Regulation Of T Cell Receptor Signaling Research Articles

Discovery of Novel PROTAC-Based HPK1 Degraders with High Potency and Selectivity for Cancer Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1, MAP4K1), a serine/threonine (SER/THR) kinase, has been identified as a negative immune regulator of T-cell receptor signaling. Deprivation of the HPK1 function suppresses tumor growth, providing an attractive strategy for cancer immunotherapy. Herein, we present a novel PROTAC-based HPK1 degrader compound DD205-291 with high selectivity and potency. DD205-291 showed a dose-dependent inhibition of SLP-76 phosphorylation and an induction of IL-2 and IFN-γ. Compared with other inhibitors, DD205-291 exhibited good efficacy and a favorable safety profile in the MC38 model. Specifically, oral administration of DD205-291 at 0.5 mg/kg in combination with anti-PD1 resulted in significant suppression with a TGI value of 91.0%. Furthermore, DD205-291 exhibited a low risk of cardiotoxicity and a wide safety window. This research effort demonstrates that DD205-291 is a promising preclinical candidate (PCC) for potential mono- and comboimmunotherapy of cancer.

HPK1 citron homology domain regulates phosphorylation of SLP76 and modulates kinase domain interaction dynamics

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed β-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.

Discovery of 5-aminopyrido[2,3-d]pyrimidin-7(8H)-one derivatives as new hematopoietic progenitor kinase 1 (HPK1) inhibitors

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling. While HPK1 is considered as a promising target for cancer immunotherapy, no small-molecule HPK1 inhibitors have been approved for cancer treatment. Herein, we report the discovery of a series of new HPK1 inhibitors with a 5-aminopyrido[2,3-d]pyrimidin-7(8H)-one scaffold. The most potent compound 9f inhibited HPK1 kinase activity with an IC50 of 0.32 nM in the time-resolved fluorescence resonance energy transfer (TR-FRET) assays, while displayed reasonable selectivity in a panel of 416 kinases. Cellular engagement of HPK1 by compound 9f was confirmed through the nano-bioluminescence resonance energy transfer (Nano-BRET) experiments. Compound 9f effectively reduced the phosphorylation of the downstream protein SLP-76 in primary peripheral blood mononuclear cells (PBMCs) and human T lymphocytic leukemia Jurkat cells. Compound 9f also enhanced the IL-2 and IFN-γ secretion in PBMCs. Furthermore, the binding mode of compound 9f with HPK1 was confirmed by the resolved cocrystal structure. Taken together, this study provides HPK1 inhibitors with a novel scaffold and clear binding mode for further development of HPK1-targeted therapeutic agents.

Highly potent, orally active novel small-molecule HPK1 inhibitor DS21150768 induces anti-tumor responses in multiple syngeneic tumor mouse models

Augmenting T-cell activity is a promising approach to enhance the efficacy of cancer immunotherapy treatment. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in immune cells and negatively regulates T-cell receptor signaling. It is reported that inhibition of the kinase function of HPK1 results in tumor growth suppression by enhancing cancer immunity. Thus, developing HPK1 inhibitors has attracted considerable attention as a future cancer immunotherapy approach. However, despite recent progress in HPK1 biology and pharmacology, various challenges still remain, such as developing HPK1 inhibitors with favorable pharmacological profiles and identifying tumor characteristics that can be applied to define susceptibility to HPK1 inhibition. Here, we present the identification and pharmacological evaluation of DS21150768, a potent small-molecule HPK1 inhibitor with a novel chemical scaffold. DS21150768 shows remarkable inhibition of HPK1 kinase activity, and in vitro studies demonstrated its potent activity to enhance T-cell function. DS21150768 is orally bioavailable and shows sustained plasma exposure, which leads to enhanced cytokine responses in vivo. We conducted a comparison of the anti-tumor efficacy of DS21150768 alone or in combination with anti-PD-1 antibody in 12 different mouse cancer cell models, and observed that the treatments suppressed tumor growth in multiple models. Furthermore, Gene Set Enrichment Analysis demonstrated significant enrichment of immune-related gene signatures in the tumor models responsive to DS21150768 treatment. Our results provide a path forward for the future development of HPK1 inhibitors and fundamental insights into biomarkers of HPK1-targeted therapy.

