Specialized dendritic cells induce tumor-promoting IL-10+IL-17+ FoxP3neg regulatory CD4+ T cells in pancreatic carcinoma

Abstract

The drivers and the specification of CD4⁺ T cell differentiation in the tumor microenvironment and their contributions to tumor immunity or tolerance are incompletely understood. Using models of pancreatic ductal adenocarcinoma (PDA), we show that a distinct subset of tumor-infiltrating dendritic cells (DC) promotes PDA growth by directing a unique T_H-program. Specifically, CD11b⁺CD103^- DC predominate in PDA, express high IL-23 and TGF-β, and induce FoxP3^neg tumor-promoting IL-10⁺IL-17⁺IFNγ⁺ regulatory CD4⁺ T cells. The balance between this distinctive T_H program and canonical FoxP3⁺ T_REGS is unaffected by pattern recognition receptor ligation and is modulated by DC expression of retinoic acid. This T_H-signature is mimicked in human PDA where it is associated with immune-tolerance and diminished patient survival. Our data suggest that CD11b⁺CD103^- DC promote CD4⁺ T cell tolerance in PDA which may underscore its resistance to immunotherapy.

Fig. 1

Distinct pancreatic ductal adenocarcinoma (PDA)-infiltrating dendritic cell (DC) promote tumor progression and regulate CD4⁺ T-cell differentiation. a CD45⁺ cells from the spleen or tumor of orthotopic PDA-bearing mice or from the spleen of control mice were gated using flow cytometry and tested for co-expression of CD11c and MHCII. CD11c⁺MHCII⁺ cells were then sub-gated and tested for expression of CD11b. Representative contour plots and quantitative data are shown. This experiment was repeated >5 times. b WT mice were made chimeric using bone marrow from CD11c.DTR mice. Cohorts were challenged with orthotopic PDA 7 weeks later. On day 12, mice began serial treatment with diphtheria toxin (DT) or phosphate-buffered saline (PBS) before sacrifice on day 25. Representative gross images of tumors and quantitative analysis of tumor volume are shown. This experiment had similar results for >5 times (scale bars = 1 µm). c–f PDA_TME DC were harvested on day 25 from tumor-bearing mice, loaded with either Ova_323–339 or Ova_257–264 peptide and administered in equal number i.p. to CD45.1 mice that had been transferred i.v. 2 days prior with equal numbers of CFSE-labeled OT-I and OT-II T cells. Parallel experiments were performed using PDA_spl. DC. On day 7 after either PDA_TME or PDA_spl DC.Ova administration, spleen and mesenteric lymph nodes (LNs) were harvested and CD45.2⁺ CD4⁺ and CD8⁺ T cells tested for co-expression of CD44 and CD62L and dilution of CFSE. c A schematic of the experimental regimen is depicted. d Quantitative results and e representative flow cytometry data for CD4⁺ T cells and f CD8⁺ T cells are shown. This experiment was repeated twice (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test), SEM

Fig. 2

PDA_TME dendritic cell (DC) exhibit a unique cytokine profile and promote IL-17 expression in naive CD4⁺ T cells. a–c Ova_323–339 peptide-pulsed PDA_TME DC and splenic DC controls were co-cultured with Ova-restricted CD4⁺ T cells at a 1:5 ratio for 5 days. Representative (a) and quantitative (b) intracellular cytokine expression in the CD4⁺ T cells are shown. Data are representative of five independent experiments using 3–6 mice per arm, showing similar results. c Concentration of IL-17A in the supernatants of co-cultures were determined. Squares and triangles denote data points from individual mice, pooled from two independent experiments. d PDA_TME DC and splenic DC controls were plated at a density of 5 × 10⁵ cells/ml without re-stimulation and supernatant cytokine concentrations were determined after 36 h in a cytometric bead array. Data are representative of experiments repeated >3 times. e Orthotopic pancreatic ductal adenocarcinoma (PDA) tumors and spleens were harvested from mice on day 25 post-implantation. Intracellular flow cytometric analysis of DC cytokine expression after 12 h of re-stimulation with LPS (1 μg/ml) is shown. Representative histograms are shown. Numbers represent median fluorescence indices (MFI). Data are representative of experiments repeated >3 times. f The frequency of IL-6, TGF-β1 and IL23p19, IL12/23p40 co-expression in PDA_TME DC and splenic DC controls were tested. Representative contour plots and quantitative data are shown (n = 5 per group). This experiment was repeated three times (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test), SEM

