Inhibits T-cell Receptor Signaling Research Articles

Antigen Priming Induces Functional Reprogramming in iNKT Cells via Metabolic and Epigenetic Regulation: An Insight into iNKT Cell-Based Antitumor Immunotherapy.

Dysfunction of intratumoral invariant natural killer T (iNKT) cells hinders their antitumor efficacy, but the underlying mechanisms and the relationship with endogenous antigen priming remain to be explored. Here, we report that antigen priming leads to metabolic reprogramming and epigenetic remodeling, which causes functional reprogramming in iNKT cells, characterized by limited cytokine responses upon restimulation but constitutive high cytotoxicity. Mechanistically, impaired oxidative phosphorylation (OXPHOS) in antigen-primed iNKT cells inhibited T-cell receptor signaling, as well as elevation of glycolysis, upon restimulation via reducing mTORC1 activation, and thus led to impaired cytokine production. However, the metabolic reprogramming in antigen-primed iNKT cells was uncoupled with their enhanced cytotoxicity; instead, epigenetic remodeling explained their high expression of granzymes. Notably, intratumoral iNKT cells shared similar metabolic reprogramming and functional reprogramming with antigen-primed iNKT cells due to endogenous antigen priming in tumors, and thus recovery of OXPHOS in intratumoral iNKT cells by ZLN005 successfully enhanced their antitumor responses. Our study deciphers the influences of antigen priming-induced metabolic reprogramming and epigenetic remodeling on functionality of intratumoral iNKT cells, and proposes a way to enhance efficacy of iNKT cell-based antitumor immunotherapy by targeting cellular metabolism.

Canagliflozin impairs T cell effector function via metabolic suppression in autoimmunity

Augmented Tcell function leading to host damage in autoimmunity is supported by metabolic dysregulation, making targeting immunometabolism an attractive therapeutic avenue. Canagliflozin, a type 2 diabetes drug, is a sodium glucose co-transporter 2 (SGLT2) inhibitor with known off-target effects on glutamate dehydrogenase and complex I. However, the effects of SGLT2 inhibitors on human Tcell function have not been extensively explored. Here, we show that canagliflozin-treated Tcells are compromised in their ability to activate, proliferate, and initiate effector functions. Canagliflozin inhibits Tcell receptor signaling, impacting on ERK and mTORC1 activity, concomitantly associated with reduced c-Myc. Compromised c-Myc levels were encapsulated by a failure to engage translational machinery resulting in impaired metabolic protein and solute carrier production among others. Importantly, canagliflozin-treated Tcells derived from patients with autoimmune disorders impaired their effector function. Taken together, our work highlights a potential therapeutic avenue for repurposing canagliflozin as an intervention for Tcell-mediated autoimmunity.

Abstract 4450: SOX4 promotes tumor development and immune evasion via disruption of zinc homeostasis between nasopharyngeal carcinoma cells and T cells

Abstract Background: Nasopharyngeal carcinoma (NPC) is a highly undifferentiated squamous malignancy with dense infiltration of dysfunctional T cells that is endemic in East and Southeast Asia. Based on multicentral single-cell RNA sequencing (scRNA-seq) analysis, we have identified SRY-Box Transcription Factor 4 (SOX4), an essential developmental transcription factor, is specifically and highly expressed by NPC cells and contributes to tumor growth and distant metastasis. SOX4 upregulation also promotes ZIP14-mediated intracellular zinc uptake, leading to competitive deprivation of free zinc in the tumor microenvironment (TME), which inhibits zinc-dependent T cell receptor (TCR) activation. Thus, our study provides experimental insights into how NPC develops and evades immune surveillance. Methods: scRNA-seq data and bulk RNA-Seq data from our lab and GEO database were integrated and analyzed on a bioinformatic basis. Immunohistochemistry staining was performed to validate the SOX4 protein level in an independent clinical cohort. Cell lines, patient-derived xenografts, and humanized mouse models were generated to evaluate the in vitro and in vivo function of SOX4. Flow cytometry was used for T-cell immunophenotyping. Immunoprecipitation was used to study protein-DNA interaction. The dual-luciferase reporter assay was utilized to demonstrate the transcriptional regulation of SOX4 on the ZIP14 promoter. qRT-PCR, immunoblotting, and immunofluorescence staining were also employed for functional studies. Result: Our transcriptome analysis showed SOX4 was frequently overexpressed by NPC cells, compared to infiltrating immune and stromal cells. Increasing SOX4 expression in normal nasopharyngeal epithelial cells enhanced proliferation, stemness, migration, and invasion. SOX4 also transcriptionally upregulated ZIP14 in NPC cells which promoted the uptake of free zinc from the TME. Thus, competitive deprivation of zinc between NPC cells and T cells in the TME inhibited TCR signaling and T-cell cytotoxicity, leading to NPC immune evasion. Conclusion: This study reveals the SOX4-ZIP14-Zinc axis as a vital mechanism for simultaneously inducing tumor development and immunosuppression in the NPC microenvironment. Therapeutic inhibition of SOX4 or ZIP14 is a new strategy for NPC patients and potentially synergizes with PD-1/PD-L1 blockades to overcome immunosuppressive signals in the TME. Citation Format: Yuma Yang, Lanqi Gong, Jie Luo, Xiaona Fang, Jiao Huang, Lu Bai, Qin Liu, Beilei Liu, Shan Liu, Jinlin Huang, Ching Ngar Wong, Baifeng Zhang, Danyang Zheng, Yu Zhang, Wei Dai, Xinyuan Guan. SOX4 promotes tumor development and immune evasion via disruption of zinc homeostasis between nasopharyngeal carcinoma cells and T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4450.

Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua

Central tolerance is achieved through positive and negative selection of thymocytes mediated by T cell receptor (TCR) signaling strength. Thus, dysregulation of the thymic selection process often leads to autoimmunity. Here, we show that Capicua (CIC), a transcriptional repressor that suppresses autoimmunity, controls the thymic selection process. Loss of CIC prior to T-cell lineage commitment impairs both positive and negative selection of thymocytes. CIC deficiency attenuated TCR signaling in CD4+CD8+ double-positive (DP) cells, as evidenced by a decrease in CD5 and phospho-ERK levels and calcium flux. We identified Spry4, Dusp4, Dusp6, and Spred1 as CIC target genes that could inhibit TCR signaling in DP cells. Furthermore, impaired positive selection and TCR signaling were partially rescued in Cic and Spry4 double mutant mice. Our findings indicate that CIC is a transcription factor required for thymic T cell development and suggests that CIC acts at multiple stages of T cell development and differentiation to prevent autoimmunity.

Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

Leukocyte-associated immunoglobulin-like receptor 1 inhibits T-cell signaling by decreasing protein phosphorylation in the T-cell signaling pathway

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1-sufficient and -deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ζ chain of T-cell receptor-associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1-induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog-sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

Pik3ip1 Is a Negative Immune Regulator that Inhibits Antitumor T-Cell Immunity.

Multiple negative regulators restrict the ability of T cells to attack tumors. This work demonstrates the role of PI3K-interacting protein 1 (Pik3ip1) in restraining T-cell responses and antitumor immunity. An anti-Pik3ip1 mAb was generated to identify the Pik3ip1 expression pattern of hematopoietic cells. Pik3ip1 -/- mice and a Pik3ip1 fusion protein were generated to investigate the effect of Pik3ip1 on T-cell-mediated antitumor immunity in MC38 and B16-F10 tumor models. Immunoblotting and confocal microscopy were used to identify inhibitory effects of Pik3ip1 on T-cell receptor (TCR) signaling. Pik3ip1 expression was quantified, and its impact on T-cell function in human tumors was measured. We demonstrated that Pik3ip1 was predominantly expressed on T cells and served as an essential rheostat for T-cell-mediated immunity. A Pik3ip1 genetic deficiency led to enhanced T-cell responsiveness upon immunization with a neoantigen. Pik3ip1 -/- mice exhibited a marked increase in antitumor immunity and were resistant to tumor growth. Furthermore, Pik3ip1 extracellular domain fusion protein enhanced MC38 tumor growth was observed. Mechanistically, we found that Pik3ip1 inhibited TCR signaling by mediating the degradation of SLP76 through Pik3ip1 oligomerization via its extracellular region. Consistent with the results from the mouse models, PIK3IP1 expression correlated with T-cell dysfunction in human tumors. Our data reveal a critical role for Pik3ip1 as a novel inhibitory immune regulator of T-cell responses and provide a potential molecular target for cancer immunotherapy.

