRNA in CD4+ T cells.

    PubMed

    Matus-Nicodemos, Rodrigo; Vavassori, Stefano; Castro-Faix, Moraima; Valentin-Acevedo, Anibal; Singh, Karnail; Marcelli, Valentina; Covey, Lori R

    2011-02-15

    CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 3' untranslated region. To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.

  16. mTOR Inhibition and Alloantigen-Specific Regulatory T Cells Synergize to Promote Long-Term Graft Survival in Immunocompetent Recipients1

    PubMed Central

    Raimondi, Giorgio; Sumpter, Tina L.; Matta, Benjamin M.; Pillai, Mahesh; Corbitt, Natasha; Vodovotz, Yoram; Wang, Zhiliang; Thomson, Angus W.

    2010-01-01

    Minimization of immunosuppression and donor-specific tolerance to MHC-mismatched organ grafts are important clinical goals. The therapeutic potential of regulatory T cells (Treg) has been demonstrated, but conditions for optimizing their in vivo function post-transplant in nonlymphocyte-depleted hosts remain undefined. Herein, we address mechanisms through which inhibition of the mammalian target of rapamycin (Rapa) (mTOR) synergizes with alloAg-specific Treg (AAsTreg), to permit long-term, donor-specific heart graft survival in immunocompetent hosts. Crucially, immature allogeneic dendritic cells (DC) allowed AAsTreg selection in vitro, with minimal expansion of unwanted (TH17) cells. The rendered Treg potently inhibited T cell proliferation in an Ag-specific manner. However, these AAsTreg remained unable to control T cells stimulated by allogeneic mature DC, – a phenomenon dependent on the release of pro-inflammatory cytokines. In vivo, Rapa administration reduced danger-associated IL-6 production, T cell proliferation and graft infiltration. Based on these observation, AAsTreg were administered post-transplant (d7) in combination with a short course of Rapa and rendered >80% long-term (>150 d) graft survival, a result superior to that achieved with polyclonal Treg. Moreover, graft protection was alloAg-specific. Significantly, long-term graft survival was associated with alloreactive T cell anergy. These findings delineate combination of transient mTOR inhibition with appropriate AAsTreg selection as an effective approach to promote long-term organ graft survival. PMID:20007530

  17. Transgenic expression of CXCR3 on T cells enhances susceptibility to cutaneous Leishmania major infection by inhibiting monocyte maturation and promoting a Th2 response.

    PubMed

    Oghumu, Steve; Stock, James C; Varikuti, Sanjay; Dong, Ran; Terrazas, Cesar; Edwards, Jessica A; Rappleye, Chad A; Holovatyk, Ariel; Sharpe, Arlene; Satoskar, Abhay R

    2015-01-01

    Cutaneous leishmaniasis, caused mainly by Leishmania major, an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major. Furthermore, T cells from L. major-susceptible BALB/c but not L. major-resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3(Tg)) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major-infected BALB/c-CXCR3(Tg) mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3(Tg) mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major.

  18. DC-Derived IL-10 Modulates Pro-inflammatory Cytokine Production and Promotes Induction of CD4+IL-10+ Regulatory T Cells during Plasmodium yoelii Infection

    PubMed Central

    Loevenich, Katharina; Ueffing, Kristina; Abel, Simone; Hose, Matthias; Matuschewski, Kai; Westendorf, Astrid M.; Buer, Jan; Hansen, Wiebke

    2017-01-01

    The cytokine IL-10 plays a crucial role during malaria infection by counteracting the pro-inflammatory immune response. We and others demonstrated that Plasmodium yoelii infection results in enhanced IL-10 production in CD4+ T cells accompanied by the induction of an immunosuppressive phenotype. However, it is unclear whether this is a direct effect caused by the parasite or an indirect consequence due to T cell activation by IL-10-producing antigen-presenting cells. Here, we demonstrate that CD11c+CD11b+CD8− dendritic cells (DCs) produce elevated levels of IL-10 after P. yoelii infection of BALB/c mice. DC-specific ablation of IL-10 in P. yoelii-infected IL-10flox/flox/CD11c-cre mice resulted in increased IFN-γ and TNF-α production with no effect on MHC-II, CD80, or CD86 expression in CD11c+ DCs. Accordingly, DC-specific ablation of IL-10 exacerbated systemic IFN-γ and IL-12 production without altering P. yoelii blood stage progression. Strikingly, DC-specific inactivation of IL-10 in P. yoelii-infected mice interfered with the induction of IL-10-producing CD4+ T cells while raising the frequency of IFN-γ-secreting CD4+ T cells. These results suggest that P. yoelii infection promotes IL-10 production in DCs, which in turn dampens secretion of pro-inflammatory cytokines and supports the induction of CD4+IL-10+ T cells. PMID:28293237

  19. Combining autologous dendritic cell therapy with CD3 antibodies promotes regulatory T cells and permanent islet allograft acceptance.

    PubMed

    Baas, Marije C; Kuhn, Chantal; Valette, Fabrice; Mangez, Claire; Duarte, Mercedes Segovia; Hill, Marcelo; Besançon, Alix; Chatenoud, Lucienne; Cuturi, Maria-Cristina; You, Sylvaine

    2014-11-01

    Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation. Copyright © 2014 by The American Association of Immunologists, Inc.

