Stefan Feske

A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.

Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (ICRAC). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

Orai1 is an essential pore subunit of the CRAC channel

Stimulation of immune cells causes depletion of Ca2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca2+ entry downstream of STIM. Combined overexpression of Orai and Stim in Drosophila cells, or Orai1 and STIM1 in mammalian cells, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs+. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.

Calcium signalling in lymphocyte activation and disease

Calcium signals in cells of the immune system participate in the regulation of cell differentiation, gene transcription and effector functions. An increase in intracellular levels of calcium ions (Ca2+) results from the engagement of immunoreceptors, such as the T-cell receptor, B-cell receptor and Fc receptors, as well as chemokine and co-stimulatory receptors. The major pathway that induces an increase in intracellular Ca2+ levels in lymphocytes is through store-operated calcium entry (SOCE) and calcium-release-activated calcium (CRAC) channels. This Review focuses on the role of Ca2+ signals in lymphocyte functions, the signalling pathways leading to Ca2+ influx, the function of the recently discovered regulators of Ca2+ influx (STIM and ORAI), and the relationship between Ca2+ signals and diseases of the immune system.

Gene regulation mediated by calcium signals in T lymphocytes.

Modulation of many signaling pathways in antigen-stimulated T and B cells results in global changes in gene expression. Here we investigate the contribution of calcium signaling to gene expression in T cells using cell lines from two severe-combined immunodeficiency patients with several cytokine deficiencies and diminished activation of the transcription factor NFAT nuclear factor of activated T cells. These T cells show a strong defect in transmembrane calcium influx that is also apparent in their B cells and fibroblasts. DNA microarray analysis of calcium entry–deficient and control T cells shows that Ca2+ signals both activate and repress gene expression and are largely transduced through the phosphatase calcineurin. We demonstrate an elaborate network of signaling pathways downstream of the T cell receptor, explaining the complexity of changes in gene expression during T cell activation.

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca2+-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-θ and PLC-γ1. T cells from Itch- and Cbl-b–deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function–associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance

Store-operated Ca2+ entry through calcium release–activated calcium channels is the chief mechanism for increasing intracellular Ca2+ in immune cells. Here we show that mouse T cells and fibroblasts lacking the calcium sensor STIM1 had severely impaired store-operated Ca2+ influx, whereas deficiency in the calcium sensor STIM2 had a smaller effect. However, T cells lacking either STIM1 or STIM2 had much less cytokine production and nuclear translocation of the transcription factor NFAT. T cell–specific ablation of both STIM1 and STIM2 resulted in a notable lymphoproliferative phenotype and a selective decrease in regulatory T cell numbers. We conclude that both STIM1 and STIM2 promote store-operated Ca2+ entry into T cells and fibroblasts and that STIM proteins are required for the development and function of regulatory T cells.

STIM1 Mutation Associated with a Syndrome of Immunodeficiency and Autoimmunity

A mutation in ORAI1, the gene encoding the pore-forming subunit of the Ca(2+)-release-activated Ca(2+) (CRAC) channel, abrogates the store-operated entry of Ca(2+) into cells and impairs lymphocyte activation. Stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum activates ORAI1-CRAC channels. We report on three siblings from one kindred with a clinical syndrome of immunodeficiency, hepatosplenomegaly, autoimmune hemolytic anemia, thrombocytopenia, muscular hypotonia, and defective enamel dentition. Two of these patients have a homozygous nonsense mutation in STIM1 that abrogates expression of STIM1 and Ca(2+) influx.

A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1–4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+–calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+–calmodulin–calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.

Biochemical and functional characterization of Orai proteins.

Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.

Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses

Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.