Fig. 3

Specific subsets of PDA_TME dendritic cell (DC) exhibit distinct phenotypic properties. a The gating strategy for subset analysis of PDA_TME and spleen DC based on expression of CD11b, CD103, and CD8α is depicted. b Frequencies of PDA_TME and spleen DC subsets based on expression of CD11b, CD103, and CD8α were analyzed. Representative contour plots and quantitative results are shown. Experiments were repeated >5 times. c Histogram plots comparing cytokine expression in distinct DC subsets are shown. Numbers adjacent to histograms represent MFI ÷ 10. Quantitative data based on MFI are shown. Dotted-line separates PDA_TME DC from splenic DC controls. Experiments were repeated three times. d The frequency of IL-6 and TGF-β1 co-expression in PDA_TME DC subsets was tested. Representative contour plots and quantitative data are shown. This experiment was repeated three times. e Enzymatic activity of ALDH in PDA_TME DC subsets was determined by the ALDEFLUOR assay. Gates were determined by respective DEAB controls (dotted histogram) for each individual sample. This experiment was repeated three times (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test), SEM

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

Fig. 5

Pancreatic ductal adenocarcinoma (PDA)-infiltrating dendritic cell (DC) subsets direct distinct CD4⁺ T-cell programs in PDA. a CFSE-labeled naive CD8⁺ or CD4⁺ polyclonal T cells isolated from the spleens of WT mice were cultured for 96 h on αCD3ε/αCD28-coated plates in the presence of conditioned media from 8-day OT-II CD4⁺ T cell/PDA_TME DC subset co-cultures. Polyclonal T cells were assessed by flow cytometry for CFSE dilution. Each individual well from the OT-II/PDA_TME DC subset co-cultures was used to generate conditioned media for a single well of polyclonal CD8⁺ and CD4⁺ T cells. Scatter plots are shown correlating the CFSE dilution of polyclonal T cells to the IL-10 expression of OT-II T cells from the respective OT-II/PDA_TME DC subset co-culture used to generate conditioned media. Linear regression was used to determine the best-fit line (solid) and 95% confidence intervals (dotted lines); p-values indicate significance of a non-zero slope, determined by an F-test. This experiment was repeated twice. b Ova_323–339 peptide-pulsed PDA_TME DC subsets and splenic DC subsets were co-cultured with Ova-restricted CD4⁺ T cells at a 1:5 ratio for 5 days. CD4⁺ T-cell expression of CD49b and CD39 were determined compared with isotype control. Representative histograms with MFIs are shown. This experiment was repeated >3 times (n = 3–6 mice). c Ova_323–339 peptide-pulsed PDA_TME DC subsets and splenic DC subsets were co-cultured with Ova-restricted CD4⁺ T cells at a 1:5 ratio for 5 days. CD4⁺ T-cell co-expression of CD39 and CD49b were determined. Representative contour plots (PDA_TME DC) and quantitative data (PDA_TME DC, PDA_Spl. DC, and Sham_Spl. DC) are shown. Experiments were repeated >3 times (n = 3–6 mice). d Tumor and spleen were harvested on day 25 from mice bearing orthotopic PDA. Tumor-infiltrating and splenic CD4⁺ T cells were assessed for expression of CD39. Representative and quantitative data are shown. This experiment was repeated >5 times. e WT mice were made chimeric using bone marrow from CD11c.DTR mice. Animals were challenged with orthotopic PDA 7 weeks later. On day 12, mice began serial treatment with diphtheria toxin (DT) or phosphate-buffered saline (PBS) before sacrifice and tumor harvest on day 25. Intra-tumoral CD4⁺ T cells were gated on flow cytometry and tested for expression of CD39, CD49b, CD73, AHR, IL-10, IL-17A, and FoxP3. This experiment was repeated >5 times (n = 3–10 mice per group). f KPC-derived tumor cells were orthotopically implanted in pancreata of WT mice admixed with either PBS or each PDA_TME DC subset previously harvested from other PDA tumors. Tumors were then harvested on day 25 and intra-tumoral CD4⁺ T cells were gated on flow cytometry and tested for expression of CD39, RORγt, and IL-10. This experiment was repeated twice (n = 5 per group). g PDA_TME DC subsets were tested for co-expression of TNFα and iNOS. Representative contour plots and quantitative data are shown. This experiment was repeated three times. h PDA_TME and PDA_spl. DC subsets were tested for expression of IL-27 compared with isotype control (data not shown). Representative contour plots and quantitative data are shown. This experiment was repeated >3 times (n = 5). i WT and IL-27R−/− mice were challenged with orthotopic PDA before sacrifice on day 25. Representative pictures of tumors and quantitative data of tumor weight are shown. Each dot represents data from a single mouse. This experiment was repeated three times (Scale bars = 1 µm). j Orthotopic PDA-bearing mice were serially treated with an iNOS inhibitor or vehicle. Tumor volume was measured at 25 days. Representative pictures of tumors and quantitative data of tumor weight are shown. Each dot represents data from a single mouse. This experiment was repeated twice (Scale bars = 1 µm). k WT and IL-27R−/− mice were challenged with orthotopic PDA before sacrifice on day 25. In addition, select cohorts of WT mice were treated with an iNOS inhibitor (n = 5 per group). CD4⁺ T-cell expression of CD39, IL-17A, IL-17F, and RORγt were determined. This experiment was repeated twice (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test), SEM

