Cytolytic CD4+ T cells express high levels of T-bet and Eomes in blood.

<p><b>(A)</b> 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>(B)</b> Frequency of perforin+ CD4+ T cells within the T-bet<sup>hi</sup>Eomes+ and T-bet<sup>dim/-</sup> population (left) and T-bet<sup>hi</sup>Eomes+ within the perforin+ or perforin- population for HIV-infected and–uninfected subjects. <b>(C)</b> Correlation between the frequency of perforin+ and T-bet<sup>hi</sup> CD4+ T cells. <b>(D)</b> Imagestream analysis on T-bet<sup>hi</sup> and T-bet<sup>dim</sup> 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-bet<sup>hi</sup> and T-bet<sup>dim</sup> CD4+ T cells are shown in the before-after graphs. <b>(E)</b> 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. <b>(F)</b> Flow plots of MIP-1α production using media (NC) and aCD3-CD28 stimulations for T-bet<sup>hi</sup> and Eomes+ CD4+ T cells, as well as correlation between the frequency of T-bet<sup>hi</sup>Eomes+ 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; ***<i>P</i> < 0.001. A non-parametric Spearman test was used for the correlations analysis. All data are derived from the North-American cohort.</p>