Simvastatin Reduces Protection and Intestinal T Cell Responses Induced by a Norovirus P Particle Vaccine in Gnotobiotic Pigs

Figures

• 

  Figure 1. Virus-specific (A,C) and non-specific (B,D) proliferating T cells in the presence or absence of simvastatin. MNCs were stimulated with P particles or PHA and cultured with BrdU. Mean frequencies of CD3+CD4+BrdU+ and CD3+CD8+BrdU+ T cells in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues were analyzed by flow cytometry (Supplementary Figure S1). Note the difference in the Y axis scale between the upper (virus-specific) and lower (non-specific) panels. An asterisk above the bars indicates a significant difference between groups for the same cell type and tissue (p < 0.05 by Kruskal-Wallis rank-sum test).

• 

  Figure 2. Total numbers of MNCs in intestinal and systemic lymphoid tissues of Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and post-challenge. Total MNC numbers were calculated based on the concentration and total volume of MNCs isolated from the tissues. Total MNC numbers plus standard errors of the means (n = 6 to 10) prechallenge and postchallenge in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues are presented. An asterisk * above the bars indicates a significant difference among groups for the same cell type and tissue at the same time point; a section sign § indicates that the numbers increased significantly following challenge in the same group (p < 0.05 by Kruskal-Wallis rank-sum test). P particles without simvastatin (P+S−), P particles with simvastatin (P+S+), Control without simvastatin (CS−), Control with simvastatin (CS+).

• 

  Figure 3. Total numbers of Th (CD3+CD4+) and CTL (CD3+CD8+) in Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and postchallenge. MNCs were gated as previously described [16] following in vitro stimulation (Supplementary Figure S2). Total numbers plus standard errors of the mean (n = 6 to 10) of CD3+CD4+ Th and CD3+CD8+ CTL in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues pre- and postchallenge are presented. See Figure 2 legend for statistical analysis and an explanation of the symbols indicating statistical significance.

• 

  Figure 4. Frequencies of Th (CD3+CD4+) and CTL (CD3+CD8+) in Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and post-challenge. MNCs were gated as previously described [16] following in vitro stimulation (Supplementary Figure S2). Data presented are mean frequencies +/− SEM (n = 6 to 10) of Th or CTL among total lymphocytes in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissue at PID 28 or PCD 7. An asterisk indicates statistical significance among groups for the same cell type at the same time point (p ≤ 0.05, Kruskal-Wallis rank-sum test).

• 

  Figure 5. Total numbers of CD25+ expressing CD4+ and CD8+ T cells in Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and postchallenge. MNCs were gated as previously described [16] and analyzed freshly on the day of cell isolation by flow cytometry (Figure S3). Total numbers plus standard errors of the means (n = 6 to 10) of CD4+CD25+FoxP3− and CD8+CD25+FoxP3− activated T cells prechallenge and postchallenge in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues are presented. See Figure 2 legend for statistical analysis and an explanation of the symbols indicating statistical significance. A number sign # indicates that the cell numbers decreased significantly following challenge in the same group (p < 0.05 by Kruskal-Wallis rank-sum test).

• 

  Figure 6. Frequencies of activated CD4+ or CD8+ T cells in Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and post-challenge. MNCs were stained freshly and gated as previously described [16] (Supplementary Figure S3). Data presented are mean frequencies +/− SEM (n = 6 to 10) of FoxP3−CD25+ cells among CD4+ or CD8+ cells in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissue at PID 28 or PCD 7. An asterisk indicates statistical significance among groups for the same cell type at the same time point (p ≤ 0.05, Kruskal-Wallis rank-sum test).

• 

  Figure 7. Total numbers of IFN-γ+ expressing CD4+ and CD8+ T cells in Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and postchallenge. MNCs were gated as previously described [16] following in vitro NoV antigen stimulation and analyzed by flow cytometry (Supplementary Figure S4). Total numbers of IFN-γ producing CD4+ and CD8+ T cells following subtraction of isotype control and mock-stimulated background numbers are calculated. Data presented are total numbers plus standard errors of the means (n = 6 to 10) prechallenge and postchallenge in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues. See Figure 2 legend for statistical analysis and an explanation of the symbols indicating statistical significance.

• 

  Figure 8. Total numbers of CD25− and CD25+ Tregs in Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and post-challenge. Tregs were gated as previously described [16] following staining freshly on the day of cell isolation (Supplementary Figure S3). Total numbers of FoxP3+CD25− and FoxP3+CD25+ Tregs plus standard errors of the means (n = 6 to 10) among total MNCs prechallenge and postchallenge in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues are presented. See Figure 2 legend for statistical analysis and an explanation of the symbols indicating statistical significance. A number sign # indicates that the cell numbers decreased significantly following challenge in the same group (p < 0.05 by Kruskal-Wallis rank-sum test).

• 

  Figure 9. Frequencies of CD25− or CD25+ Tregs in Gn pigs vaccinated with P particles with or without simvastatin feeding pre- and post-challenge. MNCs were stained freshly and gated as previously described [16] (Supplementary Figure S3). Data presented are mean frequencies +/− SEM (n = 6 to 10) of CD25− or CD25+ cells expressing FoxP3+ among total MNCs in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissue at PID 28 or PCD 7. An asterisk indicates statistical significance among groups for the same cell type at the same time point (p ≤ 0.05, Kruskal-Wallis rank-sum test).