Identification and characterization of NEMO revertant T cells in patient 2. (A) Intracellular expression of NEMO in various PBMC lineages from a healthy adult control and patient 2 were evaluated by flow cytometry. For the patient, results of the analyses performed at 2 months and 23 months are shown. Solid lines indicate staining with the anti-NEMO mAb, and dotted lines indicate the isotype control. (B) Time-course variations in the absolute count of NEMO^normal and NEMO^low T cells in patient 2. M indicates age in months. (C) TCR-Vβ repertoire analysis of the patient's CD4⁺ and CD8⁺ T cells. PBMCs from the patient and a healthy adult control were stained for the TCR-Vβ panel, CD4, CD8, and NEMO, and analyzed by flow cytometry. (D) Phenotype analysis of T cells in patient 2. PBMCs from the patient and a control were stained for the expression of NEMO, CCR7, CD45RA, and CD4 or CD8. Data shown were gated on NEMO^normal or NEMO^low CD4⁺ or CD8⁺ cells. (E-F) Cytokine production from NEMO^normal and NEMO^low T cells. PBMCs from the patient and a control were stimulated with PMA and ionomycin for 6 hours and stained for intracellular (E) IFN-γ or (F) TNF-α along with NEMO. Cells shown are gated on the CD3⁺ population.