Figure 3: Prospective prediction of neoantigen-reactive circulating CD8⁺ T cells patient PBL.

(A) Frequency of known neoantigen-reactive T cell clones in FACS-sorted T cell subsets from pt.4246 PBL. Fold change of the mean frequencies of neoantigen-reactive T cell clones between indicated groups is presented in parentheses. T-test p-value is shown between the groups. Each colored shape represents a different neoantigen TCR clone from pt.4246 PBL. (B) FACS-sorting enrichment gating of circulating CD8⁺ T cells from pt.3791 for scRNAseq. 3,290 CD39⁺CD103⁺ were sorted and mixed with 9500 bulk CD8⁺ T cells from PBL. (C) Clustering and projection of predicted neoantigen-reactive T cells from PBL, based on AUCell (red). (D) Frequency of TCR-transduced CD8⁺ cells expressing CD137 following co-culture with imDCs electroporated with patient’s TMGs (D) or mutated peptides for the corresponding TMG. (E-F) Deconvolution of TMG hits to identify specific neoantigens recognized by patient NeoTCR_PBL. (G) Summary back-projection of experimentally vetted NeoTCR_PBL cells on pt.3791 PBL UMAP. (H) Summarized mean AUC scores of ROC analysis comparing NeoTCR_PBL signature and published TIL gene-signatures for prediction of neoantigen-reactive T cells from three validation set samples (pt.3791, pt.4180, pt.4359). Random 500 gene set is shown as control (I) FACS-based enrichment and identification of neoantigen-reactive clones within circulating lymphocytes from PBL of 5 prospective patients. Neoantigen-reactive clone frequency was compared between bulk lymphocytes and within enriched sorted populations (based on Table S1). Numbers represent fold enrichment and p-value of Paired T-test. (J) Summary of the landscape of neoantigen-reactive TCR clonotypes and their cognate neoantigens shared between TIL and PBL (identified by NeoTCR_PBL signature or FACS-sorting) from all patients (total of 100 TCRs from TIL and PBL). (K). Functional avidity of 44 NeoTCR clones that were either found only in the PBL compartment (Blood), TIL compartment (Tumor), or shared between PBL and TIL (Shared). Data shown is the half-maximal reactivity for each TCR clone assessed by titrating the reactivity across a wide range of cognate mutated neopeptide concentrations relative to the wildtype peptide pulsed on autologous APCs. Also see Fig. S6–8 and Table S1.