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Background: Hematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family. It has been reported that HPK1 negatively regulates the activation of T cells. Several compounds have been developed and tested in clinical trials to target HPK1 for cancer immunotherapy. However, whether kinase inhibition is sufficient to eliminate the immunosuppressive function of HPK1, particularly in T cells, remains elusive. Methods: In this study, genetic tools were used to edit the human T lymphocyte cell line Jurkat. The activation of HPK1-null cells, HPK1-wildtype cells and HPK1-kinase-inactive cells was compared through ectopic expression of HPK1 in HPK1 knockout cells or direct HPK1 mutation. Besides genetic validation, a series of compounds that selectively target HPK1 (with or without HPK1-degradation activity) were used to assess the potential scaffold function of HPK1 in regulation of human primary T cell activation and cytotoxic activity. Results and conclusion: Augmented T-cell receptor (TCR)-induced activation in HPK1-knockout Jurkat cells was inhibited by complementation of wildtype, but not kinase-dead HPK1. HPK1 K46E-knockin and K46*-knockin Jurkat cells showed comparable levels of enhanced TCR-induced activation compared with control HPK1-wildtype Jurkat cells. Similarly, HPK1 kinase inhibitor (Compound 1) and cereblon-based (CRBN-based) HPK1 degrader (Compound 2) elicited similar degrees of maximum TCR-induced activation in primary human peripheral blood T cells. In summary, the results of this study suggested that HPK1 kinase inhibitor may be sufficient for HPK1 targeting in T cell mediated cancer immunotherapy. [ABSTRACT FROM AUTHOR]

BackgroundHematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family. It has been reported that HPK1 negatively regulates the activation of T cells. Several compounds have been developed and tested in clinical trials to target HPK1 for cancer immunotherapy. However, whether kinase inhibition is sufficient to eliminate the immunosuppressive function of HPK1, particularly in T cells, remains elusive.MethodsIn this study, genetic tools were used to edit the human T lymphocyte cell line Jurkat. The activation of HPK1-null cells, HPK1-wildtype cells and HPK1-kinase-inactive cells was compared through ectopic expression of HPK1 in HPK1 knockout cells or direct HPK1 mutation. Besides genetic validation, a series of compounds that selectively target HPK1 (with or without HPK1-degradation activity) were used to assess the potential scaffold function of HPK1 in regulation of human primary T cell activation and cytotoxic activity.Results and conclusionAugmented T-cell receptor (TCR)-induced activation in HPK1-knockout Jurkat cells was inhibited by complementation of wildtype, but not kinase-dead HPK1. HPK1 K46E-knockin and K46*-knockin Jurkat cells showed comparable levels of enhanced TCR-induced activation compared with control HPK1-wildtype Jurkat cells. Similarly, HPK1 kinase inhibitor (Compound 1) and cereblon-based (CRBN-based) HPK1 degrader (Compound 2) elicited similar degrees of maximum TCR-induced activation in primary human peripheral blood T cells. In summary, the results of this study suggested that HPK1 kinase inhibitor may be sufficient for HPK1 targeting in T cell mediated cancer immunotherapy.

Abstract Background T 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. Methods Whole-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. Results TRAIL 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. Conclusions TRAIL-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.

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LATSS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LATSS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs). [ABSTRACT FROM AUTHOR]

Background: T 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. Methods: Whole-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. Results: TRAIL 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. Conclusions: TRAIL-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. [ABSTRACT FROM AUTHOR]

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts. [ABSTRACT FROM AUTHOR]

Sustained activation of the Ca2+-release-activated Ca2+ (CRAC) channel is pivotal for effector T cell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector T cells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we identify an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of T cell receptors, orchestrates trafficking and insertion of ORAI1+ vesicles to the plasma membrane. Together, our findings suggest that T cell receptor (TCR)-stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in T cells.

