Cytolytic CD4+ T cells express high levels of T-bet and Eomes in blood.
(A) Representative flow cytometry plots of Granzyme B and perforin expression in CD4+ T cells for an HIV-infected and–uninfected subject. The distribution of Granzyme B+perforin+ (red) and Granzyme B-perforin- (blue) CD4+ T cells are shown for T-bet and Eomes expression. (B) Frequency of perforin+ CD4+ T cells within the T-bethiEomes+ and T-betdim/- population (left) and T-bethiEomes+ within the perforin+ or perforin- population for HIV-infected and–uninfected subjects. (C) Correlation between the frequency of perforin+ and T-bethi CD4+ T cells. (D) Imagestream analysis on T-bethi and T-betdim CD4+ T cells. Overlays of fluorescent channels for DAPI (nuclear) and T-bet, showing where in the cells T-bet are localized. The frequency of nuclear, nuclear/cytoplasmic and cytoplasmic localization for T-bethi and T-betdim CD4+ T cells are shown in the before-after graphs. (E) tSNE plots based on 30,000 live CD4+ T cells that were merged from three HIV-uninfected subjects with detectable cytolytic CD4+ T cells. The tSNE clustering is based on CD45RO, CD27, CCR7, T-bet, Eomes, Granzyme A, Granzyme B and perforin expression intensity. The red gate indicates the identified “effector” cluster with overlapped expression of cytolytic markers as well as T-bet and Eomes. (F) Flow plots of MIP-1α production using media (NC) and aCD3-CD28 stimulations for T-bethi and Eomes+ CD4+ T cells, as well as correlation between the frequency of T-bethiEomes+ and MIP-1α+ CD4+ T cells following aCD3-CD28 stimulations. Median and IQR are shown for all scatter plots and Mann-Whitney tests were performed to compare differences between groups; ***P < 0.001. A non-parametric Spearman test was used for the correlations analysis. All data are derived from the North-American cohort.