Abstract 1313: CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas

Abstract Background: ITK is a non-receptor tyrosine kinase that modifies T cell receptor (TCR) signaling. Mice deficient in ITK, but not resting lymphocyte kinase (RLK), exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells and secrete IFNγ. Combined disruption of ITK and RLK in mice leads to more severe T cell functional defects compared to disrupting ITK alone, and paradoxically allows for normal Th2 responses. Thus, selective pharmacologic inhibition of ITK versus RLK is necessary to inhibit Th2 responses without affecting Th1-dependent immunity. ITK is widely expressed in T cell malignancies, and activation of ITK upregulates GATA-3, a transcription factor that drives Th2 differentiation and is associated with poor survival. Here we report the discovery and characterization of CPI-818, an irreversible inhibitor of ITK. CPI-818 is highly selective for ITK over RLK allowing for an assessment of pure ITK inhibition on normal and malignant T cells. Results: CPI-818 irreversibly inhibited ITK (IC502.3 nM) with >100-fold selectivity over RLK (430 nM) and BTK (850 nM). The mechanism of ITK inhibition involves covalent binding to CYS-442 confirmed by mass spectrometry. Irreversible inhibition of ITK in vitroand in vivowas demonstrated using an active site competitive probe. CPI-818 inhibited anti-CD3/28 induced phosphorylation of ERK (T202/Y204) and PLCγ (Y783) in PMBCs, and inhibited IL2 secretion in Jurkat T cells (IC5075 nM). CPI-818 demonstrated dose dependently inhibition of TCR-induced proliferation of malignant T cells from Sezary Syndrome patients. In mice orally treatedwith CPI-818 an increase in the ratio of IFNγ/IL-4 (p<0.05) upon antigen-specific re-stimulation in an OVA transgenic adoptive transfer model was observed, consistent with Th1-skewing. Additionally, toassess the impact of CPI-818 on Th-biasing, activated human PBMCs were cultured for 6 days with CPI-818 and inhibition of IL-4 production (average 64% inhibition, n=12) was significantly greater than IFNγ production (average 19% inhibition), supportive of Th1 skewing. To assess the potential of CPI-818 to treat human T cell lymphoproliferative disorders, the safety and efficacy of CPI-818 in companion dogs with spontaneously-occurring T cell lymphomas were evaluated. CPI-818 was dosed orally at 20 mg/kg BID for 2 weeks to 5 months. Three animals were treated: 1 with peripheral T cell lymphoma and 2 with cutaneous T cell lymphoma. Full ITK occupancy in peripheral blood was confirmed using a probe assay. Evidence of anti-tumor activity was seen in all dogs including complete and partial responses. CPI-818 was well tolerated with no change in normal lymphocyte counts. These data support evaluation of CPI-818 in clinical trials in patients with T cell malignancies. Citation Format: James W. Janc, Craig M. Hill, Patrick P. Ng, Andrew N. Hoston, Antonett Madriaga, Trang P. Dao-Pick, Kitman S. Yeung, Ryan Hudson, Anne-Marie Beausoleil, Erin Bradley, Erik Verner, Douglas H. Thamm, Richard A. Miller, Joseph J. Buggy. CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1313.

Protein tyrosine phosphatase PTPN22 has dual roles in promoting pathogen versus homeostatic-driven CD8 T-cell responses.

PTPN22 (protein tyrosine phosphatase non receptor 22) encodes a tyrosine phosphatase that functions as a key regulator of immune homeostasis. In particular, PTPN22 inhibits T-cell receptor signaling and selectively promotes type I interferon responses in myeloid cells. To date, there is little information on the CD8 T-cell-intrinsic role of PTPN22 in response to a viral pathogen. We unexpectedly found that PTPN22-deficient virus-specific CD8 T cells failed to accumulate in wild-type hosts after lymphocytic choriomeningitis virus infection. Lack of PTPN22 expression altered CD8 T-cell activation and antiviral cytokine production, but did not significantly affect the composition of effector and memory cell precursors. Most significantly, in vivo, PTPN22-deficient CD8 T cells showed a profound defect in upregulating STAT-1 after lymphocytic choriomeningitis virus infection and considerably less phosphorylation of STAT-1 in response to IFN-α treatment in vitro compared with their wild-type counterparts. In stark contrast, following transfer into lymphopenic mice, CD8 T-cell expansion and central-like phenotype, was considerably increased in the absence of PTPN22. Collectively, our results suggest that PTPN22 has dual roles in T-cell clonal expansion and effector function; whereas it promotes antigen-driven responses during acute infection by positively regulating interferon signaling in T cells, PTPN22 inhibits homeostatic-driven proliferation.

