Figure 3.

Preferential accumulation of FOXP3^hiCD4^hi T cells in cART HIV follicular areas. (A) Representative examples of CD20 (red), CD4 (green), DAPI (blue) and FOXP3 (yellow) staining pattern from tonsil and control viremic and cART HIV LNs (scale bar: 30 μm). (B) Histo-cytometry immunophenotyping gating strategy used for the sequential identification of FOXP3^hiCD4^hi T cells in follicular and extrafollicular areas in a control LN (C) Representative backgating of Histo-cytometry-identified FOXP3^hiCD4^hi T cells, using Imaris software. Each sphere represents one cell. (D) Bar graph (left) demonstrating the normalised per mm² counts of extrafollicular FOXP3^hiCD4^hi T cells in tonsils (N = 5), control LNs (N = 5), 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). (E) Bar graph showing the normalised per mm² counts of follicular FOXP3^hiCD4^hi T cells in the same tissue samples. Each symbol represents one donor. (F) Bar graph with connecting lines demonstrating the normalized per mm² counts of FOXP3^hiCD4^hi T cells in follicular and extrafollicular regions in each tissue analyzed. (G) Representative immunofluorescence images showing the distribution of FOXP3^hi (green) and CD20^hi (red) cells in follicles from one cART-high-T_FH and one cART-low-T_FH tissue. The corresponding digitalized (generated by the Python distance analysis script) images are shown too. (H) Bar graph showing the calculated G function values for follicular FOXP3^hiCD4^hi T cells in cART-high T_FH (blue circles) and cART-low T_FH (blue triangles) tissues (HIV cART-high T_FH (N = 22) and HIV cART-low T_FH (N = 64)). The p values were calculated using the Mann–Whitney test and were corrected using FDR correction with q = 0.05 (Supplementary Table S2).