scholarly journals Small molecule inhibition of Csk alters affinity recognition by T cells

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Boryana N Manz ◽  
Ying Xim Tan ◽  
Adam H Courtney ◽  
Florentine Rutaganira ◽  
Ed Palmer ◽  
...  
Keyword(s):  
T Cells ◽  
Catalytic Activity ◽  
T Cell ◽  
Small Molecule ◽  
Src Family Kinases ◽  
Negative Regulator ◽  
Proliferation Inhibition ◽  
Tcr Signaling ◽  
Cell Responses ◽  
Small Molecule Inhibition

The C-terminal Src kinase (Csk), the primary negative regulator of Src-family kinases (SFK), plays a crucial role in controlling basal and inducible receptor signaling. To investigate how Csk activity regulates T cell antigen receptor (TCR) signaling, we utilized a mouse expressing mutated Csk (CskAS) whose catalytic activity is specifically and rapidly inhibited by a small molecule. Inhibition of CskAS during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation. Inhibition of CskAS enhanced activation by weak but strictly cognate agonists. Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists. Csk plays an important role, not only in basal signaling, but also in setting the TCR signaling threshold and affinity recognition.

scholarly journals 752 Novel, orally administered HPK1 inhibitors demonstrate anti-tumor efficacy and enhanced immune response

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A786-A786
Author(s):  
Stefan Chmielewski ◽  
Maciej Kujawa ◽  
Eliza Zimolag ◽  
Michal Galezowski ◽  
Andrzej Gondela ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Small Molecule ◽  
Cell Function ◽  
Adaptor Protein ◽  
Negative Regulator ◽  
Tcr Signaling ◽  
T Cell Function ◽  
Tcr Activation

BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1) is emerging as a well-renowned, druggable target for T cell-based immunotherapies. HPK1 is a member of the serine/threonine MAP4K family, predominantly expressed in hematopoietic cell lineages and shown to be a negative regulator of the T cell receptor (TCR) signaling pathway. Upon TCR activation, HPK1 is recruited to the proximity of the cell membrane and phosphorylates an adaptor protein SLP-76 at the Ser376 residue which, in turn, abrogates TCR signaling. Other studies point to a potential role of HPK1 in T cell exhaustion as well as in functional re-programming of regulatory T cells. Moreover, mounting evidence suggest that HPK1 kinase activity suppresses the immune functions of a wide range of other immune cell subsets like B cells and dendritic cells. Taken together, these observations support small-molecule HPK1 inhibitors as an attractive modality in cancer immunotherapy either as single agents or in combination with immune checkpoint inhibitors.MethodsActivity of compounds against HPK1 and selected off- and anti-targets was assessed in biochemical assays. Phosphorylation of SLP-76 was measured either by flow cytometry or TR-FRET. Jurkat and primary T cells were activated and cultured in the presence of tested compounds and immunosuppressive agents. Impact on TCR selectivity and T cell function was measured by AlphaLISA and flow cytometry. Target engagement was measured in splenocytes of mice administered orally with tested compounds followed by IP injection of aCD3 antibody. Anti-tumor efficacy of HPK1 inhibitors was assessed in a syngeneic tumor model.ResultsRyvu's proprietary small molecule HPK1 inhibitors exhibit sub-nanomolar activity against human and mouse HPK1 proteins and good selectivity against other TCR pathway kinases. Tested compounds efficiently block phosphorylation of SLP-76 upon TCR engagement. TCR selectivity of Ryvu's inhibitors, measured as a ratio between CD69 and pSer376 SLP-76 inhibition, is on par or superior to reference molecules. Tested compounds are not only able to overcome PGE-2 induced resistance following TCR activation in human PBMCs, inducing elevated IL-2 release but also affect T cell function in co-culture assay. Developed molecules have favorable PK profiles, allowing for sustained target coverage in proposed dosing regimens and demonstrate efficacy in a mammary carcinoma syngeneic model.ConclusionsRyvu has developed potent and selective HPK1 inhibitors with favorable PK and PD profiles, whose activity in vitro translates to in vivo efficacy. Further preclinical work is warranted to select a lead candidate for IND-enabling studies and subsequently clinical studies across a variety of solid tumors.

