Faculty Bibliography

Ion channels facilitate the movement of ions across the plasma and organellar membranes. A recent symposium brought together scientists who study ion channels and transporters in immune cells, which highlighted advances in this emerging field and served to chart new avenues for investigating the roles of ion channels in immunity.

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 tissue-resident 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.

Calcium signaling is fundamental to many cellular processes. An important pathway for increasing intracellular Ca2+ levels is store-operated Ca2+ entry (SOCE) regulated by stromal interaction molecule (STIM1-2), and Ca2+ channels formed by ORAI1-3 proteins. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. We present patients with Anhidrotic Ectodermal Dysplasia with Immunodeficiency (EDA-ID) caused by novel homozygous p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. A unifying feature of patients with null mutations in ORAI1 is EDA. Anhidrosis was present in patients P1-P4 and confirmed by pilocarpin iontophoresis. Patients had dry and exfoliate skin. They showed signs of heat intolerance and thermoregulatory instability characterized by several attacks of facial flushing accompanied by tachycardia, tachypnea, and hypertension. A skin biopsy showed the presence of eccrine sweat glands in the dermis demonstrating that anhidrosis is not due to a defect in sweat gland development. Recently, we reported that sweat glands require SOCE for opening of the Ca2+-activated chloride channel TMEM16A and thus chloride secretion and sweat production, pointing that anhidrosis in ORAI1-deficient patients could be functional. ORAI1-deficient patients had severe enamel defects diagnosed as hypocalcified amelogenesis imperfecta type III. In contrast, patients with EDA-ID caused by NF-kB signaling defects also have a tooth defect, which is characterized by hypodontia and conical teeth and thus is morphologically easily distinguishable from the enamel defects in ORAI1-deficient patients. ORAI1-deficient patients showed thin and brittle hair. To date, the diagnosis of EDA-ID is limited to patients with defects in NF-kB signaling who are prone to infections with mycobacteria, P.jirovecii, Candida albicans, and, most frequently, pyogenic bacteria caused by hypogammaglobulinemia and failure to mount a specific antibody response to polysaccharide antigens. In contrast, ORAI1-deficient patients are susceptible to an overlapping spectrum of pathogens, but they are also prone to viral infections, including CMV, EBV, RSV, and rotavirus. In addition, AIHA and autoimmune thrombocytopenia are also common in SOCE deficient patients but not NF-kB; instead, patients with NF-kB defects can have inflammatory bowel disease (NF-kB essential modulator colitis). Here we propose that mutations in ORAI1 that abolish SOCE constitute a new form of EDA-ID and are an important differential diagnosis of EDA-ID caused by defects in NF-kB signaling

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca²⁺ microdomains. Here, we identified a Ca²⁺ entry component involved in Ca²⁺ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca²⁺ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca²⁺ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca²⁺ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca²⁺ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca²⁺ release through RYR1, in coordination with Ca²⁺ entry through ORAI1 and STIM, rapidly increases the number of Ca²⁺ microdomains, thereby initiating spread of Ca²⁺ signals deeper into the cytoplasm to promote full T cell activation.

T cell acute lymphoblastic leukemia (T-ALL) is commonly associated with activating mutations in the NOTCH1 pathway. Recent reports have shown a link between NOTCH1 signaling and intracellular Ca²⁺ homeostasis in T-ALL. Here, we investigate the role of store-operated Ca²⁺ entry (SOCE) mediated by the Ca²⁺ channel ORAI1 and its activators STIM1 and STIM2 in T-ALL. Deletion of STIM1 and STIM2 in leukemic cells abolishes SOCE and significantly prolongs the survival of mice in a NOTCH1-dependent model of T-ALL. The survival advantage is unrelated to the leukemic cell burden but is associated with the SOCE-dependent ability of malignant T lymphoblasts to cause inflammation in leukemia-infiltrated organs. Mice with STIM1/STIM2-deficient T-ALL show a markedly reduced necroinflammatory response in leukemia-infiltrated organs and downregulation of signaling pathways previously linked to cancer-induced inflammation. Our study shows that leukemic T lymphoblasts cause inflammation of leukemia-infiltrated organs that is dependent on SOCE.

Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators. Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9-mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) CD95 deletion inhibited T cell apoptosis upon reactivation; 2) deletion of ORAI1, a Ca²⁺ release-activated channel, abolished Ca²⁺ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.

Stromal interaction molecule 1 and 2 (STIM1/2) are acting as sensors for Ca²⁺ in intracellular stores and activate store-operated Ca2+-permeable Orai channels at the plasma membrane. These proteins are expressed in many mammalian tissues and-if mutated-are responsible for various diseases (1). Store-operated Ca²⁺ entry (SOCE) after emptying internal Ca²⁺ stores by thapsigargin has already been investigated in quiescent vascular smooth muscle cells (2), but not in precapillary pulmonary arterial smooth muscle cells (PASMC) and primary lung fibroblasts. PASMC are responsible for pulmonary hypertension and vascular remodeling (3), while fibroblasts have the potential to differentiate to myofibroblasts during the development of lung fibrosis (4). Although classical transient receptor potential (TRPC) channel 1, 3 and 6 mediate receptor-operated Ca²⁺ entry (ROCE) on their own, it is still a matter of debate whether Ca²⁺ influx via SOCE is required for their activation. Therefore, we isolated PASMC from wild-type (WT) and triple deficient TRPC1/3/6-/-mice as well as STIM1/2-deficient mice and analyzed ROCE after receptor stimulation and SOCE after emptying internal Ca²⁺ stores. STIM1/2 deletion was achieved by infecting PASMC from Stim1fl/fl Stim2fl/fl mice with Cre-recombinase expressing lentiviruses. As expected SOCE was inhibited in STIM1/2-deficient PASMC and ROCE was decreased in TRPC1/3/6-deficient PASMC compared to WT cells. By contrast, SOCE was not significantly different in TRPC1/3/6-deficient PASMC and ROCE was similar in STIM1/2-deficient PASMC compared to WT cells. Initial data indicate similar results in primary lung fibroblasts. In conclusion, we demonstrated that TRPC 1/3/6 are not involved in SOCE and mediate mainly receptor operated at least in PASMC and lung fibroblasts. In the future we will investigate the role of STIM1/2-mediated SOCE in PASMC for pulmonary hypertension and vascular remodeling as well as in primary lung fibroblasts for the development of lung fibrosis

Ion channels and transporters move ions across membrane barriers and are essential for a host of cell functions in many organs. They conduct K⁺, Na⁺ and Cl^-, which are essential for regulating the membrane potential, H⁺ to control intracellular and extracellular pH and divalent cations such as Ca²⁺, Mg²⁺ and Zn²⁺, which function as second messengers and cofactors for many proteins. Inherited channelopathies due to mutations in ion channels or their accessory proteins cause a variety of diseases in the nervous, cardiovascular and other tissues, but channelopathies that affect immune function are not as well studied. Mutations in ORAI1 and STIM1 genes that encode the Ca²⁺ release-activated Ca²⁺ (CRAC) channel in immune cells, the Mg²⁺ transporter MAGT1 and the Cl^- channel LRRC8A all cause immunodeficiency with increased susceptibility to infection. Mutations in the Zn²⁺ transporters SLC39A4 (ZIP4) and SLC30A2 (ZnT2) result in nutritional Zn²⁺ deficiency and immune dysfunction. These channels, however, only represent a fraction of ion channels that regulate immunity as demonstrated by immune dysregulation in channel knockout mice. The immune system itself can cause acquired channelopathies that are associated with a variety of diseases of nervous, cardiovascular and endocrine systems resulting from autoantibodies binding to ion channels. These autoantibodies highlight the therapeutic potential of functional anti-ion channel antibodies that are being developed for the treatment of autoimmune, inflammatory and other diseases.