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

    PubMed Central

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

    2010-01-01

    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 (MDSCs) as the tolerogenic APCs capable of antigen uptake and presentation to tumor-specific Tregs. MDSC-mediated Treg induction requires arginase but is TGFβ 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 (PDE-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

  1. Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum

    PubMed Central

    Losada-Barragán, Monica; Umaña-Pérez, Adriana; Cuervo-Escobar, Sergio; Berbert, Luiz Ricardo; Porrozzi, Renato; Morgado, Fernanda N.; Mendes-da-Cruz, Daniella Areas; Savino, Wilson; Sánchez-Gómez, Myriam; Cuervo, Patricia

    2017-01-01

    Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered a primary risk factor for the development of clinical visceral leishmaniasis (VL). Protein malnutrition and infection with Leishmania infantum leads to lymphoid tissue disorganization, including changes in cellularity and lymphocyte subpopulations in the thymus and spleen. Here we report that protein malnutrition modifies thymic chemotactic factors by diminishing the CCL5, CXCL12, IGF1, CXCL9 and CXCL10 protein levels in infected animals. Nevertheless, T cells preserve their migratory capability, as they were able to migrate ex vivo in response to chemotactic stimuli, indicating that malnutrition may compromise the thymic microenvironment and alter in vivo thymocyte migration. Decrease in chemotactic factors protein levels was accompanied by an early increase in the parasite load of the spleen. These results suggest that the precondition of malnutrition is affecting the cell-mediated immune response to L. infantum by altering T cell migration and interfering with the capacity of protein-deprived animals to control parasite spreading and proliferation. Our data provide evidence for a disturbance of T lymphocyte migration involving both central and peripheral T-cells, which likely contribute to the pathophysiology of VL that occurs in malnourished individuals. PMID:28397794

  2. Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature.

    PubMed

    Kleinschnitz, Christoph; Kraft, Peter; Dreykluft, Angela; Hagedorn, Ina; Göbel, Kerstin; Schuhmann, Michael K; Langhauser, Friederike; Helluy, Xavier; Schwarz, Tobias; Bittner, Stefan; Mayer, Christian T; Brede, Marc; Varallyay, Csanad; Pham, Mirko; Bendszus, Martin; Jakob, Peter; Magnus, Tim; Meuth, Sven G; Iwakura, Yoichiro; Zernecke, Alma; Sparwasser, Tim; Nieswandt, Bernhard; Stoll, Guido; Wiendl, Heinz

    2013-01-24

    We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

  3. Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum.

    PubMed

    Losada-Barragán, Monica; Umaña-Pérez, Adriana; Cuervo-Escobar, Sergio; Berbert, Luiz Ricardo; Porrozzi, Renato; Morgado, Fernanda N; Mendes-da-Cruz, Daniella Areas; Savino, Wilson; Sánchez-Gómez, Myriam; Cuervo, Patricia

    2017-04-11

    Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered a primary risk factor for the development of clinical visceral leishmaniasis (VL). Protein malnutrition and infection with Leishmania infantum leads to lymphoid tissue disorganization, including changes in cellularity and lymphocyte subpopulations in the thymus and spleen. Here we report that protein malnutrition modifies thymic chemotactic factors by diminishing the CCL5, CXCL12, IGF1, CXCL9 and CXCL10 protein levels in infected animals. Nevertheless, T cells preserve their migratory capability, as they were able to migrate ex vivo in response to chemotactic stimuli, indicating that malnutrition may compromise the thymic microenvironment and alter in vivo thymocyte migration. Decrease in chemotactic factors protein levels was accompanied by an early increase in the parasite load of the spleen. These results suggest that the precondition of malnutrition is affecting the cell-mediated immune response to L. infantum by altering T cell migration and interfering with the capacity of protein-deprived animals to control parasite spreading and proliferation. Our data provide evidence for a disturbance of T lymphocyte migration involving both central and peripheral T-cells, which likely contribute to the pathophysiology of VL that occurs in malnourished individuals.

  4. 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.

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

    PubMed

    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 (~ 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.

  6. Passive immunization with myelin basic protein activated T cells suppresses axonal dieback but does not promote axonal regeneration following spinal cord hemisection in adult rats.

    PubMed

    Wang, Hong-Ju; Hu, Jian-Guo; Shen, Lin; Wang, Rui; Wang, Qi-Yi; Zhang, Chen; Xi, Jin; Zhou, Jian-Sheng; Lü, He-Zuo

    2012-08-01

    The previous studies suggested that some subpopulations of T lymphocytes against central nervous system (CNS) antigens, such as myelin basic protein (MBP), are neuroprotective. But there were few reports about the effect of these T cells on axon regeneration. In this study, the neonatally thymectomied (Tx) adult rats which contain few T lymphocytes were subjected to spinal cord hemisection and then passively immunized with MBP-activated T cells (MBP-T). The regeneration and dieback of transected axons of cortico-spinal tract (CST) were detected by biotin dextran amine (BDA) tracing. The behavioral assessments were performed using the Basso, Beattie, and Bresnahan locomotor rating scale. We found that passive transferring of MBP-T could attenuate axonal dieback. However, no significant axon regeneration and behavioral differences were observed among the normal, Tx and sham-Tx (sTx) rats with or without MBP-T passive immunization. These results indicate that passive transferring of MBP-T cells can attenuate axonal dieback and promote neuroprotection following spinal cord injury (SCI), but may not promote axon regeneration.

  7. Induced miR-99a expression represses Mtor cooperatively with miR-150 to promote regulatory T-cell differentiation

    PubMed Central

    Warth, Sebastian C; Hoefig, Kai P; Hiekel, Anian; Schallenberg, Sonja; Jovanovic, Ksenija; Klein, Ludger; Kretschmer, Karsten; Ansel, K Mark; Heissmeyer, Vigo

    2015-01-01

    Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4+ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4+ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs. PMID:25712478

  8. mTOR Promotes Antiviral Humoral Immunity by Differentially Regulating CD4 Helper T Cell and B Cell Responses.

