Table 1.
Antibodies.
Figure 1.
Toll like receptor signaling and mDC cytokine responses in vitro.
In vitro activation of HEK cells containing a murine TLR2/1 or TLR2/6 reporter construct in response WT (grey bars), dltX-D (black bars), or culture medium alone (white bars) (N = 6) (A). Following incubation of murine DCs with medium (white bars), WT (grey bars), or dltX-D (black bars) the release of TNFα or IL10 was determined (N = 4) (B). In addition, the IL10/TNFα ratio was calculated. Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Mann Whitney U test. *represents P-values <0.05, **represents P-values <0.01, ***represents P-values <0,001.
Figure 2.
Dendritic cell frequencies and activation in the peyer’s patches and small intestinal lamina propria.
Dendritic cells were gated based on size, granularity, and the expression of CD11c and MHC II. CD19+ B cells were excluded from analysis. Within the dendritic cell population the frequency of CD103+ or CD80+ was determined (black lines). The gates were set based on staining with an isotype control (grey lines). Representative FACS plots from the small intestinal lamina propria (SILP) and spleen are depicted (A). Tissue specific staining patterns are demonstrated for both CD103 and CD80, as shown by the FACS plots of SILP and splenic DCs (A). Dendritic cell frequencies in the Peyers Patches and SILP (N = 6) (B) following oral treatment with medium (white bars), WT (grey bars), or dltX-D (black bars). CD103+ DC frequencies in the PP and SILP (C). CD80+ DC frequencies in the PP and SILP (D). Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t- test. *represents P-values <0.05.
Figure 3.
Early activated CD4+ and CD8+ T cells, effector T cells, and FoxP3+ T cells in the Peyer’s patches, small intestinal and large intestinal lamina propria.
Within the CD4 or CD8 population the frequency of early-activated cells was determined based on the expression of CD69 (black lines). The gates were set based on staining with an isotype control (grey lines). Effector T cells were gated based on the expression of CD25 (black line) as compared to the isotype control (grey line) within the CD4 T cell population. FoxP3+ cells were excluded. FoxP3 were within the CD3+CD4+ T cell population (black line). The gate was set based on staining with an isotype control (grey line). Representative FACS plots from the small intestinal lamina propria (SILP) are depicted (A). Early activated CD4+ T cell frequencies in the PP, SILP, and LILP (N = 6) following oral treatment with medium (white bars), WT (grey bars), or dltX-D (black bars) (B). Early activated CD8+ T cell frequencies in the PP, SILP, and LILP (C). Effector T cells in the PP, SILP, and LILP (D). FoxP3+ T cell frequencies in the PP, SILP, and LILP (E). Ratio between FoxP3+ CD4+ T cells and effector T cells in the PP, SILP, and LILP (F). Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t- test. *represents P-values <0.05.
Figure 4.
Dendritic cell frequencies and activation in the spleen and mesenteric lymph nodes.
Dendritic cells were gated based on the expression of CD11c and MHC II. Within the dendritic cell population the frequency of CD103+ or CD80+ was determined. CD103+ dendritic cell frequencies in the spleen (N = 6) (A) and MLN (B) following oral treatment with medium (white bars), WT (grey bars), or dltX-D (black bars). CD80+ DC frequencies in the MLN (N = 6) (C) and spleen (D). Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t- test. *represents P-values <0.05.
Figure 5.
Early activated CD4+ and CD8+ T cells, effector T cells, and regulatory T cells in the spleen and mesenteric lymph nodes.
Within the CD4 or CD8 population the frequency of early activated cells was determined based on the expression of CD69 Effector T cells were gated based on the expression of CD25 within the CD4 T cell population. FoxP3+ cells were excluded. Regulatory T cells were gated based on the expression of FoxP3 within the CD3+CD4+ T cell population Early activated CD4+ T cell frequencies in the spleen and MLN (N = 6) following oral treatment with medium (white bars), WT (grey bars), or dltX-D (black bars) (A). Early activated CD8+ T cell frequencies in the spleen and MLN (N = 6) (B). Regulatory T cell frequencies in the spleen and MLN (C). Effector T cells in the spleen and MLN (D). Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t- test. *represents P-values <0.05.
Figure 6.
Polarized CD4+ T cell frequencies in the spleen and mesenteric lymph nodes.
Polarized CD4+ T cells were gated based on the expression of IFNγ, IL5, IL10, or IL17 within the CD3+CD4+ T cell population (top plots). The gate was set based on staining with an isotype control (bottom plots). Representative FACS plots are depicted (A). Polarized CD4+ T cell frequencies in the MLN (N = 6) (B) and spleen (C) following oral treatment with medium (white bars), WT (grey bars), or dltX-D (black bars). Polarized CD4+ T cell frequencies are depicted as the frequency of IFNγ+ cells within CD4+ T cells, IL5+ cells within CD4+ T cells, IL10+ cells within CD4+ T cells, and IL17+ cells within CD4+ T cells. Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t- test. *represents P-values <0.05.
Figure 7.
Polarized CD8+ T cell frequencies in the spleen and mesenteric lymph nodes.
Polarized CD8+ T cells were gated based on the expression of IFNγ, IL5, IL10, or IL17 within the CD3+CD8+ T cell population (top plots). The gate was set based on staining with an isotype control (bottom plots). Representative FACS plots are depicted (A). Polarized CD8+ T cell frequencies in the MLN (N = 6) (B) and spleen (C) following oral treatment with medium (white bars), WT (grey bars), or dltX-D (black bars). Polarized CD8+ T cell frequencies are depicted as the frequency of IFNγ+ cells within CD8+ T cells, IL5+ cells within CD8+ T cells, IL10+ cells within CD8+ T cells, and IL17+ cells within CD8+ T cells. Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t- test. *represents P-values <0.05.