Figure 2.. CD39 - CD69 - CD8 + TILs in infusion product are in a

(A) t-SNE plot of all CD8+ TILs from CR and NR I.P. (5 CRs, 5 NRs). (B) Frequency distribution of each cluster (1 through 8) expressed as a % of total CD8+ T cells in the I.P. for each patient. (C) Data points representing % of S.Cluster.A (top) and % of S.Cluster.B (bottom) per each patient I.P. between CRs and NRs. S.Cluster.A encompasses clusters C0, C2, C5, C6, and C7; S.Cluster.B comprises clusters C1, C3, C4, and C8. P-values by two-sided Wilcoxon rank-sum test are shown. (D) Heatmap of top 15 discriminating genes between S.Cluster.A and S.Cluster.B displayed for each cell. All discriminating genes are listed in Table S4. (E) t-SNE plot of clustered cells displayed by the top two quartiles of CD39-CD69- (DN) gene signature expression, and top two quartiles of CD39+CD69+ (DP) gene signature expression. (F) Each patient I.P. was scored by DN and DP gene signature scores and their mean scGSEA values are plotted. CRs are in red, and NRs are in black. P-values by two-sided Wilcoxon rank-sum test comparing the mean DN and DP signature scores between CRs and NRs are shown. (G) Flow cytometric analysis of inhibitory and memory markers within each subset (DN [CD39-CD69-], SP [CD39-CD69+, CD39+CD69-] and DP [CD39+CD69+] of patient I.P. in the validation set (n=38) expressed as % of each subset (parent gate). * P < 0.05 **P < 0.01 ***P < 0.001 ****P < 0.0001 by Tukeys multiple comparison test. (H) Flow cytometry of intracellular TCF7 expression for DN and DP subsets showing representative patient I.P. sample (top) and quantitation for 18 I.P. (bottom). P-value by two-sided Wilcoxon rank-sum test is shown. (I) Phenotypes of DN, SP, and DP states before and after CD3/CD28 stimulation at 48 hours showing flow cytometry plot in a representative patient sample (left) and summary of daughter cells in each state after stimulation of 6 patient I.P. (right). ***P < 0.001 by two-sided Wilcoxon rank-sum test. (J) t-SNE clusters of CD8+ TILs from Melanoma ICB cohort (15) (K) t-SNE plot colored by top two quartiles of DN and DP gene signatures, DN: red, DP: black (L) TILs from pre ICB therapy were scored by the top DN and DP gene signature scores and mean scGSEA scores are plotted. Cells from responding lesions are in red, and cells from progressing lesions are in black. Cell numbers and P-values by two-sided Wilcoxon rank-sum test are shown. (M) Clustered correlation matrix of gene signatures from other studies (Table S5) along with DN and DP scGSEA scores on pre-ICB cells from the cohort.

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