Analysis of the XL-EDA-ID patient's T cells. (A) TCR Vβ repertoire analysis of the patient's CD4⁺ and CD8⁺ cells. The patient's PBMCs were stained for the TCR Vβ panel and CD4 or CD8 as described by Kobayashi et al.²⁹  The healthy controls consisted of children 1 to 10 years old (n = 10). The values are shown as mean ± SD. (B) Naive and memory phenotypes of the patient's T cells. PBMCs from the patient and control were stained with NEMO, CCR7, CD45RA, and CD4 or CD8. Cells gated on NEMO^low or NEMO^normal of CD4 or CD8 are shown. (C) Reduced IFN-γ production of NEMO^low T cells. The patient and control PBMCs were stimulated with PMA/ionomycin/monensin for 6 hours and stained for intracellular IFN-γ along with NEMO and CD4 or CD8. Cells gated on CD4 or CD8 are shown. (D) Proliferation assay of PBMCs stimulated by an anti-CD3 mAb plus an anti-CD28 mAb. PBMCs from the patient or healthy controls were stimulated for 48 hours with an anti-CD3 mAb, an anti-CD3 mAb plus the anti-CD28 mAb ± IL-2 (100 IU/mL), PHA, or PMA/ionomycin. Data are shown as mean ± SD. (E) Reduced production of IL-2 and IFN-γ by anti-CD3 mAb plus anti-CD28 mAb. PBMCs from the patient or healthy controls were stimulated for 48 hours by the anti-CD3 mAb, anti-CD3 mAb plus anti-CD28 mAb, or PMA/ionomycin, and the supernatants were harvested for ELISA assays.