CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice

Abstract

Pancreatic cancer, one of the deadliest human malignancies, is associated with oncogenic Kras and is most commonly preceded by precursor lesions known as pancreatic intraepithelial neoplasias (PanIN). PanIN formation is accompanied by the establishment of an immunotolerant microenvironment. However, the immune contribution to the initiation of pancreatic cancer is currently poorly understood. Here, we genetically eliminate CD4+ T cells in the iKras* mouse model of pancreatic cancer, in the context of pancreatitis, to determine the functional role of CD4+ T cells during mutant Kras-driven pancreatic carcinogenesis. We show that oncogenic Kras-expressing epithelial cells drive the establishment of an immunosuppressive microenvironment through the recruitment and activity of CD4+ T cells. Furthermore, we show that CD4+ T cells functionally repress the activity of CD8+ T cells. Elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells and blocks the onset of pancreatic carcinogenesis. Thus, our studies uncover essential and opposing roles of immune cells during PanIN formation and provide a rationale to explore immunomodulatory approaches in pancreatic cancer.

Figure 1

CD4⁺ T cells are required for pancreatic tumorigenesis. A, genetic makeup of the iKras*;CD4^{− / −} mouse model. B, experimental design; n = 3–10 mice per cohort. C, hematoxylin and eosin (H&E) staining of control, iKras*, and iKras*;CD4^{− / −} pancreata 1 day, 1 week, 3 weeks, 8 weeks, and 17 weeks after pancreatitis induction. Scale bar, 50 μm. D, pathologic analysis. Data, mean ± SEM; n = 3-5 mice per cohort. The statistical difference between iKras* and iKras*;CD4^{− / −} mice at the same time point per lesion type was determined by two-tailed Student t tests. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; #, not statistically significant. Doxy, doxycycline.

Figure 2

CD4⁺T cells regulate proliferation and survival of PanIN cells. A, Ki67 immunohistochemistry staining and B,TUNEL (red) and coimmunofluorescence staining for CK19 (green), SMA (gray), and DAPI (blue) in iKras* and iKras*;CD4^{− / −} pancreata 1, 3, 8, and 17 weeks following pancreatitis. Scale bar, 25 μm. C Ki67 proliferation index in ADM/PanIN, stroma, and acinar cells and D, quantification of apoptotic cells in epithelial and stroma compartments of iKras* and Kras*;CD4^{− / −} pancreata. Data, mean ± SEM, n = 3 mice per cohort. The statistical difference between iKras* and iKras*;CD4^{− / −} mice at the same time point per cell type was determined by two-tailed Student t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3

Characterization of pancreatic immune infiltrates. Pancreatic immune cell infiltrates at 1 day and 1 week following pancreatitis induction were measured by flow cytometry to determine the percentage of CD45⁺ leukocytes (A), CD3⁺ T cells (B), CD3⁺CD8⁺ T cells (C), CD3⁺CD4⁺ T cells (D), CD3⁺CD4⁺CD25⁺FoxP3⁺ Tregs (E), CD11b⁺F4/80⁺ macrophages (F), CD11b⁺Gr-1⁺ (G), CD11b⁺Gr-1^low (H), and CD11b⁺Gr-1^high (I) immature myeloid cells. Data, mean ± SEM; the statistical difference between experimental mice was determined by Student t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4

Intrapancreatic Treg, Th17, andCD8⁺T-cell infiltrations during PanIN formation. A, percentage of Tregs out of CD3⁺CD4⁺Tcells in control and iKras* pancreata 1 day, 1 week, and 3 weeks after pancreatitis induction. B, percentage of Th17 cells out of CD3⁺CD4⁺ T cells in control and iKras* pancreata 1 day and 1 week after pancreatitis induction. Data, mean ± SEM; the statistical difference between experimental mice was determined by Student t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, FoxP3 immunohistochemistry staining in control, iKras*, and iKras*;CD4^−/− pancreata 1 day, 1 week, and 3 weeks following pancreatitis. Scale bar, 25 μm. D, quantification of CD8⁺ cells in control, iKras*, and iKras*;CD4^−/− pancreata 1 day and 3 weeks following pancreatitis induction. Data, mean ± SEM; n = 3 mice per cohort. The statistical difference between experimental mice was determined by Student t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. E, CD8 immunohistochemistry staining (scale bar, 25 μm). HPF, high-power field.

Figure 5

Enhanced CD8⁺ T-cell activity and PanIN-specific CD8⁺ T cells in iKras*;CD4^−/− Mice. A-C, intracellular cytokine staining (IFN-γ)/CD107a assay. IFN-γ and CD107a expression in CD45⁺CD8⁺CD11b^negCD11c^neg CD4^negCD19^neg cells derived from control, iKras*, and iKras*;CD4−/− pancreata was analyzed with flow cytometry at 1 day (A), 1 week (B), and 3 weeks (C) following pancreatitis induction. D, anti-PanIN CD8⁺ T-cell activity assay. GFP⁺ PanIN cells and CD3⁺CD8⁺ T cells derived from PanIN-bearing mice (pancreata and spleen) were purified by flow cytometry, and then cocultured for 4 hours. The expression of lfnγ, Gzmb, and Prf-1 was analyzed by qRT-PCR. CD3⁺CD8⁺ T cells derived from wt spleen were used as control. Data, mean ± SEM; each point indicates one sample. The statistical difference was determined by Student t tests.*, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6

CD8⁺ T-cell depletion rescues pancreatic carcinogenesis in iKras*;CD4^−/− mice. A, experimental design; n = 5–8 mice per cohort. B, hematoxylin and eosin (H&E) staining of control and iKras* pancreata 3 weeks following pancreatitis. Scale bar, 50 μm. C, H&E, PAS, Trichrome staining, Claudin 18 immunohistochemistry and D, p-ERK1/2, Ki67, cleaved caspase-3, and CD8 immunohistochemistry in control or anti-CD8-treated iKras*; CD4^−/− mice 3 weeks following pancreatitis. Scale bar, 50 μm. E, percentage of CD3⁺CD8⁺ T cells in control or anti-CD8-treated iKras*;CD4^−/− pancreata 3 weeks following pancreatitis measured by flow cytometry. Data, mean ± SEM; each point indicates one animal. F, pathologic analysis. Data, mean ± SEM; n = 3–5 mice per cohort. The statistical difference between control and anti-CD8-treated iKras*;CD4^−/− mice at the same time point per lesion type was determined by two-tailed Student t tests. *, P < 0.05; **, P < 0.01; #, not statistically significant.

Figure 7

Working model indicating that CD4⁺ T cells are required for oncogenic Kras*-induced pancreatic cancer formation and progression. A, CD4⁺ T cells contribute to pancreatic carcinogenesis by blocking the antitumor activity of CD8⁺ T lymphocytes. B, CD4⁺ T-cell loss dampens neoplastic progression by enabling CD8⁺ T-cell effector function. C, ablation of CD8⁺ T cells rescues pancreatic carcinogenesis in the CD4⁺ T cell-deficient pancreatic cancer mouse model.