Fig. 4: Neoadjuvant PD-1 blockade promotes systemic transcriptional reprogramming in MANA-specific T cells from a patient with complete pathologic response.

a, Longitudinal peripheral blood mononuclear cells were collected from complete pathologic responder MD01-005 (0% residual tumour) during treatment and in post-surgery follow up. Peripheral blood CD8⁺ T cells were sorted using FACS on the basis of expression of TCRVβ2, which corresponds to the MANA-specific CDR3 CASNKLGYQPQHF, as identified by the MANAFEST assay (Extended Data Fig. 2a). scRNA-seq–TCR-seq was performed on the sorted population from each time point. b, UMAP projection of expression profiles of 4,409 peripheral blood CD8⁺TCRVβ2⁺ T cells. c, Heat map of the top-5 differential genes, ranked by average fold change, for each T cell cluster. d, UMAP projection of MANA-specific T cells, identified via the CASNKLGYQPQHF or CASSLLENQPQHF TCRVβ CDR3, is shown for each time point. Clusters were coloured using the same colour scheme as in b. MANA-specific T cells are highlighted as triangles. e, The proportions of cells in each T cell cluster among all MANA-specific cells identified at week 2 and week 4 were compared (two-sided Fisher’s exact test and a two-sided test accounting for background cell proportion, both smaller than 0.021; Methods). f, Diffusion plot with RNA velocity for clusters in which MANA-specific T cells were detected. Cells were randomly downsampled to 100 cells (or all cells in the cluster if cluster size was smaller than 100 cells) for each cluster for visualization. g, Heat map of the top differential genes along the pseudotime trajectory from T_mem(3) to T_eff(3). h, Pseudotemporal expression of genes that significantly change along the pseudotime from T_mem(3) to T_eff(3). Red curves represent the mean temporal function estimates of the three samples from this individual (Methods). Cells with gene expression levels above the top one percentile were removed as outliers.