(A) IM prime-boosted mice received a second booster via IN route and spleen, iliac LN, medLN and lungs were examined ≥28 days post secondary boost. (B-C) Quantification of antigen-specific CD8+ T cells (B) and CD4+ T cells (C) in the indicated compartments. (D-E) Representative flow cytometry plot (D) and quantification (E) of IV-neg NP366-specific CD8+ T cell subsets in the lung. (F-G) Representative flow cytometry plot (F) and quantification (G) of IV-neg NP261-specific CD4+ T cell subsets in the lung. Data represent N = 2 independent experiments with n = 4-5 mice per group. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as determined by unpaired two-tailed Student’s t test.