Immunodeficiency due to defects in store-operated calcium entry

Abstract

Mutations in genes encoding the Calcium-Release Activated Calcium (CRAC) channel abolish calcium influx in cells of the immune system and cause severe congenital immunodeficiency. Patients with autosomal recessive mutations in the CRAC channel gene ORAI1, its activator Stromal Interaction Molecule 1 (STIM1) and mice with targeted deletion of Orai1, Stim1 and Stim2 genes reveal important roles for CRAC channels in adaptive and innate immune responses to infection and in autoimmunity. Since CRAC channels have important functions outside the immune system, ORAI1 and STIM1 deficiency are associated with a unique clinical phenotype. This review will give an overview of CRAC channel function in the immune system, examine the consequences of CRAC channel deficiency for immunity in human patients and mice and discuss genetic defects in immunoreceptor-associated signaling molecules that compromise calcium influx and cause immunodeficiency.

1. CRAC channels are essential for Ca²⁺ influx in cells of the immune system

Ca²⁺ was recognized as a T cell mitogen in the late 1960s^1-3 and has since been studied in more detail after the advent of fluorescent Ca²⁺ indicator dyes that enable measurements of intracellular Ca²⁺ levels in single cells. In resting lymphocytes, intracellular Ca²⁺ concentrations ([Ca²⁺]_i) are kept very low (~100 nM) compared to the much higher concentrations in the extracellular fluid and serum (~ 1 - 2 mM) and intracellular Ca²⁺ storage compartments such as the endoplasmic reticulum (ER, ~ 0.5 - 1 mM)⁴. Sustained or oscillatory increases in [Ca²⁺]_i may originate in Ca²⁺ influx from the extracellular space via plasma membrane Ca²⁺ channels or the release of Ca²⁺ from intracellular stores. The subsequent increase in [Ca²⁺]_i leads to the activation of a multitude of Ca²⁺ dependent proteins, enzymes and transcription factors which regulate the function and, in some instances, development of immune cells. Elevations of [Ca²⁺]_i occur following engagement of receptors on adaptive and innate immune cells including T and B cells, NK cells, mast cells, dendritic cells, neutrophils and macrophages ⁵. Immunoreceptors that generate Ca²⁺ signals include T cell, B cell, FcγR and FcεR receptors, activating NK cell receptors as well as G-protein coupled chemokine receptors ⁵. In T and B cells, antigen binding activates the protein tyrosine kinases lck, syk and ZAP-70 resulting in the phosphorylation of scaffold proteins such as LAT, SLP-76, BLNK and the recruitment of the Tec kinases Itk and Btk. Ultimately, these proximal signaling events result in the activation of phospholipase Cγ1 (in T cells) or PLCγ2 (in B cells) and the generation of inositol 1,4,5-triphosphate (InsP₃) The latter binds to InsP₃ receptors in the membrane of the endoplasmic reticulum (ER) triggering the opening of these Ca²⁺ permeable ion channels and the release of Ca²⁺ from the ER. The release of Ca²⁺ from the ER results in a transient and – at least in lymphocytes – very small increase in intracellular [Ca²⁺]_i, which may nevertheless be sufficient for some T cell responses. More importantly, however, the reduction in [Ca²⁺]_ER is the trigger mechanism for the induction of store-operated Ca²⁺ entry (SOCE), which is the predominant Ca²⁺ influx pathway in T cells and many other immune cells in response to antigen receptor engagement.

SOCE is mediated by the opening of a Ca²⁺ selective ion channel in the plasma membrane, the Ca²⁺ release activated Ca²⁺ (CRAC) channel. Ca²⁺ currents resulting from the opening of the CRAC channel were first identified in T cells and mast cells ^{6, 7} and have since been described in several other immune cells as well as cells outside the immune system ^{5, 8, 9}. The opening of CRAC channels results in sustained Ca²⁺ influx and elevations in [Ca²⁺]_i that are required for efficient stimulation of immune cells. The importance of CRAC channels for immunity to infection is emphasized by rare but very instructive patients with inherited defects in SOCE due to mutations in genes of the CRAC channel complex. These patients suffer from a severe form of immunodeficiency that is due to defects in the function, but not the development, of T cells, NK cells and potentially other immune cells ^{10, 11} as will be discussed in detail below.

2. CRAC channel function is controlled by ORAI and STIM proteins

The genes encoding the CRAC channel remained elusive until 2006 when a genomic linkage analysis in patients with inherited CRAC channel deficiency and functional RNAi screens in drosophila S2 cells resulted in the identification of ORAI1 as the gene encoding the CRAC channel located on human Chr 12q24 ^12-14. ORAI1 (also known as CRACM1), and its two homologues ORAI2 and ORAI3, represent a new class of Ca²⁺ channels that are unrelated to other ion channels. They are named after the Horae (hours) Eunomia, Dyke and Eirene in Homer's Iliad ¹⁵. All three ORAI proteins are tetraspanning plasma membrane channels with intracellular N- and C-termini and two extracellular loop regions. The first transmembrane domain (TM1) of ORAI1 contains an important negatively charged glutamate residue (E106) that was shown to function as a Ca²⁺ binding site inside the CRAC channel pore ^16-18. TM1 was later confirmed to be an alpha-helix lining the channel pore and essential for conducting Ca^{2+ 19-21}. In their active state, CRAC channels are thought to be composed of four ORAI1 proteins ^22-24. ORAI2 and ORAI3 are calcium channels with electrophysiological properties that are largely similar to those of ORAI1 when they are overexpressed in cell lines^{25, 26}, but the role of endogenous ORAI2 and ORAI3 proteins for Ca²⁺ influx is not well understood.