    PubMed

    Ye, Lilin; Lee, Junghwa; Xu, Lifan; Mohammed, Ata-Ur-Rasheed; Li, Weiyan; Hale, J Scott; Tan, Wendy G; Wu, Tuoqi; Davis, Carl W; Ahmed, Rafi; Araki, Koichi

    2017-02-15

    mTOR has important roles in regulation of both innate and adaptive immunity, but whether and how mTOR modulates humoral immune responses have yet to be fully understood. To address this issue, we examined the effects of rapamycin, a specific inhibitor of mTOR, on B cell and CD4 T cell responses during acute infection with lymphocytic choriomeningitis virus. Rapamycin treatment resulted in suppression of virus-specific B cell responses by inhibiting proliferation of germinal center (GC) B cells. In contrast, the number of memory CD4 T cells was increased in rapamycin-treated mice. However, the drug treatment caused a striking bias of CD4 T cell differentiation into Th1 cells and substantially impaired formation of follicular helper T (Tfh) cells, which are essential for humoral immunity. Further experiments in which mTOR signaling was modulated by RNA interference (RNAi) revealed that B cells were the primary target cells of rapamycin for the impaired humoral immunity and that reduced Tfh formation in rapamycin-treated mice was due to lower GC B cell responses that are essential for Tfh generation. Additionally, we found that rapamycin had minimal effects on B cell responses activated by lipopolysaccharide (LPS), which stimulates B cells in an antigen-independent manner, suggesting that rapamycin specifically inhibits B cell responses induced by B cell receptor stimulation with antigen. Together, these findings demonstrate that mTOR signals play an essential role in antigen-specific humoral immune responses by differentially regulating B cell and CD4 T cell responses during acute viral infection and that rapamycin treatment alters the interplay of immune cell subsets involved in antiviral humoral immunity. mTOR is a serine/threonine kinase involved in a variety of cellular activities. Although its specific inhibitor, rapamycin, is currently used as an immunosuppressive drug in transplant patients, it has been reported that rapamycin can also stimulate pathogen

  9. HIV-derived lentiviral particles promote T-cell independent activation and differentiation of naïve cognate conventional B2-cells in vitro.

    PubMed

    Gardt, Oliver; Grewe, Bastian; Tippler, Bettina G; Überla, Klaus; Temchura, Vladimir V

    2013-10-17

    In animal models, lentiviral particles (LP) were shown to be promising HIV vaccine candidates. Since little is known about the direct impact of LP on antigen-specific B cells, we incorporated Hen Egg Lysozyme (HEL) into LP (HEL-LP) derived from HIV to study their effect on HEL-specific, B cell receptor-transgenic B-cells (HEL(+)B-cells) in vitro. We observed preferential binding of HEL-LP to HEL(+)B-cells and their efficient internalization. HEL-LP were able to effectively cross-link B-cell receptors as indicated by the loss of surface CD62L. In the absence of CD4(+) T-cells, other activation events induced by LP in cognate naïve B-cells included increased expression of activation and co-stimulatory molecules as well as an enhanced proliferative response. Additionally, the B-cell phenotype shifted toward a germinal center pattern with further differentiation into memory and IgG3- and IgA-producing cells. The observed CD4(+) T-cell independent activation and differentiation may be due to LP-induced expression of CD40L by a subset of cognate B-cells. Thus, even in the absence of CD4(+) T-cells LP provide strong direct activation signals to cognate naïve B-cells, which may contribute to the strong humoral immune responses observed after LP immunization.

  10. Specific binding of phorbol ester tumor promoters

    PubMed Central

    Driedger, Paul E.; Blumberg, Peter M.

    1980-01-01

    [20-3H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a Kd of 25 nM; this value is very close to the 50% effective dose (ED50), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20-3H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 × 104 molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate (Ki = 2 nM), mezerein (Ki = 180 nM), phorbol 12,13-dibenzoate (Ki = 180 nM), phorbol 12,13-diacetate (Ki = 1.7 μM), phorbol 12,13,20-triacetate (Ki = 39 μM), and phorbol 13-acetate (Ki = 120 μM). The measured Ki values are all within a factor of 3.5 of the ED50 values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 μg/ml) and 4α-phorbol 12,13-didecanoate (10 μg/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate 20-acetate (100 ng/ml). These data suggest that biological responses to the phorbol esters in chicken embryo fibroblasts are mediated by this binding activity and that the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 μM), phenol (1 mM), iodoacetic acid (1.7 μM), and cantharidin (75 μM), or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 μM), retinoic acid (10 μM), or prostaglandin E2 (1 μM). These agents thus appear to act at a target distinct from that of the phorbol esters. PMID:6965793

  11. 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

  12. A CD4+ T cell line-secreted factor, growth promoting for normal and leukemic B cells, identified as thioredoxin.

    PubMed

    Rosen, A; Lundman, P; Carlsson, M; Bhavani, K; Srinivasa, B R; Kjellström, G; Nilsson, K; Holmgren, A