The RASA2 and RASA3 GTPase-activating proteins are recently recognized negative regulators of T cell activation and adhesion. RASA2 was identified as a novel immunotherapy target in a CRISPR-Cas9 screen for molecules permitting human primary T cell proliferation in the presence of inhibitors. RASA2 depletion increased antigen sensitivity in conventional and chimeric antigen receptor (CAR)-T cells and improved tumor rejection. RASA3 was identified in a CRISPR-Cas9 screen as an inhibitor of T cell receptor (TCR)-induced LFA-1-mediated ICAM-1 binding in mouse T cells. Rasa3 deficiency altered T cell adhesion, migration, and T cell-dependent antibody responses in mice. Rasa3 is highly expressed in pathogenic type 17 T helper cells and is required for the development of experimental autoimmune encephalitis in mice, suggesting that it has T cell type- and tissue-specific functions. Both RASA2/Rasa2 and RASA3/Rasa3 are rapidly repressed upon TCR stimulation of human and mouse T cells, thus promoting T cell activation. RASA3 is also inhibited by PI3K activity. Understanding the mechanisms that keep T cells in check can provide insight into targets for reinvigorating T cell function in cancer and chronic infection, or for suppressing T cell function in the context of autoimmunity and transplantation. The RASA2 and RASA3 GAPs have recently emerged as key inhibitors of T cell activation, adhesion, and migration, thereby introducing a potential new set of therapeutic targets for manipulating T cell activity. Following stimulation, the T cell receptor (TCR) and its coreceptors integrate multiple intracellular signals to initiate T cell proliferation, migration, gene expression, and metabolism. Among these signaling molecules are the small GTPases RAS and RAP1, which induce MAPK pathways and cellular adhesion to activate downstream effector functions. Although many studies have helped to elucidate the signaling intermediates that mediate T cell activation, the molecules and pathways that keep naive T cells in check are less understood. Several recent studies provide evidence that RASA2 and RASA3, which are GAP1-family GTPase-activating proteins (GAPs) that inactivate RAS and RAP1, respectively, are crucial molecules that limit T cell activation and adhesion. In this review we describe recent data on the roles of RASA2 and RASA3 as gatekeepers of T cell activation and migration. [ABSTRACT FROM AUTHOR]

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LATSS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LATSS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs).

Modulation of surface T cell antigen receptor (TCR) expression is crucial for proper T cell development and maintenance of mature T cell function at steady state and upon stimulation. We previously determined that CCDC134 (coiled-coil domain containing 134), a cytokine-like molecule that served as a potential member of the γc cytokine family, contributes to antitumor responses by augmenting CD8+ T cell-mediated immunity. Here we show that T cell-specific deletion of Ccdc134 decreased peripheral mature CD4+ and CD8+ T cells, which resulted in impaired T cell homeostasis. Moreover, Ccdc134-deficient T cells exhibited an attenuated response to TCR stimulation in vitro, showing lower activation and proliferative capacity. This was further reflected in vivo, rendering mice refractory to T cell-mediated inflammatory and antitumor responses. More importantly, CCDC134 is associated with TCR signaling components, including CD3ϵ, and attenuated TCR signaling in Ccdc134-deficient T cells via altered CD3ϵ ubiquitination and degradation. Taken together, these findings suggest a role for CCDC134 as a positive regulator of TCR-proximal signaling and provide insight into the cell-intrinsic functional consequences of Ccdc134 deficiency in the attenuation of T cell-mediated inflammatory and antitumor responses. [ABSTRACT FROM AUTHOR]

