Fig. 3

Peripheral Ki67⁺ Treg cells predict efficacy and disease relapse after anti-PD-1 therapy. a Heatmap showing phenotype of cells selected by CellCnn (n = 15,132). b T-SNE plots showing the five CD4⁺ T cell clusters we identified (left) and cells selected by CellCnn which were mainly located within the CD4⁺ Treg cluster (right). Red box indicates the CD4⁺ Treg cluster. c Box plot showing a higher frequency of cells discovered by CellCnn in patients from the relapse group than those from the relapse-free group. d Heatmaps showing comparisons of the median expression of functional markers in Treg cells from patients in the relapse-free group and the relapse group at baseline (n = 24). Bars at the top of the heatmaps represent individual samples from relapse-free group (green) and relapse group (pink). **P < 0.01, (marked in red); ns not significant. e Representative flow plots and box plots showing the frequency of Treg cells in CD4⁺ T cells (left) and Ki67⁺ Treg cells in Treg cells (right) between the relapse group and the relapse-free group compared in the aPD1-CRT arm (n = 51). f Box plots showing the frequency of Ki67⁺ Treg cells in total T cells between the relapse group and the relapse-free group compared in the CRT arm and in the aPD1-CRT arm, respectively (n = 69 and 51, respectively). P values were calculated by two-sided Wilcoxon tests in (c–f). g Kaplan–Meier curves comparing EFS improvements for anti-PD-1 plus CRT versus CRT alone in patients stratified by the median frequency of peripheral Ki67⁺ Treg cells in total T cells at baseline. P value was calculated by two-sided log-rank test. h Dynamic change of the frequency of Ki67⁺ Tregs in total T cells during treatment compared to baseline levels (in relapse-free group, n = 12 at t1−t3 and n = 10 at t4; in relapse group, n = 12 at t1−t3 and n = 8 at t4). P values were calculated by two-sided Wilcoxon signed-rank tests. Box plots represent the median (center line), the IQR (box), and the farthest data point within a maximum of 1.5 × IQR (whiskers)