ACP-196 Is a Second Generation Inhibitor of Bruton Tyrosine Kinase (BTK) with Enhanced Target Specificity

Background: Signaling through the B-cell receptor (BCR) results in cell survival and proliferation, and this pathway is critical for the progression of many B-cell malignancies, such a chronic lymphocytic leukemia (CLL). BTK, a TEC family kinase, is essential to the BCR signaling cascade. The recent approval of the first-in-class BTK inhibitor for the treatment certain B-cell malignancies, ibrutinib, has validated BTK as a clinical target. ACP-196 is a novel, second generation inhibitor of BTK with an IC50of 3 nM against purified BTK and EC50 of 8 nM in a human whole-blood CD69 B-cell activation assay. In vivo, oral administration of ACP-196 inhibits BCR-induced CD69 expression in murine splenic lymphocytes with an ED50 of 1.3 mg/kg compared with 2.9 mg/kg for ibrutinib. ACP-196 also has improved target specificity over ibrutinib with 323-,94-, 19- and 9-fold selectivity over the other TEC kinase familymembers (ITK, TXK, BMX, and TEC, respectively) and no activity against EGFR (Covey AACR, 2015). TXK and ITK are critical for T-cell receptor activation and development. ITK is also necessary for natural killer (NK)-cell mediated antibody dependent cell cytotoxicity (ADCC), which is important for the efficacy of monoclonal antibody therapy, eg, obinutuzumab. Additionally, therapeutic blockade of EGFR signaling is employed for treatment of epithelial cancers and is associated with adverse events, such as diarrhea and skin rash. Here, we describe results from signaling and functional assays to further elucidate the differential effects of ACP-196 on primary CLL cells compared with T-cells, NK-cells and epithelial cells.Methods: CLL patient samples were evaluated for pathway activation in the presence and absence of ACP-196 using phospho-protein immunoblotting. Helper T-cell (Th) skewing assays were performed using purified naive CD4+ T-cells from C57BL/6, Itk knock out (KO) and Itk/Txk double KO mice in the presence ACP-196 or ibrutinib. CD8+ T-cell activity was assessed by cytolytic activity (CTL) assays using T-cells isolated from C57BL/6 mice. The CTL response was initiated with various effector:T-cell ratios using C57BL/6 T-cells and murine BALB/c lymphoblasts. To evaluate NK-cell ADCC function, standard chromium release assays were done using purified NK-cells isolated from healthy volunteer peripheral blood mononuclear cells and cultured with obinutuzumab-coated (10 µg/mL) CLL cells in the presence and absence of ACP-196. Immunoblotting of H460 lung cancer cells for phospho-EGFR was used to evaluate the effect of ACP-196 on EGFR signaling.Results: ACP-196 treatment of primary human CLL cells showed a dose-dependent (0.01μM-1μM) decrease in p-BTK (Y223), p-ERK (T202/Y204) and p-AKT (T308) and p-IκB-α (S32), without impact on total protein levels. These findings demonstrate ACP-196 inhibits autophosphorylation of BTK as well as down-stream effectors of BCR signaling.Phospho-protein immunoblotting of primary human T-cells and Jurkat cells showed that ACP-196 does not inhibit T-cell receptor signaling molecules downstream of ITK and TXK such as IκBα, NFAT, or JunB. In in vitro helper T cell skewing assays, ACP-196, unlike ibrutinib, showed no effect on differentiation of Th subsets (Th1/Th2/Th17) or regulatory T cells (Tregs). The effect of ibrutinib on Th and Treg differentiation was similar to that observed in cells from Itk KO and Itk/Txk KO mice. In addition, ACP-196 had no effect on CD8+ T-cell viability or cytotoxicity as compared with ibrutinib-treated CD8+ T cells (46% and 36% reduction, respectively).In the in vitro ADCC assay, ACP-196 had no effect on NK cell function (chromium release of 34.8% vs 39.6% for the vehicle control). Lastly, at clinically relevant concentrations ACP-196 did not attenuate autophosphorylation of EGFR in H460 lung epithelial cells demonstrating no effect of ACP-196 on EGFR signaling.Conclusions: ACP-196 selectively inhibits BTK and BCR signaling at pharmacologic concentrations without off-target effects on T-cells, NK-cells and lung epithelial cells. ACP-196 is currently in Phase 3 trials for the treatment of CLL (ClinicalTrials.gov NCT0247568 and NCT02477696).This work was supported by the NIH NCI R01CA197870 and Acerta Pharma. DisclosuresGulrajani:Acerta Pharma: Employment. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Kaptein:Acerta Pharma: Employment. Van Lith:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment. Hamdy:Acerta Pharma BV: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ulrich:Acerta Pharma: Employment. Lannutti:Acerta Pharma: Employment. Johnson:Acerta Pharma: Research Funding.