HIV-1 latency can only be reversed in a minor fraction of human CD4+ T cells harboring genetically intact proviruses, posing a major hurdle to the elimination of the persistent reservoir. Although heterogeneous clones of latently infected cells that constitute the HIV-1 reservoir exist in a dynamic pseudo-steady state, a functional cure might be achieved by specifically targeting inducible subsets that serve as potential sources of viral rebound. Host and viral control of the transcription elongation factor P-TEFb (positive transcription elongation factor b) is a crucial rate-limiting step in the emergence of HIV-1 from latency. Therefore, comprehensively understanding its regulatory mechanisms in primary CD4+ T cells can lead to the design of more effective latency reversal agents. Sequestration of P-TEFb by 7SK snRNP (small nuclear ribonucleoprotein) is an essential feature of its regulation in primary CD4+ T cells. Both Tat and T cell signaling can remodel the ribonucleoprotein complex to facilitate the exchange of P-TEFb to proviral HIV-1. An optimal latency reversal strategy may entail HIV-1 promoter activation by either relieving epigenetic restrictions or through induction of noncanonical nuclear factor kappa B (NF-κB) signaling combined with a potent diacylglycerol mimic intended to stimulate RasGRP1-dependent biogenesis of transcriptionally active P-TEFb. Due to multiple cellular blocks in transcription, HIV-1 latency can only be reversed in a minor fraction of infected but potentially virus-producing CD4+ T cells from infected patients. Recent work shows that the biogenesis of the transcription elongation factor P-TEFb is a crucial rate-limiting step in the emergence of HIV-1 from latency. These new insights can help guide the design of more effective latency reversal agents as part of an HIV eradication strategy. Antiretroviral therapy reduces circulating HIV-1 to undetectable amounts but does not eliminate the virus due to the persistence of a stable reservoir of latently infected cells. The reservoir is maintained both by proliferation of latently infected cells and by reseeding from reactivated cells. A major challenge for the field is to find safe and effective methods to eliminate this source of rebounding HIV-1. Studies on the molecular mechanisms leading to HIV-1 latency and reactivation are being transformed using latency models in primary and patient CD4+ T cells. These studies have revealed the central role played by the biogenesis of the transcription elongation factor P-TEFb (Positive Transcription Elongation Factor b) and its recruitment to proviral HIV-1, for the maintenance of viral latency and the control of viral reactivation. [ABSTRACT FROM AUTHOR]

The intestinal tract is constantly exposed to a diverse mixture of luminal antigens, such as those derived from commensals, dietary substances, and potential pathogens. It also serves as a primary route of entry for pathogens. At the forefront of this intestinal defense is a single layer of epithelial cells that forms a critical barrier between the gastrointestinal (GI) lumen and the underlying host tissue. The intestinal intraepithelial T lymphocytes (T-IELs), one of the most abundant lymphocyte populations in the body, play a crucial role in actively surveilling and maintaining the integrity of this barrier by tolerating non-harmful factors such as commensal microbiota and dietary components, promoting epithelial turnover and renewal while also defending against pathogens. This immune balance is maintained through interactions between ligands in the GI microenvironment and receptors on T-IELs. This review provides a detailed examination of the ligands present in the intestinal epithelia and the corresponding receptors expressed on T-IELs, including T cell receptors (TCRs) and non-TCRs, as well as how these ligand-receptor interactions influence T-IEL functions under both steady-state and pathological conditions. By understanding these engagements, we aim to shed light on the mechanisms that govern T-IEL activities within the GI microenvironment. This knowledge may help in developing strategies to target GI ligands and modulate T-IEL receptor expression, offering precise approaches for treating intestinal disorders.

Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpnll) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11D61Y in T cells. We observed reduced numbers of CD8+ and increased numbers of CD4+ T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8+ T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4+ and CD8+ T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/ necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy. [ABSTRACT FROM AUTHOR]

Activated Foxp3+ regulatory T (Treg) cells differentiate into effector Treg (eTreg) cells to maintain peripheral immune homeostasis and tolerance. T cell receptor (TCR)-mediated induction and regulation of store-operated Ca2+ entry (SOCE) is essential for eTreg cell differentiation and function. However, SOCE regulation in Treg cells remains unclear. Here, we show that inositol polyphosphate multikinase (IPMK), which generates inositol tetrakisphosphate and inositol pentakisphosphate, is a pivotal regulator of Treg cell differentiation downstream of TCR signaling. IPMK is highly expressed in TCR-stimulated Treg cells and promotes a TCR-induced Treg cell program. IPMK-deficient Treg cells display aberrant T cell activation and impaired differentiation into RORγt+ Treg cells and tissue-resident Treg cells. Mechanistically, IPMK controls the generation of higher-order inositol phosphates, thereby promoting Ca2+ mobilization and Treg cell effector functions. Our findings identify IPMK as a critical regulator of TCR-mediated Ca2+ influx and highlight the importance of IPMK in Treg cell-mediated immune homeostasis. [ABSTRACT FROM AUTHOR]

Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpn11) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11D61Y in T cells. We observed reduced numbers of CD8+ and increased numbers of CD4+ T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8+ T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4+ and CD8+ T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy.

CX-5461 is a first-in-class selective RNA polymerase I inhibitor. Previously we found that CX-5461 had anti-inflammatory activities. In this study we characterized potential immunosuppressive effects of CX-5461 and explored the underlying mechanisms. Allogeneic skin transplantation model (BALB/c to C57BL/6 mice) and heterotopic heart transplantation model (F344 to Lewis rats) were used. We showed that CX-5461 was a potent inhibitor of alloimmunity which prevented acute allograft rejections. CX-5461 treatment was invariably associated with expansion of the regulatory T cell population. In vitro, CX-5461 inhibited agonists-induced T cell activation. CX-5461 consistently inhibited the expression of interferon-γ and interleukin − 2, key mediators of T cell-mediated alloimmunity. Mechanistically, CX-5461-induced immunosuppression was, at least partly, dependent on the p53-DUSP5 (dual-specificity phosphatase 5) axis and subsequent antagonism of the Erk1/2 mitogen-activated protein kinase pathway. In conclusion, our results suggest that CX-5461 is a promising candidate of a novel class of immunosuppressant which may be used as an alternative to the currently approved anti-rejection therapies.

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2-inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through the unequal activation of distinct signaling pathways, we examined Erk1/2 phosphorylation or nuclear factor of activated T cells (NFAT) and nuclear factor (NF)- κB translocation in naïve OT-I CD8+ cell nuclei. We observed the consistent digital activation of NFAT1 and Erk1/2, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength, tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed the dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC sequencing analysis also revealed that genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned the expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating the optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variations in TCR signal strength can produce patterns of graded gene expression in activated T cells. [ABSTRACT FROM AUTHOR]

Summary: Nr4a receptors are activated by T cell receptor (TCR) signaling and play key roles in T cell differentiation. Which TCR signaling pathways regulate Nr4a receptors and their sensitivities to TCR signal strength and duration remains unclear. Using Nr4a1/Nur77-GFP and Nr4a3-Timer of cell kinetics and activity (Tocky) mice, we elucidate the signaling pathways governing Nr4a receptor expression. We reveal that Nr4a1–Nr4a3 are Src family kinase dependent. Moreover, Nr4a2 and Nr4a3 are attenuated by calcineurin inhibitors and bind nuclear factor of activated T cells 1 (NFAT1), highlighting a necessary and sufficient role for NFAT1 in the control of Nr4a2 and Nr4a3, but redundancy for Nr4a1. Nr4a1-GFP is activated by tonic and cognate signals during T cell development, whereas Nr4a3-Tocky requires cognate peptide:major histocompatibility complex (MHC) interactions for expression. Compared to Nr4a3-Tocky, Nr4a1-GFP is approximately 2- to 3-fold more sensitive to TCR signaling and is detectable by shorter periods of TCR signaling. These findings suggest that TCR signal duration may be an underappreciated aspect influencing the developmental fate of T cells in vivo.

