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Activation of the Ca2+ release–activated Ca2+ (CRAC) channel is crucial for T cell functions. It was recently shown that naked cuticle homolog 2 (NKD2), a signaling adaptor molecule, orchestrates trafficking of ORAI1, a pore subunit of the CRAC channels, to the plasma membrane for sustained activation of the CRAC channels. However, the physiological role of sustained Ca2+ entry via ORAI1 trafficking remains poorly understood. Using NKD2 as a molecular handle, we show that ORAI1 trafficking is crucial for sustained Ca2+ entry and cytokine production, especially in inflammatory Th1 and Th17 cells. We find that murine T cells cultured under pathogenic Th17-polarizing conditions have higher Ca2+ levels that are NKD2-dependent than those under nonpathogenic conditions. In vivo, deletion of Nkd2 alleviated clinical symptoms of experimental autoimmune encephalomyelitis in mice by selectively decreasing effector T cell responses in the CNS. Furthermore, we observed a strong correlation between NKD2 expression and proinflammatory cytokine production in effector T cells. Taken together, our findings suggest that the pathogenic effector T cell response demands sustained Ca2+ entry supported by ORAI1 trafficking.

SRC3, a highly conserved member of the steroid receptor coactivator (SRC) family, is recruited by transcription factors to regulate cellular function. Previously, we demonstrated that SRC1, another highly conserved member of the SRC family, interacts with RORγt to regulate Th17 differentiation. However, the relationship between SRC1 and SRC3 in the regulation of Th17 cell function remains unknown. In this study, we demonstrate that mouse SRC3 interacts with RORγt in Th17 cells but not in thymocytes. In addition, Src3−/− mice exhibited defective Th17 differentiation and induction of experimental autoimmune encephalomyelitis but normal thymocyte development. Furthermore, a K313 to arginine mutation of RORγt (RORγt-K313R), which disrupts the interaction of RORγt with SRC3 but not with SRC1, impairs Th17 differentiation but not thymocyte development. These data suggest that SRC3 works with SRC1 to regulate RORγt-dependent Th17 differentiation but is not essential for RORγt-dependent thymocyte development.

Ca2+ release–activated Ca2+ channel regulator 2A (CRACR2A) is expressed abundantly in T cells and acts as a signal transmitter between TCR stimulation and activation of the Ca2+/NFAT and JNK/AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies and was shown to be one of the most sensitive targets of the widely used statin drugs. However, the physiological role of CRACR2A in T cell functions remains unknown. In this study, using transgenic mice for tissue-specific deletion, we show that CRACR2A promotes Th1 responses and effector function of Th17 cells. CRACR2A was abundantly expressed in Th1 and Th17 cells. In vitro, deficiency of CRACR2A decreased Th1 differentiation under nonpolarizing conditions, whereas the presence of polarizing cytokines compensated this defect. Transcript analysis showed that weakened TCR signaling by deficiency of CRACR2A failed to promote Th1 transcriptional program. In vivo, conditional deletion of CRACR2A in T cells alleviated Th1 responses to acute lymphocytic choriomeningitis virus infection and imparted resistance to experimental autoimmune encephalomyelitis. Analysis of CNS from experimental autoimmune encephalomyelitis–induced mice showed impaired effector functions of both Th1 and Th17 cell types, which correlated with decreased pathogenicity. Collectively, our findings demonstrate the requirement of CRACR2A-mediated TCR signaling in Th1 responses as well as pathogenic conversion of Th17 cells, which occurs at the site of inflammation.

Transcription factor retinoid acid–related orphan receptor (ROR)γt transcriptionally regulates the genes required for differentiation of Th17 cells that mediate both protective and pathogenic immunity. However, little is known about the function of posttranslational modifications in the regulation of RORγt activity. Mass spectrometric analysis of immunoprecipitated RORγt from Th17 cells identified multiple phosphorylation sites. Systematic mutation analysis of the identified phosphorylation sites found that phosphorylation of S376 enhances whereas phosphorylation of S484 inhibits Th17 differentiation. IκB kinase (IKK)α binds and phosphorylates RORγt at S376 but not S484. Knockdown of IKKα, dominant-negative IKKα, and RORγt mutants incapable of interacting with IKKα all decrease Th17 differentiation. Furthermore, nonphosophorylatable RORγt mutant (S376A) impairs whereas phosphomimetic mutant (S376E) stimulates Th17 differentiation independent of IKKα. Therefore, IKKα-dependent phosphorylation of S376 stimulated whereas IKKα-independent phosphorylation of S484 inhibited RORγt function in Th17 differentiation.

