Fig 1.
Viral titers in nasal swabs and respiratory tissues.
(A, B) Virus titers in nasal swabs and frequency of pigs positive for virus shedding at 1, 3, and 5 DPV (A) and at 1–5 DPC (B). (C–E) Virus titers in bronchoalveolar lavage fluid (BALF) (C), nasal turbinates (D), and trachea (E) at 5 DPC. ANOVA and subsequent Tukey’s adjustment was used to determine a statistically significant difference (p < 0.05), and the significance between two groups is indicated by different letters. Data are represented as mean ± SEM of log10 (TCID50/mL or TCID50/g). Symbols represent treatment groups (A, B) or individual pigs (C–E). G1 (n = 6): CD1D − / − vaccinated and challenged; G2 (n = 6): CD1D − / + vaccinated and challenged; G3 (n = 5): CD1D − / − not vaccinated and challenged; G4 (n = 5): CD1D − / + not vaccinated and challenged; G5 (n = 3): CD1D − / + not vaccinated, not challenged.
Fig 2.
Lung histopathology and CD3+ T cell localization at 5 DPC.
(A) Cumulative histological scores following assessment of the parameters shown in the materials and methods. Histologic alterations occurred at a similar degree between CD1D − /+ and CD1D − / − pigs in the vaccinated and challenged as well as the unvaccinated and challenged groups, with the latter groups showing more severe alterations (reflected by a higher cumulative score). (B) Degree of peribronchiolar/perivascular lymphocytic inflammation. (C, D) Scores for overall CD3+ T cell abundance in the lung (C) and distribution of CD3+ T cells (D), assessed by immunohistochemistry. CD3+ T cell distribution in the pulmonary parenchyma of each pig was classified into two categories: (1) scattered in pulmonary parenchyma with no defined cuffing around airways or (2) airway-centric distribution cuffing small and larger airways. ANOVA and subsequent Tukey’s adjustment was used to determine a statistically significant difference (p < 0.05), and the significance between two groups is indicated by different letters (A, B, and C). Data are represented as mean ± SEM. Symbols represent individual pigs. G1 (n = 6): CD1D − / − vaccinated and challenged; G2 (n = 6): CD1D − / + vaccinated and challenged; G3 (n = 5): CD1D − / − not vaccinated and challenged; G4 (n = 5): CD1D − / + not vaccinated and challenged; G5 (n = 3): CD1D − / + not vaccinated, not challenged.
Fig 3.
Histologic lesions (A–D) and CD3+ T cell distribution (E–H).
Histologic alterations in G1 and G2 (A and B) are similar and featured by a mild mononuclear infiltrate delimiting bronchioles (insets) and occasional expansion of the alveolar septa. In G3 and G4 (C and D), the degree of airway-centric inflammation is higher and is additionally characterized by intraluminal exudate composed of degenerate neutrophils and a hyperplastic bronchiolar epithelium with transmigrating lymphocytes and neutrophils (asterisks and insets) and regions of pulmonary atelectasis. CD3+ T cells in CD1D − / + pigs are intensely recruited around airways (F and H) regardless of vaccination status compared to CD1D − / − pigs (E and G). Immunohistochemistry for CD3 (DAB). G1: CD1D − / − vaccinated and challenged; G2: CD1D − / + vaccinated and challenged; G3: CD1D − / − not vaccinated and challenged; G4: CD1D − / + not vaccinated and challenged.
Fig 4.
Hemagglutinin (HA)-specific antibody titers measured by hemagglutinin inhibition (HI) assay and in-house isotype-specific ELISA.
(A, B) Geometric mean of HI titers against TX98 (A) and CO99 (B) in serum. (C–I) Isotype-specific (IgG, IgG1, IgG2, and IgA) antibody titers against HA in serum (C–G) and BALF (H and I) of vaccinated pigs (G1 and G2). (J) Ratio of HA-specific IgG1/IgG2 titers in serum (J). ANOVA and subsequent Tukey’s adjustment (A and B) and t-test (C–J) were used to determine a statistically significant difference (p < 0.05), and the significance between two groups is indicated by different letters. Data are represented as geometric mean ± geometric SD. Symbols represent individual pigs. G1 (n = 6): CD1D − / − vaccinated and challenged; G2 (n = 6): CD1D − / + vaccinated and challenged; G3 (n = 5): CD1D − / − not vaccinated and challenged; G4 (n = 5): CD1D − / + not vaccinated and challenged; G5 (n = 3): CD1D − / + not vaccinated, not challenged.
Fig 5.
Flow cytometric analysis of T cell frequencies in enzymatically digested lung tissue at 5 DPC.