Inert naive CD4+ T (T N) cells differentiate into functional T helper (Th) or regulatory T (Treg) cell subsets upon encountering antigens, mediating properly directed immune responses. Although all T N cells can differentiate into any of the Th and Treg cell subsets, heterogeneity exists among T N cells. By constructing reporter mice to detect ongoing T cell receptor (TCR) signaling, we identify that interleukin (IL)-1β signaling affects T N cell characteristics, independent of tonic TCR signaling, which also alters T N cell phenotypes. IL-1β reversibly attenuates the differentiation potential of T N cells toward Treg cells. IL-1β signaling is elevated in the splenic T N cells, consequently attenuating their differentiation potential toward Treg cells. Aberrant elevation of IL-1β signaling augments colitogenic activities of T N cells. T N cells in patients with colitis exhibited elevated IL-1β signaling. We demonstrate that phenotypic alteration in T N cells by IL-1β is an important mechanism in the regulation of immune responses. [Display omitted] • Effects of tonic TCR signaling on naive T cells are examined by constructing Nr4a1-dsGFP mice • IL-1β affects naive CD4+ T cell phenotypes in vivo in a tonic TCR signaling-independent manner • IL-1β attenuates differentiation potential of naive CD4+ T cells toward Tregs in the spleen • Naive CD4+ T cells in patients with colitis are under the influence of IL-1β Naive CD4+ T (T N) cells have been revealed to be a heterogeneous population. Sekiya et al. find that IL-1β signals reversibly affect T N cell phenotypes. The spleen is found to be enriched with IL-1β that is mainly produced by neutrophils, reducing the differentiation potential of splenic T N cells toward regulatory T cells. [ABSTRACT FROM AUTHOR]

Inert naive CD4 + T (T N ) cells differentiate into functional T helper (Th) or regulatory T (Treg) cell subsets upon encountering antigens, mediating properly directed immune responses. Although all T N cells can differentiate into any of the Th and Treg cell subsets, heterogeneity exists among T N cells. By constructing reporter mice to detect ongoing T cell receptor (TCR) signaling, we identify that interleukin (IL)-1β signaling affects T N cell characteristics, independent of tonic TCR signaling, which also alters T N cell phenotypes. IL-1β reversibly attenuates the differentiation potential of T N cells toward Treg cells. IL-1β signaling is elevated in the splenic T N cells, consequently attenuating their differentiation potential toward Treg cells. Aberrant elevation of IL-1β signaling augments colitogenic activities of T N cells. T N cells in patients with colitis exhibited elevated IL-1β signaling. We demonstrate that phenotypic alteration in T N cells by IL-1β is an important mechanism in the regulation of immune responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The ability of T cells to identify foreign antigens and mount an efficient immune response while limiting activation upon recognition of self and self-associated peptides is critical. Multiple tolerance mechanisms work in concert to prevent the generation and activation of self-reactive T cells. T cell tolerance is tightly regulated, as defects in these processes can lead to devastating disease; a wide variety of autoimmune diseases and, more recently, adverse immune-related events associated with checkpoint blockade immunotherapy have been linked to a breakdown in T cell tolerance. The quantity and quality of antigen receptor signaling depend on a variety of parameters that include T cell receptor affinity and avidity for peptide. Autoreactive T cell fate choices (e.g., deletion, anergy, regulatory T cell development) are highly dependent on the strength of T cell receptor interactions with self-peptide. However, less is known about how differences in the strength of T cell receptor signaling during differentiation influences the ‘function’ and persistence of anergic and regulatory T cell populations. Here, we review the literature on this subject and discuss the clinical implications of how T cell receptor signal strength influences the ‘quality’ of anergic and regulatory T cell populations.

Intravenous immunoglobulin (IVIg) therapy is widely used to treat autoimmune and infectious disorders. Despite the clinical efficacy of IVIg therapy, its precise immunosuppressive mechanisms remain unclear. Here, we provide evidence that IVIg acts directly on T cells to suppress their activation upon T cell receptor (TCR) ligation. IVIg suppressed the proliferation of murine splenocytes upon stimulation with anti-CD3 antibody and T cell-tropic mitogens. These immunosuppressive effects of IVIg were still intact against purified T cells, and the depletion of naturally-occurring regulatory T cells (nTreg) had no effect on T cell regulatory activity. Instead, we found that IVIg negatively regulated TCR signaling; IVIg co-stimulation impaired IκB degradation, nuclear translocation of the nuclear factor of activated T cells (NFAT), and the activation of mitogen-activated protein kinase (MAPK, Erk1/2). These results suggest an additional new immunosuppressive role of IVIg, which acts directly on conventional T cells to suppress the TCR signaling pathway. • Intravenous immunoglobulin (IVIg) acts directly on T cells to suppress their activation. • IVIg co-stimulation does not induce CD4+ Foxp3+ regulatory T cells. • IVIg negatively regulates T cell receptor signaling. [ABSTRACT FROM AUTHOR]

