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ORAI1 is a pore subunit of Ca2+ release-activated Ca2+ (CRAC) channels that mediate T cell receptor stimulation-induced Ca2+ entry. A point mutation in ORAI1 (ORAI1R91W) causes severe combined immunodeficiency in human patients that is recapitulated in Orai1−/− mice, emphasizing its important role in the immune cells. Here, we have characterized a novel function of ORAI1 in T cell death. CD4+ T cells from Orai1−/− mice showed robust proliferation with repetitive stimulations and strong resistance to stimulation-induced cell death due to reduced mitochondrial Ca2+ uptake and altered gene expression of proapoptotic and antiapoptotic molecules (e.g. FasL, Noxa and Mcl-1). Nuclear accumulation of NFAT (nuclear factor of activated T cells) was severely reduced in ORAI1-deficient T cells and expression of ORAI1 and a constitutively active mutant of NFAT recovered cell death. These results indicate NFAT-mediated cell death pathway as one of the major downstream targets of ORAI1-induced Ca2+ entry. By expressing various mutants of ORAI1 in wild-type and Orai1−/− T cells to generate different levels of intracellular Ca2+, we have shown that activation-induced cell death is directly proportional to the [Ca2+]i levels. Consistent with the in vitro results, Orai1−/− mice showed strong resistance to T cell depletion induced by injection of anti-CD3 antibody. Furthermore, ORAI1-deficient T cells showed enhanced survival after adoptive transfer into immunocompromised hosts. Thus, our results demonstrate a crucial role of ORAI1-NFAT pathway in T cell death and highlight the important role of ORAI1 as a major route of Ca2+ entry during activated T cell death.
Stimulation of T cell receptor (TCR) evokes Ca2+ entry via Ca2+-release-activated Ca2+ (CRAC) channels (1, 2). An increase in intracellular Ca2+ concentration ([Ca2+]i) induces proliferation and cytokine production of immune cells by activation of downstream target molecules including calmodulin (CaM), calcineurin, and the transcription factor NFAT (nuclear factor of activated T cells) (2). The Ca2+-activated CaM/calcineurin protein phosphatase complex dephosphorylates heavily phosphorylated, cytoplasmic NFAT, which in turn translocates into the nucleus and turns on various transcriptional programs. Recently, ORAI1 (CRACM1) was identified as a pore component of CRAC channels by genome-wide RNAi high throughput screens (3–6). It was also shown that human patients with a homozygous missense mutation in ORAI1 suffer from lethal, severe combined immunodeficiency (SCID) (5). Another important signaling molecule in the CRAC channel pathway, stromal interaction molecule 1 (STIM1) was identified earlier using limited RNAi screens in Drosophila and mammalian cells (7, 8). Stimulation of the TCR activates phospholipase (PLC) γ which cleaves phosphatidylinositol 4,5-bisphosphate to produce the second messenger inositol 1,4,5-trisphosphate (InsP3), which in turn binds to InsP3 receptors on the endoplasmic reticulum (ER) membrane and depletes the ER Ca2+ stores. STIM1 senses Ca2+ depletion via its EF hands and translocates into the plasma membrane-proximal regions to activate ORAI1, thereby causing a sustained increase in [Ca2+]i (7, 9, 10). This sequential activation mechanism was termed as store-operated Ca2+ entry (SOCE) since depletion of ER Ca2+ stores precedes CRAC channel activation (11). Recently, three siblings from one kindred have been identified with homozygous nonsense mutation in STIM1. These patients also suffered from SCID, further emphasizing the crucial role of CRAC channels in the immune system (12).
Recently several reports have described the immune phenotypes of ORAI1- and STIM1-deficient mice (13–18). These mice showed a defect in immune cells consistent with the SCID patients. CD4+, CD8+ effector T cells and mast cells from ORAI1-deficient mice showed a decrease in SOCE as well as cytokine production (13, 14). In addition, ORAI1 deficiency impaired Ca2+ influx and effector functions of neutrophils as well as platelets (15, 16). STIM1 deficiency also showed a pronounced decrease in SOCE and cytokine production in T cells resulting in resistance to experimental autoimmune encephalomyelitis (EAE) (17, 18). On the contrary, mice deficient in STIM2, another member of the STIM family, showed a mild defect in SOCE and correspondingly, succumbed to EAE, albeit with less severe symptoms (17). Mice lacking both STIM1 and STIM2 displayed lymphoproliferative disorders in addition to SCID phenotype and were completely resistant to EAE (18, 19). Their lymphoproliferative phenotype was attributed to a severe reduction in regulatory T cell population (18). Interestingly, SCID patients harboring mutations in STIM1 also showed enlarged lymph nodes and elevated memory T cell populations (12).
TCR signaling plays an important role in immune homeostasis for maintenance of T cell numbers and induction of cell death. Cell death induced by TCR stimulation is critical for homeostasis of peripheral T cells after antigen clearance and negative selection of autoreactive T cells in the thymus (20–22). Activated T cell death occurs through two major apoptotic pathways, the death receptor- and mitochondria-mediated pathways. Death receptor-mediated apoptosis involves the Fas ligand/Fas signaling pathway majorly regulated by NFAT (23, 24) while mitochondria-mediated cell death occurs due to loss of mitochondrial membrane potential (20). Mitochondria-mediated cell death pathway involving the Bcl-2 family members (e.g. Bcl-2 and Bcl-XL) and the BH3-only proteins (e.g. Bad, Bik, Bim, and Noxa) plays an important role in T cell death and survival as seen in isolated T cells and in animal models (20, 22, 25). Double knockout mice lacking expression of Fas and Bim show severe lymphoproliferative disorders and marked resistance to cell death, indicating an important role of both, death receptors and mitochondria in T cell death (26–28).
