Fig. 4

AMs and cDC1s are mainly responsible for boosting antigen-specific T cells in lung tissues after intranasal vaccination. a Timeline of the experiment to evaluate the role CD11c+ APCs on boosting antigen-specific T cells. WT, Batf3^−/− or CD11c-DTR mice were pre-treated with cyclophosphamide (CTX) and diphtheria toxin (DT) for lymphodepletion in all mice and CD11c+ cells depletion in CD11c-DTR mice. Activated OT-1 cells were labeled with CFSE and then transferred to mice immunized with intranasal circRNA vaccine. T cell proliferation at lung tissues was analyzed at day 3. b, c Representative flow cytometry histograms (b) and statistical results (c) of T cell proliferation with different treatments. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. n = 3 for mock vac. group; n = 4 for other groups. For LNP uptake analysis, mice received intranasal DiD-labeled LNP and lung tissues were analyzed 4 h later. d, e Representative flow cytometry histograms (d) and statistical results (e) of LNP uptake among different cell types. f Scheme of the experiment to test the potential APCs that can boost antigen-specific T cells. Mice were immunized with intranasal LNP without RNA (mock) or circRNA vaccine and 24 h later, lung tissues were collected and APCs were sorted and co-cultured with OT-1 cells for another 36 h in IFN-γ Elispot wells. (n = 3 for mock group; n = 5 for circRNA vaccine group, repeated for three times) Created with BioRender.com. g, h Representative images and statistical results of IFN-γ Elispot assay. Data were analyzed by Student’s t test. i Timeline of the experiment to explore whether vaccine can boost T cells at lung tissues. Mice were treated with FTY720 during the prime-boost process or only the boosting process or normal saline as the control group. The number of antigen-specific T cells at lung tissues was analyzed. j, k Representative plots (j) and statistical results (k) of the antigen-specific T cells with different treatments. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. All data are represented as mean ± SEM