CRAC channel regulator 2A (CRACR2A) is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca(2+)-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies and was shown to be one of the most sensitive targets of the widely used statin drugs. However, the physiological role of CRACR2A in T cell functions remains unknown. In this study, using transgenic mice for tissue-specific deletion, we show that CRACR2A promotes Th1 responses and effector function of Th17 cells. CRACR2A was abundantly expressed in Th1 and Th17 cells. In vitro, deficiency of CRACR2A decreased Th1 differentiation under non-polarizing conditions, while presence of polarizing cytokines compensated this defect. Transcript analysis showed that weakened TCR signaling by deficiency of CRACR2A failed to promote Th1 transcriptional program. In vivo, conditional deletion of CRACR2A in T cells ameliorated Th1 responses to acute lymphocytic choriomeningitis virus infection and imparted resistance to experimental autoimmune encephalomyelitis. Analysis of central nervous system from experimental autoimmune encephalomyelitis-induced mice showed impaired effector functions of both Th1 and Th17 cell types, which correlated with decreased pathogenicity. Collectively, our findings demonstrate the requirement of CRACR2A-mediated TCR signaling in Th1 responses as well as pathogenic conversion of Th17 cells, that occurs at the site of inflammation.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca2+ influx through the Ca2+ release-activated Ca2+ channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor Gö6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca2+ influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca2+ influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.

Adoptive cell transfer utilizing tumor-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against hematological malignancies, but significant clinical success has not yet been achieved in solid tumors due in part to the strong immunosuppressive tumor microenvironment. Systemic or intratumoral delivery of an immune boosting molecule to overcome local suppression has been proposed, but the full potential is limited by non-specific stimulation of tumor growth, metastasis, and angiogenesis. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is mainly mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Both in vitro and in vivo assays revealed that highly selective optical control of Ca2+ signaling in adoptively transferred CTLs was sufficient to overcome immunosuppression at the tumor site by enhancing T cell activation, IFN-γ production and antitumor cytotoxicity, leading to a significant reduction in tumor growth in mice. Together, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses only at the targeted tumor sites.

Ca2+ signalling is important for T cell stimulation and its impairment leads to immune deficiency. In T cells, Ca2+ entry is mediated by CRAC (Ca2+-release activated Ca2+) channels that consist of the pore component, Orai1 and the endoplasmic reticulum Ca2+ sensor, STIM1. We recently identified CRACR2A (CRAC channel regulator 2A) as an interacting partner of Orai1 and STIM1. Here we investigate the role of a novel isoform of CRACR2A that is predominantly expressed in immune cells. CRACR2A contains multiple functional domains including Ca2+-binding motifs and a C-terminal Rab GTPase domain. Different from small Rab GTPases, CRACR2A utilized a two-step activation mechanism of GTP binding and T cell receptor (TCR) stimulation to mediate its translocation into the immunological synapse. Defect in GTP binding or hydrolysis activity results in loss and gain of function respectively. CRACR2A played a predominant role in Ca2+ and mitogen-activated protein kinase (MAPK) signalling pathways. Furthermore, CRACR2A-deficient T cells showed impairment in Th1 differentiation. In support of these data, mice with T cell-specific deletion of CRACR2A showed a strong resistance to experimental autoimmune encephalomyelitis. Previously, small GTPase has been shown to play an important role in TCR signaling. Our study identifies the unexpected usage of a large Rab GTPase as a molecular switch to regulate TCR signalling pathways.

Orai1 is the pore subunit of the Calcium Release Activated Channels (CRAC) that activate downstream calcineurin-NFAT pathway. Loss of function mutation in Orai1 causes lethality due to severe defects in immune system, which can be rescued by bone marrow transplantation. Using a high throughput chemical library screening, we have identified a class of small molecule blockers that inhibit Orai1. One of the small molecules, compound 5D blocked the activity of a constitutively active mutant of Orai1, suggesting a direct block of ion permeation via Orai1. Primary murine Th17 cells showed highest sensitivity to block by this compound. Treatment with compound 5D not only suppressed cytokine production, but also inhibited differentiation of Th17 cells by suppression of the retinoic-acid-receptor-related orphan receptors RORα and RORγt. Exogenous expression of RORα, RORγt or a constitutive active mutant of NFAT (CA-NFAT) rescued the blocking effect, identifying the Orai1-NFAT- ROR signaling pathway to be important for Th17 differentiation. The defects in cytokine production and differentiation were recapitulated in Th17 cells from Orai1-deficient mice suggesting Orai1 as a specific target of the blockers. In vivo administration of Orai1 blockers effectively delayed the onset and reduced the severity of autoimmune disease in mice. These data indicate that derivatives of compound 5D can be used as chemical templates for development of therapeutic agents to alleviate autoimmune diseases.