Altered immune cell frequencies and impaired cytokine production by T cells of ORAI1 mutant patient. (A) CD4/CD8 T cell ratio in PBMCs of HD, patient (Pat), his father (Fa), and mother (Mo). Data are from six independent experiments. P < 0.0001. (B) Flow cytometry analysis of CD27 and CD45RA expression on CD4⁺ T cells within PBMC of an HD control and the patient. (C) Flow cytometry analysis of CD127 and CD56 expression on NK cells within PBMC of an HD and the patient. (D) Flow cytometry analysis of IL-2 production and FOXP3 expression by CD4⁺ T cells within PBMC of an HD or the patient. Cells were stimulated with PMA/ionomycin for 5 h followed by intracellular antibody staining. (E) Flow cytometry analysis of intracellular FOXP3 and surface CD45RA expression in CD4⁺ T cells within PBMC of an HD or patient. (F and G), Cytokine production by in vitro–expanded T cells stimulated with PMA/ionomycin for 4 h. Representative flow cytometry plots (left) and quantification (right). (F) IL-2 production by CD4⁺ and CD8⁺ T cells. Data are from four independent experiments. P < 0.0001. (G) IFN-γ production by CD4⁺ and CD8⁺ T cells. Data are from three independent experiments. For CD4⁺ T cells: P < 0.0001. For CD8⁺ T cells: P = 0.0232; 0.0038; 0.0055. Statistical significance was calculated using ordinary one-way ANOVA. *P <0.05, **P <0.01, ***P <0.001.