scholarly journals Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Cécile Alanio ◽  
Francesco Nicoli ◽  
Philippe Sultanik ◽  
Tobias Flecken ◽  
Brieuc Perot ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Infection ◽  
Surface Expression ◽  
Cross Sectional Study ◽  
Negative Regulator ◽  
Tcr Signaling ◽  
Cell Repertoire ◽  
Cross Sectional ◽  
Chronic Hcv

Chronic infection perturbs immune homeostasis. While prior studies have reported dysregulation of effector and memory cells, little is known about the effects on naïve T cell populations. We performed a cross-sectional study of chronic hepatitis C (cHCV) patients using tetramer-associated magnetic enrichment to study antigen-specific inexperienced CD8+ T cells (i.e., tumor or unrelated virus-specific populations in tumor-free and sero-negative individuals). cHCV showed normal precursor frequencies, but increased proportions of memory-phenotype inexperienced cells, as compared to healthy donors or cured HCV patients. These observations could be explained by low surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated TCR hyperactivation and generation of potent CD8+ T cell responses from the altered T cell repertoire of cHCV patients. In sum, we provide the first evidence that naïve CD8+ T cells are dysregulated during cHCV infection, and establish a new mechanism of immune perturbation secondary to chronic infection.

scholarly journals Trib1 regulates T cell differentiation during chronic infection by restraining the effector program

2020 ◽  
Vol 217 (5) ◽  
Author(s):  
Kelly S. Rome ◽  
Sarah J. Stein ◽  
Makoto Kurachi ◽  
Jelena Petrovic ◽  
Gregory W. Schwartz ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cell Function ◽  
T Cell Differentiation ◽  
Negative Regulator ◽  
Chronic Infections ◽  
Tcr Signaling ◽  
Viral Control ◽  
Cell Immunity

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.

scholarly journals Shp1 regulates T cell homeostasis by limiting IL-4 signals

2013 ◽  
Vol 210 (7) ◽  
pp. 1419-1431 ◽  
Author(s):  
Dylan J. Johnson ◽  
Lily I. Pao ◽  
Salim Dhanji ◽  
Kiichi Murakami ◽  
Pamela S. Ohashi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Biology ◽  
Tyrosine Phosphatase ◽  
Elevated Serum ◽  
Premature Death ◽  
Negative Regulator ◽  
T Cell Homeostasis ◽  
Tcr Signaling ◽  
Regulatory Molecule

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell–intrinsic role of Shp1, we characterized mice with a T cell–specific Shp1 deletion (Shp1fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1fl/fl CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4+ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1-deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.

scholarly journals CD6 as a potential target for treating multiple sclerosis

2017 ◽  
Vol 114 (10) ◽  
pp. 2687-2692 ◽  
Author(s):  
Yan Li ◽  
Nora G. Singer ◽  
Joy Whitbred ◽  
Michael A. Bowen ◽  
David A. Fox ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Responses ◽  
Negative Regulator ◽  
Microvascular Endothelial Cell ◽  
Cell Responses ◽  
T Cell Survival

CD6 was established as a marker of T cells more than three decades ago, and recent studies have identified CD6 as a risk gene for multiple sclerosis (MS), a disease in which autoreactive T cells are integrally involved. Nevertheless, the precise role of CD6 in regulating T-cell responses is controversial and its significance in the pathogenesis of various diseases remains elusive, partly due to the lack of animals engineered to alter expression of the CD6 gene. In this report, we found that CD6 KO mice showed decreased pathogenic T-cell responses, reduced spinal cord T-cell infiltration, and attenuated disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. CD6-deficient T cells exhibited augmented activation, but also significantly reduced survival and proliferation after activation, leading to overall decreased Th1 and Th17 polarization. Activated CD6-deficient T cells also showed impaired infiltration through brain microvascular endothelial cell monolayers. Furthermore, by developing CD6 humanized mice, we identified a mouse anti-human CD6 monoclonal antibody that is highly effective in treating established EAE without depleting T cells. These results suggest that (i) CD6 is a negative regulator of T-cell activation, (ii) at the same time, CD6 is a positive regulator of activated T-cell survival/proliferation and infiltration; and (iii) CD6 is a potential new target for treating MS and potentially other T-cell–driven autoimmune conditions.

