Journal: Nature Communications

Article Title: Unveiling conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling

doi: 10.1038/s41467-025-56773-2

Figure Lengend Snippet: 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. 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 ).

Article Snippet: Cells were then stained for 30 min on ice with a mix of cell surface antibodies: CD3-PE, CD8-SuperBright600 (Invitrogen), and CD4-AF750 (Beckman Coulter).

Techniques: In Vitro, Comparison, MANN-WHITNEY

Journal: Nature Communications

Article Title: Unveiling conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling

doi: 10.1038/s41467-025-56773-2

Figure Lengend Snippet: 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. and as shown in Supplementary Fig. . 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.

Article Snippet: Cells were then stained for 30 min on ice with a mix of cell surface antibodies: CD3-PE, CD8-SuperBright600 (Invitrogen), and CD4-AF750 (Beckman Coulter).

Techniques: Derivative Assay, Infection, Negative Control, In Vitro, Positive Control