CRAC channels are activated by the depletion of ER Ca²⁺ stores. The mechanisms underlying this activation remained as controversial as the nature of the CRAC channel itself. In 2005, stromal interaction molecule (STIM) 1 was identified as protein essential for CRAC channel activation ^{27, 28}. STIM1, and its homologue STIM2, are single-pass transmembrane proteins that are localized in the ER ²⁹. Their ER lumenal N terminus contains a pair of low affinity EF hand Ca²⁺ binding domains that sense the filling state of the ER. Mutation of acidic glutamate or aspartate residues within the EF hand results in constitutive CRAC channel activation and SOCE ³⁰. The cytoplasmic C terminus of STIM1 contains two coiled-coil domains which mediate protein interactions between STIM1 molecules to form multimeric STIM1 complexes ²⁹. The more C terminal coiled-coil domain of STIM1 is part of functional protein domain that mediates the binding of STIM1 to ORAI1 resulting in CRAC channel opening. This domain is alternatively called CAD (CRAC activating domain)³¹, SOAR (STIM-ORAI-activating region)³², OASF (ORAI1-activating small fragment)³³ and CCb9 (coiled-coil domain fragment b9)³⁴. Several other domains in the C terminus of STIM1 have also been shown to regulate SOCE and the function of other ion channels such as transient receptor potential (TRP) C1 and L-type voltage-gated Ca²⁺ channels ^35-38. Similar to STIM1, STIM2 senses [Ca²⁺]_ER and activates SOCE, albeit with different thresholds of activation and kinetics ^{39, 40}. It has therefore been suggested that STIM2 mainly regulates basal cytosolic Ca²⁺ concentrations ⁴¹.

ORAI1 and STIM1 are widely expressed in the immune system and other tissues ^{10, 42-45}. In mice, similar levels of ORAI1 protein are found in all lymphocyte populations⁴⁶. Above-average mRNA levels are, however, observed in macrophages and mast cells⁴². ORAI1 is in addition expressed at high levels in skeletal muscle and many secretory epithelia of exocrine organs such as the mammary, salivary, pancreatic and prostate glands ^{10, 43}. It is of note that the levels of ORAI1 and STIM1 in cells of the immune system are not or only moderately higher compared to those in other tissues ⁴⁴. The fact that mutations in ORAI1 and STIM1 genes in human patients mainly manifest as immunodeficiency, as will be discussed further below, is presumably due to the non-redundant function of both proteins in Ca²⁺ influx in cells of the immune system compared to other tissues.

3. The role of SOCE in immune cells

Ca²⁺ signals in cells of the adaptive and innate immune system have been associated with numerous cell functions. SOCE is the main Ca²⁺ influx pathway in lymphocytes and other immune cells following antigen stimulation via TCR, BCR or Fc receptors. Other Ca²⁺ permeable channels and pathways have, however, been described as well such as P2X receptors in mast cells and T cells ^47-50, TRPC channels and plasma-membrane expressed InsP₃ receptors in B cells ^51-53. The contribution of these channels to immune responses in vivo remains largely unclear.

Since the discovery of ORAI and STIM genes, research on Ca²⁺ influx in immune cells has focused on the role of SOCE mediated by CRAC channels. A detailed review of how SOCE regulates the function of T cells, B cells, NK cells, mast cells, neutrophils and macrophages can be found in ⁵⁴. Briefly, one of the most important roles of SOCE in all immune cells is the regulation of gene expression via the activation of Ca²⁺ dependent transcription factors such as the nuclear factor of activated T cells (NFAT) ⁵⁵, cAMP response element binding (CREB) ⁵⁶, myeloid elf-1-like factor (MEF) 2 ⁵⁷ or activating transcription factor (ATF) 2 ⁵⁸. In particular, the production of many cytokines and chemokines is regulated by SOCE ^59-61. In addition, SOCE is required for the release of cytolytic granules by CD8⁺ T cells and NK cells following stimulation through the TCR and FcγRIIIa/b (CD16), respectively ^{61, 62}. In mast cells, SOCE mediated by ORAI1 and STIM1 is essential for the production of leukotriene C4 as well as the release of histamine and serotonin containing granules in response to FcεRI crosslinking ^{63, 64}.

CRAC currents and SOCE have also been reported in other myeloid cells. In dendritic cells (DC), CRAC currents were recorded after store-depletion with thapsigargin or ATP stimulation ⁶⁵ and have been implicated in DC maturation as measured by cell surface expression of MHC class II, costimulatory molecules and chemokine receptors ^{66, 67}. More recently it was shown that in neutrophils, the production of reactive oxygen species and cell motility were dependent on ORAI1 and STIM1 expression ^68-70. Although these experiments were conducted mostly in the HL-60 neutrophil cell line, the findings are in line with older studies that demonstrated Ca²⁺ influx following crosslinking of FcγRIIa and FcγIIIb receptors, binding of chemokine receptors by IL-8 or fMLP ^71-73 and a even older work linking Ca²⁺ influx to the production of reactive oxygen species (ROS) ⁷⁴. More direct evidence for a role of SOCE in innate immune responses comes from experiments in STIM1 deficient mice, whose macrophages failed to phagocytose opsonized red blood cells in vitro and to mediate FcγR dependent autoimmune hemolytic anemia and thrombocytopenia in vivo ⁷⁵. Taken together, it is now appreciated that SOCE through CRAC channels constitutes a universal Ca²⁺ signaling pathway in cells of the immune system and likely to be involved in both adaptive and innate immune responses. The precise contributions of SOCE in different cell types to immunity in vivo still remain to be investigated.

4. CRAC channelopathy due to mutations in ORAI1 and STIM1

Even before the discovery of ORAI1 and STIM1 genes as the principal components of the CRAC channel complex, patients from three independent families with defects in SOCE and CRAC channel function had been described, who suffered from a Combined Immunodeficiency (CID) syndrome with severe infections to which the patients succumbed in their first year of life ^{10, 76-78}. All three patients later emerged to have mutations in ORAI1. ORAI1 was identified as the CRAC channel gene in part through positional cloning using DNA from one of these patient's families ¹². The clinical and cellular phenotypes of the patients will be discussed further below.

