Extended Data Fig. 6: SIV-specific CD8⁺ T cell responses are not enhanced by blockade of PD-1 and/or CTLA-4 during long-term ART.

In 5 Mamu-A^*01⁺, ICB-treated RMs, the frequency of SIV-specific cells, as determined via anti-Gag-CM9 tetramer staining with flow cytometry, were measured in (a) LN and (b) PBMC memory CD8⁺ T cells at treatment baseline (d-29 p.t.) and following ICB (d21 or 28 p.t.). The number of interferon gamma (IFN-γ) spot forming units (SFU) per 10⁶ mononuclear cells were quantified by ELISpot upon SIV-Gag stimulation in (c) PBMCs as a fold-change (d21 p.t. relative to d-29 p.t.) and (d) cross-sectionally in LN (d21 p.t). Points represent unique animals, as indicated by shape and fill, as an average of three replicates with DMSO background subtracted. All RMs are color-coded and grouped based on ICB therapy as follows with population sizes as indicated for all ELISpot analyses: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=5), pink; combined blockade (n=7), red; and BsAb (n=8), purple. From ex vivo SIV-Gag-stimulated PBMCs with unstimulated background subtracted, the distribution of cytokine co-expression was determined via flow cytometry in memory (e) CD4⁺ and (f) CD8⁺ T cells before (d-29 p.t.) and during (d21 p.t.; indicated at left) ICB administration (indicated above). Rainbow-colored inner pie wedges represent each Boolean combination of cytokine co-expression (annotated at far right); whereas, the stacked, outer concentric rings overlap with pie wedges that are positive for that cytokine (indicated at middle right): IFN-γ⁺, red; IL-2⁺, orange; TNFα⁺, green; and CD107a⁺, purple. Population sizes for all cytokine analyses are as follows: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=6), pink; combined blockade (n=7), red; and BsAb (n=8), purple. Averaged data are presented as the mean ± s.e.m., and were analyzed with a two-sided (c,d) Mann-Whitney U test or (e,f) a Permutation test.