Fig. 4: Frequencies of ISG-expressing CD8⁺ T cells in peripheral blood increase within 24 hours of anti-PD-1 therapy.

a, Cells annotated as ‘CD8⁺ T cells’ were extracted from scRNA-seq data on PBMCs (Fig. 2g; n = 30; number of samples per timepoint listed in Supplementary Data 1, D3). Four major clusters were identified. b, Genes from each bulk transcriptomic module were extracted, and Seurat’s AddModuleScore function was used to calculate module scores per cluster. Dot plots depict the proportion of cells (dot size) and the average module expression (color intensity). Cluster 0 contained the highest frequency of cells and the greatest expression of WNT signaling, TCF7 and CTNNB1 target genes and was annotated as the naive subset using the SingleR package (Monaco et al.⁶², celldex database). Clusters 1 and 2 were annotated as central and effector memory T cells (T_CM and T_EM), respectively, and cluster 3 as the ISG-expressing subset. c, Feature plots of IFNG, TNF, GZMB and PRF1 show that cells expressing these effector genes (red) are enriched in clusters 1 and 3 (T_EM and ISG subsets). d, The top panels show relative changes in the frequency of each CD8⁺ T cell cluster at C01D02 and C01D08 compared to C01D01. The bottom heatmap depicts changes in module scores, represented as the signed log(P) value (sign(fold change) × log(P value)); red indicates increased expression, and blue indicates decreased expression. e, Flow cytometry gating of CD8⁺ T cells (T_EM: CD45RA⁻CD27⁻; T_EMRA: CD45RA⁺CD27⁻) revealed significant decreases in T_CM/T_TM and increases in T_EMRA frequencies after therapy. f, HIV-specific CD8⁺ T cell proliferation was quantified by IFNγ and Ki-67 co-expression in PBMCs stimulated with an HIV Gag peptide pool. Representative gating is shown (top), and frequencies of IFNγ⁺Ki-67⁺ cells (of total CD8⁺ T cells) are plotted below, showing significant increases at C01D02. g, Genes upregulated in effector and ISG CD8⁺ T cell clusters (versus other CD8⁺ clusters) were analyzed using NicheNet to identify upstream regulators, restricted to cytokines measured in plasma. Prioritized ligands (those most correlated with target gene expression) are listed, and their regulatory potential, based on NicheNet’s reference database, is visualized in the heatmap (purple indicates higher potential). h, Frequencies of clusters 1 (T_EM) and 3 (ISG) among total CD8⁺ T cells were inversely correlated with plasma TGFβ2 and TGFβ1 concentrations, respectively (ρ and P values shown; Spearman’s correlation). i, Genes downregulated in effector and ISG CD8⁺ T cell clusters (versus other CD8⁺ clusters) were analyzed using NicheNet as in g. Prioritized ligands for downregulated targets are listed, and their regulatory potential is shown (purple indicates higher potential). Details of sample numbers for each analysis and timepoint are provided in Supplementary Data 1, D3. For d–f, Wilcoxon matched-pairs signed-rank tests were used to assess differences from C01D01. In d, *P < 0.05 and **P < 0.01. In h, Spearman’s correlation was used. Unless otherwise indicated, all statistical tests were two-sided, with multiple comparison adjustments applied where specified; otherwise, exact nominal P values are shown in the figures. Avg Exp, average expression; freq., frequency; NS, not significant.