Mutations in ORAI1

Autosomal recessive mutations in ORAI1 have been identified in three unrelated kindreds so far (Figure 2, Table 1): 1. A missense mutation in the pore-lining first transmembrane (TM) domain of ORAI1 (R91W) results in the expression of a non-functional CRAC channel¹² and abolishes SOCE in T cells, B cells, NK cells and fibroblasts of patients^{12, 61, 79}. R91 is located at the inner mouth of the CRAC channel pore and substitution of the basic Arg residue with a hydrophobic Trp was shown in silico to enhance the transmembrane probability of TM1, thus potentially interfering with the structure or mobility of TM1 in the lipid bilayer and the opening of the CRAC channel pore ⁸⁰. 2. Missense mutations in the first and third TM domains of ORAI1 (A103E, L194P) abolish protein expression, presumably by destabilizing the alpha-helical structure of the membrane domains^{10, 77}. Immundeficient patients are compound heterozygous for both mutations. 3. An insertion mutation (A88SfsX25) results in a frameshift, nonsense mediated mRNA decay and lack of ORAI1 protein expression^{10, 78}. These mutations have been described in detail elsewhere ^{10, 43, 46, 81}.

Figure 2. Mutations in ORAI1 and STIM1 genes abolish SOCE and cause immunodeficiency.

Autosomal recessive mutations in the human ORAI1 and STIM1 genes abolish CRAC channel function by interfering with ORAI1 or STIM1 mRNA expression (red), protein expression (orange) or their function (green). Protein domains in STIM1 are shown as shaded boxes (abbreviations: CC, coiled-coil domain; EFh, EF hand domain; SAM, sterile alpha motif; S/P, serine/proline rich). For details see text.

Table 1.

Mutations and clinical phenotype of ORAI1 and STIM1 deficient patients.

Gene	ORAI1			STIM1
Gene defect	R91W	A88SfsX25	A103E / L194P	E128RfsX9	1538-1 G>A
Chromosome	12q24	12q24	12q24	11q15	11q15
Inheritance	AR	AR	AR (compound het.)	AR	AR
# Patients	2	2	2	3	1
ORAI1/STIM1 expression and Ca2+ influx
mRNA / Protein	Yes / Yes	No / No	Yes / No	No / No	No*/No
SOCE / ICRAC	absent / absent	absent / absent	absent / not tested	absent / not tested	absent / not tested
Cell types affected	T cells, B cells, fibroblasts	T cells, fibroblasts	T & B cells, granulocytes, platelets, fibroblasts	Fibroblasts	B cells
Immunological Manifestations
Immunodeficiency & Infections	BCG infection, rota virus infection, Interstitial pneumonia, gastrointestinal sepsis (P1)	Recurrent pneumonia, otitis, oral and gastrointestinal candidiasis, chronic diarrhea, pyelonephritis.	Pneumonia, diarrhea since birth, CMV infection.	Bacterial infections: UTI, sepsis, pneumonia (E.coli, S.pneumoniae); Viral infections (CMV, VZV, EBV)	HHV-8 (Kaposi sarcoma)
Lymphoc. counts	Normal	Normal	Normal	Normal	Normal
Lymphoc. subsets	Normal	Normal	Normal	Foxp3+ Treg ↓	NT
T cell activation	Proliferation ↓↓ Cytokine production ↓↓	Proliferation ↓↓	Proliferation ↓↓	Proliferation ↓↓	NT
Serum Ig levels	Normal - ↑ (no specific Ab)	Normal - ↑ (no specific Ab)	Normal - ↑ (no specific Ab)	Normal (no specific Ab)	Normal
Autoimmunity & Lymphoproliferation	No	Neutropenia, Thrombocytopenia	No	AIHA, Thrombocytopenia Splenomegaly, Lymphadenopathy	AIHA, Hepatosplenomegaly, Lymphadenopathy
Extraimmunological Manifestations
Myopathy	Yes	Yes	Yes	Yes	No
Ectodermal Dysplasia	Anhydrosis Enamel dentition defect	No	Anhydrosis Enamel dentition defect	Enamel dentition defect	No
Other	No	Idiopathic encephalopathy Facial dysmorphy, posterior arch closing defect	No	No	No
Outcome	Death from sepsis 11m (P1); Survival after HSCT, now 18y (P2)	Death from pneumonia 5m (P3); Death from encephalopathy, seizures, fever 11m (P4)	Death 8m (P5); EBV-associated post-HSCT lymphoproliferative disease, survival after two HSCT, now 18y (P6)	Death from HSCT complications 9y (P7); Death from encephalitis 18m (P8); Survival after HSCT, now 9y (P9)	Death from pulmonary infection 2y (P10)
References	10, 12, 59, 61, 76, 79, 96, 98	10, 78	10, 77	11	84, 88

Abbreviations: Ab, antibody; AIHA, autoimmune hemolytic anemia; AR, autosomal recessive; BCG, Bacille Calmette-Guérin; CMV, Cytomegalo virus; I_CRAC, Ca²⁺ release activated Ca²⁺ (CRAC) channel current; GI, gastrointestinal; HSCT, hematopoietic stem cell transplantation; Ig, immunoglobulin; m, months; NT, not tested; P, patient; SOCE, store-operated Ca²⁺ entry; UTI, urinary tract infections; y, years.

^*only abnormally sliced mRNA present.