Earlier, it was noticed that T cell death mediated by increased [Ca2+]i upon TCR stimulations can be mimicked by treatment with the ionophore, ionomycin (29). In cell death induced by TCR stimulation, the relation between Ca2+ homeostasis and Bcl-2 family members such as Bax, Bak, Bcl-2, and Bcl-XL has been extensively studied (30–33). These studies indicate that ER Ca2+ homeostasis is important for T cell death by modulation of cytosolic free Ca2+, mitochondrial Ca2+ uptake, or Ca2+ entry. A relationship between Ca2+ entry and mitochondrial Ca2+ uptake in T cells has been implicated in numerous studies. T cells have been shown to accumulate Ca2+ in mitochondria upon elevation of [Ca2+]i and reversely, mitochondrial Ca2+ buffering is important for prolonged CRAC channel activity, NFAT activation, and induction of cell death (34–37). Furthermore, it was shown that in T cells, mitochondria actively translocate towards the immunological synapse, accumulate Ca2+, and prevent Ca2+-dependent inactivation of CRAC channels (38, 39). Although in vitro pharmacological studies suggest an important role of Ca2+ in cell death after TCR stimulation, the exact role of Orai1 in mitochondrial Ca2+ uptake and T cell death has not been investigated due to lack of an appropriate animal model. It is also puzzling how the same Ca2+ signaling pathway can play a critical role in various outcomes of proliferation, death, and tolerance of T cells. If the amplitude or frequency of Ca2+ entry governs the fate of T cells as proposed previously (40–42), the threshold levels of [Ca2+]i for such decisions need to be determined.
Here we investigated how different levels of Ca2+ entry influence death and survival of T cells in vitro and in vivo using Orai1−/− mice. We showed that ORAI1-deficient T cells are strongly resistant to cell death due to reduction in death receptor- and mitochondria-mediated cell death mechanisms. Based on the results from recovery experiments by expression of ORAI1 and constitutively active NFAT in Orai1−/− T cells, we determined a crucial role of the ORAI1-NFAT pathway in T cell death. Using ORAI1-deficient cells and expression of a dominant negative mutant of Orai1, we investigated how diverse levels of Ca2+ entry can influence T cell death. In support of the results from isolated T cells, Orai1−/− mice showed strong resistance to depletion of T cells upon injection of anti-CD3 antibody in vivo. In addition, survival of ORAI1-deficient T cells was enhanced after transfer into immunocompromised hosts. Our results for the first time suggest a strong correlation between elevated [Ca2+]i via ORAI1 and T cell death/survival using a genetically manipulated animal model.
Orai1−/− mice were generated as previously described (14) and re-derived using female ICR mice (Taconic) at the University of California, Los Angeles. Orai1−/− mice generated from breeding of re-derived Orai1+/− mice were verified by genotyping as well as measurement of store-operated Ca2+ entry. NOD-scid IL2Rgammanull (NSG) mice were purchased from the Jackson Laboratory (Stock No. 005557). All animals were maintained in pathogen-free barrier facilities and used in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles.
CD4+ T cells were purified from single-cell suspensions of spleens and lymph nodes of adult mice. Single-cell suspensions were prepared by mechanical disruption using cell strainer (BD Biosciences). CD4+ T cells were isolated by magnetic sorting with CD4+ beads followed by treatment with the detach beads according to the manufacturer’s instructions (Invitrogen). For effector T cell differentiation, cells were stimulated with 1 μg/ml of anti-CD3 antibody and 1 μg/ml of anti-CD28 antibody (Pharmingen) for 48 hours on a plate coated with 0.3 mg/ml of goat anti-hamster (MP Biomedicals). CD4+ effector T cells were restimulated with anti-CD3 antibody with or without anti-CD28 antibody, or with ionomycin with or without phorbol myristate acetate (PMA) for cell death analysis.
Annexin-V and 7-AAD staining (BD Biosciences) were used to detect cell death followed by flow cytometry. For cell death assay, thymocytes were stimulated with 1 μM of ionomycin and 20 nM of PMA for 16 hours and CD4+ T cell were stimulated with variable concentrations of plate-coated anti-CD3 antibody for 6 hours. Proliferation was analyzed by flow cytometric measurement of carboxy fluorescein succinimidyl ester (CFSE) dilution. Purified T cells were labeled with 5 μM CFSE (Invitrogen) at 37°C for 10 minutes followed by extensive washing with phosphate-buffered saline (PBS). CFSE-labeled T cells were stimulated with anti-CD3 and anti-CD28 for 48 hours. For in vivo transfer experiments, CFSE-labeled naïve CD4+ T cells purified from Orai1+/+ and Orai1−/− mice were injected into the tail vein of 6–8-week-old NSG mice. A week later, spleens were collected, dissociated into single cell suspensions using cell strainers, and analyzed by flow cytometry.
