Fig. 3: GNOS-PV02 drives polyfunctional antitumor neoantigen-specific T cell immunity.

a, Vaccine-induced responses assessed by IFNγ ELISpot assays without cytokine stimulation (n = 22). Cumulative magnitudes were collected from positive epitopes before and after treatment. The postvaccination response is the ‘best’ (highest magnitude) response for each patient across time points. SFU, spot-forming units. b, Total neoantigens (gray bars) and positive neoantigens before (black bars) and after (red bars) vaccination in each patient’s PTCV assessed by IFNγ ELISpot. c, Percentage of positive responding epitopes by groups. The definition of a neoantigen-specific ELISpot response can be found in Methods. d, Representative density plots (patient 22) of the T cell markers CD69, Ki67, CD107a, IFNγ and TNF upon stimulation with patient-specific PTCV epitope pools. e,f, Polyfunctionality assessed by Boolean gating of CD4⁺ or CD8⁺ cytokine-producing populations. T cell activation (CD69 and CD107a; e) and proliferation (Ki67; f) were assessed together with the double-positive expression of GZMA and perforin 1 (PRF1) to evaluate the cytolytic potential of neoantigen-reactive T cells. Four patients (patients 7, 11, 18 and 22) were analyzed in d–f. g, T cell clones expanded in the periphery and the new or expanded clones enriched in the matched tumor sample for each patient (n = 14). Total PBMC and tumor-associated T cell expansion were calculated by comparing posttreatment to pretreatment PBMC or tumor samples (differential abundance statistical analysis). h, Cumulative frequencies of peripherally expanded TCR rearrangements in tumor biopsy samples. i, Expanded clone numbers in tumor biopsy samples. j,k, TCR clonality (j) and repertoire richness (k) in tumor biopsy samples (n = 14). PD (red), SD (gray), and CR/PR (blue). Error bars correspond to the upper s.e.m. of each group. Simpson clonality reports the distribution of TCR rearrangements in a sample, in which 0 indicates an even distribution of frequencies and 1 indicates an asymmetric distribution. TCR repertoire richness reports the mean number of unique rearrangements. Lower numbers indicate focused TCR diversity. Filled symbols in c, e and f and open circles in h and i represent individual patients; the box extends from the 25th to the 75th percentile; the line inside the box is the median; and the whiskers extend from the minimum to the maximum value. Significance between groups was evaluated by a two-tailed Mann–Whitney test (c); significance within groups was evaluated by a two-tailed Wilcoxon rank test (a, h–k).