Fig 2: SARS-CoV-2 infection induces apoptosis and immune cell infiltration in the OE.a Representative hematoxylin and eosin (H&E) staining results showing histopathological changes of the OE. b Representative results of multiplex immunofluorescent detection of sustentacular cells (CK8 positive) and microvilli (Ezrin positive) of the OE. c Representative results of immunofluorescent detection of mOSNs (OMP positive) of the OE. d Apoptosis of olfactory epithelial cells (cleaved-caspase3 positive, white) after SARS-CoV-2 infection. The panels below show apoptosis of sustentacular cells (CK8 positive, yellow; indicated by cyan arrows), HBCs (CK5 positive, gold; indicated by gold arrows), mOSNs (OMP positive, green; indicated by magenta arrows), iOSN (GAP43 positive, magenta; indicated by green arrows), and olfactory nerve bundles (OMP/GAP43 positive; indicated by white arrows). e Representative multiplex immunofluorescent staining results showing infiltration of macrophages (CD68 positive, magenta), dendritic cells (CD103 positive, green), and neutrophils (Ly-6G positive, white) in the OE after infection. f Representative multiplex immunofluorescent staining results showing infiltration of CD8 cytotoxic T lymphocytes (magenta) with expression of perforin (green) and granzyme B (white) in the olfactory mucosa after infection. The framed areas are shown adjacently at larger magnifications. Scale bar, 50 µm.

Fig 3: Deletion of Smad4 in PDAC tumor cells enhanced tumor immunogenicity. A) Left, representative IHC staining images of the WT and Smad4KO PDAC tumor tissues. Right, bar graph showing the count of CD103+ cells. Scale bars = 50 µm. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01; by Mann–Whitney test. B,C) DC infiltration and activation levels in WT or Smad4KO PDAC tumors identified by FCM analysis. Data are presented as mean ± SEM; *, p < 0.05; **, p < 0.01; by Mann–Whitney test. D) DCs cocultured with tumor cells (WT or Smad4KO) for 24 h, and the percentage of CD80+ DCs was counted by FCM analysis. Three independent experiments were performed. Data are presented as mean ± SEM. ***, p < 0.001; by Mann–Whitney test. E) DCs were cocultured with PDAC-OVA (WT or Smad4KO) for 24 h, and then subjected to FCM analysis for surface expression of MHC-I-OVA (H2kb-SIIFEKL) complex. Three independent experiments were performed. Data are presented as mean ± SEM. ns, not significant; *, p < 0.05; by Mann–Whitney test. F) WT or Smad4KO PDAC cells were inoculated on Batf3 +/+ (WT) mice or Batf3 -/- mice, and then tumor growth was monitored at the indicated time points. Data are presented as mean ± SEM; n = 6 per group. ns, not significant; ***, p < 0.001; by two-way ANOVA test. G) Real-time qPCR analysis of expression levels of antigen presentation machinery molecules in WT or Smad4KO PDAC cells. Data are presented as mean ± SEM; three independent experiments were performed. ***, p < 0.001; by Mann–Whitney test. H) Western blot showing ß2M protein levels in WT or Smad4KO PDAC cells. I) FCM analysis of the cell surface expression of MHC-I and MHC-II molecules in WT or Smad4KO PDAC cells. J) B6 mice were subcutaneously inoculated with WT or Smad4KO PDAC cells expressing scrambled (Scr) or ß2m-specific shRNA (shß2m), and then tumor growth was recorded. Data are presented as mean ± SEM; n = 6 per group. ns, not significant; ***, p < 0.001; by two-way ANOVA test. K) DCs were cocultured with PDAC cells for 24 h, and the percentage of CD80+ DCs was analyzed by FCM analysis. Data are presented as mean ± SEM. ns, not significant; *, p < 0.05; **, p < 0.01; by Mann–Whitney test.

Fig 4: Intranasal immunization with the adjuvanted RBD vaccine directly activates T cells in the lungs at boost. NIH mice were immunized with PBS or PEI-adjuvanted RBD vaccine on days 0 and 14 in the presence or absence of FTY720 treatment. On day 28, immunized mice were sacrificed. Lung tissues and serum samples were collected for the analysis of lung T-cell responses and serum IgG levels, respectively. a Schedule of vaccine delivery in the presence of FTY720 during boost immunization. Quantification of lung CD4+ (b), CD4+CD44+ (c), CD8+ (d), and CD8+CD44+ (e) T cells that highly express both CD69 and CD103 after boost immunization. f Immunofluorescence analysis of lung CD4+ and CD8+ T cells with elevated CD103 expression in immunized mice on 3dpi. Green, CD4; violet, CD8; red, CD103; blue, DAPI. Scale bar, 100 µm. g RBD-specific IgG titers in the serum on day 28 in the presence or absence of FTY720 were assessed with ELISA. All the data were presented as mean ± SEM. n = 5. P values were compared to the PBS group. ns not significant

Fig 5: Intranasal immunization with adjuvanted RBD vaccine induces long-lasting lung TRM immune responses. a NIH mice were intranasally immunized with PBS, RBD or PEI-adjuvanted RBD on days 0, 7, and 21. Immunized mice were sacrificed in 1 year after prime immunization and lung tissues were harvested. Lung T-cell responses were evaluated with FCM. Representative FCM plots (b) and quantification (c) of CD8+CD44+ T cells in the lungs of immunized mice. (d) Representative FCM plots of CD69 and CD103 expression on CD8+CD44+ T cells in the lungs. Quantification of lung CD8+CD44+CD69+CD103-- (e) and CD8+CD44+CD69+CD103+ (f) T cells. Representative FCM plots (g) and quantification (h) of CD4+CD44+ T cells in the lungs of immunized mice. i Representative FCM plots of CD69 and CD103 expression on CD4+CD44+ T cells in the lungs. Quantification of lung CD4+CD44+CD69+CD103- (j) and CD4+CD44+CD69+CD103+ (k) T cells. l t-SNE maps were generated from CD3+ gated lung cells in immunized mice (3/group) and heatmap projections of CD44, CD69 or CD103 were showed on t-SNE maps. Hashed circles indicate TRM cells in the lungs. Flowjo software (V.10) was used to analyze the FCM data. All the data were presented as mean ± SEM. n = 4–5. P values were compared to PBS group (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)