Total RNA was extracted from resting and stimulated CD4+ effector T cells using TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA using oligo(dT) primers and Superscript III First-Strand cDNA synthesis kit (Invitrogen). Real-time PCR was performed using an iCycler IQ5 system (Biorad) and SYBR Green dye (Sigma) using the following primer pairs: GAPDH (GU214026 ) forward (5′-TGGAGATTGTTGCCATCAACGACCC) and reverse (5′-TAGACTCCACGACATACTCAGCACCG), Fas ligand (NM_010177) forward (5′-CTGGGTTGTACTTCGTGTATTCC) and reverse (5′-TGTCCAGTAGTGCAGTAGTTCAA), Noxa (AB041230) forward (5′-GCAGAGCTACCACCTGAGTTC) and reverse (5′-CTTTTGCGACTTCCCAGGCA), Bim (AF032461) forward (5′-GGAGATACGGATTGCACAGGA) and reverse (5′-TTCAGCCTCGCGGTAATCATT), Bok (NM_016778) forward (5′-ACATGGGGCAAGGTAGTGTC) and reverse ( 5′-GCTGACCACACACTTGAGGA), Bak (NM_007523) forward (5′-AGGTGACAAGTGACGGTGGT) and reverse (5′-AAGATGCTGTTGGGTTCCAG), Fas (NM_007987) forward (5′-TATCAAGGAGGCCCATTTTGC) and reverse (5′-TGTTTCCACTTCTAAACCATGCT), Bcl-2 (NM_009741) forward (5′-ATGCCTTTGTGGAACT A T A T G G C ) and reverse (5′-GGTATGCACCCAGAGTGATGC). Threshold cycles (CT) for all the candidate genes were normalized to the CT values for GAPDH housekeeping gene control to obtain ΔCT. Expression data were normalized to those of WT samples under resting conditions using the change-in-threshold method (2−ΔΔCT). The specificity of primers was examined by melt-curve analysis and agarose gel electrophoresis of PCR products.
For immunoblot analyses, cells were lysed in a buffer containing 50 mM Tris, pH 7.5, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM EDTA. Samples were separated on a 10% or 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and subsequently analyzed by immunoblot with relevant antibodies. Antibodies used were anti-Actin (Santa Cruz, clone I-19), anti-Bim (Santa Cruz, clone H-191), anti-Bcl-2 (Santa Cruz, clone C-2), anti-Bok (Cell signaling), anti-Bax (Cell Signaling, clone D2E11), anti-NFAT1 (purified rabbit polyclonal antibody to the ‘67.1’ peptide of NFAT1, a kind gift from Dr. Anjana Rao), and anti-Mcl-1 (Rockland). Chemiluminescence images were acquired using an Image reader LAS-3000 LCD camera (FujiFilm). Band intensities were quantified using Multi Gauge V 3.0 software (FujiFilm).
All antibodies used for flow cytometry were purchased from eBioscience and staining was performed according to the manufacturer’s instructions. The following antibodies were used for surface staining: anti-CD4 (FITC, clone GK1.5), anti-CD4 (PE, clone GK1.5), anti-CD8 (PE, clone eBioH35-17.2), anti-CD19 (PerCP-Cy5.5, clone eBio1D3), and anti-Fas Ligand (PE, clone MFL3). For Measurement of mitochondrial membrane potential, effector CD4+ T cells were stimulated with 3 μM of Ionomycin or 5 μg/ml of plate-coated anti-CD3 antibody for 24 hours and then mitochondrial mass was measured by incubating cells with 50 nM Mitotracker Deep Red (Invitrogen) for 15 min at 37 °C. For cell-cycle analysis, cells were stimulated with 1 μg/ml of anti-CD3 antibody and 1 μg/ml of anti-CD28 antibody (Pharmingen) for 24 or 48 hours in a plate coated with 0.3 mg/ml of goat anti-hamster, followed by incubation in a solution containing propidium iodide (50 nM). Effector CD4+ T cells were stimulated with 2 μg/ml of anti-CD3 antibody for 18 hours and the surface expression of Fas ligand were determined by flow cytometry using anti-FasL antibody. Samples were acquired with a FACSCalibur (BD Biosciences) and analyzed with ModiFit LT software (BD Biosciences) or FlowJo (TreeStar).
Full-length cDNA of human WT ORAI1 and mutants subcloned into bicistronic retroviral expression vector pMSCV-CITE-eGFP-PGK-Puro, which allows for simultaneous expression of Orai1, GFP and a puromycin resistance gene have been described previously (43). The cDNAs of WT NFAT1 (NFATc2) and constitutively active NFAT1 were purchased from Addgene (contributed by Dr. Anjana Rao) and subcloned into the retroviral expression plasmid as described above. For retroviral transductions, phoenix cells stably expressing gag-pol and ecotropic env (obtained from ATCC) were transfected with plasmids to produce ecotropic, replication-incompetent retrovirus using calcium phosphate transfection method. Virus-containing supernatant was collected at 2 and 3 days after transfection and CD4+ T cells were transduced twice on day 1 and day 2 after isolation in the presence of 8 μg/ml polybrene. Transduction efficiencies were evaluated by GFP expression using flow cytometry and immunoblotting.
Mice were intraperitoneally (i. p.) injected with PBS or 100 μg of anti-CD3ε (145-2C11) antibody (Bio X cell). Mice were sacrificed for analysis after 2 days of injection. Thymocytes and lymphocytes were counted and cell subset distributions were determined by flow cytometry analysis after surface staining with antibodies to CD4, CD8, or CD19. CD4+ T cells were purified from single-cell suspensions of spleens and lymph nodes of mice as described above to determine their absolute numbers.
Orai1+/+ and Orai1−/− T cells were injected into the tail vein of NOD-SCID IL2Rgammanull (NSG) mice. Before injection, isolated CD4+ T cells were transduced with retroviral vectors encoding a fusion protein of eGFP and firefly luciferase (44). Bioluminescence images were acquired at days 1, 7, 14, 21 post injections. Imaging was performed immediately after administration of D-luciferin by i.p. injection (3 mg/mouse) from a 15 mg/ml stock solution in PBS. A gray-scale surface image of each mouse was obtained by focusing at 20-cm field-of-view with 0.2 seconds exposure time. Overlapping bioluminescence images were acquired in the same field-of-view with 5 min exposure. Regions of interest (ROI) were manually selected over the whole bodies.
