Fig 2: Increased expression of the Ca2+ channel component ORAI3 in RA CD4+ T cells.a Transcriptional profiling of Ca2+ channel and Ca2+ sensor gene transcripts in CD4+ naive T cells from HC (n = 6 white bars and circles) and RA (n = 6 gray bars and squares) patients. Gene expression was normalized to β-actin. Data are presented as mean ± SEM. b ORAI3 transcripts quantified by qPCR and normalized to 18S rRNA in CD4+ naive T cells from HC (n = 22) and RA (n = 21). Data are shown as dot plots with mean ± SEM. c ORAI3 transcript levels in RA patients with CDAI activity scores <10 (n = 9) or >20 (n = 5). d, e ORAI3 transcripts in CD4+ naive T cells from RA patients dichotomized for whether they were treated (n = 5) or not treated with methotrexate (MTX) (n = 16) (d) or whether they were (n = 10) or were not (n = 11) on TNF inhibitors (TNFi, etanercept or adalimumab) (e). f ORAI3 protein levels are shown as a representative immunoblot (left) and as dot plots of densities of ORAI3 relative to β-actin of purified naive CD4+ T cells from HC (n = 10) and RA (n = 9) patients. Uncropped Western blots in Supplementary Fig. 9. g ORAI3 transcripts quantified by qPCR and normalized to 18S rRNA in CD4+ naive T cells from HC (n = 22) and gout (n = 6), systemic lupus erythematosus (SLE) (n = 7), and PsA (n = 10) patients (HC vs. gout p = 0.80; HC vs. SLE p = 0.85). Statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparison test (Fig. 2g) or with unpaired two-tailed Student’s t test (a–f). n.s. not significant. Source data are provided as a Source Data file.

Fig 3: Transcriptional control of ORAI3.a Chromatin accessibility maps of naive CD4+ T cells from RA and PsA patients and from healthy individuals were generated by ATAC-seq. Accessible regions at ORAI3 TSS region (chr16:30,960,352–1215) and downstream of TSS (chr16:30,964,994–5367). b Accessible regions were analyzed for transcription factor (TF) motifs. Indicated TFs were silenced using siRNA transfected into HEK293T cells for 48 h, non-targeting siRNA was used as a negative control. ORAI3 transcripts were quantified by qPCR and normalized to β-actin. Data are presented as mean ± SEM from three independent experiments (n = 3). Knockdown efficiency is shown in Supplementary Fig. 6. c Purified CD4+ T cells were transfected with increasing concentrations of IKAROS siRNA smart pool for 48 h. ORAI3 transcription was analyzed by qPCR and normalized to β-actin. Results are triplicates from one of two independent experiments. Data are presented as mean ± SEM. d The sequence (860 bp) upstream of ORAI3 TSS was cloned into the PGL3-basic plasmid and transfected into HEK293T cells alone or with an IKAROS-expressing plasmid. Firefly luciferase activity relative to Renilla luciferase is shown. Data are presented as mean ± SEM from three independent experiments (n = 3). e ChIP assays of CD4+ T cells using anti-IKAROS antibodies and a primer set amplifying the indicated potential binding site. Data are presented as mean ± SEM from three independent experiments (n = 3). f Flow cytometry analysis of AA-induced Ca2+ influx in IKAROS siRNA smart pool- and control siRNA-transfected CD4+ naive T cells. Histograms are representative of two experiments. g Immunoblot analysis of AA-induced ERK phosphorylation in CD4+ naive T cells transfected with IKAROS siRNA smart pool; one of two experiments. Uncropped Western blots in Supplementary Fig. 10. h Flow cytometry analysis of AA-induced CD69 expression in purified naive CD4+ T cells transfected with IKAROS siRNA smart pool. Representative histograms and data from four experiments. Data in b, c were analyzed with one-way ANOVA followed by Tukey’s multiple comparison test with adjustment. Data in d, e, and h were analyzed with unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Fig 4: Arachidonic acid acts through ORAI3 to activate T cells.a Naive CD4+ T cells were transfected with ORAI3 siRNA smart pool or control siRNA; after 48 h, Ca2+ influx was monitored over time (210 s) by flow cytometry of Fura Red with 0.1 µM AA added after the first 30 s (left). Right, knockdown efficiency. b CD4+ naive T cells transduced with lentivirus expressing ORAI3 shRNA #1, ORAI3 shRNA #2, or control shRNA were assayed for p-ERK (Thr202/Tyr204) at 0.5, 1, or 4 h after 0.3 µM AA stimulation. Uncropped Western blots in Supplementary Fig. 9. c Representative histograms of constitutive CD69 expression in Jurkat cells stably transduced with ORAI3 shRNA or control shRNA (left). Results of CD69 expression in control and ORAI3-silenced Jurkat cells from five experiments (middle, *** = p < 0.0001). The efficiency of ORAI3 silencing was confirmed by immunoblotting (right). d Immunoblot analysis of NUR77 expression in Jurkat T cell transduced with ORAI3 shRNA #1, ORAI3 shRNA #2, or control shRNA. Data represent one of two independent experiments. e Flow cytometry of AA-induced Ca2+ influx as determined from the ratio of shifts in Fura Red (at 406 and 532 nm) fluorescence in Jurkat T cells stably overexpressing ORAI3. Representative tracing of Fura Red ratios (left) and the peak calcium influxes from three experiments are shown (right). f Immunoblot of ERK phosphorylated at Thr202/Tyr204 from ORAI3-overexpressing Jurkat cells at indicated time points (0, 0.5, 1, and 4 h) after 0.3 µM AA stimulation (left) and peak p-ERK responses from three experiments are shown (right). g Flow cytometry of constitutive CD69 expression by ORAI3-overexpressing and vector control Jurkat T cells (left). CD69 expression summarized from five experiments (middle). Immunoblots documenting ORAI3 overexpression (right). Uncropped Western blots of c, d, f, g in Supplementary Fig. 10. Data are presented as mean ± SEM (a, c, e, f, g). Results in a, b, e, and f are each representative of three experiments. Data were analyzed by paired two-tailed Student’s t test (a), by one-way ANOVA followed by Tukey’s multiple comparison test (c), or by unpaired two-tailed Student’s t test (e–g). Source data are provided as a Source Data file.