Extended Data Fig. 9: Single-cell RNA sequencing analysis on T cells from baseline and progressive tumors.

a, Preparation procedure for single-cell sequencing on neoantigen-specific T cells from PT03 baseline and progressive tumors. CD103⁺ T cells were enriched using magnetic sorting from tumor digest. CD3⁺103⁺ T cells were then sorted and encapsulated for single-cell RNA-seq paired with TCR VDJ seq. To compare transcriptomic features of T cells reactive to antigens at baseline vs progression, tumor-reactive TCRs were determined using both FEST and tetramer-sorting methods. b, UMAP analysis shows T cell functional clusters for baseline (left) and progressive (right) tumor CD103⁺T cells. Overall 2781 T cells from baseline tumor and 6985 T cells from progressive tumor passed QC criteria for transcriptomic analysis. c, Distribution of T cell clones reactive to baseline (left) vs progressive tumor neoAgs (right). d, Violin plots comparing transcriptomic expression of markers linked to memory (KLF2, LEF1, IL7R), exhausted (CD69, ITGAE, LAG3, TIGIT) and effector (GZMB) T cells reactive to antigens at baseline vs progression. Baseline tumor neoAgs include those five only expressed in the baseline tumor: ASPM^I1250S, NUP133^C119Y, MEX3D^E477Q, SMAD4^D537E, BIRC6^V1860I. Progressive tumor neoAgs are the two retained neoAgs: STIP1^S45F, TUBB^T238I.