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STIM1-mediated NFAT signaling synergizes with STAT1 to control T-bet expression and TH1 differentiation

Nature Immunology volume 26, pages 484–496 (2025)Cite this article

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Abstract

Stromal interaction molecule 1 (STIM1) is critical for store-operated Ca2+ entry (SOCE) and T cell activation. T helper 1 (TH1) cells, which express T-bet (encoded by TBX21), mediate immunity to intracellular pathogens. Although SOCE is known to regulate other TH lineages, its role in Th1 differentiation remains unclear. Here, we report a patient with an intronic loss-of-function mutation in STIM1, which abolishes SOCE and causes immunodeficiency. We demonstrate that SOCE promotes nuclear factor of activated T cells (NFAT) binding to conserved noncoding sequence (CNS)-12 in the TBX21 enhancer and enables NFAT to synergize with STAT1 to mediate TBX21 expression. While SOCE-deficient CD4+ T cells have reduced expression of TBX21 in the absence of interleukin-12 (IL-12), their expression of IL-12 receptors β1 and β2 is increased, sensitizing them to IL-12 signaling and allowing IL-12 to rescue T-bet expression. Our study reveals that the STIM1-SOCE–NFAT signaling axis is essential for the differentiation of Th1 cells depending on the cytokine milieu.

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Fig. 1: STIM1 c.497+776A>G splice-site creating mutation in an immunodeficient patient.
Fig. 2: STIM1 mutation abolishes protein expression and SOCE.
Fig. 3: Transcriptional dysregulation in T cells with STIM1 c.497+776A>G mutation.
Fig. 4: SOCE regulates T-bet expression and TH1 differentiation.
Fig. 5: NFAT binds to CNS-12 to control T-bet expression.
Fig. 6: NFAT synergizes with STAT1 to induce T-bet expression.
Fig. 7: The SOCE–calcineurin–NFAT pathway suppresses IL-12 receptor expression.

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Data availability

RNA-seq and ATAC-seq data generated for this study have been deposited in the GEO database under accession numbers GSE179625 and GSE253187. Publicly available datasets that were reanalyzed for this study are: GSE64409, GSE98726, GSE67443, GSE183883, GSE207265, GSE144586, GSE204946, GSE96724, GSE92531, GSE67451, GSE93014, GSE157597 and GSE161096. Source dataare provided with this paper.

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Acknowledgements

We acknowledge technical support from the Genome Technology Center (grant no. RRID: SCR_017929), and Cytometry and Cell Sorting Laboratory (grant no. RRID: SCR_019179) at NYU Langone Health. We thank the genomics and bioinformatics cores at WCMQ for support with the WGS and initial analyses. Both cores at supported by the Biomedical Research Program (BMRP) program funded by Qatar Foundation. We thank J. Wang (NYUSOM) for providing the 3A9 cells used in this study. This study was funded by National Institutes of Health (NIH) grant nos. AI097302 and AI130143 to S.F., a grant from the Qatar Foundation to K.M. under the BMRP at Weill Cornell Medicine Qatar. Additional funding was provided by postdoctoral fellowships from the German Research Foundation (DFG) to S.K. (grant no. KA 4514/1-1) and an NIH F30 training grant no. AI164803 (to A.Y.T.).

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Author notes

  1. Sascha Kahlfuss

    Present address: Institute of Molecular and Clinical Immunology, Institute of Medical Microbiology and Hospital Hygiene, Medical Faculty, Otto-von-Guericke-University, Magdeburg, Germany

  2. Tristan E. Knight

    Present address: Kapiolani Medical Center for Women and Children, Burns School of Medicine, Honolulu, HI, USA

Authors and Affiliations

  1. Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA

    Li Zhong, Yin-Hu Wang, Sascha Kahlfuss, Miki Jishage, Maxwell McDermott, Jun Yang, Anthony Y. Tao, Ke Hu, Lucile Noyer, Dimitrius Raphael, Devisha Patel & Stefan Feske

  2. Division of Pediatric Hematology/Oncology, Children’s Hospital of Michigan, Detroit, MI, USA

    Tristan E. Knight & Meera Chitlur

  3. Central Michigan University College of Medicine, Detroit, MI, USA

    Meera Chitlur

  4. Calcium Signaling Group, Research Department, Weill Cornell Medicine, Doha, Qatar; Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA

    Khaled Machaca

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Contributions

L.Z., Y.-H.W., S.K., J.Y., L.N. and M.J. conducted experiments. L.Z., M.J., S.K., A.Y.T., M.M., D.R., D.P. and K.H., analyzed data and interpreted results. K.M. supervised WGS and interpreted results. L.Z., Y.-H.W., S.K. and S.F., designed experiments. T.E.K. and M.C. provided patient data and blood samples. L.Z. and S.F. wrote the manuscript. All authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Stefan Feske.

