Fig. 4

CD103^–CD11b⁺PDA_TME dendritic cell (DC) promote the differentiation of suppressive FoxP3^neg CD4⁺ T cells. a–e Ova_323–339 peptide-pulsed PDA_TME DC subsets were co-cultured with CFSE-labeled Ova-restricted CD4⁺ T cells for 5 days. a T-cell proliferation was determined by dilution of CFSE. Representative histograms and quantitative analysis of proliferation indices are shown. b T cells were analyzed for co-expression of CFSE and ICOS. Representative and quantitative data are shown. c CD4⁺ T cells were analyzed for co-expression of IFNγ and IL-17A. Representative contour plots and quantitative data are shown. Circles, squares, and triangles denote data points from individual mice, pooled from three independent experiments. d DC-CD4⁺ T-cell co-culture supernatant were analyzed for expression of IL-17A, IL-6, and IFNγ by cytometric bead array. e CD4⁺ T cells were analyzed for co-expression of T-bet/RORγt and FoxP3/RORγt. Representative contour plots and quantitative data are shown. Experiments were repeated three times with similar results. f KPC-derived tumor cells engineered to express Ovalbumin were implanted subcutaneously WT mice admixed with either phosphate-buffered saline (PBS) or Ova-restricted CD4⁺ T cells that had been entrained in vitro using each of the PDA_TME DC subsets or CD103^–CD11b⁺ splenic DC pulsed with Ova_323–339 peptide. Tumor volume was serially measured (n = 4–5 per group). g KPC-derived tumor cells were orthotopically implanted in pancreata of WT mice admixed with either PBS or each of the respective PDA_TME DC subsets that had been harvested from other pancreatic ductal adenocarcinoma (PDA) tumors. Representative gross images of intra-pancreatic tumors and quantitative data on tumor weight on day 25 are shown. This experiment was repeated twice (n = 5 per group; scale bars = 1 µm). h Ova_323–339 peptide-pulsed PDA_TME DC subsets were co-cultured with CFSE-labeled Ova-restricted CD4⁺ T cells for 5 days. T cells stimulated with each respective DC subset were co-stained for IL-10 and either IL-17A, IFNγ, or FoxP3. Expression of IL-10 in pan-CD4⁺ T cells and in IL-17A⁺, IFNγ⁺, and FoxP3⁺ CD4⁺ T cells was determined. Representative histogram overlays and quantitative data are shown. This experiment was repeated three times (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test), SEM