Fig. 4

Pathological regression under anti-PD-1 blockade is associated with enhanced CD4⁺ T cell cytotoxic programming and reduced serum IL-6 levels. Box plot comparing the proportions of a cytotoxic CD4⁺ T cells and b TNFRSF9⁺ Tregs among different groups. c Branched trajectory of CD4⁺ T-cell state transition inferred by Monocle. d Trajectory plot showing the pseudotime curve of CD4⁺ T cells. e Comparison of baseline serum cytokine profiles in non-pCR samples compared to pCR samples following PPCT (n = 5 per group). f Serum IL-6 levels before and after PPCT, comparing the pCR and non-pCR groups (n = 16 matched pairs per group). g CD4⁺ T cells were activated, treated with IL-6, and analyzed for cytotoxicity after 24 hours. h Proportion of cytotoxic CD4⁺ T cells after IL-6 exposure. i RNA expression levels of effector genes, characteristic markers, and transcription factors associated with cytotoxic CD4⁺ T cells after IL-6 treatment. j–n LLC tumor-bearing mice were treated with anti-PD-1 and/or anti-IL-6 antibodies (n = 5 per group). j Schematic representation of the treatment protocol. k Tumor growth curves. l Final tumor sizes. m Tumor-infiltrating cytotoxic CD4⁺ T cell proportions. n Statistical comparison of cytotoxic CD4⁺ T cell levels. P values in (a, b, k and n) were derived from one-way ANOVA, Tukey’s test; P values in (f) were derived from 2-sided paired Student’s t test; P values in (h, i) were derived from 2-sided unpaired Student’s t test; ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± SEM. BS treatment-naïve baseline; nCRT, neoadjuvant chemoradiotherapy; PPCT, preoperative pembrolizumab combined with chemoradiotherapy; PPCT-N, non-pCR following PPCT; PPCT-P, pCR following PPCT; pCR, pathological complete response