CRAC Channels and Calcium Signaling in T Cell-Mediated Immunity

Abstract

Calcium (Ca²⁺) signals play fundamental roles in immune cell function. The main sources of Ca²⁺ influx in mammalian lymphocytes following antigen receptor stimulation are Ca²⁺ release-activated Ca²⁺ (CRAC) channels. These are formed by ORAI proteins in the plasma membrane and are activated by stromal interaction molecules (STIM) located in the endoplasmic reticulum (ER). Human loss-of-function (LOF) mutations in ORAI1 and STIM1 that abolish Ca²⁺ influx cause a unique disease syndrome called CRAC channelopathy that is characterized by immunodeficiency autoimmunity and non-immunological symptoms. Studies in mice lacking Stim and Orai genes have illuminated many cellular and molecular mechanisms by which these molecules control lymphocyte function. CRAC channels are required for the differentiation and function of several T lymphocyte subsets that provide immunity to infection, mediate inflammation and prevent autoimmunity. This review examines new insights into how CRAC channels control T cell-mediated immunity.

Keywords: CRAC channel; Calcium; Lymphocyte; ORAI; STIM; T cell.

Figure 1.. Store-operated Ca²⁺ entry (SOCE) and Ca²⁺ dependent transcriptional regulation.

T cell receptor (TCR) and G protein-coupled receptor (GPCR) stimulation of T cells leads to the production of inositol-1,4,5-trisphosphate (IP₃) that binds to IP₃ receptors (IP₃Rs) located in the endoplasmic reticulum (ER) membrane. Opening of IP₃R channels results in a transient increase in cytosolic Ca²⁺ and a decrease in the ER Ca²⁺ concentration, which activates stromal interaction molecules 1 (STIM1) and STIM2. Both proteins form homodimers that undergo conformational changes and translocate to ER-plasma membrane (PM) junctions, where they bind to ORAI proteins. ORAI1 and its homologues ORAI2 and ORAI3 form hexameric complexes in the PM that constitute the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Ca²⁺ influx through CRAC channels is called store-operated Ca²⁺ entry (SOCE). It triggers various processes in T cells including the secretion of cytolytic granules and the activation of Ca²⁺-dependent enzymes including calcineurin, Erk1/2 and CaMKII, and transcription factors such as NF-kB and NFAT. Dephosphorylation of NFAT results in its translocation to the nucleus where it cooperates with other transcription factors to promote activation, differentiation and effector functions of various T helper (Th) and T regulatory (Treg) cell subsets. NFAT interaction partners and their specific functions in T cells have been reviewed in detail elsewhere [45, 46]. Note that NFAT binding to ETC genes in T cells has not been reported, indicated by “?”. pTh17, pathogenic Th17 cells.

Figure 2.. ORAI and STIM expression and SOCE-dependent control of T cell function.

(A) Expression of ORAI and STIM homologs in human and mouse T cells. mRNA expression levels in human T cells from GEO (¹, GSE107011), FANTOM5 (²) and Haemopedia (³) databases (left) and mouse T cells from Immgen (¹), ArrayExpress (², E-MTAB-2582) and Haemopedia (³) databases (right). ORAI1 is the most highly expressed ORAI homolog in human but not mouse T cells. (B) Relative contribution of ORAI and STIM proteins to SOCE in human and mouse T cells. Loss-of-function (LOF) mutations of ORAI1 and STIM1 genes all but abolish SOCE in human T cells (left). Combined genetic deletion of Orai1 and Orai2 genes or Stim1 and Stim2 genes in mouse T cells also abolishes SOCE, whereas deletion of individual genes results in partial SOCE defects (or a SOCE increase in the case of Orai2-deficient T cells).

Figure 3.. SOCE controls the development and function of murine conventional and regulatory T cells.

Thymic development of conventional CD4⁺ and CD8⁺ αβ T cells is independent of SOCE. By contrast, the development of thymic-derived, agonist-selected regulatory T (Treg) cells (and other unconventional T cell subsets including iNKT cells and CD8αα⁺ intraepithelial lymphocytes, not shown) requires SOCE. In the periphery, the differentiation of naïve CD4⁺ T cells into certain T cell subsets like T follicular helper (Tfh) cells and induced Treg (pTreg) cells requires SOCE, whereas the differentiation of Th1 cells appears to be SOCE-independent. The expression of GATA3 by Th2 cells and RORγt by pathogenic Th17 cells is dependent on SOCE, thus influencing the development of these Th subsets [43, 112]. The differentiation of thymus-derived Treg cells (tTreg) into specialized effector Treg subsets, such as T follicular regulatory (Tfr) and tissue-resident Treg cells, which regulate the GC response and maintain tissue-specific organ homeostasis, respectively, is controlled by SOCE. Various effector functions of Th cells including the production of IFNγ, IL-2, IL-4, IL-5, IL-17, GM-CSF and IL-21 cytokines, and the expression of immunosuppressive molecules by Treg cells, such as IL-10, IL-35 and granzyme B, are dependent on SOCE.

