Fig. 4. MVA s.s. generates more than half number of lung T_RM compared to intra-tracheal (i.t.) and is sufficient to protect mice against lethal respiratory challenge.

a Flow cytometric analysis of OT-I cell proliferation in draining lymph nodes at 60 h post MVA infection via s.s. CFSE-labeled naive OT-I Thy1.1⁺ cells were transferred into Thy1.2⁺ recipient mice 1 day before mice were infected with 1.8 × 10⁶ pfu MVA-Ova. b PCA of gene expression data for 19 CD8⁺ T-cell populations based on CFSE signal and different infection routes. Each dot represents an individual experiment wherein mRNA was pooled from 15 to 20 mice from 3 to 4 independent biological groups (5 mice/group). c Heatmap of differentially expressed genes selected from a pairwise comparison between s.s. and intraperitoneal (i.p.) activated T cells. d Venn diagram analysis of genes differentially expressed in pairwise comparisons between s.s., i.t., and i.p. activated T cells relative to T_N (fold change cutoff, ≥2). e qRT-PCR analysis of cell homing molecule gene expression in s.s., i.t., and i.p. activated T cells. f Flow cytometric analysis (left) and quantification (right) of lung T_RM cells at day 45 post MVA infection via indicated routes. g, h Body weight (BW) (g) and survival measurements (h) of WR-VACV re-challenged mice that were immunized previously with MVA via indicated routes 45 days earlier. OT-I WT cells were adoptively transferred into µMT mice before mice were infected with 1.8 × 10⁶ pfu MVA via indicated routes. Forty-five days later, mice were re-challenged with a lethal dose of WR-VACV by intranasal infection. DEG, differentially expressed genes. Graphs show the mean ± SD (n = 5). UI = unimmunized. ns = not significant, *p < 0.05, **p < 0.01.