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Competing interests

S.F. is a scientific cofounder and consultant of Calcimedica, and an inventor on a patent Regulators of NFAT (WO/2007/081804) related to this paper. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Clinical findings in patient with STIM1 c.497+776A>G mutation.

a, Anhidrosis of palm after exertion. b, Persistent dental hypoplasia of newly formed adult teeth of the PT at 6 years of age. c, Dilation of pupils in both eyes.

Extended Data Fig. 2 Altered gene expression in CD4+ T cells of STIM1 deficient patient.

a, Principal component analysis (PCA) of differentially expressed genes (DEGs) detected by RNA-Seq. CD4+ T cells from the patient (PT) with STIM1 c.497+776 A > G mutation and a healthy donor (HD) were left unstimulated or stimulated with anti-CD3/28 for 6 hours. b, Venn diagram of up- and down-regulated DEGs after stimulation of CD4+ T cells from HD and the PT. An adjusted P value < 0.05 and absolute log2 fold change (LFC) > 0.5 were used as cut-off. c, Gene set enrichment analysis (GSEA) of DEGs in CD4+ T cells from the PT and a HD. Normalized enrichment score (NES) and adjusted P values are as indicated for the following datasets: NFAT TF pathway (M60: PID), P < 0.01; alpha/beta T cell differentiation (GO: 0046632), P < 0.05; Hallmark IFN-gamma response (M5913), P < 0.001. d, LFC in expression levels of DEGs related to the NFAT TF pathway (M60: PID). Data are from one experiment with 2 samples per cohort and stimulation condition. e, mRNA levels of CD44, NR4A1/Nur77 and CD38 in CD4+ T cells from the PT, his father (Fa), mother (Mo) and a HD stimulated with anti-CD3/28 for 6 hours and analyzed by RNA-seq.

Source data

Extended Data Fig. 3 Attenuated IFN-γ and T-bet response in CD4+ T cells of STIM1 deficient patient.

a, mRNA levels of IFNG, IL2 and TNF in CD4+ T cells from the patient (PT), his father (Fa), mother (Mo) and a HD stimulated with anti-CD3/28 for 6 hours and analyzed by RNA-seq. b, Metascape analysis of all downregulated differentially expressed genes (DEGs) in HD, Fa, Mo vs. the PT. c, IPA upstream regulator analysis of DEGs in CD4+ T cells from the PT vs. all three controls (HD, Fa and Mo) ranked by P value and separated by upstream signaling molecules and transcription factors. Colors indicate the activation Z score. d, Epigenetic Landscape In Silico deletion Analysis (LISA) of transcriptional regulators of downregulated DEGs in T cells from the PT vs. all three controls (HD, Fa and Mo) ranked by P value. Note that LISA uses different ChIP-Seq datasets to identify transcriptional regulators; T-bet is identified as ‘T’ using GSE81881 (human Th1 cells) indicated here as T-bet*, and as Tbx21 using GSE33802 (mouse Th1 cells) indicated as Tbx21#. Fold changes were shrunk using the apeglm method. DEGs were considered significant if shrinkage of the log2 fold change (LFC) absolute value was > 0.5, and the adjusted P value < 0.05.

Source data

Extended Data Fig. 4 SOCE is required for T-bet expression in human and mouse CD4+ T cells.

a, Venn Diagram (left) of differential gene expression of potential T-bet targets in anti-CD3/28 stimulated CD4+ T cells (6 hours) from the STIM1 deficient PT and a HD (left). T-bet targets were identified by (i) T-bet binding using ChIP-Seq, (ii) positive regulation by T-bet using RNA-Seq, and (iii) reduction of H3K4me1 histone methylation in T-bet-deficient cells. Z scores of mRNA expression of T-bet target gene that fulfill all three selection criteria (right). b, GSEA analysis (left) and heatmap of DEGs (right) related to Th1 specific genes. c, Cytokine expression in CD4+ T cells from a HD and the PT restimulated with 20 nM PMA and 1 μM ionomycin for 6 hours. Bar graphs showing relative median fluorescence intensities (rel. MFI) in the patients T cells normalized to HD. d, Intracellular Ca2+ levels in HD T cells treated with DMSO or 1 μM BTP2. Cells were loaded with 2 μM Fura-2 and stimulated with 1 μM Thapsigargin (TG) in Ca2+ free Ringer solution, followed by perfusion with 2 mM extracellular Ca2+. e, T-bet protein levels in anti-CD3/28 stimulated CD4+ T cells from a HD with DMSO or 1 μM BTP2 (24 hours). Bar graphs showing relative delta MFI (rel. ΔMFI) of T-bet levels in stimulated HD T cells normalized to cells treated with DMSO. f, Intracellular Ca2+ levels in murine WT CD4+ T cells treated with DMSO or 1 μM BTP-2. g, T-bet protein levels in anti-CD3/28 stimulated CD4+ T cells from WT mice with DMSO or 1 μM BTP-2. Data are the means ± SEM for c, e and g. Data are from at least 3 independent experiments. Statistical analysis is performed by two-sided Student’s t test. * P < 0.05.

