CD8 T cells from HIV-seronegative donors. PBMCs were labeled with CD8/CD45RA/CCR7/CD27/CD28 antibodies. CD45RA⁺ CCR7⁺ naive (T_N), CD45RA⁻CCR7⁺ central memory (T_CM), and 3 dominant T_EM subpopulations, CD27⁺CD28⁺ (DP), CD27⁺CD28⁻ (CD27-single positive [27SP]), and CD27⁻CD28⁻ (DN) cells were sorted as single cells, and each individual cell was tested for the expression of 13 different mRNAs encoding effector molecules, 3 transcription factors, and Cd3e, which was used to evaluate plating efficiency. (A) Phenotypes and gates used to select different CD8 subpopulations in one HIV-seronegative donor. In some HIV-seronegative donors, a minority of CD28SP cells was found, but this subset was not studied because HIV-specific cells did not express this phenotype (data not shown). (B) In each donor, we studied 90 individual cells from each of the CD8 subpopulations. Results show expression frequencies and are the mean ± SD of 8 donors. Increased frequencies in DP to DN transition: Ifng, P = .005; Prf1, P = .01; Gzma, P = .001; Gzmb, P < .0001; Fasl, P = .005; and Tbx21, P < .002. Differences in Eomes expression between: DP and 27SP, P = .023; 27SP and DN, P = .001.