    1995-04-01

    In this study, a B cell growth stimulatory factor, constitutively secreted by a human CD4+ T cell hybridoma clone, MP6, has been purified and characterized. Serum-free 24 h culture media from MP6 cells were collected, concentrated by ultrafiltration and separated by gel chromatography. Fractions were analyzed for stimulatory activity using [3H]thymidine incorporation in normal and leukemic (B-CLL) B cells as target cells. Activity was present in a 12 kDa protein peak. Upon storage this lost activity indicating that the factor was sensitive to air oxidation, a well-known property of mammalian thioredoxins (Trxs). Treatment of the inactive fraction with dithiothreitol restored full activity. When culture medium was analyzed with a radioimmunoassay for human placenta Trx, the MP6 clone was shown to release 30-50 ng/ml per million cells during 24 h. The B cell stimulatory activity of the MP6 medium was removed by Sepharose-bound anti-human placenta Trx IgG and activity was recovered by elution from the antibodies. Furthermore, MP6 medium showed Trx activity with NADPH and Trx reductase using an insulin disulfide reduction assay. Starting from 5 l of serum-free MP6 conditioned medium, the Trx was purified approximately 100,000-fold. After gel electrophoresis banding, the material was analyzed by peptide sequencing and a full length sequence of an 104 amino acid long protein was obtained. This Trx sequence was identical to the previously published sequence of human Trx from HTLV-1 transformed T cells, adult T cell leukemia-derived factor/Trx.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin

    SciTech Connect

    Maki, Noboru; Sekiguchi, Fumiko; Nishimaki, Junichi; Miwa, Keiko; Hayano, Toshiya; Takahashi, Nobuhiro; Suzuki, Masanori )

    1990-07-01

    The recently discovered macrolide FK506 has been demonstrated to have potent immunosuppressive activity at concentrations 100-fold lower than cyclosporin A, a cyclic undecapeptide that is used to prevent rejection after transplantation of bone marrow and organs, such as kidney, heart, and liver. After the recent discovery that the cylcosporin A-binding protein cyclophilin is identical to peptidylprolyl cis-trans isomerase, a cellular binding protein for FK506 was found to be distinct from cyclophilin but to have the same enzymatic activity. In this study, the authors isolated a cDNA coding for FK506-binding protein (FKBP) from human peripheral blood T cells by using mixed 20-mer oligonucleotide probes synthesized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys-Phe-Asp, reported for bovine FKBP. The DNA isolated contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence were identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the DNA sequence isolated represents the gene coding for FKBP. This result suggests that two catalytically similar proteins, cyclophilin and FKBP, evolved independently. In Northern blot analysis, mRNA species of {approx}1.8 kilobases that hybridized with human FKBP cDNA were detected in poly(A){sup +} RNAs from brain, lung, liver, and placental cells and leukocytes. Induction of Jurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not affect the level of FKBP mRNA.

  14. The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells.

    PubMed

    van Leeuwen, J E; Paik, P K; Samelson, L E

    1999-10-01

    T-cell receptor (TCR) engagement results in the activation of Src family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosphorylation of multiple cellular substrates including the complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of growth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negative regulatory phosphotyrosine. We recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Moreover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletion of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways. Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncogenic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of transcription factors.

  15. Human T-Lymphoid Progenitors Generated in a Feeder-Cell-Free Delta-Like-4 Culture System Promote T-Cell Reconstitution in NOD/SCID/γc−/− Mice

    PubMed Central

    Reimann, Christian; Six, Emmanuelle; Dal-Cortivo, Liliane; Schiavo, Andrea; Appourchaux, Kevin; Lagresle-Peyrou, Chantal; de Chappedelaine, Corinne; Ternaux, Brigitte; Coulombel, Laure; Beldjord, Kheira; Cavazzana-Calvo, Marina; Andre-Schmutz, Isabelle

    2012-01-01

    Abstract Slow T-cell reconstitution is a major clinical concern after transplantation of cord blood (CB)-derived hematopoietic stem cells. Adoptive transfer of in vitro-generated T-cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T-cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Culture of human CD34+ CB cells in this new DL-4 system enabled the in vitro generation of large amounts of T-cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc−/− (NSG) mice, DL-4 primed T-cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αβ T cells that subsequently left the thymus and accelerated T-cell reconstitution. T-cell reconstitution was even faster and more robust when ex vivo-manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro-generated human T-cell progenitors to accelerate T-cell reconstitution and also introduces a feeder-cell-free culture technique with the potential for rapid, safe transfer to a clinical setting. PMID:22689616

  16. 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

  17. Adhesion to fibronectin promotes the activation of the p125FAK/Zap‐70 complex in human T cells

    PubMed Central

    Bearz, A; Tell, G; Formisano, S; Merluzzi, S; Colombatti, A; Pucillo, C

    1999-01-01

    The β1 integrins are a family of heterodimeric adhesion receptors involved in cell‐to‐cell contacts and cell‐to‐extracellular matrix interactions. Through their adhesive role, integrins participate in transduction of outside/inside signals and contribute to trigger a multitude of cellular events such as differentiation, cell activation, and motility. The fibronectin integrin receptors, α4β1 and α5β1, can function as costimulatory molecules in T‐cell receptor (TCR)‐dependent T‐cell activation. In the current study the Jurkat T‐cell line was used as a model system to investigate the TCR‐independent role of cell adhesion to fibronectin in the activation of Zap‐70, a central molecule in the signalling events in T cells. Upon adhesion to plastic immobilized fibronectin but not to bovine serum albumin (BSA) the phosphorylation of p125FAK, a protein kinase that localizes to focal adhesion sites, was induced. Moreover, clustering of fibronectin receptors led to the detection of a p125FAK/Zap‐70 complex. Finally, while the complex between fak‐B, another protein kinase localized to focal adhesion sites, and Zap‐70 was detected in cells plated either on BSA or on fibronectin, the formation of the p125FAK/Zap‐70 complex appeared specifically induced following fibronectin‐mediated integrin clustering. These data suggest the existence of a high degree of specificity when the members of the β1 integrin family mediate signalling pathways in T cells. PMID:10594689

  18. 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

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

    PubMed

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

    2016-01-25

    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.

