Figure 4.

Single-dose intranasal, but not intramuscular, immunization induces trained airway macrophages

(A) Experimental schema.

(B) Left, t-SNE maps were generated from concatenating CD45⁺ CD11b⁺/CD11c⁺ gated BAL mononuclear cells from 12 individual animals (3 per group of route/vaccine condition). Analysis was performed utilizing default FlowJo V.10 software settings. Right, heatmap projections of CD11c, CD11b, MHC II, Siglec-F, Ly6G, or Ly6C on t-SNE maps. Hashed circle indicates an MHC-II-high population.

(C) Overlap of populations arising after i.m. or i.n. Tri:HuAd (red) or i.m. or i.n. Tri:ChAd (blue). Hashed circles indicate a unique MHC-II-high population induced following i.n. immunization.

(D) MFI of MHC II expression on AM (left) and IM (right) in BAL at 8 weeks post-immunization.

(E) Experimental schema.

(F) Left, t-SNE maps were generated from concatenating CD45⁺ CD11b⁺/CD11c⁺ gated BAL mononuclear cells from 12 individual animals (3 per group of route/vaccine condition). Analysis was performed utilizing default FlowJo V.10 software settings. Right, heatmap projections of CD11c, CD11b, MHC II, Siglec-F, Ly6G, or Ly6C on t-SNE maps. Hashed circle indicates an MHC-II-high population.

(G) Overlap of populations arising after i.n. Tri:HuAd (red) or Tri:ChAd (blue) or empty vector equivalent controls. Hashed circles indicate a unique MHC-II-high population induced following i.n. immunization with either Tri:HuAd or Tri:ChAd.

(H) MFI of MHC II expression on AM (left) and IM (right) in BAL at 4 weeks post-immunization.

Data presented in (D and H) represent mean ± SEM. Data are representative of 2 independent experiments, n = 3–6 mice/group. Statistical analysis for (D and H) were one-way ANOVA with Tukey’s multiple comparisons test. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Alveolar macrophage (AM), interstitial macrophage (IM), and median fluorescence intensity (MFI).