Only individuals homozygous or compound heterozygous for the mutations described above present with immunodeficiency and the non-immunological symptoms of CRAC channelopathy (Figure 3), whereas heterozygous individuals (parents, siblings) are healthy. Their T cells (and other cell types tested) have normal SOCE at physiological extracellular Ca²⁺ concentrations indicating that monoallelic expression of ORAI1 is sufficient for SOCE, T cell function and immunity to infection. The same is true for patients with mutations in STIM1 that will be discussed below. It is noteworthy, however, that heterozygosity for the ORAI1-R91W mutations reduces CRAC channel currents by ~ 50% ⁸². SOCE is also reduced by ~ 50% but only at subphysiological (< 1.2 mM) extracellular Ca²⁺ concentrations ^{12, 82}. This is in contrast to cells from individuals heterozygous for null mutations (A103E, L194P, A88Sfx25) which have normal SOCE¹⁰, suggesting that the R91W mutation has a dominant negative effect on CRAC channel function. This is plausible as the mutant protein subunits are likely to be assembled into the tetrameric ORAI1 channel complex^22-24. Experiments using concatenated tetramers of wildtype and mutant (R91W) ORAI1 proteins confirmed this dominant negative effect⁸³. One mutant subunit was sufficient to reduce CRAC currents by ~ 50%, whereas two completely abolished channel function. The less severe effect of heterozygously expressed ORAI1-R91W proteins on CRAC channel function may be explained by a decreased incorporation of mutant subunits into the tetrameric ORAI1 complex in heterozygous individuals unlike in concatenated tetramers, where the ratio of wildtype to mutant subunits is fixed at 50%.

Figure 3. Synopsis of the clinical phenotype in CRAC channelopathy.

Autosomal recessive mutations in ORAI1 and STIM1 are associated with a unique clinical phenotype that is characterized by (1) severe immunodeficiency (despite normal leukocyte numbers), (2) autoimmune cytopenias and hepatosplenomegaly / lymphadenopathy, (3) congenital, global muscular hypotonia and partial iris hypoplasia, (4) ectodermal dysplasia (anhidrosis and hypocalcified amelogenesis imperfecta, type III). Muscular hypotonia correlates histologically with a predominance of slow twitch (type I) and atrophy of fast twitch (type II) muscle fibers. Upper panel: H&E stain of a muscle biopsy from a patient with ORAI1-R91W mutation; lower panel: immunofluorescence using anti-ORAI1 and anti-MHC fast antibodies¹⁰. Common infections and pathogens in ORAI1 and STIM1 deficient patients are listed on the left (for details see Table 1). For a list of abbreviations see Table 1.

Mutations in STIM1

Inherited mutations in STIM1 that impair SOCE and cause immunodeficiency are equally rare as those in ORAI1. To date, four patients from two families have been reported, who are homozygous for autosomal recessive mutations in STIM1 that abolish mRNA and protein expression (Figure 2, Table 1) ^{11, 84}. Patients from a third family have recently been identified which lack SOCE due to a missense mutation in STIM1, but the molecular mechnism that causes the defect in Ca²⁺ influx in cells from these patients has not been resolved yet ⁶¹. Three patients from one family were homozygous for an adenine insertion in exon 3, which results in a frameshift, premature stop codon and termination at position 136 of the protein sequence (E128RfsX9). A predicted 15 kDa N-terminal STIM1 fragment, however, was not observed. Instead, STIM1 mRNA levels were greatly reduced and STIM1 protein expression was not detectable in the patients’ cells suggestive of nonsense mediated mRNA decay ¹¹. STIM1 protein expression was also abolished in a patient from another family whose B cells lacked SOCE ⁸⁴. She was homozygous for a splice site mutation in exon 8 of STIM1 (1,538-1 G>A) and lacked normally spliced, full-length STIM1 mRNA transcripts. Neither full-length nor truncated STIM1 proteins (representing abnormal STIM1 splice products) were detectable in the patient's B cells. It is noteworthy that expression not only of wildtype STIM1 but also STIM2 was able to rescue SOCE in fibroblasts from one of the STIM1 deficient patients ¹¹ suggesting that STIM1 and STIM2 have overlapping functions in CRAC channel activation. Nevertheless, even the above average endogenous mRNA expression levels of STIM2 in human CD4⁺ and CD8⁺ T cells, CD56⁺ NK cells and CD19⁺ B cells compared to other cell types⁴² appear to be insufficient to compensate for a loss of STIM1 expression and prevent immunodeficiency in STIM1 deficient patients.

The complete loss of SOCE in ORAI1 and STIM1 deficient patients and their severe (and to a large degree overlapping) clinical phenotype indicate that STIM1 and ORAI1 are the major protagonists in CRAC channel function and SOCE in human immune cells. By contrast, ORAI2, ORAI3 and STIM2 appear to play only a minor role as their endogenous expression was unable to mediate SOCE and to prevent the patients’ immunodeficiency. The physiological role of ORAI2, ORAI3 and STIM2 for CRAC channel function and Ca²⁺ influx in human lymphocytes remains to be resolved. It has been speculated that ORAI2 is important for SOCE in murine T cells ⁶⁴, but evidence for such a role in human T cells is missing. Above average mRNA expression levels of ORAI2 and ORAI3 were observed in human CD19⁺ B cells and CD14⁺ monocytes, respectively⁴², suggesting their possible involvement in SOCE in these cells. STIM2 is required for murine T cell function, as T cells from mice with conditional deletion of the Stim2 gene have a significant defect in sustaining Ca²⁺ influx, which results in impaired cytokine production in vitro and partial protection of Stim2 deficient mice from T-cell mediated autoimmune diseases ^85-87. The role of STIM2 in SOCE and immunity in humans needs to be investigated. Taken together, existing data from ORAI1 and STIM1 deficient patients and gene-targeted mice indicate that ORAI1 and STIM1 are the predominant CRAC channel proteins in human and murine T cells, B cells, NK cells and potentially other immune cells.

5. Clinical phenotype of CRAC channelopathy

Autosomal recessive mutations in ORAI1 and STIM1 are the cause of a unique disease syndrome we termed CRAC channelopathy. It is characterized by severe immunodeficiency, autoimmunity, muscular dysplasia and ectodermal dysplasia (Figure 3, Table 1). The clinical disease spectrum in ORAI1 and STIM1 deficient patients is largely identical, indicating that both genes act – more or less exclusively – in the same signaling pathway. Although the number of patients with mutations in ORAI1 and STIM1 genes observed to date is small, ORAI1 deficient patients appear to have more severe disease since they die in the first year of life without hematopoietic stem cell transplantation (HSCT)¹⁰. By contrast, most STIM1 deficient patients survived the first year of life without treatment^{11, 84}, which may also explain the higher overall incidence of autoimmunity in these individuals.