  20. 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

  1. 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.

  2. Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Vella, Jennifer L.; Reis, Isildinha M.; De la fuente, Adriana C.; Gomez, Carmen; Sargi, Zoukaa; Nazarian, Ronen; Califano, Joseph; Borrello, Ivan

    2015-01-01

    Purpose Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play a key role in the progression of head and neck squamous cell carcinoma (HNSCC). On the basis of our preclinical data demonstrating that phosphodiesterase-5 (PDE5) inhibition can modulate these cell populations, we evaluated whether the PDE5 inhibitor tadalafil can revert tumor-induced immunosuppression and promote tumor immunity in patients with HNSCC. Experimental Design First, we functionally and phenotypically characterized MDSCs in HNSCCs and determined, retrospectively, whether their presence at the tumor site correlates with recurrence. Then, we performed a prospective single-center, double-blinded, randomized, three-arm study in which patients with HNSCC undergoing definitive surgical resection of oral and oropharyngeal tumors were treated with tadalafil 10 μg/day, 20 μg/day, or placebo for at least 20 days preoperatively. Blood and tumor MDSC and Treg presence and CD8+ T-cell reactivity to tumor antigens were evaluated before and after treatment. Results MDSCs were characterized in HNSCC and their intratumoral presence significantly correlates with recurrence. Tadalafil treatment was well tolerated and significantly reduced both MDSCs and Treg concentrations in the blood and in the tumor (P < 0.05). In addition, the concentration of blood CD8+ T cells reactive to autologous tumor antigens significantly increased after treatment (P < 0.05). Tadalafil immunomodulatory activity was maximized at an intermediate dose but not at higher doses. Mechanistic analysis suggests a possible off-target effect on PDE11 at high dosages that, by increasing intracellular cAMP, may negatively affect antitumor immunity. Conclusions Tadalafil seems to beneficially modulate the tumor micro- and macro-environment in patients with HNSCC by lowering MDSCs and Tregs and increasing tumor-specific CD8+ T cells in a dose-dependent fashion. PMID:25320361

  3. Tim-1(+) B cells suppress T cell interferon-gamma production and promote Foxp3 expression, but have impaired regulatory function in coronary artery disease.

    PubMed

    Gu, Xiao-Long; He, Huan; Lin, Lin; Luo, Guo-Xin; Wen, Yan-Fei; Xiang, Ding-Cheng; Qiu, Jian

    2017-10-01

    Atherosclerosis and its associated coronary artery disease (CAD) represent another chronic low-grade inflammatory disorder. Regulatory B cells (Bregs) possess essential functions in maintaining peripheral tolerance and inhibiting pathogenic inflammation through IL-10. Here, we investigated one subset of Bregs, Tim-1(+) B cell, and its role in atherosclerosis and CAD patients. In healthy individuals, IL-10-producing B cells were predominantly found in the Tim-1(+) B cells. Upon stimulation of the B cell receptor (BCR) and Toll-like receptor 9 (TLR-9) by anti-BCR antibodies and CpG, respectively, the Tim-1(+) B cells could further upregulate IL-10 expression. In contrast, the Tim-1(+) B cells were present at normal frequency in CAD patients, but showed impaired capacity to upregulate IL-10 with or without BCR + CpG stimulation. The stimulated Tim-1(+) B cells from healthy individuals also suppressed expression of interferon gamma (IFN-γ), an atherogenic cytokine in T cells, in an IL-10-dependent fashion, and strongly promoted the expression of Foxp3 in naive CD4(+) CD45RO(-) T cells. In contrast, the Tim-1(+) B cells from CAD patients were unable to suppress IFN-γ secretion, and only minimally increased the expression of Foxp3 in naive CD4(+) CD45RO(-) T cells. Despite this, the frequency of Tim-1(+) B cells in the atherosclerotic lesions from CAD patients was inversely correlated with the frequency of IFN-γ-expressing T cells. Together, these results demonstrated that CAD patients presented an inflammatory disorder in regulatory B cells, which could be used as a therapeutic target. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  4. CD40 promotes MHC class II expression on adipose tissue macrophages and regulates adipose tissue CD4+ T cells with obesity.

    PubMed

    Morris, David L; Oatmen, Kelsie E; Mergian, Taleen A; Cho, Kae Won; DelProposto, Jennifer L; Singer, Kanakadurga; Evans-Molina, Carmella; O'Rourke, Robert W; Lumeng, Carey N

    2016-06-01

    Obesity activates both innate and adaptive immune responses in adipose tissue, but the mechanisms critical for regulating these responses remain unknown. CD40/CD40L signaling provides bidirectional costimulatory signals between antigen-presenting cells and CD4(+) T cells, and CD40L expression is increased in obese humans. Therefore, we examined the contribution of CD40 to the progression of obesity-induced inflammation in mice. CD40 was highly expressed on adipose tissue macrophages in mice, and CD40/CD40L signaling promoted the expression of antigen-presenting cell markers in adipose tissue macrophages. When fed a high fat diet, Cd40-deficient mice had reduced accumulation of conventional CD4(+) T cells (Tconv: CD3(+)CD4(+)Foxp3(-)) in visceral fat compared with wild-type mice. By contrast, the number of regulatory CD4(+) T cells (Treg: CD3(+)CD4(+)Foxp3(+)) in lean and obese fat was similar between wild-type and knockout mice. Adipose tissue macrophage content and inflammatory gene expression in fat did not differ between obese wild-type and knockout mice; however, major histocompatibility complex class II and CD86 expression on adipose tissue macrophages was reduced in visceral fat from knockout mice. Similar results were observed in chimeric mice with hematopoietic Cd40-deficiency. Nonetheless, neither whole body nor hematopoietic disruption of CD40 ameliorated obesity-induced insulin resistance in mice. In human adipose tissue, CD40 expression was positively correlated with CD80 and CD86 expression in obese patients with type 2 diabetes. These findings indicate that CD40 signaling in adipose tissue macrophages regulates major histocompatibility complex class II and CD86 expression to control the expansion of CD4(+) T cells; however, this is largely dispensable for the development of obesity-induced inflammation and insulin resistance in mice. © Society for Leukocyte Biology.

