Extended Data Fig. 9: Profiling of mouse T cells in response to anti-PD1 or RIPR-PD1 treatment.

a, Mice inoculated with MC38 tumour cells were treated with PBS, anti-PD1 or RIPR-PD1(RMP), 200 μg, every 3 days starting at day 5 post tumour inoculation. On day 12 T cells from spleen (Spl), peripheral and distal lymph nodes (pLN, dLN) and tumour infiltrating lymphocytes (TILs) were collected and analysed by flow cytometry. b, Quantification of tumour weight. c, Quantification of PD-1⁺ CD4⁺ T cells in TILs. d–f, Quantification of the fraction of positive CTLA-4 (d), TIM-3 (e) and LAG3 (f) from TILs for CD4⁺ T cells. g, h, Quantification of the fraction of CD62L^lowCD44^high effector memory CD8⁺ T cells isolated from the spleen (g) or tumour (h). i, Quantification of the fraction of CXCR3-positive cells for splenic CD8⁺ cells. Analysis of CD4⁺ cells showed increased fraction of CD137 (4-1BB) (j) and CXCR3 (k) in TILs. l, Quantification of effector memory and PD-1⁺ cells in tumour-free mice. Mice were treated with PBS, anti-PD1 or RIPR-PD1 every 3 days from day 0 to day 6 and T cells were isolated and analysed on day 7. m–r, CD8⁺ and CD4⁺ T cells were analysed on day 7 for CD44, CD62L (m, n; CD8⁺, spleen) and PD-1 expression in the spleen (o, p) and lymph nodes (mLN; q, r). s, Quantification of T_reg cells in tumour-free mice. Tumour-free FoxP3–GFP mice were treated with PBS, anti-PD1 or RIPR-PD1 (200 μg every 3 days) from day 0 to day 6, n = 3 mice per group. T cells were analysed on day 7. Quantification of the fraction of FoxP3–GFP-positive cells (gated on the CD3⁺CD4⁺ population) for cells isolated from the spleen. All data are mean ± s.d. from n = 5 mice (b–k, m–q) or n = 3 (s) representative from 2 independent experiments. For representative gating strategy, see Supplementary Fig. 2.

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