5.1. Immunodeficiency

The immunodeficiency in CRAC deficient patients is characterized by recurrent severe infections with viral, bacterial, fungal and, in one case, mycobacterial pathogens (Table 1)^{10, 11, 84}. Patients are frequently affected by herpes virus infections including those with cytomegalovirus (CMV), Epstein-Barr Virus (EBV) and varizella zoster virus. Infections with Streptococcus pneumoniae, Escherichia coli and other bacteria have caused otitis media, pneumonia, gastroenteritis, urinary tract infections, meningitis and sepsis. In addition, CRAC deficient patients suffered from infections with Toxoplasma gondii, Candida albicans and Bacille-Calmette Guerin (BCG), an attenuated vaccination strain of Mycobacterium bovis ⁷⁶. While most patients suffered from repeated infections with multiple classes of pathogens, one STIM1 deficient patient was unusual, as she did not have recurrent severe infections. She did, however, develop disseminated Kaposi sarcoma (KS) two years of age due to infection with human herpes virus 8 (HHV8)^{84, 88}. She died from severe pulmonary infection three months after the beginning of antitumor therapy. The severity of infections, their time of onset in the first year of life and the spectrum of pathogens in CRAC deficient patients is similar to those with Severe Combined Immunodeficiency (SCID) who have a impaired T and/or B cell development and immunity. In contrast to SCID patients, T and B cell development is normal in CRAC deficient patients. Their immunodeficiency is due to the impaired function of T cells^{11, 59, 76, 77}, NK cells⁶¹ and most likely other cells of the innate immune system.

The immunodeficiency in CRAC deficient patients is severe because of the 10 patients with confirmed or strongly suspected mutations in ORAI1 and STIM1 genes, only three (one patient with ORAI-R91W, ORAI1-A103E/L194P and STIM1-E128RfsX9 mutation each)⁸¹, have survived after HSCT, whereas five succumbed to infections < 2 years of age and two died from treatment complications. Like in SCID, infections in ORAI1 and STIM1 deficient patients respond poorly to treatment with antibiotics or intravenous immunoglobulin (IVIg) substitution and HSCT is the only curative therapy.

5.2. Autoimmunity

Lymphoproliferation and autoimmune cytopenias were observed in all STIM1 deficient patients known to date^{11, 84}. Besides lymphadenopathy and hepatosplenomegaly, the patients suffered from Coombs-positive hemolytic anemia and thrombocytopenia associated with autoantibodies against platelet glycoproteins Ib/IX¹¹. The most likely cause of the patients’ autoimmunity is the reduced number of CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg) that was observed in the blood of a STIM1 deficient patient¹¹. Treg cells are essential for maintaining immunological tolerance to self-antigens and their development was severely impaired in mice with T-cell specific deletion of Stim1 and Stim2 genes⁸⁶. In these mice, the numbers of Treg cells in the thymus and secondary lymphoid organs were reduced to ~ 10% of the levels found in wildtype controls. In addition, the suppressive function of the residual Stim1/Stim2 deficient Treg cells was severely impaired. These mice develop a phenotype similar to those of STIM1 deficient patients which includes lymphadenopathy, splenomegaly, autoantibodies and inflammatory infiltration of the lung with eosinophils, basophils and lymphocytes, colitis, dermatitis and blepharitis⁸⁶. By contrast, mice with T-cell specific deletion of Stim1 alone or knockin mice homozygous for the non-functional Orai1-R93W mutation have normal Treg numbers and partially impaired Treg function in vitro ^{82, 86, 89} .

An alternative explanation for the autoimmunity in STIM1 deficient patients and Stim1/Stim2 deficient mice is an aberrant selection of autoreactive T cells and/or B cell during their development in the thymus and bone marrow due to altered TCR signaling thresholds in the absence of Ca²⁺ influx. The T cell receptor repertoire (analyzed using monoclonal antibodies against the TCR β chain variable regions Vβ3, Vβ8, Vβ13-6, Vβ13-17, and Vβ21-3) in a patient lacking STIM1 expression, however, was normal ¹¹, and similar observations were made in Stim1/Stim2 deficient mice.

In contrast to STIM1 deficient patients, autoimmunity and lymphoproliferation have so far only been observed in one out of six ORAI1 deficient patients who presented with autoimmune thrombocytopenia and neutropenia ¹⁰. The cause of the discrepancy between ORAI1 and STIM1 deficient patients is not known, partially owing to the fact that blood samples from ORAI1 deficient patients to analyze the number of Foxp3⁺ Treg cells were not available. A simple explanation for the lower incidence of autoimmunity in ORAI1 compared to STIM1 deficient patients may be their early mortality. Analysis of a larger patient cohort will be necessary to determine whether STIM1 may have a gene-specific role in the development of autoimmunity.

Despite the reduced numbers of Treg cells, the autoimmunity in STIM1 deficient patients and Stim1/Stim2 deficient mice is less pronounced than that observed in patients with IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome or scurfy mice which lack Treg cells due to mutations in the human and mouse genes encoding FOXP3. This is most likely due to the fact that FOXP3⁺ Treg cells are not completely absent in STIM1 deficient compared to IPEX patients⁹⁰. In addition, effector functions of potentially autoreactive T and B cells are also impaired in the absence of STIM1 and SOCE, thereby attenuating the severity of autoimmune disease. Stim1 deficient (and to a lesser degree also Stim2 deficient) mice were protected from developing autoimmune CNS inflammation in a murine model of multiple sclerosis^{85, 87} and autoimmune colitis⁸².