1 × 106 T Cells were loaded with 1 μM Fura 2-AM for 30 min at 25°C and attached to poly-L-lysine–coated coverslips for 15 min. Intracellular [Ca2+]i measurements were performed using essentially the same methods as recently described (45). Briefly, cells were mounted on a RC-20 closed-bath flow chamber (Warner Instrument Corp., Hamden, CT) and analyzed on an Olympus IX51 epifluorescence microscope with Slidebook (Intelligent Imaging Innovations, Inc.) imaging software. Fura-2 emission was detected at 510 nm with excitation at 340 and 380 nm and emission ratio (340/380) was acquired at every 5-s interval after background subtraction. For each experiment, 50–60 individual T cells were analyzed using OriginPro (Originlab) analysis software. [Ca2+]i was estimated from the relation [Ca2+]i = K* (R-Rmin) (Rmax-R)−1. K*, Rmin, and Rmax were independently measured in control cells. For mitochondrial Ca2+ measurements, cells were loaded at 22–23 °C for 45 min with 10 μM rhod-2/AM in culture medium, washed with fresh medium, electroporated as reported (39) to remove the cytosolic rhod-2, washed with fresh medium twice, stored at room temperature for 10 min, and used immediately. Rhod-2 fluorescence was background subtracted and normalized to the initial fluorescence values.
CD4+ T cells were cultured in nonpolarizing conditions until day 5 and stimulated for the indicated time with 10 nM PMA plus various concentrations of ionomycin at a density of 1 × 105 cells per well in a volume of 200 μl in 96-well plates. After stimulation, cells were attached to poly-L-lysine-coated 384-well plates in triplicates by centrifugation for 3 min at 200g. Cells were fixed with 2% paraformaldehyde, stained with anti-NFAT1 antibody and FITC-conjugated anti-rabbit (secondary antibody), and counterstained with the DNA-intercalating dye DAPI (4,6-diamidino-2-phenylindole). Images were acquired with a 20x objective using ImageXpress automated imaging system and analyzed by MetaXpress software (Molecular Devices). Nuclear translocation was assessed by calculation of a correlation of the intensity of NFAT1 staining and DAPI staining. T cells were considered to have nuclear NFAT1 when over 80% of the FITC–anti-NFAT1 staining coincided with the fluorescence signal from DAPI. Each data point represents an average of at least 450 individual cells per well.
Statistical analysis was carried out using two-tailed Student’s t-test. Differences were considered significant when p values were <0.05.
To determine how reduced SOCE by ORAI1 deficiency influences T cell proliferation, first we examined the number of Orai1+/+ and Orai1−/− naïve and effector CD4+ cells after stimulation with plate-coated anti-CD3 and anti-CD28 antibodies. Six days after stimulation, the number of Orai1−/− T cells did not show a significant difference with that of wild-type (WT) T cells (Fig. 1A, 1st stimulation). However, after re-stimulation, the number of Orai1−/− effector T cells showed a robust increase while that of WT effector T cells increased modestly (Fig. 1A, 2nd stimulation). Furthermore, ORAI1-deficient T cells continuously responded to repetitive stimulations and expanded robustly while WT T cells disappeared, possibly by undergoing cell death (Fig. 1A). ORAI1-deficient T cells were capable of expanding up to at least twelve stimulations that we have tested (data not shown). Because the number of T cells is the summation of cell death and proliferation rate, we measured both the rate of proliferation and cell death in Orai1+/+ and Orai1−/− effector T cells. The measurements of the CFSE dilution rate together with cell cycle analysis showed a slightly enhanced proliferation rate and cell cycle progression of Orai1−/− T cells especially at the early stages of 48 and 72 hours post stimulation (Fig. 1B and Suppl. Fig. 1A). Surprisingly, we observed an almost twofold increase in Orai1−/− live cell populations as determined by side and forward scatter after stimulation, suggesting that Orai1−/− T cells may exhibit resistance to cell death (Suppl. Fig. 1B). To directly measure cell death, we differentiated naïve CD4+ T cells to effector cells, and treated them with various concentrations of plate-coated anti-CD3 antibody for 6 hours. Orai1−/− effector T cells showed a marked decrease in Annexin V+ or 7-AAD+ populations as compared with WT cells at all the concentrations of anti-CD3 antibody tested (Fig. 1C). As previously reported for human T cells lacking functional Orai1 (5), ORAI1-deficient T cells showed a marked decrease in SOCE depending on gene dosage, with heterozygous cells showing intermediate levels of SOCE when compared with Orai1+/+ and Orai1−/−cells (Suppl. Fig. 2A and B) (14). Together, these results suggest that the robust increase in the numbers of Orai1−/− effector T cells responding to repetitive stimulations is predominantly due to strong resistance to cell death with minor contributions from the increased proliferation rate.
