Ca2+ signals regulate the function of many immune cells and promote immune responses to infection, cancer, and autoantigens. Ca2+ influx in immune cells is mediated by store-operated Ca2+ entry (SOCE) that results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels. The CRAC channel is formed by three plasma membrane proteins, ORAI1, ORAI2, and ORAI3. Of these, ORAI1 is the best studied and plays important roles in immune function. By contrast, the physiological role of ORAI3 in immune cells remains elusive. We show here that ORAI3 is expressed in many immune cells including macrophages, B cells, and T cells. To investigate ORAI3 function in immune cells, we generated Orai3-/- mice. The development of lymphoid and myeloid cells in the thymus and bone marrow was normal in Orai3-/- mice, as was the composition of immune cells in secondary lymphoid organs. Deletion of Orai3 did not affect SOCE in B cells and T cells but moderately enhanced SOCE in macrophages. Orai3-deficient macrophages, B cells, and T cells had normal effector functions in vitro. Immune responses in vivo, including humoral immunity (T cell dependent or independent) and antitumor immunity, were normal in Orai3-/- mice. Moreover, Orai3-/- mice showed no differences in susceptibility to septic shock, experimental autoimmune encephalomyelitis, or collagen-induced arthritis. We conclude that despite its expression in myeloid and lymphoid cells, ORAI3 appears to be dispensable or redundant for physiological and pathological immune responses mediated by these cells.

The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides chloride, VRAC transports other molecules, for example, immunomodulatory cyclic dinucleotides (CDNs) including 2′3′cGAMP. Here, we identify LRRC8C as a critical component of VRAC in T cells, where its deletion abolishes VRAC currents and RVD. T cells of Lrrc8c−/− mice have increased cell cycle progression, proliferation, survival, Ca2+ influx and cytokine production—a phenotype associated with downmodulation of p53 signaling. Mechanistically, LRRC8C mediates the transport of 2′3′cGAMP in T cells, resulting in STING and p53 activation. Inhibition of STING recapitulates the phenotype of LRRC8C-deficient T cells, whereas overexpression of p53 inhibits their enhanced T cell function. Lrrc8c−/− mice have exacerbated T cell-dependent immune responses, including immunity to influenza A virus infection and experimental autoimmune encephalomyelitis. Our results identify cGAMP uptake through LRRC8C and STING–p53 signaling as a new inhibitory signaling pathway in T cells and adaptive immunity. Concepcion et al. show that the volume-regulated anion channel LRRC8C mediates the transport of cGAMP in T cells, resulting in a noncanonical STING–p53-dependent suppression of Ca2+ influx, T cell proliferation and cytokine production.

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca(2+)influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca(2+)channelORAI1. We here identify patients with a novel mutation inSTIM1 (p.L374P) that abolished Ca(2+)influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion ofSTIM1 in all immune cells enhanced susceptibility to mucosalC. albicansinfection, whereas T cell-specific deletion ofSTIM1 impaired immunity to systemicC. albicansinfection.STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo andHIF1 alpha signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles ofSTIM1 in regulating transcription and metabolic programs in non-pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell-mediated inflammatory diseases.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissueresident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

Pathogenic Th17 cells play important roles in many autoimmune and inflammatory diseases. Their function depends on T cell receptor (TCR) signaling and cytokines that activate signal transducer and activator of transcription 3 (STAT3). TCR engagement activates stromal interaction molecule 1 (STIM1) and calcium (Ca2+) influx through Ca2+-release-activated Ca2+ (CRAC) channels. Here, we show that abolishing STIM1 and Ca2+ influx in T cells expressing a hyperactive form of STAT3 (STAT3C) attenuates pathogenic Th17 cell function and inflammation associated with STAT3C expression. Deletion of STIM1 in pathogenic Th17 cells reduces the expression of genes required for mitochondrial function and oxidative phosphorylation (OXPHOS) but enhances reactive oxygen species (ROS) production. STIM1 deletion or inhibition of OXPHOS is associated with a non-pathogenic Th17 gene expression signature and impaired pathogenic Th17 cell function. Our findings establish Ca2+ influx as a critical regulator of mitochondrial function and oxidative stress in pathogenic Th17 cell-mediated multiorgan inflammation.

It is widely assumed that inositol trisphosphate (IP3) and ryanodine (Ry) receptors share the same Ca2+ pool in central mammalian neurons. We now demonstrate that in hippocampal CA1 pyramidal neurons IP3- and Ry-receptors are associated with two functionally distinct intracellular Ca2+ stores, respectively. While the IP3-sensitive Ca2+ store refilling requires Orai2 channels, Ry-sensitive Ca2+ store refilling involves voltage-gated Ca2+ channels (VGCCs). Our findings have direct implications for the understanding of function and plasticity in these central mammalian neurons.

Ca2+ signals were reported to control lipid homeostasis, but the Ca2+ channels and pathways involved are largely unknown. Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca2+ channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 or ORAI1 show a similar phenotype, suggesting a cell-intrinsic role for SOCE in the regulation of lipid metabolism. SOCE is crucial to induce mobilization of fatty acids from lipid droplets, lipolysis, and mitochondrial fatty acid oxidation. SOCE regulates cyclic AMP production and the expression of neutral lipases as well as the transcriptional regulators of lipid metabolism, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and peroxisome proliferator-activated receptor α (PPARα). SOCE-deficient cells upregulate lipophagy, which protects them from lipotoxicity. Our data provide evidence for an important role of SOCE in lipid metabolism.

Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrientsensing pathway. We propose that SOCE controls a critical "metabolic checkpoint'' at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses.

Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is critical for lymphocyte function and immune responses. CRAC channels are hexamers of ORAI proteins that form the channel pore, but the contributions of individual ORAI homologues to CRAC channel function are not well understood. Here we show that deletion of Orai1 reduces, whereas deletion of Orai2 increases, SOCE in mouse T cells. These distinct effects are due to the ability of ORAI2 to form heteromeric channels with ORAI1 and to attenuate CRAC channel function. The combined deletion of Orai1 and Orai2 abolishes SOCE and strongly impairs T cell function. In vivo, Orai1/Orai2 double-deficient mice have impaired T cell-dependent antiviral immune responses, and are protected from T cell-mediated autoimmunity and alloimmunity in models of colitis and graft-versus-host disease. Our study demonstrates that ORAI1 and ORAI2 form heteromeric CRAC channels, in which ORAI2 fine-tunes the magnitude of SOCE to modulate immune responses.

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription- factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.