In T cells, T cell receptor (TCR) signaling initiates downstream transcriptional mechanisms for T cell activation and differentiation. Foxp3-expressing regulatory T cells (Treg) require TCR signals for their suppressive function and maintenance in the periphery. It is, however, unclear how TCR signaling controls the transcriptional program of Treg. Since most of studies identified the transcriptional features of Treg in comparison to naïve T cells, the relationship between Treg and non-naïve T cells including memory-phenotype T cells (Tmem) and effector T cells (Teff) is not well understood. Here, we dissect the transcriptomes of various T cell subsets from independent datasets using the multidimensional analysis method canonical correspondence analysis (CCA). We show that at the cell population level, resting Treg share gene modules for activation with Tmem and Teff. Importantly, Tmem activate the distinct transcriptional modules for T cell activation, which are uniquely repressed in Treg. The activation signature of Treg is dependent on TCR signals and is more actively operating in activated Treg. Furthermore, by using a new CCA-based method, single-cell combinatorial CCA, we analyzed unannotated single-cell RNA-seq data from tumor-infiltrating T cells, and revealed that FOXP3 expression occurs predominantly in activated T cells. Moreover, we identified FOXP3-driven and T follicular helper-like differentiation pathways in tumor microenvironments, and their bifurcation point, which is enriched with recently activated T cells. Collectively, our study reveals the activation mechanisms downstream of TCR signals for the bifurcation of Treg and Teff differentiation and their maturation processes.

Preprogrammed IL-17-producing γδ T cells constitute a poorly understood class of lymphocytes that express rearranged antigen receptors but appear to make little use of them. γδT17 cells were first characterized as tissue-resident sentinels with innate effector function. However, ongoing research continues to reveal unexpected complexity to this unusual subset, including phenotypic plasticity, memory-like activity and unique migratory behavior. Despite these advances, at the core of γδT17 cell biology remain fundamental gaps in knowledge: Are γδT17 cells truly innate or has the importance of the T cell receptor been overlooked? How unique are they among IL-17-producing lymphocytes? How similar are these cells between mice and humans? We speculate that answering these unresolved questions is key to successful manipulation of γδ T cells in clinical settings.

In T cells, T cell receptor (TCR) signaling initiates downstream transcriptional mechanisms for T cell activation and differentiation. Foxp3-expressing regulatory T cells (Treg) require TCR signals for their suppressive function and maintenance in the periphery. It is, however, unclear how TCR signaling controls the transcriptional program of Treg. Since most of studies identified the transcriptional features of Treg in comparison to naïve T cells, the relationship between Treg and non-naïve T cells including memory-phenotype T cells (Tmem) and effector T cells (Teff) is not well understood. Here, we dissect the transcriptomes of various T cell subsets from independent datasets using the multidimensional analysis method canonical correspondence analysis (CCA). We show that at the cell population level, resting Treg share gene modules for activation with Tmem and Teff. Importantly, Tmem activate the distinct transcriptional modules for T cell activation, which are uniquely repressed in Treg. The activation signature of Treg is dependent on TCR signals and is more actively operating in activated Treg. Furthermore, by using a new CCA-based method, single-cell combinatorial CCA, we analyzed unannotated single-cell RNA-seq data from tumor-infiltrating T cells, and revealed that FOXP3 expression occurs predominantly in activated T cells. Moreover, we identified FOXP3-driven and T follicular helper-like differentiation pathways in tumor microenvironments, and their bifurcation point, which is enriched with recently activated T cells. Collectively, our study reveals the activation mechanisms downstream of TCR signals for the bifurcation of Treg and Teff differentiation and their maturation processes. [ABSTRACT FROM AUTHOR]