In addition to regulation of peripheral T cell homeostasis, Ca2+ signaling and TCR stimulation-mediated cell death plays a major role during negative selection in the thymus (42, 46–48). To measure cell death of thymocytes, we stimulated Orai1+/+ and Orai1−/− thymocytes with phorbol myristate acetate (PMA) and ionomycin (Fig. 2A). Ionomycin or anti-CD3 antibody alone did not induce robust cell death in thymocytes (data not shown), consistent with previous reports that costimulation is also required for negative selection (47). The live cell populations determined by forward and side scatter was significantly higher in stimulated Orai1−/− thymocytes (Fig. 2A). In addition, apoptotic cell population of Orai1−/− thymocytes as judged by annexin-V+ or 7-AAD+ cells was was markedly reduced after stimulation (Fig. 2B). Previously, a very marginal difference in single-positive (SP) CD4 and CD8 T cell populations was observed in young, 6–8 week-old Orai1−/− and STIM1−/− mice (13, 14, 49). Consistent with these results, we observed a minor difference in the thymi of 6–8 week-old Orai1+/+ and Orai1−/− mice (Fig. 2C). However, significantly higher populations of CD4+ and CD8+ SP cells and a lower percent of double-positive (DP) cells appeared in the thymi from Orai1−/− mice older than 12 weeks (Fig. 2C). These results suggest that Orai1-mediated Ca2+ entry is dispensable for development of T cells in the thymus; however, some of the thymocytes may escape cell death and accumulate with age. Our results in Fig. 2A, B, and C are consistent with earlier observations indicating that Ca2+ entry in thymocytes set up a threshold for negative selection (42, 46, 48), although the defects in Orai1−/− mice are not strong enough to influence T cell development in young mice. We conclude that ORAI1 plays a major role in stimulation-induced cell death of peripheral T cells, and also affects to a lesser extent, cell death of thymocytes.
To elucidate the mechanism of strong resistance to cell death in Orai1−/− effector T cells (Fig. 1), we examined functions of major signaling pathways involved in induction of cell death including mitochondrial Ca2+ uptake and the downstream transcriptional events. Ca2+ accumulates in mitochondria upon elevation of [Ca2+]i in T cells and reversely, Ca2+ buffering by mitochondria is important for sustained CRAC channel activity suggesting a reciprocal relationship between CRAC channels and mitochondria (34–37). To examine mitochondrial functions in Orai1−/− T cells upon stimulation, we stained ionomycin or anti-CD3 antibody-stimulated Orai1+/+ or Orai1−/− T cells with Mitotracker-Deep Red 633, a dye that binds to actively respiring mitochondria. In WT cells, stimulation with ionomycin and anti-CD3 antibody increased the Mitotrackerlow cell population, indicative of cells lacking functional mitochondria (Fig. 3A). In contrast, this Mitotrackerlow population was substantially reduced in Orai1−/− cells possibly due to reduced accumulation of Ca2+ in the mitochondria (Fig. 3A).
Previous studies have reported that location of mitochondria close to the site of Ca2+ entry is important to maintain sustained Ca2+ entry via CRAC channels in T cells (38, 39). Interestingly ORAI1 also translocates into the immunological synapse (IS) resulting in localized Ca2+ influx at the IS (50, 51). Since Orai1 is a critical component of the CRAC channels, these results suggest that Ca2+ entry via Orai1 may serve as a route for mitochondrial Ca2+ uptake in T cells. To investigate this hypothesis, we measured mitochondrial Ca2+ accumulation in Orai1+/+ and Orai1−/− cells. While control Orai1+/+ T cells showed a robust increase in mitochondrial [Ca2+] upon store depletion, we observed minimal Ca2+ uptake by mitochondria from Orai1−/− T cells (Fig. 3B). Ca2+ ions accumulated in the mitochondria were from the extracellular medium, since we did not observe any mitochondrial Ca2+ accumulation in Ca2+-free external solutions (Fig. 3B, bar graph). These data indicate that in T cells, ORAI1 plays a crucial role in mitochondrial Ca2+ accumulation and thereby mitochondria-mediated cell death pathway.
Death receptor-mediated Fas/FasL signaling pathway plays a critical role in T cell death after stimulation (20–22, 52–54) and it has been shown that expression of FasL is regulated by the Ca2+-NFAT pathway using NFAT-deficient cells and microarray analysis of ionomycin-treated primary T cells (23, 55). In addition, non-death receptor-mediated cell death pathways involving Bcl-2 family members (e.g. Bcl-2 and Bcl-XL) and the BH3-only proteins (e.g. Bad, Bik, Bim, and Noxa) are also critical for survival and death of T cells (20, 22, 25). We hypothesized that reduced cell death of Orai1−/− T cells may involve altered gene expression of key apoptotic molecules affecting the death receptor-or mitochondria-mediated cell death pathways. Indeed, deficiency of ORAI1 dramatically decreased the mRNA expression levels of FasL, not Fas, as judged by measurements of FasL transcripts, which is consistent with the decrease of surface expressed FasL proteins in Orai1−/− T cells (Fig. 4A). However, lower expression of FasL in Orai1−/− T cells may not be the sole contributor for their high resistance to cell death since T cells from Faslpr mice lacking expression of Fas, therefore harboring disabled Fas/FasL signaling, did not show the same growth pattern upon repetitive stimulation as observed in Orai1−/− T cells (Suppl. Fig. 3). These results suggest that the impact of ORAI1 deficiency can be broader than just a decrease in expression of Fas ligand. Hence, we examined expression levels of various molecules known to be involved in T cell death. Our analysis showed that the mRNA expression levels of a proapoptotic factor Noxa was severely impaired in Orai1−/− T cells while that of Bim was mildly influenced (Fig. 4B). The transcript levels of other proapoptotic molecules such as Bok and Bak also showed reduced expression in Orai1−/− T cells while the mRNA expression levels of the antiapoptotic molecule, Bcl-2 was not affected. Correspondingly, the protein levels of these proapoptotic molecules including Bok and Bax were also reduced in Orai1−/− effector T cells while Bim was marginally increased in the absence of ORAI1 (Fig. 4C). The protein levels of Noxa could not be tested due to lack of the antibody that can detect the murine Noxa protein (56). Among antiapoptotic candidates, Mcl-1 protein levels were increased in ORAI1-deficient T cells while Bcl-2 levels remained unaffected, which is consistent with the mRNA analyses (Fig 4C). Recently, it was proposed that the ratio of Noxa to Mcl-1 is important for setting up a threshold for cell death induced by TCR stimulation (56). T cells lacking Noxa (Pmaip1−/−) show a survival advantage in vitro resulting in an increase in the number of effector and memory T cells in vivo (56). Our results show a novel role of ORAI1-mediated Ca2+ entry in regulating the ratio of Noxa to Mcl-1 (Fig. 4B and C). In summary, our data suggest that reduced [Ca2+]i in Orai1−/− T cells after stimulation decreased cell death and enhanced cell survival by influencing multiple cell death pathways including FasL expression and the ratio of Noxa to Mcl-1.
