Extended Data Fig. 6 |. Analysis of tumour-associated T cells from patient 7.

a, Expression levels (log₂(TPM/10 + 1)) of selected marker genes for regulatory, cytotoxic and naive/memory T cell phenotypes; and expression of co-inhibitory receptors and effector cytokines based on scRNA-seq of tumour-associated CD4⁺ and CD8⁺ T cells from the relapse specimen of patient 7 (top). The average levels of these gene sets were used to define the expression scores of these signatures (bottom). b, Expression levels of selected marker genes for all tumour-associated CD3⁺ T cells from patient 7, including those unable to be resolved as CD4 or CD8 based on scRNA-seq. ND, not determined. c, Expression program associated with cytotoxicity in CD8⁺ (x axis) and CD4⁺ (y axis) T cells. For CD8, we compared all CD8⁺ T cells to non-T_reg CD4⁺ T cells. For CD4, we divided the non-T_reg CD4⁺ T cells by their average expression of a predefined cytotoxic signature (PRF1, NKG7, GZMK, GZMA and CST7) into three groups (low, medium and high) and compared the high to the low groups. The expression log₂ ratios of all expressed genes for these two comparisons (CD8 and CD4) are shown. To identify significant differences, we performed a one-sided permutation test, which shuffled the assignments of cells to groups 100,000 times and defined P values based on the number of times the shuffled log₂ ratios are higher (or lower for negative effects) than the observed log₂ ratios. P values were adjusted by Bonferroni correction and significant genes were defined as those with an adjusted P value below 0.05 and a fold change above 2 (Supplementary Table 10). Significant genes are shown as red dots; significant genes that have also been identified previously⁹ are shown in blue font. d, Multiplex immunofluorescence demonstrates increase in CD8⁺PD-1⁺ cell infiltration at relapse in patients who did not receive dexamethasone during vaccine priming (red). Infiltrates were determined by enumerating the mean number of CD8⁺PD-1⁺ cells in 20× fields. The number of fields evaluated per sample was: 4 fields for relapse samples from patients 7 and 8; 5 fields for initial and relapse samples from patient 3, relapse samples from patient 5 and initial samples from patient 8; and 6 fields for initial and relapse samples from patient 4, and initial samples from patients 5 and 7. Data are mean ± s.e.m. P values are two-sided and based on model F-tests. e, Enumeration of the SOX2⁺PD-L1⁺ cells in matched initial and relapse tumour sections, evaluated by multiplex immunofluorescence. Infiltrates were determined by enumerating the mean number SOX2⁺PD-L1⁺ cells in 20× fields as in d. Data are mean ± s.e.m.