Extended Data Fig. 2: Downregulation of CTL IFNAR1 by Treg and adenosine.

a WT or SA OT-1 cells were first co-cultured with or without iTregs for 24 h (Treg:OT-1 = 1:3) and then combined with MC38OVA-luc cells at indicated E:T ratios. Killing of MC38OVA-luc cells was analyzed using luciferase assay as described in Methods. Data are shown as mean ± SEM (n = 3 co-cultures.) Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. *P = 0.0288. b Tumor associated Tregs were isolated from MC38 WT tumors and cocultured with OT-1 cells for 24 h (Treg: OT-1 = 1:3). Then analysis of IFNAR1 levels on OT-1 CTL as well as killing of MC38OVA-luc cells (co-cultured at ratio E:T = 10:1) was performed as described in Methods. Data are shown as mean ± SEM (WT and SA IFNAR1, n = 3 co-cultures; WT and SA lysis%, n = 6 co-cultures Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test. c Representative flow cytometry analysis of levels of IFNAR1 on the surface of CD3⁺CD8⁺ T cells treated with or without adenosine (Ado, 1 mM) prostaglandin E2 (PGE2, 1 μg/ml) or tumor growth factor-beta (TGF-β, 5 ng/ml) for 2 h. d Lysis of MC38OVA-luc cells co-cultured in vitro with OT-1 cells pretreated or not with adenosine (Ado, 1 mM for 24 h) at indicated E:T ratios. Data are shown as mean ± SEM (n = 3 co-cultures). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001. e Schematic of crosses to generate the Ifnar1^ΔCD8 mice. f Genotyping analysis of Ifnar1^ΔCD8 mice by PCR. g Validation of Ifnar1 ablation in CD8⁺ cells using the flow cytometry analysis of IFNAR1 levels on the surface of CD4⁺ and CD8⁺ T cells in Ifnar1^+/+ and Ifnar1^ΔCD8 mice. It is representative of n = 3 independent experiments.

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