Supplementary Figure 5: T_FH,T_H17 and T_H22 cytokine expression by HIV-specific CD4⁺ T cells compared to CMV-specific CD4⁺ T cells in CPs and ECs and in the absence of CD40 blockade and CD40L pre-gating.

(a) Comparison of CXCL13 protein expression in the supernatant of CD8-depleted PBMCs of CPs versus ECs by two-tailed Mann-Whitney test. The cells were stimulated with a Gag-peptide pool for 48 h and CXCL13 protein concentration was measured by ELISA. Bars represent median with +/- interquartile range (n= 8 CPs, 7 ECs). (b,c) Statistical comparison of frequency of PD-1/TIGIT subpopulations in (b) CXCL13 mRNA⁺ or (c) IL21 mRNA⁺ HIV-specific CD4⁺ T cells in CPs compared to ECs by two-tailed Mann-Whitney test (b,c; n = 6 CPs, 5 ECs). Bars represent median with +/- interquartile range. (d) Correlation of quantification of p24-specific antibodies in plasma by Biolayer Interferometry (BLI) binding analysis or ELISA (two-tailed Spearman correlation test, n = 12 CPs, 15 ECs). Semi-quantitive scale for anti-p24 antibody binding index: -, -/+, +, ++, +++ symbols correspond to peak values at <0.2, 0.2-0.3 0.3-0.5, 0.5-0.8, >0.8 nm, respectively (two-tailed Spearman correlation test, n=12 CPs, 15 ECs). (e,f) Correlation of weighted Mean Fluorescence Intensity for IL22 and IL17F detected by RNA-Flow-FISH cytometry to protein concentration detected by a magnetic bead-based assay (Luminex) (n= 6 CPs, 6 ECs) (two-tailed Spearman correlation). (g) Detection of IL-17A protein in the supernatant of CD8-depleted PBMCs 48 h after stimulation with a Gag-peptide pool by Luminex. Bars represent median with interquartile range and statistical comparison was performed by two-tailed Mann-Whitney test (n = 8 CPs, 8 ECs). (h) SPICE analysis of phenotyping of IL22 mRNA⁺ and IL17F mRNA⁺ antigen-specific CD4⁺ T cells from the CP group. Pie charts represent median percentages of CCR6/CXCR3 subpopulations (n= 6 CPs, 6 ECs). (i,j) Correlation of IL22 and IL17F mRNA levels assessed by RT-qPCR (Fluidigm) with the protein expression of the activation markers HLA-DR and CD38 on total unstimulated CD8⁺ T cells by two-tailed Spearman correlation (n = 11 CPs, 6 VCs, 12 ECs). (k,l) Correlation of bacterial diversity (Shannon Entropy) with the protein expression of the activation markers HLA-DR and CD38 on total unstimulated CD4⁺ and CD8⁺ T cells by two-tailed Spearman correlation (n = 8 CPs, 6 ECs). (m) Hierarchical clustering of Morisita-Horn dissimilarity indexes illustrates that samples largely segregate by disease status based on Genus TPM values (n= 12 CPs, 8 ECs).