T_reg suppression assay from fluorescent activated cell sorting purified T_reg cells in CD3G-mutated patients. (A) CD25^hiCD127^lo T_reg sort purification strategy from control (left) and patient (right) samples. Intranuclear FOXP3 staining was performed on CD25^loCD127^hi T_eff cells (solid blue) and CD25^hiCD127^lo T_reg cells (red line). (B) Representative plots of Cell Trace Violet dilution in experiments in which CD4⁺ CD25^low CD127^hi T_eff cells from controls were either left unstimulated (solid blue) or stimulated with anti-CD3 + anti-CD28 + anti-CD2, in the absence (solid gray) or presence (red line) of T_reg cells from controls (left) or from CD3G-mutated patients (right). (C) Cumulative percentage of dividing cells in T_reg suppression assay, normalized to the no T_reg group within each individual experiment. T_reg function was tested in 4 patients (P2-P5) and 4 controls. Results are expressed as mean ± standard deviation. *P < .05; **P < .01; ***P < .001.