Source data

Extended Data Fig. 5 Regulation of TBX21 expression by CNS-12.

a, Analysis of chromatin accessibility of Tbx21 locus in murine CD4+ T cell by DNase I hypersensitive sites sequencing (DNase-seq). Cells were stimulated with 20 ng/ml phorbol myristate acetate (PMA) and 2 μM calcium ionophore (CaI) for 4 hours (GSE67451). b, Analysis of chromatin accessibility of Tbx21 locus in murine CD8+ T cells by ATAC-Seq. Cells were stimulated with 10 ng/ml PMA and 0.5 μM ionomycin (Iono) or left unstimulated (resting) for 2 hours with or without 2 μM cyclosporin A (CsA) (GSE93014). c, Analysis of chromatin accessibility, RNA polymerase II (pol II) and H3K27ac binding in TBX21 gene locus in human CD4+ and CD8+ T cells. ATAC-seq data of human naive CD4+ T cells with or without anti-CD3/CD28 for 1 day (Top, GSE161096). ATAC-seq data of human naïve CD8+ T cells with or without anti-CD3/CD28 for 2 days (Middle, GSE212699). ChIP-Seq analysis of Pol II and H3K27ac binding to the TBX21 gene locus in human T cells activated with anti-CD3/CD28 Dynabeads for 24 hours (Bottom, GSE183883). d, Analysis of NFATc2 binding to CNS-12 of Tbx21 in CD4+ T cells from WT and Stim1/2CD4 mice by ChIP-qPCR. T cells were stimulated with anti-CD3/CD28 and cultured for 5 days, then restimulated with 20 nM PMA and 1 μM ionomycin for 1 hour followed by ChIP-qPCR. e, ChIP-Seq analysis of p300, H3K27ac and JUNB binding to the Tbx21 gene locus in mouse CD4+ T cells. Murine CD4+ T cell stimulated with anti-CD3/CD28 antibodies for 5 days (Top, GSE207265) or with 20 ng/ml phorbol myristate acetate (PMA) and 2 μM calcium ionophore (CaI) for 4 hours (Bottom, GSE67443). The statistical significance of differences between ChIP-seq peaks of test and control groups was calculated using MACS2 v2.1.1., and for ATAC-seq peaks using the DESEQ2 package. ***P < 0.001; **P < 0.01; *P < 0.05.

Source data

Extended Data Fig. 6 Effects of NFAT and STAT1 on Tbx21 transcription.

a, Two-step model of T-bet expression. b-c, Representative flow cytometry plots (left) and relative median fluorescence intensities (Rel. MFI, right) of T-bet (b), and percentage of T-bet+ cells (c). Murine WT CD4+ T cells were pretreated with DMSO or 1 μM FK506 for 30 min and then stimulated with anti-CD3/CD28 antibodies with or without exogenous IL-12 (10 ng/ml) or IFN-γ (100 ng/ml) and/or anti-IL-4 antibodies (5 ug/ml) for 3-4 days. The bar graph in (b) shows relative MFI of T-bet normalized to T-bet levels in DMSO-treated CD4+ T cells without exogenous IL-12 or IFN-γ. d, T-bet protein levels in CD4+ T cells from a HD and the PT with STIM1 mutation stimulated with anti-CD3/CD28 for 4 days with or without exogenous IFN-γ (100 ng/ml). Shown are relative delta MFI (Rel. ΔMFI) levels of T-bet normalized to T-bet in HD T cells without IFN-γ. e-f, Tbx21 mRNA (e) and T-bet protein (f) levels in mouse WT CD4+ T cells pretreated with anti-IFN-γ antibodies or left untreated for 1 hour followed by anti-CD3/CD28 stimulation for 24 hours (RT-qPCR) or 3-4 days (flow cytometry). The bar graph in (f) shows relative MFI of T-bet normalized to T-bet levels in DMSO-treated CD4+ T cells. g, ChIP-seq analysis of H3K27ac binding to the Tbx21 locus in mouse CD4+ T cells. Cells were activated by anti-CD3/CD28 stimulation either under neutral conditions (nc, with anti-IFN-γ and anti-IL-4 antibodies) or treatment with 100 ng/ml IFN-γ for 3 days (GSE96724). h, ChIP-Seq analysis of H3K27ac binding to the Tbx21 locus in murine naïve CD4+ T cells, Th1, Th2 and Th17 cells (GSE144586). Data are the means ± SEM for b to f. Data are from at least 3 independent experiments. Statistical analysis is performed by two-sided Student’s t test. *** P < 0.001; **P < 0.01; *P < 0.05.

