Extended Data Fig. 9: CD28 signalling promotes exhausted T cell fitness in lymph nodes.

(a–e) Naïve congenically marked (CD45.1⁺) P14 cells were adoptively transferred into wildtype (CD45.2⁺) mice, which were then infected with LCMV-Docile and subjected to B7 blockade using anti-CD80/86 antibodies treatment starting on d18 p.i. Endogenous antigen-responsive PD-1⁺ as well as P14 cells were analysed on d25 p.i. using flow cytometry. (a) Flow cytometry plots and quantification showing the frequencies of Ki67⁺ cells among endogenous T_PEX cells in the spleen and lymph nodes (LN) on d25 p.i. (n = 15). (b) Flow cytometry plots and quantification showing IFN-γ and TNF production of from spleen and LN-derived P14 cells after incubation with gp33 peptide ex vivo (Untreated: n = 20; Anti-B7: n = 16). (c) Flow cytometry plots and quantification showing the frequencies of intermediate Ly108^intCX3CR1⁺ cells among P14 T cells in the spleen and LN on d25 p.i. (Untreated: n = 20; Anti-B7: n = 12). (d) Flow cytometry plots and quantification showing the frequencies of Ki67⁺CX3CR1⁺ cells among endogenous antigen-responsive CD8⁺ T cells in the spleen and LN on d25 p.i. (n = 15). (e) Flow cytometry plots and quantification showing the frequencies of intermediate Ly108^intCX3CR1⁺ cells among endogenous antigen-responsive CD8⁺ T cells in the spleen and LN on d25 p.i. (n = 18). (f) Flow cytometry plots and quantification showing the surface expression of CD28 among P14 T_PEX and T_EX cells in the spleen and LN on d25 p.i. (n = 8). Flow cytometry plots are representative. Dots in graphs (a–f) represent individual mice; bars represent median. Quantification and statistics derive from one-way ANOVA (a–f) and based on all data points across at least two (f) or three (a–e) independent experiments.