The diverse functions of the PD1 inhibitory pathway.

p110γ and p110δ Phosphoinositide 3-Kinase Signaling Pathways Synergize to Control Development and Functions of Murine NK Cells

Lag3 and PD-1 are immune checkpoints that regulate T cell responses and are current immunotherapy targets. Yet how they function to control early stages of CD4+ T cell activation remains unclear. Here, we show that the PD-1 and Lag3 pathways exhibit layered control of the early CD4+ T cell activation process, with the effects of Lag3 more pronounced in the presence of PD-1 pathway co-blockade (CB). RNA sequencing revealed that CB drove an early NFAT-dependent transcriptional profile, including promotion of ICOShi T follicular helper cell differentiation. NFAT pathway inhibition abolished CB-induced upregulation of NFAT-dependent co-receptors ICOS and OX40, whilst unaffecting the NFAT-independent gene Nr4a1. Mechanistically, Lag3 and PD-1 pathways functioned additively to regulate the duration of T cell receptor signals during CD4+ T cell re-activation. Phenotypic changes in peripheral blood CD4+ T cells in humans on anti-Lag3 and anti-PD-1 combination therapy revealed upregulation of genes encoding ICOS and OX40 on distinct CD4+ T cell subsets, highlighting the potential translational relevance of our findings. Our data therefore reveal that PD-1 and Lag3 pathways converge to additively regulate TCR signal duration and may preferentially control NFAT-dependent transcriptional activity during early CD4+ T cell re-activation.

Impaired hepatitis B vaccine responses during chronic hepatitis C infection: Involvement of the PD-1 pathway in regulating CD4+ T cell responses

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

BackgroundIn spite of advancements in treatment of ankylosing spondylitis (AS), its pathogenesis remains incompletely understood. The current evidence suggests that AS is both an autoinflammatory and autoimmune disease involving innate...

Hematopoietic progenitor kinase 1 (HPK1) has been shown to act as a negative regulator of T cell receptor signaling and of subsequent effector T cell function. Inhibiting HPK1 to enhance T cell activity has emerged as a promising strategy for cancer immunotherapy. Upon T cell receptor engagement, the kinase domain of HPK1 is activated and targets components of the T cell receptor (TCR) signaling pathway for degradation, including SLP76. However, the molecular events connecting HPK1 kinase activity to observed T cell functions downstream of proximal TCR signaling are not well understood. Through transcriptional profiling of HPK1-kinase-dead (HPK1-KD) versus wild-type CD8+ T cells following an acute, attenuated Listeria monocytogenes infection, we observed increased expression of many genes associated with T cell activation and effector function, including changes in expression of several key transcription factors and their targets. Upon activation, HPK1-KD T cells, as well as T cells treated with our small-molecule HPK1 inhibitor, produce higher levels of a wide range of effector cytokines in vitro and in vivo. Treatment of mice with the HPK1 inhibitor also inhibited tumor growth in several syngeneic tumor models. These data expand our understanding of the role of HPK1 in inhibiting CD8+ T cell effector programs, and, importantly, the impact of HPK1 inhibitors on T cell function. Citation Format: Rachel Y. Ames, Rongqi Zhao, Parker Mace, Adam Grant, Guorui Xie, Heather Milestone, Molly Grandcolas, Eva Fang, Nicolae Kiosea, Stephen Wong, Martin Brovarney, Scott Jacobson, Danilo Nebalasca, Jorge Arguello, Blanca Gomez, Hiranmayee Kandala, Michelle Y. Ko, Lan Nguyen, Omar Robles, Grant Shibuya, Anton A. Shakhmin, Parcharee Tivitmahaisoon, Vi-Anh Vu, Ashkaan Younai, Mikhail Zibinsky, Babu Subramanyam, Daniel Poon, Mohsen Sabouri Ghomi, David J. Wustrow, Paul D. Kassner, George E. Katibah, Dirk G. Brockstedt. HPK1 inhibits CD8+ T cell effector gene expression following T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2654.

The emergence of novel biophysical approaches has revealed that mechanosensing at the immunological synapse is critical in regulating T cell activation and function. To investigate the role of the mechanosensitive cation channel, Piezo1, on T cell function, we generated T cell specific Piezo1 knockout mice (P1cKO). Previously we demonstrated that Piezo1 restrains inflammatory Th1 and Th17 polarization in vitro. However, Piezo1 deficiency in T cells ameliorated disease progression in primary autoimmune mouse models, while completely ablating disease phenotype in adoptive transfer autoimmune mouse models. Therefore, we hypothesized that Piezo1 deficiency in T cells increases T cell receptor (TCR) signaling and activation leading to increased T cell exhaustion and apoptosis. Preliminary studies of early TCR signaling suggest that P1cKO CD4+ T cells have increased phosphorylated ZAP-70 (pZAP-70) compared to wildtype, and Piezo1 activation by Yoda-1 conversely decreased pZAP-70. After 3 days of TCR stimulation P1cKO CD4+ T cells similarly expressed activation markers CD25, CD69, and CD95 compared to wildtype, but had an increased expression of exhaustion markers LAG-3, PD-1, and TIGIT. Cell tracking in vivo revealed that P1cKO effector CD4+ T cells expand rapidly after transfer but fail to persist long term. Taken together, our data suggests that Piezo1 plays an important role during early T cell activation that ultimately restrains hyper inflammatory CD4+ T cell responses.