scholarly journals Yap suppresses T cell function and infiltration in the tumor microenvironment

2019 ◽  
Author(s):  
Eleni Stampouloglou ◽  
Anthony Federico ◽  
Emily Slaby ◽  
Stefano Monti ◽  
Gregory L. Szeto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
T Cell Activation ◽  
Cell Biology ◽  
Cell Activation ◽  
Cell Function ◽  
T Cell Responses ◽  
Negative Regulator ◽  
Cell Responses

ABSTRACTA major challenge for cancer immunotherapy is sustaining T cell activation and recruitment in immunosuppressive solid tumors. Here we report that Yap levels are sharply induced upon activation of CD4+ and CD8+ T cells and that Yap functions as an immunosuppressive factor and inhibitor of effector differentiation. Loss of Yap in T cells results in enhanced T cell activation, differentiation and function, which translates in vivo to an improved ability for T cells to infiltrate and repress tumors. Gene expression analyses of tumor-infiltrating T cells following Yap deletion implicates Yap as a mediator of global T cell responses in the tumor microenvironment and as a key negative regulator of T cell tumor infiltration and patient survival in diverse human cancers. Collectively, our results indicate that Yap plays critical roles in T cell biology, and suggest that inhibiting Yap activity improves T cell responses in cancer.

scholarly journals Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis

2018 ◽  
Author(s):  
Judith F. Ashouri ◽  
Lih-Yun Hsu ◽  
Steven Yu ◽  
Dmitry Rychkov ◽  
Yiling Chen ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Autoimmune Arthritis ◽  
Receptor Signaling ◽  
Tcr Signaling ◽  
Autoreactive T Cells

AbstractHow pathogenic CD4 T cells in Rheumatoid Arthritis (RA) develop remains poorly understood. We used Nur77—a marker of T cell antigen receptor (TCR) signaling—to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into IL-17 producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic auto-antigen exposure in vivo. The enhanced autoreactivity was associated with upregulation of IL-6 cytokine signaling machinery, which might in part be attributable to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)—a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFPhi CD4 T cells from SKGNur mice were hyper-responsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice, SOCS3 expression was similarly downregulated in RA synovium. This suggests that, despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6 which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA.Significance StatementHow arthritis-causing T cells trigger rheumatoid arthritis (RA) is not understood since it is difficult to differentiate T cells activated by inflammation in arthritic joints from those activated through their TCR by self-antigens. We developed a model to identify and study antigen-specific T cell responses in arthritis. Nur77—a specific marker of TCR signaling—was used to identify antigen-activated CD4 T cells in the SKG arthritis model and patients with RA. Nur77 could distinguish highly arthritogenic and autoreactive T cells in SKG mice. The enhanced autoreactivity was associated with increased IL-6-receptor-signaling, likely contributing to their arthritogenicity. These data highlight a functional correlate between Nur77 expression, arthritogenic T cell populations, and heightened IL-6 sensitivity in SKG mice with translatable implications for human RA.

scholarly journals TET2 Regulates CD8+ T Cell Differentiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1423-1423
Author(s):  
Shannon A. Carty ◽  
Mercy Gohil ◽  
Gary A Koretzky ◽  
Martha S Jordan
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Differentiation ◽  
Tcr Signaling ◽  
Cell Responses ◽  
And Function

Abstract Regulation of DNA methylation is critical for proper T cell differentiation and function. Antigen-specific CD8+ T cells undergo global remodeling of DNA methylation following viral infection, suggesting that DNA methylation may direct antigen-specific T cell responses. TET2 is a member of the Ten-Eleven-Translocation (TET) family, which converts 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates ultimately leading to DNA demethylation. How TET2 regulates T cell differentiation and function is unknown. Here we demonstrate that TET2 expression is regulated by TCR signaling in primary murine T cells. Furthermore, using a novel flow cytometric assay to measure 5hmC levels on a single cell basis, we find that TCR signaling also regulates TET activity as evidenced by a rapid increase in global 5hmC levels after TCR stimulation that is blunted in TET2-deficient T cells. To determine the role of TET2 in T cell responses, we generated mice deficient in TET2 in the T cell compartment (TET2fl/flCD4Cre+) mice. These mice develop grossly normal thymic and peripheral T cell subsets. Given the regulation of TET2’s expression and activity by TCR stimulation, we used a murine model of acute viral infection, specifically LCMV-Armstrong, to test if TET2 regulates antigen-specific T cell responses in vivo. Following viral challenge, TET2fl/flCD4Cre+ mice have similar antigen-specific CD8+ T cell expansion and effector responses but exhibit significantly enhanced memory CD8+ T cell differentiation compared to control mice. These data demonstrate that TET2 plays a critical role in directing CD8+ T cell differentiation and function. Studies are ongoing to identify specific TET2 regulated genes important in the development of CD8+ T cell memory. Disclosures No relevant conflicts of interest to declare.

