Fig. 4: ARF-derived peptide (ARFP)-specific T cell responses are mediated by CD4⁺ and CD8⁺ T cells exhibiting polyfunctional cytokine profiles.

a Representative gating scheme for the identification of ARFP-specific T cell responses. Upper panel, gatings for G02-ARFP-specific CD4⁺ (left) and CD8⁺ (right) T cell responses are shown for the EC-3 PLWH. Lower panel, gatings for Env01-specific T cell responses as for G02. Env01 epitope is a known immunodominant peptide from HIV-Env. CD8⁺ CD4^- T cell populations were pre-gated on CD8⁺ CD3⁺ T cells, lived cells, and doublets were excluded. Gates for each cytokine/chemokine were set based on the negative controls (cells loaded with DMSO-containing medium, NS: Not stimulated) NS_ARF and NS_CDS for the assessment of G02-ARFP- and Env01-specific T cell responses, respectively. A figure exemplifying the gating strategy is provided in Supplementary Fig. 13.b Heatmap of ARFP recognized by CD4⁺ or CD8⁺ T cells from PLWH summarizing the ICS assays. Yellow and green rectangles are used to indicate CD4⁺ and both CD4⁺ and CD8⁺ T cell positive responses in ICS, as defined in (a), respectively. NA not applicable (i.e., not tested), NS not significant. c Percentage of CD4⁺ or CD8⁺ T cell responses among ARFP-specific T cell responses after in vitro stimulation with ARFP at the cohort level. Proportion of ARFP- and CDS-specific CD4⁺CD3⁺ (d) or CD8⁺CD3⁺ (e) T cells secreting 1 to 5 cytokines simultaneously. The numbers on the top indicate the percentage of polyfunctional T cells secreting at least three cytokines. ARFP-specific T cell responses from all tested donors are combined. f Comparison of the percentage of polyfunctional T cells secreting at least three cytokines among ARFP- and CDS-specific CD4⁺CD3⁺ T cells in the EC and ART patient groups. g Same as (f) among ARFP- and CDS-specific CD8⁺CD3⁺ T cells in the EC group. Each dot represents one peptide-specific T cell response. The lines correspond to the median responses. A two-sided Mann–Whitney test with Dunn’s comparison was applied. ns: not significant. Data for YSL-I- and YSL-II-specific T cell responses are integrated in the graphs from (d–g).