Ca ²⁺ flux in patient primary skin fibroblasts, primary T cells, and gene-edited cell lines expressing patient protein variants. (A) RT-qPCR for ITPR1, ITPR2, and ITPR3 transcripts in primary skin fibroblasts obtained from three HDs and three patients (P1, P2, and P3). Results shown are relative to beta-actin (ACTB) expression are representative of two independent experiments and are not statistically significant via multiple unpaired t tests with Holm–Sidak correction for multiple comparisons. (B) Western blot for ITPR3 and GAPDH in primary skin fibroblasts from three HDs and three patients (P1, P2, and P3); representative of two independent experiments. (C–J) The ratiometric Ca²⁺ indicator Indo-1 was used to measure cytoplasmic Ca²⁺ concentration. (C) Ca²⁺ flux after stimulation with anti-CD3 plus F(ab′)2 in thawed pre-HCT primary T cells from P1 (blue) and from a HD (grey) in Ca²⁺-free media and upon addition of CaCl₂. This single, independent experiment was analyzed separately from the data shown in Fig. 4 A due to poor cell viability. (D) Ca²⁺ flux after stimulation with anti-CD3/F(ab′)2 in primary T cells from P4 (blue) and from a HD (grey) in Ca²⁺-containing media and upon addition of ionomycin at 240 s; representative of three independent experiments; not significant, Mann–Whitney U test. (E) Ca²⁺ flux after stimulation with ionomycin in Ca²⁺-free media and upon addition of CaCl₂ in CTLs from P2 and P4 (light blue) versus one HD; representative of two independent experiments. (F) Ca²⁺ flux in fibroblasts from four HDs (grey) versus P1, P2, and P3 (blue) stimulated with ATP; traces are shown in I and graph of the AUC of the peaks in II. Graphs represent the mean of three separate experiments and error bars indicate SEM. (G) Ca²⁺ flux in WT (red) and ITPR3 KO Jurkat T cells (light grey), and ITPR3 KO cells stably transduced with WT ITPR3 (dark grey) after stimulation with (I) anti-CD3/F(ab′)2 or (II) thapsigargin in Ca²⁺-free media and upon addition of CaCl₂; representative of two independent experiments. (H) Ca²⁺ flux upon addition of CaCl₂ in Ca²⁺-free media in (H) Jurkat ITPR3KO T cells expressing WT or variant ITPR3 proteins; (I) shows representative plots of ITPR3 WT (left) and variant expressing (right) cells either pre-stimulated with anti-CD3 and addition of F(ab′)2 (at timepoint A) followed by addition of CaCl₂ (at timepoint B; darker line), or CaCl₂ alone (at timepoint A; paler line); (II) shows a comparison of Ca²⁺ influx upon addition of CaCl₂ alone (i.e., without agonist stimulation) in cells expressing WT ITPR3 or the various ITPR3 variant proteins; representative of two independent experiments. (I) Ca²⁺ flux upon addition of CaCl₂ in Ca²⁺-free media in CTLs from P2, P4, and one HD; representative of two independent experiments. (J) Ca²⁺ flux in ex vivo differentiated T cells from P5 and from one HD stimulated with anti-CD3/F(ab′)2 in (I) single positive CD4⁺ and (II) single positive CD8⁺ cells; representative of two independent experiments. Source data are available for this figure: SourceData FS1.