Preprogrammed IL-17-producing γδ T cells constitute a poorly understood class of lymphocytes that express rearranged antigen receptors but appear to make little use of them. γδT17 cells were first characterized as tissue-resident sentinels with innate effector function. However, ongoing research continues to reveal unexpected complexity to this unusual subset, including phenotypic plasticity, memory-like activity and unique migratory behavior. Despite these advances, at the core of γδT17 cell biology remain fundamental gaps in knowledge: Are γδT17 cells truly innate or has the importance of the T cell receptor been overlooked? How unique are they among IL-17-producing lymphocytes? How similar are these cells between mice and humans? We speculate that answering these unresolved questions is key to successful manipulation of γδ T cells in clinical settings. [ABSTRACT FROM AUTHOR]

ObjectiveTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell apoptosis by transducing apoptosis signals after interacting with its receptor (TRAIL-R). Although the actual biological role of TRAIL remains to be elucidated, recent accumulating evidence implies that TRAIL regulates immune responses and immune cell homeostasis via an apoptosis-independent pathway, suggesting a novel immune-regulatory role of TRAIL in autoimmune diseases. The purpose of this study is to address the immune-regulatory role and molecular mechanism of TRAIL in regulating T cell activation in autoimmune diseases.DesignTRAIL was administered to mice to induce experimental autoimmune encephalomyelitis (EAE), and to evaluate its impact on neuroinflammation and disease activity. The effects of TRAIL on neuroantigen [myelin oligodendrocyte glycoprotein (MOG)35–55]-activated T cell proliferation and cytokine production were investigated. TRAIL-treated MOG35–55-activated splenic Th17 cells were further adoptively transferred into Rag1 KO mice to induce passive EAE. Gene expression profiles of CD4+ T cells from EAE mice treated with TRAIL were analyzed by RNA sequencing and transcriptome analysis.ResultsTRAIL suppressed autoimmune encephalomyelitis and inhibited T cell reactivity to neuro-antigen in murine EAE, and the effects were dependent on TRAIL-R signaling. Moreover, TRAIL directly inhibited activation of MOG35–55-activated CD4+ T cells, resulting in suppression of neuroinflammation and reduced disease activity in adoptive transfer-induced EAE. Furthermore, TRAIL-R signaling inhibited phosphorylation of proximal T cell receptor (TCR)-associated tyrosine kinases in activated CD4+ T cells. Importantly, TRAIL/TRAIL-R interaction downregulated TCR downstream signaling genes in RNA sequencing and transcriptome analysis.ConclusionTRAIL/TRAIL-R interaction regulates CD4+ T cell activation in autoimmune inflammation and directly suppresses T cell activation via inhibiting TCR signaling, suggesting that TRAIL-R serves as a novel immune checkpoint in T cell responses.