Figure 4.. SOCE controls the proliferation and clonal expansion of CD4⁺ and CD8⁺ T cells.

Ca²⁺ mediated activation of calcineurin and NFAT induces, directly or indirectly, the expression of several transcription factors such as IRF4, Myc and HIF1α, which together with NFAT regulate the expression of glucose transporters and glycolytic enzymes that support the metabolic switch of activated mouse and human T cells, their entry into the cell cycle and proliferation [41].

Figure 5.. SOCE regulates the function of pathogenic Th17 cells.

The differentiation of murine Th17 cells depends on cytokine signaling via STAT3 which together with SOCE regulates the expression of Th17 cytokines such as IL-17A, IL-17F, GM-CSF and molecules that are associated with a pathogenic Th17 cell signature [125, 134]. SOCE also controls the expression of nuclear-encoded mitochondrial genes in pathogenic Th17 cells that form the electron transport chain (ETC) and promote oxidative phosphorylation (OXPHOS) [92].

Figure 6.. Immunity to viral infection requires SOCE in both CD8⁺ and CD4⁺ T cells.

In mouse and human CD8⁺ T cells, SOCE controls the secretion of cytolytic granules and the expression of Fas ligand (FasL) and inflammatory cytokines in an NFAT-dependent manner. Strong suppression of SOCE results in the impaired differentiation of murine CD8⁺ cytotoxic effector T cells and interferes with the maintenance of LCMV-specific CD8⁺ memory T cells. The latter depends on SOCE in CD4⁺ T cells and their expression of CD40L. In the absence of SOCE, memory CD8⁺ T cell responses are impaired and acute viral infections become chronic [38].

Figure 7.. SOCE in CD4⁺ T cells controls the germinal center (GC) reaction and humoral immunity.

SOCE regulates the expression of IRF4 and BATF that initiate the differentiation of murine CD4⁺ T cells into T follicular helper (Tfh) cells and their migration into GCs by upregulating CXCR5 expression. NFAT, together with Bcl-6, controls the induction of PD-1, ICOS and CD40L as well as the production of the B cell-stimulating cytokines IL-4 and IL-21 by Tfh cells, which collectively help GC B cells to undergo class-switch recombination and affinity maturation of their antigen receptors and to differentiate into memory B cells and plasma cells [42].

Figure 8.. Apoptosis in T cells is regulated by SOCE.

Ca²⁺ influx triggers activation-induced cell death (AICD) in murine T cells by inducing FasL and TRAIL expression. In addition, SOCE regulates cell-intrinsic apoptosis mechanisms by regulating the expression of the Bcl-2 family members Bok, Bak and Noxa and by facilitating Ca²⁺-dependent cytochrome C release from mitochondria [29].

Figure 9.. SOCE is essential for immune tolerance by controlling the development and function of Treg cells.

SOCE in T cells is crucial for the generation of ‘agonist-selected’ T regulatory (Treg) cells in mice during their development in the thymus [33, 59]. Inactivation of SOCE in tTreg cells after their thymic development revealed additional roles of CRAC channels in mature Treg cells. Whereas SOCE is largely dispensable for the maintenance of Treg cells in peripheral lymphoid organs, the differentiation tTreg cells into tissue-resident Treg cells and/or their homing into the parenchyma of non-lymphoid organs in mice is strictly dependent on CRAC channels [40]. Similarly, the differentiation of tTreg cells into T follicular regulatory (Tfr) cells and their migration into B cell follicles requires SOCE. Absence of tissue-resident Treg and Tfr cells in mice that completely lack SOCE specifically in Treg cells results in the production of autoantibodies, the accumulation of self-reactive T cells and multiorgan autoimmunity.

Figure 10.. Distinct SOCE requirements of different T cell subsets and T cell-mediated immune responses.

The production of inflammatory cytokines such as IL-17A, IL-17F, GM-CSF and IFNγ by murine pathogenic Th17 and Th1 cells requires strong Ca²⁺ signals because partial inhibition of SOCE by genetic deletion of Orai1 or Stim1 attenuates cytokine production and Th17/Th1-mediated diseases such as EAE and IBD. By contrast, the function of murine CD4⁺ Tfh and CD8⁺ T cells, which are required for humoral and cellular immunity to infection, respectively, are less dependent on SOCE as only combined deletion of Orai1/Orai2 or Stim1/Stim2 (and thus SOCE) abolishes their function. Similarly, effector Treg cell functions are less dependent on SOCE as defects are observed only when SOCE is almost completely abolished. These distinct SOCE requirements of murine T cell subsets provide a compelling rationale to postulate the existence of a therapeutic window for the treatment of Th1/Th17-cell mediated autoimmune diseases in human patients through partial inhibition of SOCE without interfering with the ability of Tfh and CD8⁺ T cells to provide immunity to infection or Treg cells to maintain immune tolerance.