ALPS DNT lack T-bet expression. (A) Representative plots showing expression of Eomes and T-bet in ALPS and control DNT (gate: CD3⁺TCRαβ⁺CD4⁻CD8⁻), and in control CD8⁺TEMRA cells (gate: CD3⁺CD8⁺CCR7⁻CD45RO⁻). (B) T-bet messenger RNA was quantified by RT-PCR using sorted DNT, CD8⁺CD45RA⁺, and CD8⁺CD45RA⁻ T-cell populations. CD8⁺CD45RA⁻ memory cells served as a positive control because the proportions of TEMRA cells were too low. Representative data from 1 of 4 analyzed ALPS-FAS patients are shown. (C) CD4⁺TEMRA, CD8⁺+TEMRA, and DNT were gated and analyzed for Eomes and T-bet expression in combination with CD28 to distinguish between “DNT-like” (CD28+) and “normal” (CD28⁻) TEMRA cells. (D) T-bet and Eomes levels were compared between ALPS DNT, control DNT, CD4⁺CD28⁺TEMRA, and CD8⁺CD28⁺TEMRA from 5 ALPS patients and CD8⁺TEMRA from 5 healthy donors. The great majority of control CD8⁺TEMRA were CD28⁻. MFI, mean fluorescence intensity; ns, not significant. **P < .01.