Figure 1.

Similar in situ cell density of T_FH cells in viremic and cART PLWH LNs. (A) Representative examples of CD20 (red), CD4 (green), DAPI (blue), CD57 (gray) and PD1 (cyan) staining pattern from tonsil and control viremic and cART HIV LNs (scale bar: 30 μm). (B) The Histo-cytometry gating scheme used for the identification of T_FH cell subsets based on their expression of PD1 and CD57 is shown. F and EF areas were manually identified based on the density of the CD20 signal, and the relevant cell counts were extracted for specific tissue localities. (C) Histo-cytometry-identified PD1^hiCD57^hi T_FH cells were backgated to the original image using Imaris software. Each sphere represents one cell. (D) Bar graph (left) demonstrating the cell density (normalized per mm² counts) of follicular PD1^hi T_FH cells in tonsils (N = 5), control LNs (N = 5), viremic HIV (N = 12) and cART HIV LNs (N = 20). Viremic and cART were further subdivided based on their T_FH counts (HIV vir-high T_FH (N = 6), HIV vir-low-T_FH (N = 6), HIV cART-high-T_FH (N = 6) and HIV cART-low-T_FH (N = 14)). Each symbol represents one donor. The p values were calculated using the Mann–Whitney test and were corrected using FDR correction with q = 0.05 (Supplementary Table S2). Dot graph (right) shows the distribution of PD1^hi cell densities among the samples. Each symbol represents a follicle. (E) Bar graph demonstrating the normalized per mm² numbers of follicular PD1^hiCD57^hi T_FH cells in the same groups/subgroups of the samples. (F) Linear regression analysis to address the correlation between PD1^hi and PD1^hiCD57^hi absolute follicular counts between different subgroups. Each symbol represents a follicle. R-squared and p-values are displayed on graphs. (G) Linear regression analysis (lower) to address the correlation between normalized follicular PD1^hi counts and blood viral load—pVL. Dot graph (upper) showing the pVL differences (as a log scale) between the viremic HIV subgroups.