Inhibition of IL2-Inducible T-Cell Kinase (ITK)-Mediated Chemoresistance By Ibrutinib in T-Cell Lymphoproliferative Disorders

Background: Most (≈ 95%) T-cell lymphomas (TCL) express an intact T-cell receptor (TCR), suggesting that malignant T cells, like their B-cell counterparts, may benefit from antigen-receptor signaling. TCR engagement culminates in the activation of pathways required for T-cell proliferation and survival. The Tec family kinase and BTK homologue ITK is required for optimal TCR-dependent signaling. Therefore, we sought to understand whether TCR activation promotes chemotherapy resistance in TCL, and whether this may be overcome upon inhibition of ITK.Methods: TCL cell lines, mouse models and primary patient specimens were utilized. TCR signaling was engaged by anti-CD3/CD28 beads. ITK and GATA3 were inhibited by lentiviral-mediated shRNA knockdown and by ibrutinib, an ITK inhibitor. The chemoresistance of TCL cells was investigated in vitro and in vivo.Results: We have previously shown by gene expression profiling, proliferation, cytokine release, and signaling pathway analysis that TCR signaling remains intact in TCL [Blood, 2014, 124(21), 2959]. To further investigate the effect of TCR on chemoresistance, T8ML1 (a PTCL, NOS cell line) and primary cells from TCL patients (n=4) were treated with either vincristine or romidepsin in vitro. For T8ML1, the viability of cells treated with either vincristine or romidepsin increased by 2.9+/-0.14 fold or 1.4 +/- 0.035 fold respectively (p<0.01) upon TCR engagement. The enhanced viability of T8ML1 by TCR engagement under either vincristine or romidepsin treatment was abolished by shRNA-mediated ITK knockdown and significantly inhibited by ibrutinib (p<0.01). Similarly for primary TCL patient cells, the viability of cells treated with romidepsin increased by 2.4 +/- 0.35 fold upon TCR engagement, which was also significantly inhibited by ibrutinib treatment (p<0.01). To study the mechanism of TCR signaling in TCL chemoresistance, downstream targets of TCR signaling (NFκB, GATA3) were examined. Upon TCR engagement, NFκB activity increased by 1.7 +/- 0.22 fold in T8ML1 and 1.8 +/- 0.36 fold in primary cells from TCL patients (p<0.01) as shown by DNA-binding and nuclear localization. The activation of NFκB in T8ML1 and primary TCL patient cells was significantly inhibited by ibrutinib (p<0.01). We and others have previously shown that GATA3 identifies a distinct subset of PTCL, NOS that is characterized by inferior progression-free survival following anthracycline-based chemotherapy. [Blood, 2014, 123 (19), 3007-3015; Blood, 2014, 123(19), 2915-2923]. Furthermore, GATA-3 regulates the homeostatic survival of normal T cells following TCR engagement. In addition to NFκB, GATA3 protein increased by 3.0 +/- 0.45 fold in T8ML1 (p<0.01) and by 3.6 +/- 3.0 fold in primary TCL patient cells (p<0.03) upon TCR engagement, which was also inhibited by ibrutinib treatment. Furthermore, in T8ML1 cells, GATA3 upregulation by TCR engagement was abolished by shRNA-mediated ITK knockdown. To study the effect of GATA3 on chemoresistance, GATA3 was knocked down by lentiviral-mediated shRNAs in TCL cell lines (T8ML1, H9 and MyLa). The viability of TCL lines following GATA3 knockdown decreases by 2-4 fold (p<0.01) following treatment with either vincristine, romidepsin or 4-hydroxycyclophosphamide. In comparison to tumor xenografts generated fromTCL lines transduced with a non-targeting shRNA, GATA3 deficient tumor xenografts were significantly more sensitive to vincristine alone or combined vincristine/cyclophosphamide (p<0.01).Conclusions: TCR engagement promotes resistance to chemotherapy in T-cell lymphomas in an ITK- and GATA-3-dependent manner. Furthermore, chemotherapy resistance following TCR engagement is significantly impaired by ibrutinib. Therefore, ibrutinib may warrant further investigation in the T-cell lymphomas. DisclosuresOff Label Use: We used ibrutinib to inhibit T-cell receptor signaling and discussed its clinical implication in T-cell lymphomas..

