Extended Data Fig. 2

(A) Exemplary dot plot of surface PD-1 and IgG4 staining at baseline and post-therapy of one patient undergoing dual therapy (left); representative stainings of PD-1 and Ki-67 on CD8⁺ T cells (right) (B) CD8⁺ T cells from 10 patients (adju n = 2, mono n = 4, dual n = 4) analyzed in one batch were projected by UMAP embedding and clustered by FLOWSOM algorithm. As in Fig. 1d, 8 clusters were identified and frequencies of cluster 2-8 in patient samples across therapy regimens and time points are indicated. For (C–H), ICB therapy-treated patients within different treatment groups (adjuvant (adju) (n = 11), palliative (mono) (n = 15), and combination therapy with anti-CTLA-4 (n = 20)) were included. (C) Scatter plots depict frequencies of populations gated for different expression levels of perforin and granzyme B expression on PD-1⁺CD8⁺ T cells (Perforin^int adju P = 0.2695, mono P = 0.0246, dual P = 0.0032; GZMB^int adju P = 0.2061, mono P = 0.5995, dual P = 0.0002; Perforin^hi adju P = 0.1475, mono P = 0.3028, dual P = 0.1769; GZMB^hi adju P = 0.5195, mono P = 0.0189, dual P = 0.8480). (D) Frequencies of IL-2 (adju P = 0.7002, mono P = 0.8672, dual P = 0.6742) and CCL3/4 (adju P = 0.5566, mono P = 0.8672, dual P = 0.1991) produced by PD-1⁺CD8⁺ T cells after PMA/ ionomycin stimulation are indicated. (E) Scatter plots depicting frequencies of indicated markers within Ki-67⁺CD8⁺ T cells (Perforin^int adju P = 0.2783, mono P = 0.0215, dual P = 0.0037; GZMB^int adju P = 0.5771, mono P = 0.0706, dual P < 0.0001; Perforin^hi adju P = 0.7002, mono P = 0.4131, dual P = 0.3683; GZMB^hi adju P = 0.4648, mono P = 0.5245, dual P = 0.8408). (F) Cytokine production (IFN-γ adju P = 0.7002, mono P = 0.1395, dual P = 0.0010; TNF adju P = 0.0420, mono P = 0.6698, dual P = 0.0002; CCL3/4 adju P = 0.7646, mono P = 0.5830, dual P = 0.0441) and functional exhaustion score (FES) of Ki-67⁺CD8⁺ T cells across therapy regimens and time points (adju P = 0.4131, mono P = 0.2166, dual P < 0.0001). (G) Scatter plots depict frequencies of populations expressing indicated markers of PD-1^-CD8⁺ T cells (Ki-67: adju P = 0.1445, mono P = 0.9012, dual P = 0.0005; CD38^hi: adju P = 0.0830, mono P = 0.1102, dual P < 0.0001; CD39: adju P = 0.2783, mono P = 0.7615, dual P = 0.0006; LAG3: adju P = 0.4785, mono P = 0.3591, dual P = 0.0066; CD127: adju P = 0.6211, mono P = 0.5614, dual p = 0.0004) (H) Fold change of Ki-67, CD38^hi, CD39, LAG-3, CD127, IFN-γ, TNF, IL-2, GZMB^int, GZMB^hi, Perforin^int, Perforin^hi PD-1⁺CD8⁺ T cells (++) vs. PD-1^-CD8⁺ T cells (+-) after ICB therapy within different treatment groups. Where applicable, samples pre-therapy are represented in open circles and post-therapy in open squares in scatter plots. Bar and error bar represent median and 95% CI in scatter plots. Statistical significance was determined by two-sided paired Wilcoxon signed-rank test (B–G) or two-sided Kruskall-Wallis Test (H) (ns = non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).