5.3. Non-immunological symptoms

CRAC channelopathy is associated with a unique combination of immunological and nonimmunological symptoms that is pathognomonic and facilitates the diagnosis of this PID. The non-immunological symptoms include congenital, generalized muscular hypotonia, a dental enamel calcification defect (amelogenesis imperfecta type III) and an inability to sweat (anhidrosis) that is presumably due to impaired sweat gland function^{10, 11, 81}. These defects have been discussed in detail elsewhere and will only briefly be discussed here^{43, 46}. The muscular dysplasia in ORAI1 deficient patients is characterized histologically by an atrophy of type II muscle fibers¹⁰. It constitutes a severe clinical problem in patients who survived after HSCT and are now 9-17 years old^{10, 11, 61}. Besides resulting in the partial immobilization of ORAI1 deficient patients, a muscular hypotonia of the respiratory musculature impairs expectoration, mucus clearance and resulted in the development of chronic pulmonary disease. A similar nonprogressive muscular hypotonia as well as partial iris hypoplasia were observed in STIM1 deficient patients, although the muscle biopsy and electromyography did not reveal abnormalities ^{11, 81}.

The muscular dysplasia in CRAC channel deficient patients is consistent with an important role of SOCE in the function and potentially the development of skeletal muscle ^{43, 91, 92}. This notion is supported by the defective function of skeletal muscle fibers from Stim1^−/− mice, which fatigued rapidly upon repeated stimulation and showed a decrease in tetanic force ⁹³. Skeletal muscle isolated from Stim1 deficient mice also showed a reduction in muscle fiber diameter and a mitochondriopathy⁹³. By contrast, skeletal muscle from Orai1 knockin mice (in which the wildtype Orai1 gene was replaced with a mutated version that encodes for the non-functional ORAI1-R93W protein that is analogous to ORAI1-R91W in human patients) did not show structural abnormalities⁸². Skeletal muscle function was not tested in these mice. The reason for the discrepancy between Stim1 and Orai1 deficient mice is not clear. Taken together, CRAC channels emerge to have important roles in the refilling of SR calcium stores, contraction and potentially also the differentiation of skeletal muscle fibers.

6. Cellular defects underlying immune dysregulation in CRAC deficiency

The immunodeficiency in CRAC channel deficient patients and mice is due to impaired activation of T cells ^{59, 76, 77, 82}, NK cells ⁶¹ and potentially other innate immune cells (see section 3 above).

The immunodeficiency in ORAI1 and STIM1 deficient patients most closely resembles that observed in CID patients with defects in T-cell mediated immunity. In contrast to SCID patients, T cell development (and that of most other lymphoid and myeloid lineages for that matter), is normal in the absence of SOCE ^{10, 11, 77, 78, 82} with the notable exception of regulatory T cells⁸⁶. The numbers of CD4⁺ and CD8⁺ T cells, CD16⁺ CD56⁺ NK cells and CD19⁺ or CD20⁺ B cells in SOCE deficient patients were largely normal ^{10, 11}. Normal development of T, B and NK cells has also been observed in mice lacking Orai1, Stim1, Stim2 and Stim1/Stim2 gene expression ^{63, 64, 82, 86, 89}. In addition, the T cell receptor repertoire (as measured by the distribution of Vβ chains) in STIM1 deficient patients was comparable to that in healthy controls ¹¹. These findings are surprising, as Ca²⁺ signals had been observed in immature T cells in response to pre-TCR stimulation and had been implicated in the development and positive selection of T cells in the thymus^{94, 95}. In addition, many immunodeficiencies that are due to defects in T or B cell signaling molecules are associated with defects in SOCE and lymphocyte development (see section 7). Since Ca²⁺ influx following TCR and BCR stimulation is predominantly if not exclusively mediated by ORAI1 and STIM1 (see section 4) and neither patients nor mice lacking expression of these genes have defects in T or B cell development^{11, 12, 60, 64, 82, 89}, the role of Ca²⁺ signals in lymphocyte development has to be revisited. It is possible that Ca²⁺ influx following pre-TCR stimulation is not required for T cell development. Alternatively, the release of Ca²⁺ from intracellular Ca²⁺ stores (which is largely intact in T cells lacking ORAI1 or STIM1 expression^{12, 86}) may be sufficient to promote T cell development. Another possibility is that STIM-independent, non-store operated Ca²⁺ channels mediate Ca²⁺ influx in immature T cells.

By contrast, SOCE is essential for T cell function. This notion is supported by the spectrum of pathogens (Table 1) that causes infections in SOCE deficient patients (which includes viruses and C. albicans, immunity to which depends on CD8⁺ T cells and Th17 cells, respectively), the defects in T cell function observed in SOCE deficient human and mouse T cells^{59, 76, 82, 89} and the important role of ORAI1, STIM1 and STIM2 in T cell mediated immune responses identified using gene-targeted mice ^{82, 89}. In human T cells, SOCE deficiency results in a severe defect in T cell proliferation in response to stimulation with anti-CD3 antibodies, recall antigens (tetanus toxoid, PPD, candidin) and mitogens (PHA, ConA and PMA plus ionomycin) ⁷⁶. SOCE regulates the expression of hundreds of genes in CD8⁺ T cells including those encoding cytokines, chemokines and cell surface receptors ⁵⁹. Prominent among the genes that require SOCE for their expression are the cytokines IL-2, IL4, IL-10, IFN-γ, TNF-α and the chemokines CCL3 (MIP-1α), CCL4 (MIP-1β) or lymphotactin ^{59, 76}. A similar defect in cytokine production by T cells was observed in mice with targeted deletion of Orai1, Stim1 or Stim2 ^{64, 82, 86, 89}. This defect transcends the boundaries of helper cell (Th) subsets as the production of IFNγ by Th1 cells, IL-4 and IL-10 by Th2 cells as well as IL-17A, IL-17F and IL-22 by Th17 cells was impaired in Orai1 and Stim1 deficient mice ^{64, 82, 86, 89}. T-cell mediated immune responses in vivo also required SOCE. ORAI1 deficient patients lacked delayed-type hypersensitivity responses following intraepidermal challenge with recall antigens and T-cell dependent antibody production ^{76, 77}. Orai1 knockin mice lacking expression of functional ORAI1 had impaired T-cell mediated immune responses in vivo. They failed to mount a T-cell dependent hypersensitivity response following epicutaneous application of a contact allergen and showed impaired rejection of HLA mismatched skin allografts⁸². In addition, mice lacking Stim1 or Stim2 selectively in T cells or in all hematopoietic cells were protected from T-cell mediated autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and inflammatory bowel disease ^{85, 87}.