A20 is a negative regulator of both NF-κB and JNK signaling following T cell receptor stimulation in humans

Abstract A20 is an NF-κB-induced, dual function ubiquitin editing enzyme that negatively regulates NF-κB signaling induced by TNF and other innate immune receptors, constraining inflammation. Indeed, A20 haploinsufficiency drives a severe autoinflammatory disease in humans. Although A20 serves a similar negative feedback function for T cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun. We employed an extensive set of mutant expression constructs to better elucidate how A20 regulates TCR signaling output in Jurkat T cells. Intriguingly, A20 knockout cells showed enhanced, sustained NF-κB and JNK signaling after stimulation, which was reversed upon wild-type (WT) A20 complementation. A20 deubiquitinase function was entirely dispensable for NF-κB and JNK regulation, whereas zinc finger domains that confer E3 ligase activity (ZnF4) and linear ubiquitin-binding capacity (ZnF7) were required. Abolishing MALT1-dependent proteolytic cleavage of A20 did not affect suppressive function, although neither N- nor C-terminal A20 cleavage fragments retained regulatory capacity. We also found that CRISPR-mediated deletion of A20 adversely affects the survival and expansion of primary human T cells. Ongoing studies will determine how A20 deletion affects human T cell effector responses after stimulation, and decipher how dysregulated NF-κB and/or JNK signaling contributes to T cell-intrinsic abnormalities that perturb adaptive immune homeostasis.

Abstract 5083: Investigating the development and therapeutic potential of intra-tumoral CXCR6+ effector/effector memory T cells (TEFF/EM) in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype that makes up 10-20% of all breast cancer cases. Because of the higher risk of metastasis and recurrence post-treatment, there is a need to develop novel therapeutics to address these challenges. In previously published work, we demonstrated that CXCL16 traps CXCR6+PD-1+ effector/effector memory (TEFF/EM) within the primary tumor and that these T cells have anti-metastatic function that can be harnessed when released into peripheral sites with intra-tumoral administration of anti-CXCL16 prior to surgical resection. However, whether CXCL16 blockade can be used in combination with conventional checkpoint targets and the developmental route of this CXCR6+ TEFF/EM is not known. To develop a novel neoadjuvant therapy, we use the spontaneously metastasizing murine TNBC model, 4T1. Here, we report that anti-4-1BB, and not anti-PD-1, when used in combination with blockade of CXCL16 can prolong survival post-surgical resection. Using anti-4-1BB in combination with CRISPR-generated Cxcl16-KO 4T1 model, we show that both deficiency of CXCL16 within the tumor microenvironment (TME) and anti-4-1BB contribute synergistically to survival advantage. To study the development of this population of T cells, we use the Py8119 murine TNBC model. We definitively show that CXCR6+PD-1+ TEFF/EM is uniquely generated within the primary tumor. Co-expression of CXCR6 and PD-1 indicates that T cell receptor (TCR) signaling is a potential regulator of the development of this TEFF/EM sub-population. Analyses of our previously published scRNA-seq dataset reveals Lat2 to be the top gene involved in TCR signaling. Tumors growing on Lat2-KO animals show a significant increase in tumor size accompanied by significant reduction of CD44high/mid CXCR6+PD-1+ TEFF/EM. However, competitive transfer of naïve WT and Lat2-KO T cells into Tcra-KO recipients led to comparable levels of CXCR6+ TEFF/EM. Surprisingly, transferred Lat2-KO T cells failed to engraft within the primary tumor relative to WT T cells. Future work will address if this engraftment defect is due to expansion/survival defects. Therefore, regulation of TCR signaling within the TME via LAT2 potentially represents a mechanism in which primary tumors trap T cells, promoting their exhaustion. Importantly, this can be harnessed through combinatorial activation and blockade of 4-1BB and CXCL16 respectively. Citation Format: Bryan Jian Wei Lim, Xiao-Fan Wang, Qi-Jing Li. Investigating the development and therapeutic potential of intra-tumoral CXCR6+ effector/effector memory T cells (TEFF/EM) in triple-negative breast cancer. [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 5083.

Expression of Non-T Cell Activation Linker (NTAL) in Jurkat Cells Negatively Regulates TCR Signaling: Potential Role in Rheumatoid Arthritis.

