Extended Data Fig. 5: Effect of STAT inhibition and CTLA-4 blockade on IL-21 signaling.

(A) Workflow of cytokine (IL-21: 80 ng/ml)/ anti-CTLA-4/ and inhibitor treatment (PerV:1 mM, Gando:1 µM, Stattic: 5 µM, Tofa: 100 nM, Upada: 1 µM), flow cytometric staining and data analysis (n = 5). (B) Representative histograms of flow cytometric data showing pSTAT1, pSTAT3, and pSTAT5 expression in PD-1⁺CD8⁺ T cells with or without anti-CTLA-4 treatment. (C) Median fluorescence intensity (MFI) and frequencies (%) of pSTAT1 (MFI: unstim. versus IL-21 P = 0.0034, IL-21 versus IL-21+Tofa P = 0.0447, IL-21 versus IL-21+Upada P = 0.0374; frequencies: unstim. versus IL-21 P = 0.0028, IL-21 versus IL-21+Upada P = 0.0311), pSTAT3 (MFI: unstim versus IL-21 P = 0.0146, DMSO vs. IL-21 P = 0.0098, IL-21 versus IL-21+Upada P = 0.0011; frequencies: DMSO versus IL-21 P = 0.0065, IL-21 versus IL-21+Upada P = 0.0007), and pSTAT5 (MFI: IL-21 versus IL-21+Gando P = 0.0374; frequencies: IL-21 vs. IL-21+Gando P = 0.0447) on PD-1⁺CD8⁺ T cells with anti-CTLA-4 treatment. (D) Heatmaps of the MFI of pSTATs on PD-1⁺CD8⁺ T cells and PD-1^-CD8⁺ T cells. pSTATs expression was scaled by Z-score. (E) Comparison of MFI of pSTAT1, pSTAT3, and pSTAT5 gated on PD-1⁺CD8⁺ T cells with or without anti-CTLA-4 treatment. (F) MFI and frequencies of pSTAT1, pSTAT3 and pSTAT5 on PD-1⁺CD8⁺ T cells with anti-CTLA-4 or iplimumab treatment. Two-sided Friedman analysis was performed in (C). Two-sided pairedWilcoxon signed-rank test was performed in (E). P values are indicated (P * < 0.05, P ** < 0.01, P *** < 0.005, P **** < 0.001). Error bars indicate mean ± SEM. Gando, Gandotinib; JAK, janus kinase; PerV, pervanadate; pSTATs, phosphorylated-STATs; Tofa, Tofacitinib citrate; Upada, Upadacitinib. Schematic in A was created using BioRender.com.