T cells with heterozygous ITPR3 variants are impaired in their ability to express NF-κB and NFAT-dependent genes, proliferate, and upregulate metabolic enzymes in response to stimulation. (A) Graphs showing the percentage of CD25⁺ cells in CD4⁺ (left) and CD8⁺ T cells (right) stimulated with anti-CD3/CD28 compared with unstimulated for P2, P4, and two HDs across two independent experiments. (B) Nuclear translocation of NFAT after TCR engagement in CTLs from P2, P4, and two HDs in three independent experiments. The left panel shows representative images of (I) P185 target cells (yellow) and stimulated CD8⁺ CTLs (purple), (II–IV) intracellular staining for NFAT (blue) and nuclear staining with DAPI (red), and (V) a composite image illustrating the mildly reduced nuclear NFAT translocation in P4 compared to HD; scale bar = 5 μm. The right panels show the ratios between nuclear NFAT and cytoplasmic NFAT measured in MFI in unstimulated and stimulated CTLs from P2, P4 and the HDs in all three experiments; ns, non-significant, **P <0.01 and ***P <0.001, one-way ANOVA with Šídàk’s multiple comparisons test. (C) Genes activated by NF-κB and NFAT are downregulated in patient T cells compared with HD following anti-CD3/CD28 stimulation for 4 or 12 h (I) Graph showing normalized enrichment scores (NES) for NF-κB and NFAT transcription factor (TF) binding motifs within 5 Kb of the transcriptional start sites of genes significantly upregulated in HD versus patient T cells after 4 h of stimulation; filled symbols denote NF-κB motif analysis: square=bergman_dif_Rel, circle=homer_GGGGGAATCCCC_NFkB-p50_p52, triangle=transfac_pro_M01223; empty symbols denote NFAT motif analysis: square=taipale_NFATC1_full_NATGGAAANWWWWTTTYCMN_repr, circle=taipale_NFATC1_full_TTTTCCATGGAAAA_repr, triangle=cisbp_M5658. (II and III) Heatmaps showing expression level of genes significantly upregulated in HD versus patient T cells following stimulation that contain TF binding motifs for (II) NF-κB and (III) NFAT. (D) Bar chart showing proliferation of PBMCs from P1 (pre-HCT), P2 (pre-HCT), P4, and P5 (pre-HCT) versus HDs after stimulation with anti-CD3, PHA, or PMA and ionomycin. Proliferation was assessed by measuring the incorporation of titrated (³H) thymidine and quantified as counts per minute (cpm) over background across six independent experiments. Bars represent the median values and lines the interquartile range; *P < 0.05, **P < 0.01, t test with Sidak-Bonferonni correction for multiple comparisons. (E) Graphs showing expression (gMFI) of metabolic enzymes in CD4⁺ (left) and CD8⁺ T cells (right) stimulated with anti-CD3/CD28 compared with unstimulated cells for P2, P4, and two HDs across two independent experiments. (F) Gene set enrichment analyses of differentially expressed genes identified by RNA-sequencing of patients (P2, P4, and P5) and HD T cells (n = 3) stimulated with anti-CD3/28. Enrichment scores (ES) and P values are displayed for the following gene sets: (I) fatty acid metabolism, (II) tricyclic acid (TCA) enzyme complex, (III) mitochondrial biogenesis, (IV) mitochondrial genes, (V) protein import into the mitochondrial matrix, and (VI) bound by FOXP3.