The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum.

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.

The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4⁺ T cells.

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

SRC2 controls CD4+ T cell activation via stimulating c-Myc-mediated upregulation of amino acid transporter Slc7a5.

T cell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ T cell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in T cells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C. rodentium) infection. Adoptive transfer of naive CD4+ T cells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/ recipients. Further, CD4+ T cells from SRC2fl/fl/CD4Cre mice display defective T cell proliferation, cytokine production, and differentiation both in vitro and in vivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for T cell activation. Slc7a5 fails to be up-regulated in CD4+ T cells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ T cells to induce EAE. Therefore, SRC2 is essential for CD4+ T cell activation and, thus, a potential drug target for controlling CD4+ T cell-mediated autoimmunity.

Ca2+ Signaling Augmented by ORAI1 Trafficking Regulates the Pathogenic State of Effector T Cells.

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 Is a Cofactor for RORγt in Th17 Differentiation but Not Thymocyte Development.

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.

SRC1 promotes Th17 differentiation by overriding Foxp3 suppression to stimulate RORγt activity in a PKC-θ-dependent manner.

Th17 cells are major players in multiple autoimmune diseases and are developmentally contingent on reciprocal functionality between the transcription factor Retineic acid receptor-related orphan nuclear receptor gamma (RORγt) and Forkhead box protein P3 (Foxp3). Here we deciphered a previously unappreciated role of Steroid receptor coactivator 1 (SRC1) in defining the lineage decision for the development of Th17 versus induced T-regulatory (iTreg) cells. We demonstrate that SRC1 functions as a critical coactivator for RORγt in vivo to promote the functional dominance of RORγt over Foxp3 and thus establishing an unopposed Th17 differentiation program. In the absence of SRC1, T cell polarization resulted in decreased IL-17+ and increased Foxp3+ cells during both in vitro differentiation and in vivo development of experimental autoimmune encephalomyelitis. Mechanistically, T cell receptor (TCR) signaling molecule protein kinase C theta (PKC-θ)-mediated phosphorylation of SRC1 is important for inducing enhanced RORγt-SRC1 interaction, stable DNA binding, and resultant IL-17A transcription. Furthermore, phospho-SRC1-mediated recruitment of CARM1 induced prominent asymmetric dimethylation of H3R17 while preventing repressive H3K9 trimethylation and hence further modifying the IL-17 locus for optimal transcription. Moreover, binding of phospho-SRC1 to RORγt displaced bound Foxp3, leading to prompt degradation of the dissociated Foxp3 via a ubiquitin-proteosomal pathway and hence reversing the inhibitory action of Foxp3 on RORγt activity. Thus, SRC1 acts as a crucial molecular mediator to integrate positive PKC-θ-dependent TCR signals to induce peak RORγt activity and establish phenotypic dominance of Th17 over the iTreg pathway.

Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

Histone Lys-specific demethylases (KDMs) play a key role in many biological processes through epigenetic mechanisms. However, the role of KDMs in inflammatory responses to oral bacterial infection is poorly understood. Here, we show a novel regulatory role of KDM3C in inflammatory responses to oral bacterial infection. KDM3C expression is transiently suppressed in human and mouse macrophages exposed to LPS from Porphyromonas gingivalis (Pg LPS). Loss of KDM3C in both human and mouse macrophages led to notable induction of proinflammatory cytokines in response to Pg LPS stimulation. Also, KDM3C depletion led to strong induction of p65 phosphorylation and accelerated nuclear translocation in cells exposed to Pg LPS. Kdm3C knockout (KO) in mice led to increased alveolar bone destruction upon induction of experimental periodontitis or pulp exposure compared with those of the wild-type (WT) littermates. The Kdm3C KO mice also revealed an increased number of osteoclasts juxtaposed to the bony lesions. We also confirmed enhanced osteoclastogenesis by bone marrow-derived macrophages isolated from the Kdm3C KO compared with the WT controls. These findings suggest an anti-inflammatory function of KDM3C in regulating the inflammatory responses against oral bacterial infection through suppression of NF-κB signaling and osteoclastogenesis.-Lee, J. Y., Mehrazarin, S., Alshaikh, A., Kim, S., Chen, W., Lux, R., Gwack, Y., Kim, R. H., Kang, M. K. Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

CRACR2A-Mediated TCR Signaling Promotes Local Effector Th1 and Th17 Responses.

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.

ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

RATIONALE: Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. OBJECTIVE: We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. METHODS AND RESULTS: Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. CONCLUSIONS: Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.

Regulation of Th17 Differentiation by IKKα-Dependent and -Independent Phosphorylation of RORγt.

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.

Junctophilin-4, a component of the endoplasmic reticulum-plasma membrane junctions, regulates Ca2+ dynamics in T cells.

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.

Ubiquitination of RORγt at Lysine 446 Limits Th17 Differentiation by Controlling Coactivator Recruitment.

The transcription factor retinoid acid-related orphan receptor γ t (RORγt) directs the differentiation of Th17 cells. Th17 cells mediate pathological immune responses responsible for autoimmune diseases, including psoriasis and multiple sclerosis. Previous studies focused on RORγt target genes and their function in Th17 differentiation. In this study, we assessed posttranscriptional regulation of RORγt and identified a functional ubiquitination site, K446. Mutation of K446 to arginine to prevent ubiquitination greatly enhanced recruitment of steroid receptor coactivator 1 (SRC1), a coactivator critical for RORγt activity. Correspondingly, the K446 to arginine mutation potentiated Th17 differentiation. We also showed that ubiquitin-specific protease (USP)15 interacted with RORγt, removed ubiquitin from K446, and stimulated RORγt activity by enhancing coactivator SRC1 recruitment. Knockdown of USP15 or expression of inactive USP15 impaired Th17 differentiation, suggesting a positive role for USP15-mediated deubiquitination of RORγt in Th17 differentiation. Therefore, ubiquitination of K446 limits RORγt-mediated Th17 differentiation by inhibiting the recruitment of coactivator SRC1. Our study will inform the development of treatments that target RORγt ubiquitination pathways to limit Th17-mediated autoimmunity.