In contrast to the defect in T cell activation following TCR stimulation, T cell from patients with mutations in ORAI1 and STIM1 have an activated phenotype with increased numbers of HLA-DR⁺, CD45RO⁺ or CD29⁺ T cells^{10, 11}. A similar increase in activated CD44^hi CD62L^lo T cells (both CD4⁺ and CD8⁺) is observed in T cells from Orai1, Stim1 and especially Stim1/Stim2 deficient mice ^{82, 86}. Such TCR and SOCE independent T cell activation might also contribute to the autoimmune lymphoproliferative phenotype present in ORAI1 and STIM1 deficient patients.

The susceptibility to viral infections observed in ORAI1 and STIM1 deficient patients points to an important role of SOCE in antiviral immune responses by CD8⁺ T cells and NK cells. SOCE in human and mouse CD8⁺ T cells and NK cells is mediated by ORAI1 and STIM1 ^{59, 61, 82}. Consequently, CD8⁺ T cells and NK cells from ORAI1 deficient patients and mice lacked expression of TNFα and IFNγ ^{61, 82, 96}. In addition, both CD8⁺ T cells and NK cells from ORAI1 and STIM1 deficient patients showed severe defects in cytotoxic granule exocytosis and in cell-mediated cytotoxicity of tumor cells ⁶¹. Similar defects were observed in SOCE deficient CD8⁺ T cells and NK cells isolated from Orai1 knock-in mice ⁸². Taken together, these findings suggest that SOCE is critical for cytotoxic function of CD8⁺ T cells and NK cells in antiviral (and potentially also antitumor) immune responses and that the increased susceptibility to infections in SOCE deficient patients is likely to be due to defects in both adaptive and innate immunity.

Despite the reported role of SOCE in B cell activation⁹⁷, severely impaired Ca²⁺ influx in B cells from ORAI1 and STIM1 deficient patients and mice does not seem to result in an overt B cell defect. The development and maturation of B cells was normal in ORAI1 or STIM1 deficient patients^{10, 11} and mice with conditional deletion of Stim1 and Stim2 genes in B cells⁶⁰ or knock-in mice expressing a non-functional form of Orai1⁸². Likewise, B cells are able to differentiate into Ig producing plasma cells and to produce antibodies in the absence of SOCE. Serum IgG, IgM and IgA levels were essentially normal in ORAI1 and STIM1 deficient patients^{11, 98}. ORAI1 deficient patients did, however, lack antigen-specific antibody responses after vaccination against tetanus, diphteria and poliovirus^{10, 76, 78}, suggesting that the function of follicular helper T cells may require SOCE⁷⁶. In contrast to ORAI1 deficient patients, Stim1^−/− mice showed a normal humoral immune response to immunization with keyhole limpet hemocyanin (KLH); serum titers of anti-KLH antibodies before and after booster immunizations were similar in Stim1 deficient and wildtype mice⁸⁹. Similar observations were made in mice with B-cell specific deletion of both Stim1 and Stim2 genes, which showed normal primary and memory antigen-specific antibody responses, normal affinity maturation and had similar numbers of germinal center B cells compared to wildtype mice ⁶⁰. Collectively, these data show that – in contrast to humans – SOCE in murine T cells and B cells is dispensable for antigen-specific antibody responses⁸⁹. The cause for the discrepancy between humans and mice is not known.

7. Other immunodeficiencies associated with defects in SOCE

Mutations in ORAI1 and STIM1 genes are not the only inherited gene defects that are associated with impaired SOCE and immunodeficiency. Inborn errors in immunity to infection can also result from mutations in molecules transmitting signals from the T cell or B cell receptor that regulate – among other pathways – the release of Ca²⁺ from ER stores and thus SOCE. Since most of these molecules are specifically expressed in T and B cells, the resulting immunodeficiency is largely limited to these cell types in contrast to the more universal role of SOCE, ORAI1 and STIM1 in many cell types within the immune system and beyond. Examples for gene defects that affect SOCE and immune function are mutations in the zeta-chain-associated protein kinase 70 (ZAP-70) in T cells ^99-101, the IL-2-inducible T cell kinase (ITK) in T cells ¹⁰², Bruton's tyrosine kinase (BTK) ^103-105 in B cells and the B cell linker protein (BLNK, also known as SLP-65) in B cells¹⁰⁶.

Loss-of-function mutations in ZAP-70 cause combined immunodeficiency (CID) and are associated with impaired development of CD8⁺ T cells and defective activation of CD4⁺ T cells ^99-101. Ca²⁺ influx was found to be significantly impaired in ZAP-70 deficient T cells after TCR stimulation but could be rescued – in contrast to patients with mutations in ORAI1 or STIM1 – by direct depletion of ER Ca²⁺ stores and thus activation of SOCE with ionomycin ¹⁰⁰. Whereas SOCE was abolished in T cells isolated from the peripheral blood of ZAP-70 deficient patients, thymocytes showed only a partial defect in Ca²⁺ influx. The difference has been explained by the higher expression levels of the src kinase Syk in immature versus mature human T cells and its role in compensating for the loss of ZAP-70 function¹⁰⁷.