Conserved Motifs within Hepatitis C Virus Envelope (E2) RNA and Protein Independently Inhibit T Cell Activation.

T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp’s contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.

Constitutive AP-1 Activity and EBV Infection Induce PD-L1 in Hodgkin Lymphomas and Posttransplant Lymphoproliferative Disorders: Implications for Targeted Therapy

Programmed cell death ligand 1 (PD-L1) is a molecule expressed on antigen-presenting cells that engages the PD-1 receptor on T cells and inhibits T-cell receptor signaling. The PD-1 axis can be exploited by tumor cells to dampen host antitumor immune responses and foster tumor cell survival. PD-1 blockade has shown promise in multiple malignancies but should be directed toward patients in whom it will be most effective. In recent studies, we found that the chromosome 9p24.1 amplification increased the gene dosage of PD-L1 and its induction by JAK2 in a subset of patients with classical Hodgkin lymphoma (cHL). However, cHLs with normal 9p24.1 copy numbers also expressed detectable PD-L1, prompting analyses of additional PD-L1 regulatory mechanisms. Herein, we utilized immunohistochemical, genomic, and functional analyses to define alternative mechanisms of PD-L1 activation in cHL and additional EBV(+) lymphoproliferative disorders. We identified an AP-1-responsive enhancer in the PD-L1 gene. In cHL Reed-Sternberg cells, which exhibit constitutive AP-1 activation, the PD-L1 enhancer binds AP-1 components and increases PD-L1 promoter activity. In addition, we defined Epstein-Barr virus (EBV) infection as an alternative mechanism for PD-L1 induction in cHLs with diploid 9p24.1. PD-L1 was also expressed by EBV-transformed lymphoblastoid cell lines as a result of latent membrane protein 1-mediated, JAK/STAT-dependent promoter and AP-1-associated enhancer activity. In addition, more than 70% of EBV(+) posttransplant lymphoproliferative disorders expressed detectable PD-L1. AP-1 signaling and EBV infection represent alternative mechanisms of PD-L1 induction and extend the spectrum of tumors in which to consider PD-1 blockade.

Phenotypic characterization of mice carrying a PTPN22 single nucleotide polymorphism associated with autoimmunity in humans (47.13)

Abstract Type 1 diabetes (T1D) is a strongly heritable autoimmune disease due to the destruction of pancreatic β-cells by self-reactive T-cells. The third strongest genetic locus for T1D involves a single-nucleotide polymorphism (SNP) causing an arginine(R) to triptophan(W) substitution at codon 620 (620R&amp;gt;W) in PTPN22, the gene encoding the tyrosine phosphatase LYP, an important negative regulator of T-cell receptor (TCR) activation. However, the cellular effects of the 620R&amp;gt;W risk allele remain controversial, as different studies report either a gain or a loss of function in its ability to inhibit TCR signaling. To study this variant in an animal model, we created a knock-in mouse by introducing the 620R&amp;gt;W substitution in, the murine LYP orthologue, PEP encoded by Ptpn22. The knock-in analyzed on the autoimmune-resistant C57Bl/6 background showed a mild but significant increase in CD4+ helper T-cell activation and proliferation upon weak TCR activation. Conversely, upon strong TCR stimulation, the tendency seemed to be reversed as the 620W allele showed a slightly decreased responsiveness compared to the 620R allele. Phenotypic analysis of other immune cellular compartments, including memory T cells, showed no significant difference between genotypes. There was no evidence of autoimmunity in this genetic background. This dependence of allelic effect on the strength of the TCR signal may reconcile previous observations and indicate a complex role of R620&amp;gt;W in autoimmunity.

Immunology in the heart of the High Tatra mountains

Immunology in the heart of the High Tatra mountains

The effects of membrane compartmentalization of csk on TCR signaling

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.