  5. Angiotensin II Promotes the Development of Carotid Atherosclerosis in Type 2 Diabetes Patients via Regulating the T Cells Activities: A Cohort Study

    PubMed Central

    Wang, Kai; Jin, Feng; Zhang, Zhanpu; Sun, Xiaochuan

    2016-01-01

    Background Specific T cell phenotype has been reported to potentially contribute to the development of angiotensin II (Ang II)-induced several vascular disorders. Type 2 diabetes mellitus (T2DM) is intimately associated with cardiovascular disease. The present study aimed to investigate the relationship between T cell phenotypes and Ang II in T2DM patients combined with carotid atherosclerosis (CA). Material/Methods This study was performed on 50 patients with T2DM in our hospital. Based on the presence of CA, they were divided into CA group (presence of CA, n=30) or T2DM group (absence of CA, n=20). Additionally, 10 healthy participants were selected as controls. Basic characteristics of all participants were collected and recorded. Peripheral blood mononuclear cells (PBMCs) isolated from patients and controls with or without Ang II and Ang II receptor blocker (ARB) treatment were used to detect Th1, Th2, and Th17 cell proportions, mRNA levels of T-bet, GATA3, and RORγt as well as the expression of IFN-γ, IL-4, and IL-17 by flow cytometry, ELISA, and Real-Time PCR. Results Ang II levels were notably higher in patients in the CA group than those in the T2DM and control group (p<0.05). Th1 and Th17 positive cells, mRNA levels of T-bet and RORγt as well as the expression of IFN-γ and IL-17 were significantly increased in the CA group compared with the T2DM group and control group (p<0.05). Moreover, the activities of T cells and related cytokines were significantly increased of healthy controls after Ang II treatment (p<0.05), while these changes were notably weakened by ARB treatment (p<0.05). Conclusions Ang II promotes the development of CA in T2DM patients by regulating T cells activities. PMID:27782101

  6. The T cell response to allelic determinants on Igh-1-encoded IgG2a antibodies: dual recognition examined by using antigenic antibodies with binding affinity for Ia molecules.

    PubMed

    Hedrick, S M; Schwartz, R H

    1983-04-01

    In this report we examine the effects of binding antigen directly to Ia molecules during immunization and stimulation of T cells in culture. To accomplish this we characterize the T cell response to Igh-1j-encoded IgG2a antibodies in C57BL/10 H-2 congenic mice. In contrast to the response to the closely related Igh-1a-encoded IgG2a antibodies, high responder mice were (C57BL/10 X B10.A)F1 hybrids, and the important alleles appeared to map to I-Ab and I-Ak. C57BL/10 and B10.A(5R) strains were low responders (I-Ab), and B10.A and B10.A(4R) strains were nonresponders (I-Ak). The interest in using monoclonal IgG2a antibodies as T cell antigens was that different V-region-encoded binding specificities could be associated with the same IgG2a antigenic determinant. In our interpretation of the Langman and Cohn dual binding site model of the T cell receptor, the response to anti-Ia antibody should have some of the same characteristics as that to an allogeneic Ia molecule; in particular, it should be non-H-2-restricted. Although the response to anti-Ia antibody had several interesting properties, no difference in the restriction specificity was found for the T cell response to anti-Ia antibodies relative to unreactive IgG2a antibodies. Our conclusion is that T cell receptors must bind both Ia and antigen molecules for activation to occur. In a separate set of experiments, the T cell response resulting from immunization with anti-Ia antibody was examined. Because the antigen was shown to be associated with the Ia molecule during immunization, experiments were designed to detect an alteration or lack of H-2 restriction in the resulting T cell population. A long-term T cell line induced after such immunization showed no qualitative differences from a T cell line produced with unreactive IgG2a antibody. These results demonstrate that simply attaching a foreign antigen to a restriction element is not sufficient to lead to unrestricted T cell activation. This in turn suggests the

  7. 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.

  8. CREB-2, a Cellular CRE-Dependent Transcription Repressor, Functions in Association with Tax as an Activator of the Human T-Cell Leukemia Virus Type 1 Promoter

    PubMed Central

    Gachon, Frederic; Peleraux, Annick; Thebault, Sabine; Dick, Joelle; Lemasson, Isabelle; Devaux, Christian; Mesnard, Jean-Michel

    1998-01-01

    The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent. PMID:9733879

  9. Fucoidan can function as an adjuvant in vivo to enhance dendritic cell maturation and function and promote antigen-specific T cell immune responses.

