Fig. 2: TRTpred applications for discovery of immune repertoires and validation of MixTRTpred.
a, Richness (top) and clonality (bottom) of inferred tumor-reactive (+/−) clones using TRTpred in n = 5 cohorts: internal data (melanoma n = 14); ref. 14 (melanoma, n = 4); ref. 11 (GI, n = 5); ref. 8 (n = 1 melanoma, n = 2 breast and n = 12 GI); ref. 12 (lung, n = 4). Patients are color-coded according to the cancer type. Metrics are displayed in logarithmic scale and statistics were performed using a one-tailed t-test. b, Proportion of inferred TRT CD8+ T cells in melanoma (n = 19) and other solid tumors (n = 23, as described in a). Statistics were performed using a one-tailed Wilcoxon test. c, Sequential multiplexed immunohistochemistry of patient 1 with hematoxylin (red) and CD8 (yellow) staining. Upper panel, whole-tissue section; lower panel (white rectangle), magnified image. Scale bars, 500 μm and 50 μm, for the whole-tissue section and magnified images, respectively. This is a representative experiment among n = 5 independent patients with melanoma. d,e, Cumulative frequency of inferred high-avidity (d) or tumor-reactive (e) CD8+ clones identified in microdissected areas of stroma and tumor in n = 5 independent patients with melanoma (patients 1–5). Statistics were performed using a pairwise one-tailed Wilcoxon test. f, TRTpred results depicting the distance matrix of the top 20 ranked tumor-reactive among high structural avidity clones. The five clones selected are the ones with the highest tumor-reactive score in each cluster (TCRs 1–5; Supplementary Table 6), defined by hierarchical clustering. g, In vitro validation of the tumor reactivity of the five TCRs (TCRs 1–5) predicted through MixTRTpred through CD137 upregulation assay (mean of n = 2 biologically independent replicates). The color code corresponds to that of panel f. h,i, IL-2 NOG mice were subcutaneously engrafted with tumor cells from patient 14 followed by adoptive transfer of TCR-transduced primary CD8+ T cells. h, Tumor-bearing mice received 5 × 106 CD8+ T cells transduced (day 11) with TCR1, TCR3 or TCR5 (Supplementary Table 6). i, Mice were adoptively transferred with infusion products containing 1 × 106 total CD8+ T cells transduced (day 11) with TCR1 or TCR3 or TCR5 (single TCRs) or with a pool of 1 × 106 total CD8+ T cells transduced with the three TCRs (TCR cocktail, 0.33 × 106 CD8+ T cells transduced with each TCR). In h and i, 5 × 106 CD8+ untransduced T cells were transferred as control. Data show mean ± s.e.m. of n = 3–5 biologically independent replicates. In box plots, the boxes represent the median and the interquartile range (IQR), while the whiskers extend to 1.5 times the IQR. ID, identification; Irr Ctrl, irrelevant control; Mel, melanoma; Pan, pan-cancer; GI, gastrointesinal.
