Fig 2: 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, SIRPa, and TGF-ß) or a Th17 signature (ROR?t, 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 CD3e 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 CD3e 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 ROR?t 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 ROR?t 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).

Fig 3: IPCEF impairs migration of pathogenic Th17 cells(A) Th17 cells were polarized as indicated, followed by transduction with RV-Mock or RV-IPCEF. RV+ cells were then sorted, reactivated, and plated on a Transwell for 48 h to determine the number of cells migrating toward a CCL20 gradient. n = 3 technical replicates per group.(B) The frequency of CCR6 expression in Th17 cells, as indicated, was assessed by flow cytometry, and CCR6+ frequencies were pooled for analysis.(C–E) Th17 or iTreg cells were transduced and plated on a Transwell, as described in (A). The number of cells migrating toward CCL17 (C), CCL1 (D), or CCL19 (E) was then assessed. **p < 0.01, ***p < 0.001, ****p < 0.0001 (2-way ANOVA). Data are representative of 3 independent experiments and are presented as means ± SDs.

Fig 4: Tpres cells are enriched in Treg and Tresp cells in Th17(A) Experimental setup. After purification by cell sorting, AND Tpres cells are incubated for 3–6 days in the absence or presence of naive AND Tresp cells.(B) ELISA measurement of IL-2 and TNF-a concentrations in supernatants of Tpres cells cultured for 3 days in the presence or absence of Tresp cells. The bar graphs show the means ± SEMs of triplicates.**p < 0.01 (2-tailed unpaired Student’s t test).(C) Expression of CCR6, CD25, and Foxp3 by Tpres and Tresp cells from the experiment in (B), measured by flow cytometry. Bar graphs represent means ± SEMs (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 (2-tailed unpaired Student’s t test).(D) Generation of FoxP3+CD25+ T cells within AND Tpres and Tresp cell populations. After overnight incubation with MCCp-loaded BMDCs and purification, Tpres were cultured alone (Tpres alone) or together with naive AND Tresp cells (Tpres+Tresp) for 6 days and then analyzed by flow cytometry for Foxp3 and CD25 expression. In parallel, AND T cells were cultured uninterrupted for 6 days with MCC-loaded BMDCs (DC+Tpres). Data represent the means ± SEMs of biological triplicates. **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA test).(E) Tpres and Tresp cells were stained with surface CD25 and intracellular IL-17A, Foxp3, ROR?t, or IFN?. Two-color contour plots are on the left and quantification in the bar plots to the right. **p < 0.01; ****p < 0.0001 (2-tailed unpaired Student’s t test).(F) Quantification via qRT-PCR of mRNA expression of sorted Tpres, Tresp cells, and naive AND T cells. Data are presented as the means ± SEMs of n = 2–4 biological replicates normalized to the mean values of naive cells (set as 1). *p < 0.05; **p < 0.01 (2-tailed unpaired Student’s t test).

Fig 5: Abundance of DCs and antigen determines Treg versus Th17 response in vivo(A) Experimental setup of the immunization protocol with different numbers of DCs.(B) OT2 CD4 response to antigen was evaluated by measuring the expression of the CD44 activation marker as a function of the dose of OVAp-loaded DCs. Bar graphs show the means ± SEMs (n = 3 mice per condition; unpaired 2-tailed Student’s t test; **p < 0.01).(C) Two-color contour plots showing percentages of Foxp3+CD25+ Treg cells and IL-17A+CCR6+ Th17 cells within the CD45.2+CD4+ OT2 cell population in the function of the dose of DC APCs. Bar plots show the means ± SEMs (n = 3 mice per condition; unpaired 2-tailed Student’s t test; *p < 0.05; ***p < 0.001).(D) Experimental setup of intraperitoneal (i.p.) infection with increasing number of plaque-forming units (PFUs) of MVA-OVA.(E) CD8 T cell response to the virus was evaluated by measuring the percentage of I-Ab-OVAp tetramer+ cells within the CD8+ T cell population. Graph shows the means ± SEMs (n = 2–4 mice per condition; unpaired 2-tailed Student’s t test; **p < 0.01).(F) Quantification of the percentage of Foxp3+CD25+ Treg and IL-17A+CCR6+ Th17 within the splenic endogenous CD4 T cell population. Graphs show the means ± SEMs (n = 2–4 mice per condition; unpaired 2-tailed Student’s t test; *p < 0.05; **p < 0.01; ***p < 0.001).