Extended Data Figure 5. Peripheral T cell exhaustion, reinvigoration, CD8/Treg ratio, and tumor PD-L1 predict response to RT + immune checkpoint blockade.

a) Heat map showing the relative proportions of PD-1⁺ CD8 T cells that are Ki67⁺GzmB⁺ or Eomes⁺ and the CD8/Treg ratio for each sample (columns) subtracted from the average values of untreated controls. Black hatches indicated CR and treatment with RT + anti-CTLA4 (C4) +/− anti-PD-L1 (P1). From these data, a multivariable RF predictor for CR was developed. Boxplot shows bootstrap distributions of variable importance scores (more predictive variables have higher values), and of b) minimal depth (MD), a statistic to measure predictiveness. Bar plot shows % bootstrap models for which the MD for the indicated variable was significant. Bootstrap mean +/− SD for the out-of-bag prediction error rate is listed on top. c) Probability of CR vs. change (treated vs. untreated control) in CD8/Treg ratio for mice with a high (blue dots) or low (red dots) change in % PD1⁺ splenic CD8 T cells that are Eomes⁺. d) Heat map similar to (a) except using T cells from peripheral blood. e) Percent peripheral blood PD-1⁺ CD8 T cells that are Eomes⁺ vs. Ki67⁺GzmB⁺ after RT + checkpoint blockade. Values are subtracted from average of untreated controls. Each circle represents a mouse. Probability of CR (proportional to circle size), prediction error rate, and quadrant boundaries are estimated from the RF model. f) Representative contour plots examining splenic CD8 T cells from B16-F10 or Res 499 tumors for PD-1 and Eomes (top), followed by examination of the PD-1⁺Eomes⁺ subset for Ki67 and GzmB (bottom). g) Ratios of PD-1⁺Eomes⁺ splenic CD8 T cells that are Ki67⁺GzmB⁺ (reinvigorated) compared to Ki67⁻GzmB⁻ (exhausted) from mice with Res 499 tumors.