ITK is a tyrosine kinase of the Tec family that is activated by TCR stimulation and recruited into a complex with the adaptor protein SLP-76, where it mediates the phosphorylation of PLCγ and production of InsP₃. Accordingly, SOCE in response to TCR stimulation was significantly reduced in Itk^−/− mice¹⁰⁸. These mice had reduced numbers of mature thymocytes¹⁰⁹ and CD4⁺ T cells in the periphery¹⁰⁸. The only two known ITK deficient patients, by contrast, had normal T cell numbers, which were mostly CD45RO⁺ memory T cells¹⁰². By contrast, both ITK deficient patients and mice lacked NKT cells, potentially explaining the severe immune dysregulation and susceptibility to EBV infection observed in the patients¹⁰². Unfortunately, SOCE has not been measured in the patients’ T cells.

In B cells, mutations in the Tec kinase BTK and the scaffold protein BLNK in human patients are the cause of X-linked and autosomal recessive agammaglobulinemia, respectively¹⁰⁶. BLNK is phosphorylated by the tyrosine kinase Syk following stimulation of the BCR in mature and the pre-BCR in immature B cells. This results in the recruitment of BTK and PLCγ2 into a trimolecular complex with BLNK and the phosphorylation of PLCγ2 by BTK⁹⁷. Not surprisingly then, Ca²⁺ influx in pre-B cells isolated from the bone marrow of BTK deficient patients was severely impaired following crosslinking of the μ-heavy chain¹¹⁰. Similarly, crosslinking of the BCR on B cells isolated from BLNK deficient mice failed to induce SOCE¹¹¹. Since signaling through the pre-BCR complex is required for survival of immature B cells, B cell development in BTK and BLNK deficient patients and mice is compromised at the transition of immature to mature B cells^{106, 111}. The ensuing failure to produce antibodies in these patients results in an increased susceptibility to bacterial infections.

Defects in additional signaling molecules in mouse T and B cells have been associated with impaired SOCE and immune dysregulation. The conditional deletion of PLCγ1 in mouse T cells, for instance, abolishes SOCE¹¹², whereas a partial defect in Ca²⁺ influx is observed in bone marrow derived mast cells from Slp76^−/− and Lat^−/− mice ^{113, 114}. Taken together, defects in many immunoreceptor-associated signaling molecules can affect SOCE in T and B cells, and it is tempting to speculate that impaired Ca²⁺ signals contribute to the immune dysregulation observed in patients and mice lacking these proteins.

8. Conclusion

Inborn errors of CRAC channel function in patients with autosomal recessive mutations in the CRAC channel gene ORAI1 and that of its activator STIM1 are defined by a unique immunodeficiency syndrome. Its phenotype provides essential information about the physiological function of CRAC channels. In combination with studies in Orai1, Stim1 and Stim2 deficient mice, these patients have established an important role for ORAI1 and STIM1 in adaptive and innate immune responses to infection, in skeletal muscle function and ectodermally derived tissues such as teeth and sweat glands (Figure 3). CRAC channels have important functions in additional tissues and cell types, although no overt clinical pathologies have been observed in ORAI1 and STIM1 deficient patients yet that indicate an abnormal function of, for instance, pancreatic acinar, smooth muscle or endothelial cells ⁴³. It is possible that CRAC channels play a somewhat redundant role in these cell types and that disease phenotypes only emerge under conditions when other, potentially compensating Ca²⁺ signaling pathways start to fail. Strong evidence in favor of a critical role for SOCE outside the immune system comes from mice with genetic deletion of Orai1 and Stim1 which are perinatally lethal ^{63, 64, 75, 82, 86, 93}. Since deletion of both genes results in a similar phenotype, it is likely that the cause of death is related to impaired SOCE and not an exclusive, non-SOCE related function of ORAI1 or STIM1 proteins. The cause of death in these mice is unclear but occurs too early to be explained by immune dysregulation. Additional research will also have to address the physiological function of ORAI2 and ORAI3 in vivo, which is poorly defined. Important clues may come from the genetic deletion of these genes in mice or the discovery of patients with mutations in ORAI2 and ORAI3 genes. Both genes may have functions within the immune system that need to be explored. SOCE has been implicated in the function of many innate immune cells and it will be important to understand how impaired SOCE in these cells contributes to the immunodeficiency of ORAI1 and STIM1 deficient patients. Finally, the fact that the clinical phenotype in these patients is relatively limited to the immune system and studies in mice showing that SOCE is required for T-cell dependent autoimmune responses ^{82, 85, 87} suggest that CRAC channels may be a good drug target for immune modulation in the context of inflammation. Given the almost ubiquitous expression of ORAI and STIM genes, the observation of CRAC currents in many cell types and the perinatal lethality of Orai1 and Stim1 deficient mice, more research is, however, required to understand the physiological role of SOCE in vivo.

Figure 1. Store-operated Ca²⁺ entry (SOCE) through CRAC channels and its functions in immune cells.

SOCE in T, B, NK and mast cells, neutrophils and macrophages is induced by engagement of immunoreceptors such as the TCR, BCR, Fcγ and Fcε receptors and activating NK cell receptors. The activation of PLCγ1 and PLCγ2 results in the production of InsP₃ and the release of Ca²⁺ from ER Ca²⁺ stores via the opening of InsP3 receptor channels. The resulting decrease of the [Ca²⁺]_ER is sensed by STIM1 and STIM2, which translocate to ER-plasma membrane junction where they bind to and activate ORAI1 CRAC channels. The resulting sustained Ca²⁺ influx activates Ca²⁺ regulated enzymes such as calcineurin and is required for the transcription of many genes (especially cytokine genes regulated by the Ca²⁺/calcineurin dependent transcription factor NFAT). SOCE is necessary for the degranulation and cytotoxic function of CD8⁺ T cells and NK cells, phagocytosis by macrophages, and production of reactive oxygen species in neutrophils. Abbreviations: CRAC, Ca²⁺ release activated Ca²⁺ channels; ER, endoplasmic reticulum; InsP3, inositol-1,4,5-trisphosphate; NFAT, nuclear factor of activated T cells; PLC, phospholipases C; STIM, stromal interaction molecule.