Figure 6.
The DC:T cell ratio determines Treg versus Th17 differentiation in vitro
(A) Graphical representation of working hypothesis on the effect of professional APC:T cell ratio on CD4 differentiation.
(B) Generation of Treg and Th17 cells upon 6-day co-culture of 2.5 × 106 AND T cells with varying numbers of MCCp-loaded BMDCs. Treg versus Th17 differentiation was determined according to the expression of CD25, Foxp3, IL-17A, and CCR6 markers. Two-color contour plots show Th17 and Treg differentiation under the optimal DC doses. Bar graphs show the total number of cells with the Treg and Th17 phenotype (means ± SEMs of triplicate datasets; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; 2-tailed unpaired Student’s t test).
(C) Heatmap representation of genes differentially transcribed in conditions of 1, 10, or 100 AND T cells per well. Plotted genes correspond to those that have been associated with either Treg or Th17 signatures after GSEA analysis (Figure S9). Color-coded relative number of reads per gene is indicated in the scale bars to the right. Statistical analysis of expression differences was carried out by a 2-tailed paired Student’s t test. p values are considered significant if <0.05.
(D) qRT-PCR analysis of genes associated with a Treg signature (Foxp3, SIRPα, and TGF-β) or a Th17 signature (RORγt, IL-17A, and IL-17F) after mRNA extraction from AND T cell and MMCp-loaded BMDC co-cultures at indicated DC:T cell ratios. Bar plots show the means ± SEMs of n = 3–9 biological replicas. mRNA expression was normalized to the expression of the T cell-restricted CD3ε gene. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (1-way ANOVA test).
(E) Differentiation of AND T cells from WT and Rhog−/− mice according to the DC:T cell ratio after 6 days of co-culture. Bar plots show the mean ± SEM of n = 3–9 biological replicas. mRNA expression is shown relative to that of CD3ε used to normalize for T cell number. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (2-tailed unpaired Student’s t test).
(F) Co-expression of RORγt and Foxp3 in Rhog−/− AND T cells co-cultured for 6 days with MCCp-loaded BMDCs at a 1:10 DC:T cell ratio. The graph shows the percentage of AND WT and Rhog−/− CD4+IL-17A+ cells that co-express RORγt and Foxp3 upon co-culture at different DC:T cell ratios. Data are shown as means ± SEMs of triplicate cultures. ∗p < 0.05 (2-tailed paired Student’s t test).