So far, our data suggest that Ca2+ entry via ORAI1 induces T cell death by influencing the transcription levels of multiple apoptotic genes and this event can be mediated by transcription factors that are activated by elevated [Ca2+]i (e.g. NFAT). To examine a role of NFAT in ORAI1-mediated cell death, first we measured the nuclear accumulation of NFAT in ORAI1-deficient T cells. In WT cells, most of the NFAT proteins translocated into the nuclei after treatment with 1 μM of ionomycin (Fig. 5A, top panels). Even at the lowest concentration of ionomycin that we tested (0.2 μM), translocation was observed albeit with less efficiency. However, treatment of ORAI1-deficient T cells with even high concentrations of ionomycin (1 μM), resulted in reduced levels of nuclear NFAT (Fig. 5A, bottom panels). As seen in Fig. 5B, within 30 minutes of ionomycin (1 μM) treatment, only about 50% of ORAI1-deficient cells showed nuclear NFAT while more than 80% of WT cells showed nuclear NFAT. Even after 2 hours, a majority of the WT cells (65%) retained nuclear NFAT, but only a small fraction of ORAI1-deficient cells (~25%) showed nuclear NFAT (Fig. 5B). These results were validated by immunoblotting to detect de-phosphorylated NFAT in Orai1+/+ and Orai1−/− cells after stimulation with anti-CD3 antibody and ionomycin (Fig. 5C). We observed a robust dephosphorylation of NFAT after stimulation with anti-CD3 antibody (10 μg/ml) and ionomycin (1 μM) in WT cells, which was dramatically reduced in ORAI1-deficient cells. In consistence with reduced NFAT translocation, ORAI1-deficient T cells showed a substantial reduction in production of IL-2 and IFN-γ after stimulation with anti-CD3 and anti-CD28 antibodies (Suppl. Fig. 4). Together, these results suggest a close correlation between ORAI1-mediated Ca2+ entry and activation of NFAT, and the resistance of Orai1−/− T cells to cell death can be partially attributed to reduced translocation of NFAT.
Next, to examine if NFAT activity was directly responsible for resistance to cell death observed in ORAI1-deficient cells, we measured cell death upon expression of WT and constitutive active mutant of NFAT. Constitutive active NFAT with alanine substitutions of twelve key serine/threonine residues exists in a dephophorylated form and resides in the nucleus (23). WT T cells showed robust cell death upon stimulation with anti-CD3 antibody (Fig. 5D, left). Overexpression of WT-NFAT marginally enhanced the levels of cell death, while expression of the constitutively active (CA)-NFAT induced cell death even under resting conditions, which was pronounced after stimulation. In ORAI1-deficient cells, while expression of WT-NFAT did not enhance cell death, CA-NFAT induced cell death under both resting as well as stimulated conditions (Fig. 5D, right). These results strongly suggest that NFAT plays a major role in the cell death pathways induced by Ca2+ entry via ORAI1.
Next, we sought to determine the correlation between intracellular Ca2+ and induction of cell death by expressing various mutants of Orai1 in WT and ORAI1-deficient T cells. The goals of these experiments were twofold. First, we wanted to verify whether expression of ORAI1 in ORAI1-deficient T cells can rescue their reduced cell death phenotype. Second, we wanted to examine whether a further reduction in SOCE in ORAI1-deficient cells can enhance their resistance to cell death. We chose ORAI1E106Q, a pore mutant of ORAI1 that has a dominant negative effect on SOCE in T cells (6) and ORAI1R91W, a mutant identified in SCID patients that results in inactive CRAC channels (5). Expression of ORAI1E106Q and ORAI1R91W strongly inhibited SOCE in WT T cells (Fig. 6A, left panel). These data showed that ORAI1R91W in addition to ORAI1E106Q also has a strong suppression effect on the endogenous CRAC channel activity when expressed in WT T cells. In ORAI1-deficient T cells, expression of WT ORAI1 recovered SOCE, while that of ORAI1E106Q and ORAI1R91W further suppressed the residual SOCE in these cells, most likely by multimerizing with other ORAI proteins (Fig. 4A, right panel). Next, we examined the effect of expression of these mutants on cell death. As expected, in WT T cells, expression of ORAI1R91W reduced cell death upon anti-CD3 stimulation (Fig. 6B, top two panels). Interestingly in ORAI1-deficient T cells, expression of WT ORAI1 recovered cell death to the levels similar to WT T cells, while expression of ORAI1R91W further suppressed cell death (Fig. 6B, bottom two panels). These results indicate a direct correlation between intracellular Ca2+ accumulation via ORAI1 and resistance to cell death, with reduction in SOCE proportionally enhancing resistance to cell death.
