Extended Data Fig. 2: Direct antigen delivery into the lung is essential for mucosal recall responses, which are comparable between male and female C57BL/6 mice.

a, Schematic of the experimental setup showing i.m. primed C57BL/6 mice with mRNA-LNPs (day 0) were i.m. boosted with mRNA-LNPs or i.n. or i.t. or i.p. boosted with an unadjuvanted SARS-CoV-2 spike protein at day 14 post-priming, followed by lung, BALF, serum collection at day 7 post-boosting. b, Number of extravascular IV^–CD44⁺Tet⁺CD4⁺ T cells and IV^–CD44⁺Tet⁺CD8⁺ T cells of naïve C57BL/6J mice (D0, n = 7); i.m. primed+i.m. boosted mice (D21^P+B, n = 5); i.m. primed+i.n. boosted mice (D21^P+S(i.n.), n = 5); i.m. primed+i.t. boosted mice (D21^P+S(i.t.), n = 5); i.m. primed+i.p. boosted mice (D21^P+S(i.p.), n = 5) at day 21 post-priming analyzed by flow cytometry as in a. c, Representative flow cytometry plots and quantification of the number of total and RBD-specific IgA⁺ ASCs in the lung of mice as in b. d, ELISA measurements of SARS-CoV-2 spike protein S1 subunit-specific IgA and IgG in the BALF and serum from mice as in b. e, Schematic of the experimental setup showing female and male C57BL/6J mice were i.m. primed with mRNA-LNPs at day 0, i.n. boosted with an unadjuvanted spike protein at day 14, followed by lung, BALF, and serum collection at day 21 post-priming. f, Number of extravascular CD44⁺Tet⁺CD4⁺ T cells, CD44⁺Tet⁺CD8⁺ T cells, RBD⁺CD38⁺GL7^-IgM^-IgD^- CS B cells, total IgA⁺ ASCs, and RBD⁺IgA⁺ ASCs in the lung of naïve female C57BL/6 mice (D0^Female, n = 4); P + S female (D21^P+S(Female), n = 5) and male (D21^P+S(Male), n = 5) mice at day 21 post-priming analyzed by flow cytometry as in e. g, ELISA measurements of SARS-CoV-2 S1-specific IgA and IgG in the BALF and serum from mice as in f. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (b,c,d,f,g). Tukey’s multiple comparisons were performed; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were pooled from two (b,c,d,f,g) independent experiments and representative (f,g) of an experiment. Values shown as zero represent the absence of detectable cells (b,c,f).

Source data