Figure 9. In the absence of Tregs, IL-2 blockade induces TOX-mediated CD8⁺ TIL exhaustion, promoting MC38 tumor growth.

(A) Experimental schema depicts the administration of IL-2–blocking Abs (S4B6-1 and JES6-1) or matched isotype Abs in MC38 tumor–bearing WT and KO mice (2 × 10⁶ MC38 cells/mouse, s.c.; n = 6–8/group). (B) Representative flow cytometric plots and summary graphs show the frequencies of Tcf1^–TOX⁺ and Tim3⁺Lag3⁺ cells within CD44^hiCD62L^loCD8⁺ TILs from day-11 MC38 tumors grown in WT and KO mice (n = 6–7/group). (C) Growth curves of MC38 tumors grown in WT and KO mice that received IL-2 blockade (group 4; 120 μg for each Ab) or isotype Ab treatment (group 1). Tumor sizes were measured. (D) Spectral flow cytometric analysis of CD8⁺ TILs isolated from day-11 MC38 tumors grown in WT and KO mice treated with IL-2 blockade (group 4) or isotype Ab (group 1). Cluster 11 (CD44^hiCD62L^loTcf1^–TOX⁺PD-1⁺Lag3⁺Tim3⁺CD8⁺ subset), shown as circles, is highlighted in green. Expression distribution of the indicated markers is also shown in the plots on the right. (E) Heatmap of the indicated markers by cluster. (F) edgeR analysis indicates significant differences in CD8⁺ TIL clusters between KO and WT groups (cluster 11 is highlighted). (G) Significant differences in cluster 11 enrichment among the indicated groups. Green indicates low abundance of cluster 11; purple indicates high abundance of cluster 11. Results are representative of 3 independent experiments. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (IL-2 blockade vs. isotype). A 2-tailed Student’s t test was performed for comparisons of different experimental groups (G) where multiple comparisons were not performed; for analyses where multiple comparisons were performed, a 1-way ANOVA with Šidák’s multiple-comparison correction was performed for comparison of treatment groups across genotypes (B); a 1-way ANOVA analysis with Dunnett’s multiple-comparison correction was performed for comparison within groups for treatments compared with the respective isotype (B). Tumor growth were curves were analyzed by repeated-measures 2-way ANOVA (C).