T cells from Orai1−/− mice showed a strong resistance to cell death upon stimulation in vitro. To examine if this was the case in vivo, we injected anti-CD3 antibody into Orai1+/+ and Orai1−/− mice and examined depletion of T cells. In the lymph nodes of Orai1+/+ mice, CD4+ population decreased upon injection of anti-CD3 antibody, however, the depletion was much less in Orai1−/− mice (Fig. 7A and B). Depletion of CD8+ cells in control mice was more pronounced than CD4+ cells and even CD8+ cells survived better in Orai1−/− mice after anti-CD3 antibody injection (Fig. 7B). The number of thymocytes in anti-CD3 antibody-injected Orai1+/+ control mice reduced markedly (25% of that in PBS-injected mice) while that in Orai1−/− mice decreased less (50% of that in PBS-injected mice) (Fig. 7C), recapitulating in vitro resistance to cell death (Fig. 2A and B). These results further support the idea that ORAI1-mediated cell death upon stimulation can be a common mechanism for CD4+ and CD8+ T cells in the peripheral lymphoid organs as well as thymocytes.
To further test the hypothesis that reduced cell death of Orai1−/− T cells may benefit T cell survival in vivo, we transferred Orai1+/+ and Orai1−/− T cells into immunocompromised NOD-SCID IL2Rgammanull (NSG) mice. After transfer of the same number of CFSE-labeled cells (3 × 106) into NSG recipients by intravenous injection, we detected only 0.3% of the total splenocytes of the recipient mice as Orai1+/+ CFSE-positive cells after a week while threefold more Orai1−/− CFSE-positive cells were detected (0.9% of total splenocytes; Fig. 7D). These results were confirmed independently by non-invasive imaging of transferred Orai1+/+ and Orai1−/− CD4+ T cells expressing firefly luciferase. Under identical conditions of isolation, transduction, and transfer, Orai1−/− T cells showed enhanced survival over a period of three weeks when compared with Orai1+/+ T cells (Fig. 7E). Taken together, these results suggest that deficiency of ORAI1 antagonizes cell death and improves survival of T cells in vitro and in vivo.
Ca2+ ions play a pivotal role in cell proliferation and death by controlling a plethora of signaling pathways (40). Although in vitro pharmacological studies suggest a direct role of Ca2+ in cell death after TCR stimulation, the exact route of Ca2+ entry and the detailed molecular mechanism have not been investigated due to a lack of the molecular identity and an appropriate animal model. Here we reaffirmed the important role of [Ca2+]i for T cell death using Orai1−/− T cells and expression of the dominant negative mutants of ORAI1 in WT T cells. In addition, we demonstrated that ORAI1-NFAT pathway is crucial for activation of the downstream cell death programs in effector T cells, since overexpression of CA-NFAT could completely reconstitute cell death in ORAI1-deficient T cells. T cells lacking ORAI1 showed better survival than controls, in vitro upon TCR stimulations (Fig. 1), and in vivo after injection of anti-CD3 antibody and transfer to immunocompromised hosts (Fig. 7). Furthermore, thymocytes lacking ORAI1 showed increased resistance to cell death in vitro (Fig. 2) and in vivo after anti-CD3 antibody injection (Fig. 7C), indicating a possible role of ORAI1 in thymocyte death. In support of these data, Orai1−/− thymocytes showed a significantly higher population of SP cells particularly in mice aged more than 12–16 weeks (Fig. 2). Only marginal differences were observed in the thymi from 6–8 week-old Orai1−/− mice, consistent with the previous analyses of the same aged Orai1−/− or STIM1−/− mice (Fig. 2) (13, 14, 49). These results suggest that the increase in SP population in aged Orai1−/− mice may be caused by accumulation of the survived thymocytes with age by escaping cell death, rather than a defect in T cell development. Our results showing a resistance of ORAI1-deficient thymocyte to cell death are consistent with earlier reports that the [Ca2+]i levels determine cell death or maturation of thymocytes, depending on avidity of antigen-TCR interactions and the strength of costimulation (e.g. CD28) (42, 46–48). Together, these results suggest that high [Ca2+]i through ORAI1 may be commonly used to control the negative selection process in the thymus and to maintain the size of the T cell pool by playing a negative role in T cell survival.
Our and other groups’ observations showed that CRAC channels are crucial for the function of effector T cells such as cytokine production (Suppl. Fig. 4) (13, 14). These data together with the SCID symptoms of human patients harboring mutations in ORAI1 or STIM1 genes demonstrate a positive role of CRAC channels in the immune response (5, 12, 57). Here we showed that ORAI1 plays a bona fide role in stimulation-induced cell death further emphasizing the role of ORAI1 in the diverse functions of effector T cells in addition to cytokine production (Fig. 1). So far, none of the data from the patients and mice harboring deletion or mutations of Orai1 and STIM1 genes indicates any severe defect in development or homing of T cells in the peripheral lymphoid organs. However, these results do not rule out the role of Ca2+ signaling in T cell development or homing because it is still possible that Ca2+ plays a role via entering through alternate routes (e.g. ORAI2, ORAI3, or other non-store-operated Ca2+ channels) instead of ORAI1. In support of this idea, reduction of SOCE in ORAI1-deficient naïve T cells was much less than that in effector T cells and ORAI2 instead of ORAI1 was abundantly expressed in naïve T cells as shown by mRNA expression analysis (13, 14).