T lymphocytes are key players in adaptive immune responses through the recognition of peptide antigens through the T Cell Receptor (TCR). After TCR engagement, a signaling cascade is activated, leading to T cell activation, proliferation, and differentiation into effector cells. Delicate control of activation signals coupled to the TCR is needed to avoid uncontrolled immune responses involving T cells. It has been previously shown that mice deficient in the expression of the adaptor NTAL (Non-T cell activation linker), a molecule structurally and evolutionarily related to the transmembrane adaptor LAT (Linker for the Activation of T cells), develop an autoimmune syndrome characterized by the presence of autoantibodies and enlarged spleens. In the present work we intended to deepen investigation into the negative regulatory functions of the NTAL adaptor in T cells and its potential relationship with autoimmune disorders. For this purpose, in this work we used Jurkat cells as a T cell model, and we lentivirally transfected them to express the NTAL adaptor in order to analyze the effect on intracellular signals associated with the TCR. In addition, we analyzed the expression of NTAL in primary CD4+ T cells from healthy donors and Rheumatoid Arthritis (RA) patients. Our results showed that NTAL expression in Jurkat cells decreased calcium fluxes and PLC-γ1 activation upon stimulation through the TCR complex. Moreover, we showed that NTAL was also expressed in activated human CD4+ T cells, and that the increase of its expression was reduced in CD4+ T cells from RA patients. Our results, together with previous reports, suggest a relevant role for the NTAL adaptor as a negative regulator of early intracellular TCR signaling, with a potential implication in RA.

PTPN22 interacts with EB1 to regulate T-cell receptor signaling.

The PTPN22 gene encoding the Lyp/Pep protein tyrosine phosphatase is a negative regulator of T-cell receptor (TCR) signaling. Recent studies have shown that phosphorylation of end-binding protein 1 (EB1) is associated with the TCR activation. In this study, using 2-hybrid and mass spectrometry analyses, we identified EB1 as a protein associated with PTPN22. Furthermore, we discovered that EB1 specifically bound to the P1 domain of PTPN22 by competing with CSK, and the variant PTPN22-R620W does not affect the association with EB1, which is instrumental with respect to the regulation of TCR signaling. In addition, PTPN22 dephosphorylates EB1 at tyrosine-247 (Y247), which decreases the expression of the T-cell activation markers CD25 and CD69 and the phosphorylation levels of the TCR molecules ZAP-70, LAT, and Erk, leading to the eventual downregulation of the transcription factor NFAT and reduced the levels of secreted IL-2. The findings of this study provide new insights into the TCR signaling and the T-cell immune response, which are important for clarifying the mechanism of PTPN22-related autoimmune diseases.

CD5 signalosome coordinates antagonist TCR signals to control the generation of Treg cells induced by foreign antigens

CD5 is characterized as an inhibitory coreceptor with an important regulatory role during T cell development. The molecular mechanism by which CD5 operates has been puzzling and its function in mature T cells suggests promoting rather than repressing effects on immune responses. Here, we combined quantitative mass spectrometry and genetic studies to analyze the components and the activity of the CD5 signaling machinery in primary T cells. We found that T cell receptor (TCR) engagement induces the selective phosphorylation of CD5 tyrosine 429, which serves as a docking site for proteins with adaptor functions (c-Cbl, CIN85, CRKL), connecting CD5 to positive (PI3K) and negative (UBASH3A, SHIP1) regulators of TCR signaling. c-CBL acts as a coordinator in this complex enabling CD5 to synchronize positive and negative feedbacks on TCR signaling through the other components. Disruption of CD5 signalosome in mutant mice reveals that it modulates TCR signal outputs to selectively repress the transactivation of Foxp3 and limit the inopportune induction of peripherally induced regulatory T cells during immune responses against foreign antigen. Our findings bring insights into the paradigm of coreceptor signaling, suggesting that, in addition to providing dualistic enhancing or dampening inputs, coreceptors can engage concomitant stimulatory and inhibitory signaling events, which act together to promote specific functional outcomes.