Fig. 6

The suppressive program in CD4⁺ T cells blunting antitumor immunity in human pancreatic ductal adenocarcinoma (PDA) is distinct from other human cancers. a Data from PDA patients in the TCGA dataset were stratified on the basis of RNAseq gene expression (PolyA⁺ IlluminaHiSeq). Kaplan–Meier survival data comparing the highest and lowest patient quartiles (with the exception of Il17a and Il17f) are represented as HR with 95% confidence intervals, calculated using the log-rank (Mantel–Cox) method. Statistical comparisons for Il17a and Il17f represent patients with positive expression of each respective gene versus patients with zero expression. HR values <1 indicate increased OS, whereas values >1 indicate decreased OS for each gene. The developed “validation signature” indicates the gene expression sum of the 7 Tr1-associated genes subtracted by 3 Treg-associated genes (ΣTr1–ΣT_REG). b Kaplan–Meier survival curve, plotting the highest (red) and lowest (blue) quartiles of PDA patients from the TCGA database stratified by expression of the ΣTr1–ΣT_REG signature, with tick marks indicating censored patients. P-value was determined using the log-rank (Mantel–Cox) method. c Kaplan–Meier survival curve, plotting the highest (red) and lowest (blue) quartiles of melanoma patients from the TCGA dataset, and analyzed with the same methods as in b. d, e The highest (red) and lowest (blue) quartiles of PDA (d) and melanoma (e) patients stratified by the ΣTr1–ΣT_REG validation signature were analyzed for cd3e and cd8a gene expression; p-values were determined by unpaired t-test with Welch’s correction. f, g Plots represent prf1 and gzmb gene expression in the tumors of patients from the highest (red) and lowest (blue) quartiles of the ΣTr1–ΣT_REG validation signature from PDA patients (f) and melanoma patients (g), using RNAseq data from the TCGA database. All dot plots were analyzed with unpaired t-tests with Welch’s correction. h Kaplan–Meier survival curve, plotting the highest (red) and lowest (blue) quartiles of PDA patients from the TCGA database stratified by expression of three-gene T_REG signature, with tick marks indicating censored patients. P-value was determined using the log-rank (Mantel–Cox) method. i Plots represent cd3e, cd8a, gzmb, and prf1 gene expression in the tumors of patients from the highest (red) and lowest (blue) quartiles of the three-gene T_REG signature from PDA patients. All dot plots were analyzed with unpaired t-tests with Welch’s correction (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)