The role of CRAC channels in T cell proliferation is more complex than in cytokine production or cell death because it differs significantly in various mouse models. In the current work, we showed that Orai1−/− T cells do not have a defect in proliferation (Fig. 1). Instead, the absence of ORAI1 enhanced the proliferation rate at the early stage of activation. A recent report examining T cell functions of knockin mice harboring a point mutation at position 93 (ORAI1R93W) to recapitulate the phenotype observed in SCID patients (ORAI1R91W), showed markedly reduced proliferation of Orai1R93W/R93W effector T cells (58). It is known that ORAI1 can form heteromultimers with other Orai family members (6), and human ORAI1R91W mutant has been shown to function in a dominant negative manner in an ORAI1-STIM1 overexpression system (59). We also observed suppression of SOCE in WT and Orai1−/− T cells upon expression of ORAI1R91W (Fig. 6A). Therefore, it is possible that the levels of SOCE in T cells isolated from Orai1R93W/R93W mice are much lower than those of Orai1−/− T cells, which may not reach the minimum requirement of [Ca2+]i to promote proliferation of T cells. Consistent with these observations, STIM1-deficient T cells showed normal proliferation with marginal differences in vitro and in vivo after immunization, while T cells lacking both STIM1 and STIM2 proliferate to a much lesser extent (18, 19). Together, these results suggest that T cell proliferation does not require high [Ca2+]i levels and the threshold of [Ca2+]i necessary for T cell proliferation seems to be much lower than that for cytokine production or cell death.
A direct correlation between the [Ca2+]i levels and activated T cell death was further verified by recovery experiments in ORAI1-deficient T cells. Expression of ORAI1 in ORAI1-deficient T cells almost completely recovered normal cell death levels in addition to SOCE (Fig. 6). Interestingly, we were able to suppress T cell death in WT T cells by expression of the dominant negative mutant of Orai1, ORAI1E106Q and ORAI1R91W (Fig. 6B and data not shown). Expression of ORAI1R91W in ORAI1-deficient T cells further suppressed the residual levels of SOCE and cell death induced by stimulation (Fig. 6B). Together, these results indicate that the degree of stimulation-induced T cell death is directly proportional to [Ca2+]i levels regulated via ORAI1. Furthermore, our data supports a model in which excessive Ca2+ entry mediated by ORAI1 supports death programs mediated by multiple cell death pathways including a transcriptional event mediated by NFAT, and non-transcriptional events of mitochondrial Ca2+ uptake. This conclusion is supported by the following observations. First, Orai1−/− T cells showed reduced expression of proapoptotic molecule, FasL that is known to be regulated by Ca2+-NFAT signaling. Second, our results showed [Ca2+]i-mediated regulation of the ratio of Noxa to Mcl-1, that is recently identified to set a threshold for T cell death (Fig. 4) (56). Finally, Orai1−/− T cells showed severely reduced mitochondrial Ca2+ uptake and thereby reduced mitochondria-mediated cell death (Fig. 3). Since the NFAT family of transcription factors are direct downstream targets of Ca2+-CaM/calcineurin pathway, our results together with those of NFAT family-deficient mice suggest that excessive [Ca2+]i turns on cell death transcriptional programs, many of which are regulated by NFAT (23, 55). It is also known that predominant accumulation of nuclear NFAT can turn on transcriptional programs of a status of T cell tolerance termed anergy (23). ORAI1-deficient T cells were not anergic because proliferation and cell cycle progression were actively induced upon repetitive stimulations (Fig. 1A). Therefore, the threshold levels of [Ca2+]i for anergy induction may be higher than that observed in Orai1−/− T cells. Future studies in Orai1−/− T cells examining the expression levels or activities of anergy-inducing factors including E3 ubiquitin ligases (Cbl-b, Itch, or GRAIL) and caspase 3 that cleaves/degrades TCR signaling molecules, and the zinc finger transcription factor Ikaros that suppresses IL-2 transcription, would validate the role of ORAI1 in anergy (60, 61).
The rise in [Ca2+]i triggered by TCR stimulation plays a pivotal, positive role in T cell activation, cytokine production and proliferation. However, symptoms of the patients with nonfunctional CRAC channels, especially those with mutations in STIM1 gene are perplexing because in addition to a defect in activation of immune cells as exemplified by SCID symptoms, they also display lymphoproliferative disorders demonstrated by lymphoadenopathy, splenomegaly, and elevated memory T cell population (12). In addition to the proposed mechanism of reduced regulatory T cell population (18), our observations of resistance to cell death can serve as an alternate explanation for the lymphoproliferative symptoms of SCID patients. Our findings demonstrate a crucial role of high [Ca2+]i in diverse immune tolerance mechanisms such as negative selection, induction of cell death, and T cell survival. Our and other groups’ results point towards different threshold levels of [Ca2+]i to trigger various functions of T cells including proliferation, anergy, or death. A comprehensive study of animal models with various levels of CRAC channel activity can facilitate further understanding of how different amplitudes of Ca2+ entry influence diverse activities of T cells.
We thank Dr. Talal Chatila for critical comments on the manuscript; Drs. Ram Raj Singh, Steven Bensinger, David Brooks, and Dong-Sung An for sharing animals and active discussions; and Drs. Caius Radu, David Stout and Seungmin Hwang for help with bioluminescence imaging. We also thank Drs. Anjana Rao, Robb MacLean, Peng Zhao, Peipei Ping, David Liem, and Reuben Kim for sharing antibodies and reagents.
This work was supported by the National Institute of Health grants AI-083432, AI-088393 (to Y.G.), and a fellowship from the American Heart Association (to S.S.).
The authors declare no competing financial interests.