Abstract IA18: The mechanistic basis of cancer immunotherapy

Abstract The advent of new approaches to the immunotherapy of cancer has caused a dramatic shift not only in the treatment of cancer but also in our understanding of cancer biology. The rapid rate of progress in the clinic, however, has outpaced our understanding of the basic mechanistic features that underlie the therapeutic advances. This is most notable in the case of “checkpoint” inhibitors, such as antibodies to the negative regulatory axis defined by PD-1 and PD-L1. While blocking the interaction of PD-L1 with PD-1 is often assumed to reverse the process of T-cell exhaustion, there is little direct evidence for this interpretation, an incomplete definition of what is meant by “exhaustion,” and a poor understanding of how PD-1 regulates T-cell activity. Starting with observations made in the clinic, we have used biochemical reconstitution together with in vivo analysis in mice to illuminate key features of the PD-L1/PD-1 axis, demonstrating that it functions primarily to regulate T-cell function by controlling signaling by costimulators such as CD28. Conditional knockout experiments also revealed that dendritic cell B7.1 plays a uniquely important role in activating CD28. Distinct from costimulatory molecules, entirely different regulatory mechanisms appear to regulate T-cell receptor signaling. Finally, we have begun to evaluate the origin of T cells that engage antitumor responses, results that further emphasize a need for continuous priming of antitumor T cells to maintain a potent anticancer response. Citation Format: Ira Mellman. The mechanistic basis of cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA18.

MicroRNAs in Tumor Exosomes Drive Immune Escape in Melanoma.

MicroRNAs (miRNA), small noncoding RNAs that regulate gene expression, exist not only in cells but also in a variety of body fluids. These circulating miRNAs could enable intercellular communication. miRNAs are packaged in membrane-encapsulated vesicles, such as exosomes, or protected by RNA-binding proteins. Here, we report that miRNAs included in human melanoma exosomes regulate the tumor immune response. Using microscopy and flow cytometry, we demonstrate that CD8+ T cells internalize exosomes from different tumor types even if these cells do not internalize vesicles as readily as other immune cells. We explored the function of melanoma-derived exosomes in CD8+ T cells and showed that these exosomes downregulate T-cell responses through decreased T-cell receptor (TCR) signaling and diminished cytokine and granzyme B secretions. The result reduces the cells' cytotoxic activity. Using mimics, we found that miRNAs enriched in exosomes-such as Homo sapiens (hsa)-miR-3187-3p, hsa-miR-498, hsa-miR-122, hsa-miR149, and hsa-miR-181a/b-regulate TCR signaling and TNFα secretion. Our observations suggest that miRNAs in melanoma-derived exosomes aid tumor immune evasion and could be a therapeutic target.

UBASH3B Is a Novel Prognostic Biomarker and Correlated With Immune Infiltrates in Prostate Cancer.

Background: UBASH3B (STS1) is an important gene that negatively regulates T-cell receptor signaling in activated T-lymphocytes that involved in cancers. However, the function of UBASH3B in prostate cancer (PCa) and the correlation between UBASH3B and tumor-infiltrating immune cells still remain unclear.Methods: Real-time PCR and immunohistochemistry were applied to detect mRNA and protein expression of UBASH3B in PCa patients and benign prostate hyperplasia patients (BPH). Clinical features of patients with PCa were recorded and Kaplan Meier curve was subsequently plotted. Based on mRNA expression of UBASH3B, patients with PCa from TCGA database were divided into low-UBASH3B-expression group and high-UBASH3B-expression group for construct lncRNA-miRNA-mRNA network and analyzing GO and KEGG pathways. Single gene analysis method was performed by using GSEA to interpret gene expression data in PCa. The PPI network was constructed using STRING and the correlation between UBASH3B and tumor-infiltrating immune cells was analyzed by TIMER and CIBERSORT.Results: The mRNA and protein expression of UBASH3B were upregulated in PCa. The abundant expression of UBASH3B is associated with poor prognosis in PCa. The subnetwork of UBASH3B contains three lncRNAs (MIAT, LINC01297, MYLK-AS1) and four miRNAs (hsa-miR-200a-3p, hsa-miR-455-5p, hsa-miR-192-5p, hsamiR- 215-5P). The mRNA expression of UBASH3B was involved in 28 KEGG pathways. GSEA analysis showed that 18 hallmark gene sets were significantly enriched in high-UBASH3B-expression, whereas 1 gene set was enriched in low-UBASH3B-expression. PPI network revealed a tightly interaction between UBASH3B and LCP2 (an immune related gene). TIMER and CIBERSORT database indicated that UBASH3B was correlated with 11 types of tumor-infiltrating immune cells (naïve B cell, memory B cells, resting CD4+ memory T cell, activated CD4+ memory T cell, regulatory T cell, activated NK cell, M2 macrophages, resting dendritic cells, activated dendritic cells, resting mast cells, neutrophils).Conclusions: In conclusion, UBASH3B may be a novel potential prognostic biomarker and is associated with tumor-infiltrating immune cells in tumor microenvironment, suggesting UBASH3B as a potential target for future treatment of PCa.

