PMC

Mean frequencies of HRV-specific IFN-γ producing CD4+ and CD8+ T cells among proliferating and non-proliferating T cells in pigs vaccinated with AttHRV3x. Data are presented as mean frequency±standard error of the mean (n = 4). Black bars, ileum, hatched bars, spleen; and white bars, blood. Asterisks denote significant difference between BrdU+ and BrdU− IFN-γ producing T cells for the same tissue (Kruskal–Wallis test, p < 0.05).

Detection of HRV-specific IFN-γ producing CD4+CD8+ double positive T cells by intracellular staining and flow cytometry. After gating on the CD3+CD4+CD8+ subpopulation similarly to , IFN-γ PE and forward scatter dot plots were drawn within the CD4+CD8+ double positive T cell subpopulation. The frequencies of IFN-γ+CD4+CD8+ (labeled on the dot plot) are among CD3+ MNC that were stimulated in vitro with HRV antigen, PHA (positive control), or medium (background control), respectively.

Mean frequency of total CD4+ and CD8+ T cells in VirHRV infected or AttHRV3x vaccinated pigs and the controls. Frequencies of CD3+CD4+ and CD3+CD8+ T cells were analyzed among the MNC that were cultured for 17 h with medium-only. Data are presented as mean frequency ± standard error of the mean (n = 5–8). Black bars, VirHRV group; hatched bars, AttHRV3x; and vertical lined bars, mock-inoculated controls. Different letters on top of bars indicate significant differences among groups for the same cell type and tissue (Kruskal–Wallis test, p < 0.05).

Examples of HRV-specific IFN-γ producing CD4+, CD8+ and CD4+CD8+ T cell responses in ileum of gnotobiotic pigs at PID 28. Ileal MNC was from pigs infected with VirHRV, or vaccinated with AttHRV-2/6VLP, AttHRV3x or mock-inoculated controls. MNC were stimulated in vitro with HRV antigen for 17 h. A representative dot plot was shown for each inoculation group. The responses in AttHRV2x group (not shown) were similar to AttHRV3x group. The numbers at the upper right corners of dot plots of panels (A) and (B) are the frequencies of IFN-γ+CD4+ or IFN-γ+CD8+ T cells. The numbers in the rectangles in dot plots of panel (C) are the frequencies of IFN-γ+CD4+CD8+ double positive T cells. ND, undetermined.

HRV-specific IFN-γ producing T cell responses in Gn pigs. Data are presented as mean frequency±standard error of the mean (n = 4–8). The frequencies are calculated by subtracting the frequencies detected in medium-only stimulated MNC from HRV antigen-stimulated MNC. Black bars, VirHRV1× group; white bars, AttHRV-2/6VLP group; light hatched bars, AttHRV3x; heavy hatched bars, AttHRV2x group; and vertical lined bars, mock-inoculated controls. Different letters on top of bars indicate significant differences in frequencies among groups for the same cell type and tissue. Asterisks denote significant difference when compared to ileum; and Δ indicates significant difference when compared to blood for the same cell type in the same group (Kruskal–Wallis test, p < 0.05).

Detection of HRV-specific IFN-γ producing CD4+ and CD8+ T cells by intracellular staining and flow cytometry. Stained cells were gated first for mononuclear cell (MNC) populations based on forward scatter and side scatter profile followed by gating on CD3+ T cells. Then a bivariate dot plot was drawn to define CD4+, CD8+ and CD4+CD8+ subpopulations within CD3+ T cells (A). Dot plots in (B) depict IFN-γ producing CD4+ and CD8+ T cells among CD3+ MNC from spleen of a HRV-infected gnotobiotic pig. The MNC were stimulated in vitro with purified HRV antigen, PHA (positive control), or medium (background control). The frequencies of IFN-γ+CD4+ or IFN-γ+CD8+ cells are labeled on the upper right corner of each dot plot. Quadrants are defined by using MNC stained with isotype-matched irrelevant antibodies (C).