Phenotypic, cytolytic and transcriptional differences between LN and blood CD4+ T cells in HIV-infected and -uninfected individuals.

<p><b>(A)</b> Flow cytometry plots (HIV-infected CP) and scatter plots for naïve and memory subsets of LN and peripheral blood (PB) CD4+ T cells in HIV-infected and -uninfected subjects. <b>(B)</b> Flow cytometry plots (HIV-infected CP) showing the lack of T-bet^hiEomes+ CD4+ T cells in LNs. Corresponding scatter plots demonstrating the frequency of T-bet^hi cells of memory (non-naïve) CD4+ T cells (top) and frequency of Eomes+ cells of T-bet^hi CD4+ T cells (bottom) for matched LN and PB. <b>(C)</b> Flow plots (HIV-infected CP) showing the lack of Granzyme B+perforin+ CD4+ T cells in LNs and scatter plots with the frequency of LN and PB perforin+ cells of memory CD4+ T cells (top). Frequencies of Granzyme B+ cells of perforin+ CD4+ T cells (bottom) for matched LN and PB. <b>(D)</b> Flow plots (HIV-infected CP) and scatter plots showing the distribution of CD27+ cells within the Granzyme B+ CD4+ T cell compartment for matched LN and PB. <b>(E)</b> The distribution of different populations in the tSNE space is based on 30.000 live CD4+ T cells that were merged from LN and PB from a HIV-infected CP with detectable levels of cytolytic cells in the PB and LN Tfh cells. The tSNE clustering is based on CD45RO, CD27, CCR7, T-bet, Eomes, Granzyme B, perforin, CXCR5 and PD-1 expression on gated bulk CD4+ T cells. The naïve cluster (green) is based on high CCR7 and low CD45RO intensity; the Tfh cluster (red) on high intensity of PD-1 and CXCR5; and the effector cluster (orange) on high T-bet and perforin expression intensity. After separating out the merged LN and PB single CD4+ T cell data, a lack of Tfh cells was apparent in PB and effector CD4+ T cells in the LN (lower right tSNE plots). Median and IQR are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between two unmatched groups, and Wilcoxon matched-pairs single rank tests between matched samples; *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001. All data-points are derived from the North-American and Mexico cohort.