scholarly journals DLGH1 Is a Negative Regulator of T-Lymphocyte Proliferation

2007 ◽  
Vol 27 (21) ◽  
pp. 7574-7581 ◽  
Author(s):  
Linda M. Stephenson ◽  
Bénédicte Sammut ◽  
Daniel B. Graham ◽  
Joaquim Chan-Wang ◽  
Karry L. Brim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Receptor ◽  
Suppressor Gene ◽  
Negative Regulator ◽  
Tcr Signaling ◽  
Mammalian Embryo ◽  
T Lymphocyte Proliferation ◽  
Discs Large

ABSTRACT Discs large homolog 1 (DLGH1), a founding member of the membrane-associated guanylate kinase family of proteins containing PostSynaptic Density-95/Discs large/Zona Occludens-1 domains, is an ortholog of the Drosophila tumor suppressor gene Discs large. In the mammalian embryo, DLGH1 is essential for normal urogenital morphogenesis and the development of skeletal and epithelial structures. Recent reports also indicate that DLGH1 may be a critical mediator of signals triggered by the antigen receptor complex in T lymphocytes by functioning as a scaffold coordinating the activities of T-cell receptor (TCR) signaling proteins at the immune synapse. However, it remains unclear if DLGH1 functions to enhance or attenuate signals emanating from the TCR. Here, we used Dlgh1 gene-targeted mice to determine the requirement for DLGH1 in T-cell development and activation. Strikingly, while all major subsets of T cells appear to undergo normal thymic development in the absence of DLGH1, peripheral lymph node Dlgh1 −/− T cells show a hyper-proliferative response to TCR-induced stimulation. These data indicate that, consistent with the known function of Discs large proteins as tumor suppressors and attenuators of cell division, in T lymphocytes, DLGH1 functions as a negative regulator of TCR-induced proliferative responses.

scholarly journals Regulation of T cell-dendritic cell interactions by IL-7 governs T-cell activation and homeostasis

Blood ◽  
2009 ◽  
Vol 113 (23) ◽  
pp. 5793-5800 ◽  
Author(s):  
Manoj Saini ◽  
Claire Pearson ◽  
Benedict Seddon
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Responses ◽  
Tcr Signaling ◽  
Cell Responses

Abstract Interleukin-7 (IL-7) plays a central role in the homeostasis of the T-cell compartment by regulating T-cell survival and proliferation. Whether IL-7 can influence T-cell receptor (TCR) signaling in T cells remains controversial. Here, using IL-7–deficient hosts and TCR-transgenic T cells that conditionally express IL-7R, we examined antigen-specific T-cell responses in vitro and in vivo to viral infection and lymphopenia to determine whether IL-7 signaling influences TCR-triggered cell division events. In vitro, we could find no evidence that IL-7 signaling could costimulate T-cell activation over a broad range of conditions, suggesting that IL-7 does not directly tune TCR signaling. In vivo, however, we found an acute requirement for IL-7 signaling for efficiently triggering T-cell responses to influenza A virus challenge. Furthermore, we found that IL-7 was required for the enhanced homeostatic TCR signaling that drives lymphopenia-induced proliferation by a mechanism involving efficient contacts of T cells with dendritic cells. Consistent with this, saturating antigen-presenting capacity in vivo overcame the triggering defect in response to cognate peptide. Thus, we demonstrate a novel role for IL-7 in regulating T cell–dendritic cell interactions that is essential for both T-cell homeostasis and activation in vivo.

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