Variant ITPR3 proteins impair Ca ²⁺ flux in T cells. (A–C) In primary T cells: Ca²⁺ flux after stimulation with (A) anti-CD3 plus F(ab′)2 and (B) thapsigargin (Thap) in Ca²⁺-free media and after addition of CaCl₂. The left panels show representative examples of Ca²⁺ flux in primary T cells from P4 (blue) versus one HD (grey). The right panels show the ratio between the peak (100–150 s) versus baseline (0–30 s) in P2 (pre-HCT), P4 (untransplanted), and P5 (pre-HCT) versus 10 HDs. Independent replicates are included for two patients (n = 2 for P2 and P4) and three HDs. The lines show the median and the interquartile range. Triangles and circles distinguish between frozen and fresh samples, respectively (the HD indicated with an open triangle is the mother of P2, who carries the R1850Q polymorphism). *P < 0.05, Mann–Whitney U test. (C) Baseline cytosolic Ca²⁺ levels in primary patient T cells, expressed as fold change over HD. Data represent 12 HD and 12 patient samples (P2, P4, and P5), pooled across four independent experiments; **P <0.01, paired T test. (D–E) In CTLs: Ca²⁺ flux after stimulation with (D) anti-CD3/F(ab′)2 (left panel) and thapsigargin (right panel) in Ca²⁺-free media and after addition of CaCl₂ in CTLs from P2 and P4 (blue) versus one HD (grey). This is representative of four independent experiments. (E) Ca²⁺ flux after stimulation with ionomycin in Ca²⁺-free media in CTLs from P2 and P4 (light blue) versus one HD; representative of two independent experiments. **P < 0.01, Welch’s T test. (F–H) In Jurkat T cell lines: (F) western blots for ITPR1, ITPR2, ITPR3, and GAPDH from protein extracts from WT Jurkat T cells, ITPR3 KO Jurkat T cells, and stably transduced ITPR3 KO Jurkat T cells expressing WT ITPR3 or the A196T, I2506N, and R2524C proteins; representative of two independent experiments. (G–H) The left panels show representative Ca²⁺ flux after stimulation with (G) anti-CD3 and F(ab’)2 or (H) thapsigargin in one ITPR3 KO Jurkat T cell clone (light grey) and in stably transduced ITPR3 KO Jurkat T cells (derived from the same ITPR3 KO clone) expressing WT ITPR3 (dark grey) or the A196T, I2506N, and R2524C protein variants (shades of blue). The right panel shows the ratio between the peak versus baseline in the ITPR3 KO clone and in transduced KO cells expressing WT (three clones) or variant ITPR3 proteins (three clones for A196T, two clones for I2506N, and three clones for R2524C) in five independent experiments. ns: non-significant, *P < 0.05, **P = 0.01, and ***P < 0.01, Mann–Whitney U test. Source data are available for this figure: SourceData F4.