Synergistic role of diacylglycerol kinases α and ζ in T cell development and self-tolerance (137.37)

Abstract Signal for the T cell receptor (TCR) plays critical roles in T cell development and function. TCR signaling can induce positive or negative selection of thymocytes, induces activation or anergy of mature T cells, and induce conventional T cell activation or regulatory T cells to suppress immune function. The mechanisms that modulate TCR signaling to direct these distinct outcomes have been poorly understood. Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA) through phosphorylation. In mammals, ten DGK isoforms have been identified. Using both a gain of function and a loss of function approaches, we have found that DGK ( and ( are important regulators for T cells. Enhanced DGK activity inhibits TCR signaling and T cell maturation. Deficiency of either DGK( or ( causes T cells hyperresponsive to TCR stimulation and resistant to anergy induction. Loss of both DGK( and ( in mice results in impairment of positive selection, spontaneous T cell activation, impairment of regulatory T cell function, and autoimmunity. We further demonstrate that DGKs function both as signal terminator by inhibiting DAG-mediated signaling and as signaling initiator by generating PA.

T Cell Adhesion Primes Antigen Receptor-Induced Calcium Responses through a Transient Rise in Adenosine 3′,5′-Cyclic Monophosphate

It is well established that sustained increases in cyclic AMP (cAMP) such as those triggered by forskolin inhibit T cell activation. We describe here an unexpected phenomenon: in T cells, a transient cAMP increase triggered by the interaction with a dendritic cell strongly potentiates T cell receptor (TCR) signaling. We discovered this effect by examining the molecular basis of the adhesion-dependent sensitization of T cells. T cell adhesion caused extracellular-signal-regulated kinase (ERK) activation, which was necessary for the sensitization process. T cell sensitization could be mimicked in suspended cells by the uncaging of caged cAMP upon ultraviolet illumination. Calcium responses occurring in T cells upon interaction with dendritic cells were strongly inhibited when protein kinase A activation was blocked. Thus, whereas sustained cAMP increases are well known to inhibit TCR signaling, transient cAMP increases occurring physiologically upon formation of an immunological synapse facilitate antigen detection.

Lateral Compartmentalization of T Cell Receptor Versus CD45 by Galectin-N-Glycan Binding and Microfilaments Coordinate Basal and Activation Signaling

Lateral compartmentalization of membrane proteins into microdomains regulates signal transduction; however, structural determinants are incompletely understood. Membrane glycoproteins bind galectins in proportion to the number (i.e. NX(S/T) sites) and degree of GlcNAc branching within attached N-glycans, forming a molecular lattice that negatively regulates T cell function and autoimmunity. We find that in resting T cells, partition of CD45 inside and T cell receptor (TCR)/CD4-Lck/Zap-70 outside microdomains is positively and negatively regulated by the galectin lattice and actin cytoskeleton, respectively. In the absence of TCR ligands, the galectin lattice counteracts F-actin to retain CD45 in microdomains while concurrently blocking TCR/CD4-Lck/Zap-70 partition to microdomains by preventing a conformational change in the TCR that recruits Nck/Wiscott Aldrich Syndrome (WASp)/SLP76/F-actin/CD4 to TCR. The counterbalancing activities of the galectin lattice and actin cytoskeleton negatively and positively regulate Lck activity in resting cells and CD45 versus TCR clustering and signaling at the early immune synapse, respectively. Microdomain-localized CD45 inactivates Lck and inhibits TCR signaling at the early immune synapse. Thus, the galectin lattice and actin cytoskeleton interact on opposing sides of the plasma membrane to control microdomain structure and function, coupling basal growth signaling with thresholds to activation.

Enhanced T cell responses due to diacylglycerol kinase zeta deficiency.

Much is known about how T cell receptor (TCR) engagement leads to T cell activation; however, the mechanisms terminating TCR signaling remain less clear. Diacylglycerol, generated after TCR ligation, is essential in T cells. Its function must be controlled tightly to maintain normal T cell homeostasis. Previous studies have shown that diacylglycerol kinase zeta (DGKzeta), which converts diacylglycerol to phosphatidic acid, can inhibit TCR signaling. Here we show that DGKzeta-deficient T cells are hyperresponsive to TCR stimulation both ex vivo and in vivo. Furthermore, DGKzeta-deficient mice mounted a more robust immune response to lymphocytic choriomeningitis virus infection than did wild-type mice. These results demonstrate the importance of DGKzeta as a physiological negative regulator of TCR signaling and T cell activation.