Source data

Extended Data Fig. 7 Regulation of Il12rb1 and Il12rb2 genes.

a-b, ChIP-Seq analysis of HDAC1, 4 and 7 binding to Il12rb1 (a) and Il12rb2 (b) loci in mouse Th17 cells (GSE92531). Boxes highlight potential gene regulatory regions. c, Analysis of HDAC1 binding to the promoter of Il12rb1 by ChIP-qPCR using mouse CD4+ T cells. Cells from WT mice were stimulated with anti-CD3/CD28 for 5 days, incubated with DMSO or 1 μM FK506 for 30 min and restimulated with 20 nM PMA and 1 μM ionomycin for 1 hour. d, Il12rb1 and Il12rb2 mRNA levels in CD4+ T cells from WT or Stim1/2CD4Cre mice that were cultured with DMSO or 100 nM TSA for 24 hours in the presence of 10 ng/ml IL-12. e, Analysis of chromatin accessibility, T-bet binding and GATA3 binding to the Il12rb2 locus. Top two rows show ATAC-Seq data from CD4+ T cells of WT OTII (WT) and Orai1/2CD4 (Orai1fl/fl Orai2−/− Cd4Cre) OT-II mice that had been injected into TCRα−/− host mice followed by infection with the PR8-OVA strain of influenza A virus (IAV) for 8 days. The middle two rows show ATAC-Seq data of CD8+ T cells from WT mice that were left untreated or pretreated with 2 μM cyclosporin A (CsA) for 15 mins and stimulated with 10 ng/ml PMA and 0.5 μM ionomycin (iono) or left unstimulated (resting) for 2 hours (GSE93014). Bottom rows show ChIP-Seq data from naïve mouse CD4+ T cells and Th1 cells (GSE204946). Boxes highlight potential gene regulatory regions. The table in e (right) summarizes information about chromatin accessibility and TF binding to the Il12rb2 locus. Data in c-d are the means ± SEM; statistical analysis was performed by two-sided Student’s t test. ***P < 0.001; **P < 0.01; *P < 0.05.

Source data

Extended Data Fig. 8 Model of SOCE- and NFAT-dependent T-bet expression and Th1 differentiation.

In CD4+ T cells, TCR engagement activates STIM1 to induces SOCE through ORAI1 Ca2+ channels. Increased intracellular Ca2+ activates NFAT to promote the production of IFN-γ, which binds to the IFN-γ receptor (IFNGR) and activates STAT1. Moreover, NFAT directly binds to CNS-12 of TBX21. STAT1 and NFAT synergize to induce T-bet expression and thus, Th1 differentiation. NFAT also binds to IL12RB1 and IL12RB2 genes and inhibits their expression, likely by acting as a partner of HDAC proteins (IL12RB1) and other negative regulators (IL12RB2). Lack of SOCE following TCR stimulation impairs T-bet expression when IL-12 is not present, but sensitizes T cells to IL-12 signaling by enhancing the expression of IL-12Rβ1 and IL-12Rβ2, thus promoting TBX21 expression and Th1 differentiation when IL-12 is available. Created with BioRender.com.

Extended Data Table 1 Clinical phenotypes of a patient with STIM1 c.497+776A>G mutation
Full size table
Extended Data Table 2 Immunologic profile of a patient with STIM1 c.497+776A>G mutation
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Supplementary information

Supplementary Information

Supplementary Note, Figs. 1–5 and Tables 1–6.

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Supplementary Data Table 1

qPCR primers.

Supplementary Data 2

Source data for supplementary figures.

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Source Data Figs. 1–7 and Extended Data Figs. 2–7

Statistical source data in one file.

Source Data Figs. 1 and 2

Unprocessed western blots.

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Zhong, L., Wang, YH., Kahlfuss, S. et al. STIM1-mediated NFAT signaling synergizes with STAT1 to control T-bet expression and TH1 differentiation. Nat Immunol 26, 484–496 (2025). https://doi.org/10.1038/s41590-025-02089-8

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