While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR) signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR–CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Objective: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell apoptosis by transducing apoptosis signals after interacting with its receptor (TRAIL-R). Although the actual biological role of TRAIL remains to be elucidated, recent accumulating evidence implies that TRAIL regulates immune responses and immune cell homeostasis via an apoptosis-independent pathway, suggesting a novel immune-regulatory role of TRAIL in autoimmune diseases. The purpose of this study is to address the immune-regulatory role and molecular mechanism of TRAIL in regulating T cell activation in autoimmune diseases. Design: TRAIL was administered to mice to induce experimental autoimmune encephalomyelitis (EAE), and to evaluate its impact on neuroinflammation and disease activity. The effects of TRAIL on neuroantigen [myelin oligodendrocyte glycoprotein (MOG)35-55]-activated T cell proliferation and cytokine production were investigated. TRAIL-treated MOG35-55-activated splenic Th17 cells were further adoptively transferred into Rag1 KO mice to induce passive EAE. Gene expression profiles of CD4+ T cells from EAE mice treated with TRAIL were analyzed by RNA sequencing and transcriptome analysis. Results: TRAIL suppressed autoimmune encephalomyelitis and inhibited T cell reactivity to neuro-antigen in murine EAE, and the effects were dependent on TRAIL-R signaling. Moreover, TRAIL directly inhibited activation of MOG35-55-activated CD4+ T cells, resulting in suppression of neuroinflammation and reduced disease activity in adoptive transfer-induced EAE. Furthermore, TRAIL-R signaling inhibited phosphorylation of proximal T cell receptor (TCR)-associated tyrosine kinases in activated CD4+ T cells. Importantly, TRAIL/TRAIL-R interaction downregulated TCR downstream signaling genes in RNA sequencing and transcriptome analysis. Conclusion: TRAIL/TRAIL-R interaction regulates CD4+ T cell activation in autoimmune inflammation and directly suppresses T cell activation via inhibiting TCR signaling, suggesting that TRAIL-R serves as a novel immune checkpoint in T cell responses. [ABSTRACT FROM AUTHOR]

Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin ‘foci’. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

Although the Associative (linked) Recognition of Antigen (ARA) model for a Self (S)–Nonself (NS) discrimination, now over 50 years old, is built on a solid conceptual and experimental base, two unsettled questions remain. 1. How is ARA accomplished for T–T interactions? 2. How does the immune system get started? In examining these questions, unanticipated aspects of the ARA Model itself had to be reconsidered. This essay spells out these problems and suggests possible solutions. [ABSTRACT FROM AUTHOR]

Immune aging is best known for its immune defects that increase susceptibility to infections and reduce adaptive immune responses to vaccination. In parallel, the aged immune system is prone to autoimmune responses and many autoimmune diseases increase in incidence with age or are even preferentially encountered in the elderly. Why an immune system that suboptimally responds to exogenous antigen fails to maintain tolerance to self-antigens appears to be perplexing. In this review, we will discuss age-associated deviations in the immune repertoire and the regulation of signaling pathways that may shed light on this conundrum [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor 2 (TLR2) serves as a co-stimulatory receptor for human T cells by enhancing T cell receptor (TCR)-induced cytokine production and proliferation. However, it is unknown where signals from the TCR and TLR2 converge to enhance T cell activation. To address this gap, we examined changes in TCR-induced signaling following concurrent TLR2 activation in human T cells. Both proximal TCR-mediated signaling and early NFκB activation were not enhanced by TCR andTLR2 co-activation, potentially due to the association of TLR2 with TLR10. Instead, TLR2 co-induction did augment Akt and Erk1/Erk2 activation in human T cells. These findings demonstrate that TLR2 activates distinct signaling pathways in human T cells and suggest that alterations in expression of TLR2 co-receptors may contribute to aberrant T cell responses. [Copyright &y& Elsevier]

Lactic acidosis is common to most solid tumors and has been found to affect infiltrating immune cells. Here we document effector phase inhibition of cytotoxic T cells (CTLs) involving complete blockage of cytokine production and partial impairment of lytic granule exocytosis. Lactic acidosis impaired TCR-triggered phosphorylation of JNK, c-Jun and p38, while not affecting MEK1 and ERK. The select targeting of signaling proteins involved in IFNγ production (JNK/c-Jun, p38) without affecting those jointly used in cytokine regulation and granule exocytosis (MEK1/ERK) explains the observed split effect of lactic acidosis on the CTL responses. CTL inhibition by lactic acidosis showed fast dynamics with immediate onset and reversion. Functional recovery by neutralizing the extracellular pH despite continuous presence of lactate holds promise that CTL activity can be improved in the milieu of solid tumors with appropriate anti-acidosis treatment, thereby increasing the efficacy of adoptive T cell therapy. [ABSTRACT FROM AUTHOR]