AIMP1 regulates TCR signaling and induces differentiation of regulatory T cells by interfering with lipid raft association

In addition to a role in translation, AIMP1 is secreted to affect various immune cells, such as macrophages, dendritic cells, B cells, and natural killer cells. However, the direct effects of AIMP1 on T cells have not yet been reported. In this study, we investigated whether AIMP1 could modulate T cell responses directly. Results revealed that AIMP1 significantly inhibited T cell receptor (TCR)-dependent activation and proliferation of CD4 T cells, as well as decreased TCR stimuli-induced Ca2+ influx in CD4 T cells. In addition, microscopic analysis revealed that lipid raft association in response to TCR engagement was significantly reduced in the presence of AIMP1, and the phosphorylation of PLCγ and PI3K was also down-regulated in CD4 T cells by AIMP1. Furthermore, AIMP1 specifically enhanced the differentiation of regulatory T (Treg) cells, while it had no effect on T helper type 1 (Th1), type 2 (Th2), and type 17 (Th17) cell differentiation. Collectively, these results indicate that AIMP1 affects T cells directly by down-regulating TCR signaling complex formation and inducing Treg cell differentiation in CD4 T cells.

TMEM16F regulates bystander TCR-CD3 membrane binding at the immunological synapse

Abstract The signaling cascade induced by T cell receptor (TCR) triggering has been extensively studied, but the steps separating peptide recognition from TCR phosphorylation remain elusive. In resting T cells, the TCR CD3ɛ and ζ chains dynamically associate with the plasma membrane (PM) through electrostatic interactions with anionic lipids such as phosphatidylserine (PS). Upon ligand recognition, the CD3 chains dissociate from the PM, enabling robust signaling initiation. However, the mechanisms regulating PM dissociation remain obscure. Recent studies have highlighted the role of TMEM16F, a PS-specific scramblase, in regulating TCR signaling, but the molecular steps involved in this activity have not been identified. To study the role of TMEM16F in regulating TCR activation, we knocked-down its expression using shRNA targeting. Our results show that TMEM16F knockdown inhibited PS redistribution, which significantly reduced TCR activation. To showcase TMEM16F activity in relation to TCR signaling, we transduced T cells with a mutant form of the scramblase that constitutively redistributes PS. TMEM16F-mutant T cells showed a significant increase in proximal TCR signaling. This was directly linked with an increase in PS redistribution upon TCR engagement, which enabled the dissociation of bystander CD3ɛ chains and their participation in signaling. Our results show that PS redistribution by way of TMEM16F activity is critical for TCR signaling. Our results also establish our ability to modulate PS redistribution by targeting TMEM16F activity in order to regulate T cell activation. These findings may ultimately lead to novel approaches enabling the modulation of TCR and other immune receptor activity in T cell-based immunotherapies.

Abstract A196: Systematic identification of markers and drug targets on exhausted CD8+ T-cells

Abstract CD8+ T-cells are responsible for detection and killing of hosT-cells expressing non-self proteins. Upon antigen persistence, as in cancer or chronic viral infection, antigen-specific CD8+ T-cells lose their effector function, a phenomenon known as T-cell exhaustion. This process is concomitant with increased surface expression of negative regulatory proteins such as PD-1 and CTLA-4. Blockade of these molecules has become a widespread and successful clinical strategy for the treatment of malignancies. Unfortunately, not all tumor types are responsive to checkpoint molecule blockade, nor do all patients with susceptible tumor types exhibit clinical responses. These failures highlight the need for identification of additional negative regulators on exhausted, tumor-specific CD8+ T-cells. Here, we perform a systematic approach to identify surface proteins expressed on exhausted murine and human CD8+ T-cells. Using RNA-sequencing data from antigen-specific CD8+ T-cells from the mouse lymphocytic choriomeningitis virus (LCMV) infection model as well as data from human tumor-infiltrating CD8+ T-cells, we show high cross-species concordance of genes encoding membrane proteins in T-cell exhaustion. These expression profiles not only include many known checkpoint molecules such as PD-1, but also several proteins that negatively regulate T-cell receptor signaling but are largely unexplored in the process of CD8+ T-cell exhaustion. We validate these results performing by flow cytometry on both mouse and human CD8+ T-cells, including on T-cells obtained from resected human tumor tissue. To substantiate the importance of these markers, we further explore the function of one identified surface protein, CD101, in the murine LCMV infection model. We demonstrate that CD101 is induced on antigen-specific of CD8+ T-cells weeks after infection, in contrast with the early expression of canonical exhaustion markers such as PD-1. We show that antigen-specific CD101+ cells arise from CD101- precursors and that this differentiation process results in a loss of effector function, cytokine production, and proliferative capacity with simultaneous large transcriptomic changes. Together, these results demonstrate that exhaustion-associated surface protein expression patterns are largely conserved between humans and mice and provide a comprehensive catalog of phenotypic markers and potential drug targets for CD8+ T-cell-directed immunotherapy. Citation Format: Will H. Hudson, Julia L. Gensheimer, Haydn T. Kissick, Rafi Ahmed. Systematic identification of markers and drug targets on exhausted CD8+ T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A196.

