Fig. 6: Experimental schedule to analyze the fate of functional antigen-specific CD8⁺ T_RM upon unrelated secondary virus infection.

A The survival of previously induced IAV-specific T_RM was examined upon secondary inflammatory events by subsequent infection with the unrelated RSV (1 × 10⁶ PFU) or by LPS treatment (10 µg) on day 56 after priming. Three weeks after the challenge, lymphocytes were isolated from lung tissues. One set of initially primed mice remained without secondary exposure (- RSV, - LPS, respectively) and served as the control group. B Body weight was measured for nine days following the RSV challenge, and values represent percentages of the initial weight on day 0. C Antigen-specific CD8⁺ T cells were identified by pentamer staining. Total numbers of HA_533-541-, NP_147-155-, and M2_82-90-specific tissue-resident CD8⁺ T cells are shown. D Frequencies of non-circulating CD8⁺ T cells that are at least positive for the single expression of either CD107a, IFNγ, IL-2, or TNFα were assessed after in vitro restimulation. E Immunofluorescence staining of lung tissues from initially rAd-immunized or H1N1-infected mice reinfected with RSV on day 35 and collected an additional 35 days after challenge. Tissue sections were stained with anti-B220 (green), RSV M2_82-90 pentamer (yellow), and IAV NP_147-155 pentamer (red). Nuclei are shown in blue (Hoechst33342). Images were acquired on a Leica SP5X laser scanning confocal microscope using a 40x oil objective and are representatives from a collection of different slices of three mice per group (n = 3). Scale bars in each image are 50 µm. C, D Each data point represents an individual mouse, and bars represent the mean of the group mean ± SEM per group (n = 6 mice per group, only in (D) one sample in the group rAd (-RSV) was lost due to technical issues). Numbers above the columns show fold change between unchallenged (- RSV) and challenged (+ RSV) mice. Statistical significances were analyzed (B) by two-way ANOVA followed by Tukey’s multiple comparison test, or (C, D) by one-way ANOVA followed by Tukey’s multiple comparison test ((B): *, p < 0.05 H1N1 + RSV vs. naïve + RSV; (C, D); *, p < 0.1; ***, p < 0.001; ****, p < 0.001). (A) Created in BioRender. Tenbusch, M. (2025) https://BioRender.com/g83h450.