Immunological Disorders: Regulation of Ca2+ Signaling in T Lymphocytes.

Engagement of T cell receptors (TCRs) with cognate antigens triggers cascades of signaling pathways in helper T cells. TCR signaling is essential for the effector function of helper T cells including proliferation, differentiation, and cytokine production. It also modulates effector T cell fate by inducing cell death, anergy (nonresponsiveness), exhaustion, and generation of regulatory T cells. One of the main axes of TCR signaling is the Ca2+-calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway. Stimulation of TCRs triggers depletion of intracellular Ca2+ store and, in turn, activates store-operated Ca2+ entry (SOCE) to raise the intracellular Ca2+ concentration. SOCE in T cells is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which have been very well characterized in terms of their electrophysiological properties. Identification of STIM1 as a sensor to detect depletion of the endoplasmic reticulum (ER) Ca2+ store and Orai1 as the pore subunit of CRAC channels has dramatically advanced our understanding of the regulatory mechanism of Ca2+ signaling in T cells. In this review, we discuss our current understanding of Ca2+ signaling in T cells with specific focus on the mechanism of CRAC channel activation and regulation via protein interactions. In addition, we will discuss the role of CRAC channels in effector T cells, based on the analyses of genetically modified animal models.

Orai1 mediates osteogenic differentiation via BMP signaling pathway in bone marrow mesenchymal stem cells.

Orai1 is a pore-subunit of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel that mediates Ca(2+) influx in most non-excitable cells via store-operated Ca(2+) entry (SOCE) mechanism. We previously demonstrated that Orai1 is involved in mediating osteogenic potential of mesenchymal stem cells (MSCs), but the underlying mechanism of this function remains unknown. Here, we report that Orai1 mediates osteogenic differentiation via bone morphogenic protein (BMP) signaling pathway in bone marrow MSCs (BMSCs). In osteogenic conditions, BMSCs derived from wild-type mice underwent osteoblastic differentiation and induced mineralization as demonstrated by increased alkaline phosphatase activity and alizarin red S staining, respectively. The expression of Runx2, a master regulator of osteoblast differentiation, and osteogenic differentiation markers were markedly increased in wild-type BMSCs under osteogenic conditions. In contrast, osteogenic conditions failed to induce such effects in BMSCs derived from Orai1-deficient (Orai1(-/-)) mice, indicating that Orai1 is, in part, necessary for osteogenic differentiation of MSCs. We also found that BMP2 successfully induced phosphorylation of Smad1/5/8, the immediate effector molecules of BMP signaling, in wild-type BMSCs, but failed to do so in Orai1(-/-) BMSCs. Downstream target genes of BMP signaling pathway were consistently increased by osteogenic conditions in wild-type BMSCs, but not in Orai1(-/-) BMSCs, suggesting a novel molecular link between Orai1 and BMP signaling pathway in the osteogenic differentiation process. Further functional studies demonstrated that activation of BMP signaling rescues osteogenic differentiation capacity of Orai1(-/-) BMSCs. In conclusion, Orai1 regulates osteogenic differentiation through BMP signaling, and the Orai1-BMP signaling may be a possible therapeutic target for treating bone-related diseases.

Calcium signaling via Orai1 is essential for induction of the nuclear orphan receptor pathway to drive Th17 differentiation.

Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1).

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.

Protein kinase D orchestrates the activation of DRAK2 in response to TCR-induced Ca2+ influx and mitochondrial reactive oxygen generation.

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 Ca(2+) influx through the Ca(2+) release-activated Ca(2+) 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 Ca(2+) 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 Ca(2+) influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.

ORAI1 deficiency impairs activated T cell death and enhances T cell survival.

ORAI1 is a pore subunit of Ca(2+) release-activated Ca(2+) channels that mediate TCR stimulation-induced Ca(2+) entry. A point mutation in ORAI1 (ORAI1(R91W)) causes SCID in human patients that is recapitulated in Orai1(-/-) mice, emphasizing its important role in the immune cells. In this study, we have characterized a novel function of ORAI1 in T cell death. CD4(+) T cells from Orai1(-/-) mice showed robust proliferation with repetitive stimulations and strong resistance to stimulation-induced cell death due to reduced mitochondrial Ca(2+) uptake and altered gene expression of proapoptotic and antiapoptotic molecules (e.g., Fas ligand, Noxa, and Mcl-1). Nuclear accumulation of NFAT was severely reduced in ORAI1-deficient T cells, and expression of ORAI1 and a constitutively active mutant of NFAT recovered cell death. These results indicate NFAT-mediated cell death pathway as one of the major downstream targets of ORAI1-induced Ca(2+) entry. By expressing various mutants of ORAI1 in wild-type and Orai1(-/-) T cells to generate different levels of intracellular Ca(2+), we have shown that activation-induced cell death is directly proportional to the intracellular Ca(2+) concentration levels. Consistent with the in vitro results, Orai1(-/-) mice showed strong resistance to T cell depletion induced by injection of anti-CD3 Ab. Furthermore, ORAI1-deficient T cells showed enhanced survival after adoptive transfer into immunocompromised hosts. Thus, our results demonstrate a crucial role of the ORAI1-NFAT pathway in T cell death and highlight the important role of ORAI1 as a major route of Ca(2+) entry during activated T cell death.