Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder.

CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient's T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells.

CRAC Channel Controls the Differentiation of Pathogenic B Cells in Lupus Nephritis.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channel is the main Ca2+ influx pathway in lymphocytes and is essential for immune response. Lupus nephritis (LN) is an autoimmune disease characterized by the production of autoantibodies due to widespread loss of immune tolerance. In this study, RNA-seq analysis revealed that calcium transmembrane transport and calcium channel activity were enhanced in naive B cells from patients with LN. The increased expression of ORAI1, ORAI2, and STIM2 in naive B cells from patients with LN was confirmed by flow cytometry and Western blot, implying a role of CRAC channel in B-cell dysregulation in LN. For in vitro study, CRAC channel inhibition by YM-58483 or downregulation by ORAI1-specific small-interfering RNA (siRNA) decreased the phosphorylation of Ca2+/calmodulin-dependent protein kinase2 (CaMK2) and suppressed Blimp-1 expression in primary human B cells, resulting in decreased B-cell differentiation and immunoglobulin G (IgG) production. B cells treated with CaMK2-specific siRNA showed defects in plasma cell differentiation and IgG production. For in vivo study, YM-58483 not only ameliorated the progression of LN but also prevented the development of LN. MRL/lpr lupus mice treated with YM-58483 showed lower percentage of plasma cells in the spleen and reduced concentration of anti-double-stranded DNA antibodies in the sera significantly. Importantly, mice treated with YM-58483 showed decreased immune deposition in the glomeruli and alleviated kidney damage, which was further confirmed in NZM2328 lupus mice. Collectively, CRAC channel controlled the differentiation of pathogenic B cells and promoted the progression of LN. This study provides insights into the pathogenic mechanisms of LN and that CRAC channel could serve as a potential therapeutic target for LN.

Impact of Diverse Ion Channels on Regulatory T Cell Functions.

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

CRAC Channels and Calcium Signaling in T Cell-Mediated Immunity.

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.

Differential expression of CRAC channel in alloxan induced Diabetic BALB/c mice.

Objectives: CRAC (Calcium Release Activated Calcium) channel is one of the most important channels regulating calcium influx and has been involved in many autoimmune diseases. The contribution of CRAC channel in the pathogenesis of Type 1 Diabetes (T1D) has not been described much. Thus, we aimed to study the expression of CRAC channel and inflammatory cytokines like IL-1ß (Interleukin -1ß) and TNF-α (Tumor Necrosis Factor-α) in the spleen-derived cytotoxic T cells, Bone marrow monocytes (BMM) and macrophages differentiated from BMM in the alloxan induced T1D mice.Materials and methods: BALB/c mice treated with alloxan and vehicle control for 12 and 24 h. Spleen derived T cells; Bone marrow derived monocytes were isolated from the control and diabetic BALB/c mice as well as macrophages differentiated from the control and diabetic BMM.Results: We observed increased expression of CRAC channel components like STIM1 (Stromal Interaction Molecule), ORAI1 and ORAI2 and inflammatory cytokines like IL-1ß and TNF-α in the spleen derived cytotoxic T cells and Macrophages differentiated from BMM as well as the downregulated expression of the same and CRAC channel in BMM of 12 and 24 h alloxan induced BALB/c mice.Conclusions: This study suggests that differential expression of CRAC channel correlated with the expression of inflammatory cytokines, thus CRAC channel might be responsible for the increased production of inflammatory cytokines in the alloxan induced T1D mice.

Focus Issue: The ins and outs of ORAI in immune cells.

This Focus Issue highlights research into cell-specific regulation of store-operated calcium entry through the ORAI/STIM channel complex. Understanding the properties of these channels and how ORAI activity is regulated will lead to a better molecular view of immune cell function and diseases involving the immune system.

A calcium-redox feedback loop controls human monocyte immune responses: The role of ORAI Ca2+ channels.

In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.