    PubMed

    Jin, Jun-O; Zhang, Wei; Du, Jiang-Yuan; Wong, Ka-Wing; Oda, Tatsuya; Yu, Qing

    2014-01-01

    Fucoidan, a sulfated polysaccharide purified from brown algae, has a variety of immune-modulation effects, including promoting antigen uptake and enhancing anti-viral and anti-tumor effects. However, the effect of fucoidan in vivo, especially its adjuvant effect on in vivo anti-tumor immune responses, was not fully investigated. In this study, we investigated the effect of fucoidan on the function of spleen dendritic cells (DCs) and its adjuvant effect in vivo. Systemic administration of fucoidan induced up-regulation of CD40, CD80 and CD86 expression and production of IL-6, IL-12 and TNF-α in spleen cDCs. Fucoidan also promoted the generation of IFN-γ-producing Th1 and Tc1 cells in an IL-12-dependent manner. When used as an adjuvant in vivo with ovalbumin (OVA) antigen, fucoidan promoted OVA-specific antibody production and primed IFN-γ production in OVA-specific T cells. Moreover, fucoidan enhanced OVA-induced up-regulation of MHC class I and II on spleen cDCs and strongly prompted the proliferation of OVA-specific CD4 and CD8 T cells. Finally, OVA immunization with fucoidan as adjuvant protected mice from the challenge with B16-OVA tumor cells. Taken together, these results suggest that fucoidan can function as an adjuvant to induce Th1 immune response and CTL activation, which may be useful in tumor vaccine development.

  10. Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum.

    PubMed Central

    Jakobsen, P H; Hviid, L; Theander, T G; Afare, E A; Ridley, R G; Heegaard, P M; Stuber, D; Dalsgaard, K; Nkrumah, F K

    1993-01-01

    The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living in an area with a high rate of transmission of malaria. Lymphocytes from a large proportion of the Ghanaian blood donors proliferated in response to the RAP-1 peptide, unlike those of Danish control blood donors, indicating that this sequence contains a malaria-specific T-cell epitope broadly recognized by individuals living in an area with a high transmission rate of malaria. Most of the donor plasma samples tested contained immunoglobulin G (IgG) and IgM antibodies recognizing the merozoite proteins, while only a minority showed high IgG reactivity to the synthetic peptides. PMID:8418048

  11. Oxidized multiwalled carbon nanotubes as antigen delivery system to promote superior CD8(+) T cell response and protection against cancer.

    PubMed

    de Faria, Paula Cristina Batista; dos Santos, Luara Isabela; Coelho, João Paulo; Ribeiro, Henrique Bücker; Pimenta, Marcos Assunção; Ladeira, Luiz Orlando; Gomes, Dawidson Assis; Furtado, Clascídia Aparecida; Gazzinelli, Ricardo Tostes

    2014-09-10

    Properties like high interfacial area with cellular membranes, unique ability to incorporate multiple functionalization, as well as compatibility and transport in biological fluids make carbon nanotubes (CNTs) useful for a variety of therapeutic and drug-delivery applications. Here we used a totally synthetic hybrid supramolecule as an anticancer vaccine formulation. This complex structure comprises CNTs as delivery system for the Cancer Testis Antigen named NY-ESO-1, allied to a synthetic Toll-Like Receptor agonist. The CNT constructs were rapidly internalized into dendritic cells, both in vitro and in vivo, and served as an intracellular antigen depot. This property favored the induction of strong CD4(+) T as well as CD8(+) T cell-mediated immune responses against the NY-ESO-1. Importantly, the vaccination significantly delayed the tumor development and prolonged the mice survival, highlighting the potential application of CNTs as a vaccine delivery system to provide superior immunogenicity and strong protection against cancer.

  12. Rapid Copper Acquisition by Developing Murine Mesothelioma: Decreasing Bioavailable Copper Slows Tumor Growth, Normalizes Vessels and Promotes T Cell Infiltration

    PubMed Central

    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. PMID:24013775

  13. Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival1

    PubMed Central

    Chen, Qian; Davidson, Todd S.; Huter, Eva N.; Shevach, Ethan M.

    2009-01-01

    TLRs are a class of conserved pattern recognition receptors that are used by cells of the innate immune system. Recent studies have demonstrated the expression of TLRs on both human and mouse T cells raising the possibility that TLRs play a direct role in adaptive immunity. TLR2 is activated primarily by bacterial wall components including peptidoglycan and lipoproteins. Several studies have shown that mouse regulatory T (Treg) express TLR2 and claimed that engagement of TLR2 by synthetic ligands reversed their suppressive function. In contrary, enhancement of Treg function was observed following engagement of TLR2 on human Treg. We have re-examined the expression and function of TLR2 on mouse Treg purified from Foxp3-GFP knock in mice. TLR2 ligation by TLR2 agonist, the synthetic bacterial lipoprotein (BLP) Pam3CSK4, enhanced the proliferative responses of both conventional T cells and Treg in response to TLR stimulation in the absence of APC. Treatment of Foxp3+ Treg with Pam3CSK4 did not alter their suppressive function in vitro or in vivo and did not reduce their level of Foxp3 expression. An additional effect of TLR2 stimulation of Treg was induction of Bcl-xL resulting in enhanced survival in vitro. Treatment of mice with the TLR2 agonist enhanced the antigen-driven proliferation of Treg in vivo, but did not abolish their ability to suppress the development of EAE. Development of methods to selectively stimulate TLR2 on Treg may lead to a novel approaches for the treatment of autoimmune diseases. PMID:19748987

  14. Engagement of TLR2 does not reverse the suppressor function of mouse regulatory T cells, but promotes their survival.

