Figure 3.

Chronic virus infection skews memory bystander phenotype and function. (A) Experimental approach. Adoptive transfer of CD45.1⁺ OT-I CD8⁺ T cells into congenic CD45.2 WT C57BL/6 mice, followed by priming with VV-OVA. On at least day 30 after priming, the mice were infected with LCMV docile at an intermediate (10⁴ ffu) or high (10⁶ ffu) dose. The experiment was terminated 30 d after LCMV infection. (B) Memory bystander CD8⁺ T cells in the spleen, lung, and blood. Flow cytometry plots pregated on live CD8⁺ T cells from spleen are depicted. Representative FACS plots are shown for CD45.1⁺ bystander T cells (memory only, + LCMV intermediate, and + LCMV high) and their expression of KLRG1 and IL-7Rα. Right: Quantification of percentages of KLRG1⁺ IL-7Rα⁻ OT-I T cells and KLRG1⁻ IL-7Rα⁺ OT-I T cells shown for memory only (white, uninfected), + LCMV intermediate dose (gray), and + LCMV high dose (black) in the spleen and lung. FSC-H, forward scatter height. (C) Representative flow cytometry plots of cytokine production and quantification of IFNγ, IL-2, and TNFα production by memory bystanders from memory only (white, uninfected), + LCMV intermediate dose (gray), and + LCMV high dose (black) in the lung after 6 h of restimulation with the cognate antigen. (D) Left: Experimental approach. Mice were infected with a high-dose LCMV docile. 30 d after infection, memory bystander CD8⁺ T cells were adoptively transferred into chronically infected mice. Bystander T cells were analyzed 30 d after transfer. Right: Quantification of percentages of KLRG1⁺ IL-7Rα⁻ OT-I T cells and KLRG1⁻ IL-7Rα⁺ OT-I T cells shown for memory only (white, uninfected) and + LCMV (black) in the spleen and lung. (B–D) One representative experiment out of two is shown with four mice per group. Statistical analysis was performed using the unpaired two-tailed Student’s t test: ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars show SEM.