Fig. 5: MS-based identification of ARF-derived HLA-presented peptides and polyfunctional T cells responses.

a List of CDS- and ARF-derived peptides identified in HIV_NL4-3-infected primary CD4⁺ T cells. b T cell responses specific to YSL-ARF-derived peptide in PBMCs of PLWH. IFNγ responses of donors EC-1 and EC-3 upon YSL-peptide stimulation, on day 12. POOL (+), YSL-I, and YSL-II correspond to cells loaded with a pool of peptides containing YSL-I and YSL-II peptides, or only with YSL-I and YSL-II peptides, respectively. POOL (−) (white bar): negative control using cells loaded with DMSO-containing medium. Data were presented as mean values of technical triplicates ± SD. Positive response: number of rough IFNγ⁺ spots per well >20 and two times higher than the negative control. SAT: saturated well. c Functionality of YSL-specific CD4⁺ T cell response from EC-3 donor. Following a 12-day in vitro expansion, ICS was performed for MIP-1β, IFNγ, IL2, CD107a, and TNFα after stimulation with YSL-I (top panel) or an Env-derived peptide (MHC-II_env01, bottom panel), used as a positive control. FACS gatings were performed as in Fig. 4 and as shown in Supplementary Fig. 13. Right panel, the left pie charts illustrate the percentage of activated (yellow) and non-activated (blue) T cells following YSL- or Env-peptide stimulation, upper and lower lines, respectively. The polyfunctional profile of activated YSL- or Env-specific T cells are shown in the right pie charts. Boolean analysis was conducted to determine the percentage of cells secreting 1, 2, 3, 4, or 5 cytokines simultaneously (red, green, purple, blue, and orange, respectively). Polyfunctional profiles of YSL-I- (d) and YSL-II-specific (e) CD4⁺ T cell responses. As in (c), Left pie charts: percentage of activated (yellow) and non-activated (blue) T cells following peptide stimulation. Right pie charts: polyfunctionality of activated T cells, for all responding donors (codes are indicated on the top). Boolean analysis determined the percentage of cells secreting one or five cytokines simultaneously. The same FACS gating as in (c). f Heatmap summarizing YSL-I and YSL-II peptide-specific CD4⁺ T cell responses in all tested PLWH. NA not applicable, NS tested but not significant.