(A) CD3+ T cells as a proportion of lung lymphocytes. (B) αβ T cells as a proportion of lung lymphocytes. (C) CD8αβ+ T cells as a proportion of CD3+ T cells. ANOVA and subsequent Tukey’s adjustment was used to determine a statistically significant difference (p < 0.05), and the significance between two groups is indicated by different letters. Data are represented as mean ± SEM. Symbols represent individual pigs. G1 (n = 6): CD1D − / − vaccinated and challenged; G2 (n = 6): CD1D − / + vaccinated and challenged; G3 (n = 5): CD1D − / − not vaccinated and challenged; G4 (n = 5): CD1D − / + not vaccinated and challenged; G5 (n = 3): CD1D − / + not vaccinated, not challenged.
Fig 6.
Single-cell transcriptomic analysis of vaccinated and IAV-infected CD1D − /− and CD1D − / + pig lungs.
(A) UMAP visualization of pig lung cells. (B) The average frequency of each cell type is presented for each group. Significance (p < 0.05) was determined by t-test in each cell type. (C) Bar graphs displaying the number of upregulated and downregulated differentially expressed genes (DEGs) in G1 compared to G2 pigs. (D–E) Ingenuity pathway analysis (IPA) of DEGs in lymphocytes (D) and myeloid cells (E). The y-axis shows the top pathways identified in each cell type within the threshold −2.0 < Z > 2.0. Dot size indicates significance [−log10(P value)], and dot color saturation reflects the z-score. G1 (n = 4): CD1D − / − vaccinated and challenged; G2 (n = 4): CD1D − / + vaccinated and challenged.
Fig 7.
Immune receptor profiling of pulmonary αβ T cells and B cells.
(A) UMAP plot showing the alignment of single TRA, single TRB, and paired TRA + TRB cells. (B) UMAP plot of T/NK/ILC cell clusters with paired TRA + TRB, which were used for downstream analysis. (C) Number of cells expressing 1, 2, or ≥3 clones identified by CDR3β sequence. (D) Box plot showing the proportion of cells expressing ≥3 clones across vaccinated groups (G1 and G2). Each dot represents an individual animal. Statistical significance was assessed by t-test. (E) Box plot showing the average length of CDR3β amino acid sequences by vaccinated groups (G1 and G2). Each dot represents the mean CDR3β length of a sample. Statistical significance was assessed by t-test. (F) Proportion of cells expressing the top 5 expanded CDR3β sequences in each animal. (G) Cells expressing the CDR3β sequence CASSSGGSETQYF. (H) UMAP plot showing the alignment of cells expressing single IGK, IGL, IGH, and paired IGL/IGK + IGH chains. (I) UMAP plot of B cells with paired IGL/IGK + IGH, which were used for downstream analysis. (J) Box plot of the proportion of cells expressing ≥3 clones (combined IGL/IGK and IGH CDR3) in vaccinated groups (G1 and G2). Each dot represents an individual animal. Significance was determined by t-test. (K–L) Box plots of the proportion of cells expressing IGHG (K) and IGHA (L) in the vaccinated groups (G1 and G2). Each dot represents an individual animal. G1 (n = 4): CD1D − / − vaccinated and challenged; G2 (n = 4): CD1D − / + vaccinated and challenged.
Fig 8.
Immune receptor profiling of pulmonary γδ T cells.
(A) UMAP plot showing the alignment of single TRG, single TRD, and paired TRG + TRD cells. (B) UMAP plot of cells with paired TRG + TRD, which were used for downstream analysis. (C) Sankey-plot showing the number of cells expressing TRG and TRD VJC gene segments, as well as their recombination patterns. (D) Number of cells expressing 1, 2, or ≥3 clones identified by CDR3γ sequence. (E) Proportion of cells expressing the top 5 expanded CDR3γ sequences in each animal. (F–H) Proportion of cells expressing TRGV (F), TRGJ (G), and TRGC (H) segments by γδ T cell subtype. (I) Box plot of the proportion of cells expressing ≥3 clones in G1 and G2 groups. Each dot represents an individual animal. G1 (n = 4): CD1D − / − vaccinated and challenged; G2 (n = 4): CD1D − / + vaccinated and challenged.
Fig 9.
Cellular responses to TX98 and CO99 measured by ELISpot assays.
(A, B) Interferon-γ (IFN-γ) production by PBMCs collected at −1, 14, 20, and 26 DPV (5 DPC) after incubation with live TX98 (A) and CO99 (B) virus. (C, D) IFN-γ production by lung cells collected at 5 DPC after incubation with live TX98 (C) and CO99 (D) virus. (E, F) Interleukin-2 (IL-2) production by the same PBMCs after incubation with live TX98 (E) and CO99 (F) virus. ANOVA and subsequent Tukey’s adjustment was used to determine a statistically significant difference (p < 0.05), and the significance between two groups is indicated by different letters. Data are represented as mean ± SEM. Symbols represent individual pigs. G1 (n = 6): CD1D − / − vaccinated and challenged; G2 (n = 6): CD1D − / + vaccinated and challenged; G3 (n = 5): CD1D − / − not vaccinated and challenged; G4 (n = 5): CD1D − / + not vaccinated and challenged; G5 (n = 3): CD1D − / + not vaccinated, not challenged.