Extended Data Fig. 10: Immune checkpoint blockade induces T_PEX and CX3CR1⁺ T_EX cell proliferation and egress from lymph nodes.

(a) Naïve congenically marked (CD45.1⁺) P14 CD8⁺ T cells were adoptively transferred to naïve wildtype (CD45.2⁺) mice, which were subsequently infected with LCMV-Docile. A single dose of anti-PD-L1 was given on d26 p.i. with or without subsequent daily FTY720 treatment. CD8⁺ T cells were analysed using flow cytometry on d30 p.i. (a) Representative flow cytometry plots and quantification of Ki67⁺ cells among the P14 T_PEX cells in the spleen and lymph nodes (LN) (Ctrl: n = 17; ICB/FTY720: n = 24). (b–e) Naïve wildtype mice were infected with LCMV-Docile. A single dose of anti-PD-L1 was given on d26 p.i. with or without subsequent daily FTY720 treatment. Endogenous CD8⁺ T cells in the blood, spleen and pooled LN were analysed using flow cytometry on d30 p.i. (b) Schematic of the experimental setup. (c) Quantification showing the numbers of endogenous CX3CR1⁺ and CX3CR1⁻ T_EX cells (Ctrl: n = 10; ICB: n = 14; ICB/FTY720: n = 18) in 100 µL of peripheral blood on d30 p.i. (d) Representative flow cytometry plots and (e) quantification showing the frequencies of endogenous CX3CR1⁺ (left – Ctrl: n = 24; ICB: n = 14; ICB/FTY720: n = 24) and proliferating CX3CR1⁺ (right – Ctrl: n = 19; ICB: n = 9; ICB/FTY720: n = 18) cells in the spleen and LN on d30 p.i. (f, g) Naïve congenically marked (CD45.1⁺) P14 CD8⁺ T cells were adoptively transferred to naïve wildtype (CD45.2⁺) mice, which were subsequently infected with LCMV-Docile. A single dose of anti-PD-L1 was given on d26 p.i. with or without subsequent daily FTY720 treatment. CD8⁺ T cells were analysed using flow cytometry on d30 p.i. (f) Representative flow cytometry plots and (g) quantification showing the numbers of P14 and endogenous T_PEX cells in peripheral blood (Ctrl: n = 10; ICB: n = 14; ICB/FTY720: n = 16). Dots in graphs (a, c, e, g) represent individual mice; bars represent median. Quantification and statistics derive from multiple t tests (b) and one-way ANOVA (c, e, g) and based on all data points across at least two (c, g) or three (a, e) independent experiments.