BackgroundT cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation.MethodsWhole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells.ResultsTRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation.ConclusionsTRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

Dependence of T Cell Antigen Recognition on T Cell Receptor-Peptide MHC Confinement Time

Chronic lymphocytic leukemia (CLL) remains incurable despite treatment advances, and a major challenge is that biomarkers that predict response and resistance to current therapies are lacking. We report that activated and proliferating malignant CLL B cells in circulation express PD-1, a protein normally expressed in T cells. PD-1 expression is absent in circulating B cells from healthy controls and nonmalignant B cells from patients with CLL. Circulating PD-1+ CLL cells are found in all treatment naïve patients, regardless of immunoglobulin heavy-chain variable region gene mutation status or cytogenetic abnormalities. PD-1+ CLL cells are transcriptionally distinct compared to PD-1- CLL cells and upregulate genes associated with cell activation, proliferation, and B cell receptor (BCR) and toll-like receptor (TLR) signaling. Indeed, ex vivo stimulation of the BCR and TLR9 readily increased PD-1 expression in CLL cells from treatment-naïve patients within 24 h, an effect that was blocked by Bruton's tyrosine kinase inhibitors (BTKi). More importantly, patients initiating BTKi therapy experienced profound reductions in circulating PD-1+ CLL cell numbers within 1 mo, which is in line with reduction in Ki-67+ CLL cells. Elevated percentages of circulating PD-1+ CLL cells also preceded a clinical diagnosis of disease progression in patients receiving BTKi. Thus, our findings indicate that PD-1 expression is a potential biomarker to identify proliferating CLL cells in vivo and will be useful to predict response and resistance to BTKi. In addition, eliminating PD-1+ CLL cells with depleting anti-PD-1 monospecific or bispecific antibodies should be explored as a potential therapeutic strategy.

Programmed cell death-1 (PD-1) is an inhibitory receptor highly expressed on T cells specific for viral and tumor antigens. TGF-β1 affects the tumor microenvironment and high serum levels are associated with both malignancy and pathogenesis in some chronic viral infections. However, the effect of TGF-β1 on PD-1 expression is unknown. PD-1 expression requires nuclear factor of activated T cells (NFATc1) activation through T cell receptor (TCR) signaling. We found that TGF-β1 signaling increases NFATc1-induced pdcd-1 mRNA and PD-1 expression on human peripheral CD4+ and CD8+ T cells. Smad2 and Smad3 are phosphorylated following TGF-β Receptor I binding and ultimately mediate gene transcription. We found that inhibition of TGFβRI kinase activity or Smad3 phosphorylation blocked TGF-β1-dependent PD-1 enhancement on T cells. Similarly, when compared to TCR stimulation alone, TGF-β1 increased PD-1 expression on murine wild-type (WT) but not on Smad3 knock-out (KO) CD4+ T cells. In contrast, Smad2 KO CD4+ T cells maintain increased PD-1 expression in the presence of TGF-β1, demonstrating a specific role for Smad3. We show that Smad3 increases pdcd-1 transcription by directly binding to a proximal region of the human PD-1 promoter and stabilizing NFATc1 binding. In addition to TGF-β1’s previously known effects on T cell function, our findings suggest that TGF-β1 mediates suppression via PD-1 upregulation. Smad3 may represent an additional target in therapeutic modulation of PD-1.