    PubMed

    Chen, Qian; Davidson, Todd S; Huter, Eva N; Shevach, Ethan M

    2009-10-01

    TLRs are a class of conserved pattern recognition receptors that are used by cells of the innate immune system. Recent studies have demonstrated the expression of TLRs on both human and mouse T cells raising the possibility that TLRs play a direct role in adaptive immunity. TLR2 is activated primarily by bacterial wall components including peptidoglycan and lipoproteins. Several studies have shown that mouse regulatory T (Treg) cells express TLR2 and claimed that engagement of TLR2 by synthetic ligands reversed their suppressive function. In contrary, enhancement of Treg function was observed following engagement of TLR2 on human Treg. We have reexamined the expression and function of TLR2 on mouse Treg purified from Foxp3-GFP knock-in mice. TLR2 ligation by TLR2 agonist, the synthetic bacterial lipoprotein Pam3CSK4, enhanced the proliferative responses of both conventional T cells and Treg in response to TLR stimulation in the absence of APC. Treatment of Foxp3+ Treg with Pam3CSK4 did not alter their suppressive function in vitro or in vivo and did not reduce their level of Foxp3 expression. An additional effect of TLR2 stimulation of Treg was induction of Bcl-x(L) resulting in enhanced survival in vitro. Treatment of mice with the TLR2 agonist enhanced the Ag-driven proliferation of Treg in vivo, but did not abolish their ability to suppress the development of experimental autoimmune encephalomyelitis. Development of methods to selectively stimulate TLR2 on Treg may lead to a novel approaches for the treatment of autoimmune diseases.

  15. Zinc Binding and Dimerization of Streptococcus pyogenes Pyrogenic Exotoxin C Are Not Essential for T-Cell Stimulation

    DTIC Science & Technology

    2003-03-14

    MS and, if needed, by an N-terminal amino acid sequencing . The final purity was greater than 95% as judged by LC-MS. The recombinant HLA - DR1 protein...the MHCII HLA - DR1 indicates a potentially lower affinity zinc coordination site, present at the interface with the receptor (6), where one of the...receptor binding were performed on a Biacore 3000 (Biacore Inc., Piscataway, NJ). In a typical experiment, a solution of recombinant HLA - DR1 in 10 mM

  16. Effects of chlorella on activities of protein tyrosine phosphatases, matrix metalloproteinases, caspases, cytokine release, B and T cell proliferations, and phorbol ester receptor binding.

    PubMed

    Cheng, Fong-Chi; Lin, Atsui; Feng, Jin-Jye; Mizoguchi, Toru; Takekoshi, Hideo; Kubota, Hitoshi; Kato, Yoko; Naoki, Yo

    2004-01-01

    A Chlorella powder was screened using 52 in vitro assay systems for enzyme activity, receptor binding, cellular cytokine release, and B and T cell proliferation. The screening revealed a very potent inhibition of human protein tyrosine phosphatase (PTP) activity of CD45 and PTP1C with 50% inhibitory concentration (IC(50)) values of 0.678 and 1.56 microg/mL, respectively. It also showed a moderate inhibition of other PTPs, including PTP1B (IC(50) = 65.3 microg/mL) and T-cell-PTP (114 microg/mL). Other inhibitory activities and their IC(50) values included inhibition of the human matrix metalloproteinases (MMPs) MMP-1 (127 microg/mL), MMP-3 (185 microg/mL), MMP-7 (18.1 microg/mL), and MMP-9 (237 microg/mL) and the human peptidase caspases caspase 1 (300 microg/mL), caspase 3 (203 microg/mL), caspase 6 (301 microg/mL), caspase 7 (291 microg/mL), and caspase 8 (261 microg/mL), as well as release of the cytokines interleukin (IL)-1 (44.9 microg/mL), IL-2 (14.8 microg/mL), IL-4 (49.2 microg/mL), IL-6 (34.7 microg/mL), interferon-gamma (31.6 microg/mL), and tumor necrosis factor-alpha (11 microg/mL) from human peripheral blood mononuclear cells. Chlorella also inhibited B cell proliferation (16.6 microg/mL) in mouse splenocytes and T cell proliferation (54.2 microg/mL) in mouse thymocytes. The binding of a phorbol ester, phorbol 12,13-dibutyrate, to its receptors was also inhibited by Chlorella with an IC(50) of 152 microg/mL. These results reveal potential pharmacological activities that, if confirmed by in vivo studies, might be exploited for the prevention or treatment of several serious pathologies, including inflammatory disease and cancer.

  17. Macrophages in T cell/histiocyte rich large B cell lymphoma strongly express metal-binding proteins and show a bi-activated phenotype.

    PubMed

    Hartmann, Sylvia; Tousseyn, Thomas; Döring, Claudia; Flüchter, Patricia; Hackstein, Holger; Herreman, An; Ponzoni, Maurilio; de Wolf-Peeters, Chris; Facchetti, Fabio; Gascoyne, Randy D; Küppers, Ralf; Steidl, Christian; Hansmann, Martin-Leo

    2013-12-01

    Abundant macrophage infiltration in tumors often correlates with a poor prognosis. T cell/histiocyte rich large B cell lymphoma (THRLBCL) is a distinct aggressive B cell lymphoma entity showing a high macrophage content. To further elucidate the role of tumor-associated macrophages in THRLBCL, we performed gene expression profiling of microdissected histiocyte subsets of THRLBCL, nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), Piringer lymphadenitis, sarcoidosis, nonspecific lymphadenitis and monocytes from peripheral blood. In a supervised principal component analysis, histiocytes from THRLBCL were most closely related to epithelioid cells from NLPHL, with both types of cells expressing genes related to proinflammatory and regulatory macrophage activity. Moreover, histiocytes from THRLBCL strongly expressed metal-binding proteins like MT2A, by which histiocytes of