Figure 6. CD49a-expressing subpopulations are not inhibited by PD-1, LAG-3, or TIM-3 and display a TRM-like phenotype.

(A, D, E, F, H) Nur77-GFP mice were implanted SC with BRPKp110. (A, D, H) Mice were either left untreated or (E, F) IP injected with a checkpoint blockade inhibitor cocktail 48 hours prior to harvest on day 14. (A, E, F, H, G) CD3⁺CD8⁺ cells were analyzed for Nur77-GFP, CD49a, CD49b and/or CD69 expression immediately or (D) after culture with CD3/CD28 beads for 12 hours. Nur77 gMFI was normalized to CD8⁺ T cells in non-draining lymph nodes (NDLN). %Nur77 on CD8⁺ T cells is after subtraction of values from NDLN in the same mice (Supplementary Fig. S7A). Intracellular IFNγ and TNFα expression was determined on (B, F) CD3⁺CD8⁺ cells immediately after harvest, 4–6 hours after IV injection with Brefeldin A, or (C) after culture with CD3/CD28 beads for 4–6 hours in presence of Brefeldin A. Marker expression of subpopulations was compared with repeated-measures one-way ANOVA and Tukey’s multiple comparisons tests (A-F). Differences between treatment groups within each subpopulation was tested with a Welch’s corrected T-test (E, F). CD69⁺ and CD69^neg subsets within the integrin-expressing subpopulations were compared with a paired T-test (H). A, n=12, 3 independent experiments; B, n=5; C, n=13, 2 independent experiments; D, n=4; E/F, n=7/group, 2 independent experiments; G, n=15, 3 independent experiments; H, n=8/group, 2 independent experiments. Bar graphs and error bars indicate mean +/− SD. Factors of significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.