CD25-Dependent Feedback Control of the B-Cell Receptor and Its Oncogenic Mimics in B-Cell Malignancies

Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells.Interleukin-2 (IL2) functions as essential T-cell growth factor. IL2 signals through β- and γ-, but not α-chains (CD25) of its heterotrimeric receptor. CD25-deficiency causes lymphoproliferation and autoimmunity, however, its mechanistic role is unclear. Our experiments based on genetic mouse models and engineered patient-derived B-cell leukemia and lymphoma xenografts revealed that CD25 expressed on B-cells is not an IL2 receptor chain, but in fact binds the B-cell receptor (BCR) to regulate its activity. Suggesting IL2-independent functions, defects in CD25-/-B-cells were not replicated in IL2-deficient mice. CD25 bound the BCR but not IL2Rβ- and IL2Rγ-chains. IL2Rβ- and IL2Rγ-chains can pair with other chains to form receptors for different cytokine-ligands. However, CD25 represents the first example of a cytokine receptor chain that binds to the BCR for negative feedback regulation.Likewise, in T-cells, CD25 had a bifunctional role and either functioned as IL2 receptor chain or as negative feedback regulator of T-cell receptor signaling. CD25-function was regulated by cell-membrane translocation, which required phosphorylation of its cytoplasmic tail at S268 (see schematic, left). In a family with monogenic autoimmunity, a mutation immediately preceding S268 compromised CD25-surface translocation, which was restored by homology-directed repair of the S268 motif. CD25-interactome analyses identified PKCd as critical effector molecule downstream of CD25 to mediate B-cell selection during normal B-cell development and calibrate oncogenic BCR signaling in B-cell tumors.In B-cell malignancies, BCR-dependent survival and proliferation signals are often substituted by oncogenic BCR-mimics (e.g. BCR-ABL1, JAK2, BRAFV600E, LMP2A, CD79B mutations; see schematic, right). Accordingly, we identified CD25 surface-expression as biomarker of oncogenic BCR-signaling and predictor of poor clinical outcomes. CD25-/-B-cell leukemia failed to initiate fatal disease in transplant recipients. Owing to imbalances of oncogenic BCR-signaling and p53-checkpoint activation, CD25-/- B-cell leukemia failed to initiate fatal disease in transplant recipients. In patient-derived xenograft models of drug-resistant B-cell malignancies, treatment with a CD25-specific antibody drug-conjugate (ADCT-301) extended survival of transplant recipients or eradicated disease. These findings identified CD25 as previously unrecognized feedback regulator of oncogenic BCR-signaling and provide a rationale for therapeutic targeting of CD25 in refractory B-cell malignancies. DisclosuresForman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Weinstock:Genentech/Roche, Monsanto: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Travera: Equity Ownership; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties. Uzel:Novartis: Research Funding.

Hematopoietic Progenitor Kinase-1 Structure in a Domain-Swapped Dimer

Enhancement of antigen-specific Tcell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of Tcell receptor signaling, which dampens Tcell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented Tcell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.