Butyrophilin (BTN) proteins are members of the B7 immunoglobulin superfamily and exhibit well-characterized immunomodulatory functions in mammals. We have recently identified BTN1A1 as an immune checkpoint protein prominently upregulated in response to acute inflammatory insults. Further in vitro and in vivo assays have validated BTN1A1 as an immune checkpoint target, particularly for patients refractory to anti-PD-1/PD-L1 antibody treatment. We have also developed a humanized antibody targeting human BTN1A1, hSTC810, which is expected to enter into Phase I clinical trials in the first quarter of 2022. In this study, a cell microarray from Retrogenix (Whaley Bridge, UK) was used to identify binding partner(s) for the extracellular domain of human BTN1A1. Through this screening approach, we found that BTN1A1 binds to galectin-1 (Gal1), galectin-9 (Gal9), and neuropilin 2 (NRP2). These three putative binding partners could specifically bind to wild-type BTN1A1 but not to this protein's unglycosylated (2NQ) form. Of these three targets, immunoprecipitation and Biacore binding assays revealed that Gal9 exhibited the greatest affinity for human BTN1A1, followed by Gal1, with respective KD values of 22.7 nM and 1.88 μM - an 83-fold difference. Gal9 binding to human BTN1A1 was dependent on BTN1A1 glycosylation status and required the carbohydrate recognition domain (CRD) of Gal9. As Gal9 is a known PD-1-binding protein, the KD of Gal9 for PD-1 was additionally assessed and found to be 19.7 nM. These results thus predicted the potential formation of BTN1A1/Gal9/PD-1 complexes. Consistent with these predictions, immunoprecipitation assays performed using cells expressing Myc-tagged versions of these three proteins demonstrated the formation of BTN1A1/Gal9, PD-1/Gal9, and BTN1A1/Gal9/PD-1 complexes. CRISPR-mediated BTN1A1 knockout in Jurkat T cells induced both PD-1 expression and T cell activation. BTN1A1 also suppressed T cell receptor (TCR) signaling in Jurkat cells, and the addition of exogenous recombinant Gal9 protein further blunted such BTN1A1-mediated TCR-signaling downregulation. Such downregulation was not observed in PD-1 knockout Jurkat cells. Together with the observation that BTN1A1 does not bind to PD-1 directly, the data suggest that BTN1A1 suppresses T cell activation by interacting with PD-1 through Gal9. As high Gal9 expression levels are correlated with poor prognosis in multiple cancers, our results highlight this BTN1A1-Gal9-PD-1 axis as a novel therapeutic target for immunotherapeutic drug development. (1) Chung EM, Bong YS, Kim YS, Park A, You YO, Sharma A, Lin SH, Lee YJ, Jung H, Yoo SS. BTN1A1: a novel immune checkpoint for cancer immunotherapy beyond the PD-1/PD-L1 axis. Cancer Res 2021;81(13_Suppl): Abstract nr 1643. Citation Format: Ezra M Chung, Young-Seung Kim, Chunai Wu, Andrew H Park, Hyunjin Jung, Stephen S Yoo. The immune checkpoint protein BTN1A1 suppresses T cell activation through interactions with Gal9 and PD-1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA019.

CD72 is a 45-kDa B cell transmembrane glycoprotein that has been shown to be important for B cell activation. However, whether CD72 ligation induces B cell activation by delivering positive signals or sequestering negative signals away from B cell receptor (BCR) signals remains unclear. Here, by comparing the late signaling events associated with the mitogen-activated protein kinase pathway, we identified many similarities and some differences between CD72 and BCR signaling. Thus, CD72 and BCR activated the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase. Both CD72- and BCR-mediated ERK and JNK activation required protein kinase C activity, which was equally important for CD72- and BCR-induced B cell proliferation. However, CD72 induced stronger JNK activation compared with BCR. Surprisingly, the JNK activation induced by both BCR and CD72 is Btk independent. Although both CD72 and BCR induced Btk-dependent ERK activation, CD72-mediated proliferation is more resistant to blocking of ERK activity than that of BCR, as shown by the proliferation response of B cells treated with PD98059 and dibutyryl cAMP, agents that inhibit ERK activity. Most importantly, CD72 signaling compensated for defective BCR signaling in X-linked immunodeficiency B cells and partially restored the proliferation response of X-linked immunodeficiency B cells to anti-IgM ligation. These results suggest that CD72 signals B cells by inducing BCR-independent positive signaling pathways.

The earliest event in T cell receptor(TCR) signaling is the activation of the Src family kinase(SFK) Lck. Phosphorylation of a conserved tyrosine in the C-terminal tail of Lck inhibits its kinase activity and is reciprocally regulated by the protein tyrosine kinase, C-terminal Src kinase(Csk), and the protein tyrosine phosphatase, CD45. Using a chemical-genetic approach, our lab has generated a novel analog-sensitive dominant inhibitory allele of Csk(CskAS) that can be specifically inhibited by a small molecule. Surprisingly, inhibition of the CskAS allele leads to rapid T cell activation in the absence of any TCR stimulation, indicating that Lck activity is dynamically regulated by an equilibrium between Csk and CD45 activities and that there is an adaptive response to the reduced basal signaling imposed by the dominant inhibitory CskAS. Moreover, mutating the SH2 and SH3 domains of CskAS results in reduced T cell activation upon inhibition of CskAS, suggesting that protein-protein interactions are important for this ligand-independent activation. We have identified Dok-1(downstream of tyrosine kinases-1), an adaptor protein that inhibits tyrosine kinase signaling, as a candidate in controlling the adaptive response responsible for ligand-independent T cell activation. Dok-1 is hyperphosphorylated and interacts with Csk via its SH2 domain upon CskAS inhibition. We suggest that Dok-1 is part of a negative feedback loop that acts to dampen TCR signals.

BackgroundProgrammed death 1 (PD-1) is an immune checkpoint receptor expressed by activated T-cells. Activation of the PD-1 pathway reduces T cell activation and inflammation. Targeting PD-1 in cancer can result...