Development of a screening strategy for new modulators of T cell receptor signaling and T cell activation

Activation of the T cell receptor (TCR) leads to the generation of a network of signaling events critical to the developmental decision making and activation of T cells. Various experimental approaches continue to identify new signaling molecules, adaptor proteins, and other regulators of TCR signaling. We propose a screening strategy for the identification of small molecules affecting TCR signaling based on the uncoupling of TCR stimulation from cellular responses in developing thymocytes. We demonstrate that this strategy successfully identifies inhibitors of kinases already shown to act downstream of TCR engagement, as well as new inhibitors. The proposed strategy is easily scalable for high throughput screening and will contribute to the identification of new druggable targets in T cell activation.

Prognostic value of the expression of immunomodulatory targets in surgically resected lung adenocarcinoma.

173 Background: Therapies targeting immune checkpoints have recently shown encouraging activity in patients with non-small cell lung cancer. T-cell activation is controlled by the balance of co-stimulatory molecules that act positively in concert with T-cell receptor (TCR) signaling induced by antigen stimulation and co-inhibitory molecules (immune checkpoint molecules) that negatively regulate TCR signaling. Methods: We performed a Cap Analysis of Gene Expression (CAGE) analysis to quantify the gene expression of PD-L1, PD-L2, co-inhibitory molecules (CTLA-4, PD-1, TIM-3, BTLA, VISTA and LAG-3) and co-stimulatory molecules (CD28, OX40, GITR, CD317, CD27 and HVEM) using RNA extracted from 71 frozen tumor tissue samples of surgically resected lung adenocarcinomas, including 35 EGFR-mutated, 11 KRAS-mutated and 25 wild-type tumors. The correlations between the expression of the above immunomodulatory targets and the overall survival were analyzed. None of patients received immunotherapy. Results: Lung adenocarcinomas in this study were divided into two groups: tumors showing high expression of almost all immunomodulatory targets and those with low expression. The expression of CTLA-4, PD-1 and PD-L1 was not prognostic. The prognosis of patients with tumors showing high expression of TIM-3 was significantly worse than that of those with low expression, regardless of the EGFR and KRAS mutation status. The prognosis of patients with tumors showing high expression of VISTA was significantly better than that of those with low expression, regardless of the EGFR and KRAS mutation status. The prognosis of patients with tumors showing high expression of LAG-3 was significantly worse than that of those with low expression among overall patients. However, high expression of LAG-3 was an unfavorable prognostic factor in patients with EGFR-mutated or wild-type tumors, and it was a favorable prognostic factor in patients with KRAS-mutated tumors. Conclusions: Lung adenocarcinomas may be divisible into immunogenic and nonimmunogenic tumors. The prognostic value differs according to the immunomodulatory target and driver oncogene status.

The protein tyrosine phosphatase, nonreceptor type 22-1858C->T (rs2476601) polymorphism is not a genetic risk factor for systemic lupus erythematosus in Indian Tamils

Background: Systemic lupus erythematosus (SLE), a systemic autoimmune disease, occurs due to disruption of immune homeostasis against self-antigens. The etiology of SLE is complex and multiple genetic factors contribute to disease susceptibility and clinical phenotypes. Protein tyrosine phosphatase, nonreceptor type 22 (PTPN22) is a lymphoid-specific phosphatase that negatively regulates T-cell receptor signaling and is responsible for the maintenance of T-cell homeostasis. Genetic aberrations affecting the function of PTPN22 result in the proliferation of autoreactive T-cells and development of autoimmune diseases. Methods: We carried out a case–control genetic study to analyze the association of PTPN22 R620W polymorphism (rs2476601) with disease susceptibility and clinical and autoantibody profile in Indian Tamils with SLE. Three hundred SLE patients satisfying the 1997 revised American College of Rheumatology classification criteria for SLE were enrolled in the study. Disease activity was measured using the SLE Disease Activity Index. We recruited 460 age-, sex-, and ethnicity-matched individuals without a family history of autoimmune diseases as control population. Genomic DNA was extracted from the blood sample by salting-out method. The PTPN22-1858C->T (rs2476601) polymorphism was screened by polymerase chain reaction-restriction fragment length polymorphism. Results: The frequency of the ancestral allele “C” was similar in both cases and controls (99.3% and 99.8%, respectively) and the mutant allele “T” was less frequent in South Indian Tamil population; it did not influence clinical or serological phenotypes. Conclusion: Our findings suggest that the PTPN22 (rs2476601) polymorphism is less frequent and did not confer a risk for lupus or its associated clinical or serological phenotypes in South Indian Tamils.