IL35-Producing B Cells Promote the Development of Pancreatic Neoplasia

Abstract

A salient feature of pancreatic ductal adenocarcinoma (PDAC) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent establishment of a protumorigenic microenvironment. Here, we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia and PDAC lesions as well as in oncogenic Kras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic Kras was significantly compromised in B-cell-deficient mice (μMT), and this growth deficiency could be rescued by the reconstitution of a CD1d(hi)CD5(+) B-cell subset. The protumorigenic effect of B cells was mediated by their expression of IL35 through a mechanism involving IL35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL35-producing CD1d(hi)CD5(+) B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B-cell/IL35 axis as a therapeutic target.

Significance: This study identifies a B-cell subpopulation that accumulates in the pancreatic parenchyma during early neoplasia and is required to support tumor cell growth. Our findings provide a rationale for exploring B-cell-based targeting approaches for the treatment of pancreatic cancer.

Figure 1. B cells infiltrate mouse and human pancreatic neoplasia and promote growth of Kras^G12D-PDEC in vivo

(A) Immunohistochemical detection of B cells in human (CD20 staining) and mouse (B220 staining) in pancreata from hPanIN (20 patient samples), p48^Cre (control, 5 mice), KC (10 mice), or KRas^G12D-PDEC (9 mice) orthotopic lesions, as indicated. Inset, B cells in the parenchyma of an adjacent tissue section detected by immunofluorescence using anti-CD19 (green) and DAPI (blue). Representative images are shown. Scale bars, 100μm. (B) Hematoxylin and eosin (H&E) staining and immunohistochemical staining for CD20, CXCL13 and vimentin from serial sections in a representative sample of human pancreatic cancer containing PanIN lesions. Inset, sections of human PanIN lesions were stained by immunofluorescence (CXCL13, red; vimentin, green; and DAPI, blue). Scale bars, 100μm; inset 7.5μm. (C) Serial sections of a KC mouse pancreas were stained by immunohistochemistry with CXCL13 or immunofluorescence (n=10; CXCL13, red; vimentin, green; and DAPI, blue). A representative image is shown. Scale bars, 100μm and 12 μm (inset). (D) Expression of CXCL13 mRNA in cellular subsets isolated from pancreata of KC mice. Error bars indicate SD. (n=6) (E) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRas^G12D-PDEC implantation into WT or μMT mice were stained with H&E or anti-GFP antibody. Where indicated, μMT mice were reconstituted with WT B cells 2 days prior to orthotopic implantation. Representative images are shown. Scale bars, 100μm. (F) Graph depicts quantification of the data in (E) and indicates the average fraction of GFP⁺ signal per field of view (FOV; 10 FOV per animal; n=12 WT, n=14 μMT, n=9 μMT+WT B cell animals). Error bars indicate SD; P values were determined by Student's t-test (unpaired, two-tailed); p value: ***<0.001.

Figure 2. CD1d^highCD5⁺ B cells are expanded in pancreatic neoplasia and are functionally important for sustaining growth of Kras ^G12D-PDEC in vivo

(A) Quantification of flow cytometric analysis of plasma cells from spleens, mesenteric lymph nodes (MLN), and pancreata of p48^Cre (control) or KC mice. Cells were analyzed for the presence of markers CD19, B220 and CD138 (n=5 p48^Cre, n=5 KC). (B) Quantification of flow cytometric analysis of immune cells from pancreata of p48^Cre (control) mice, KC mice (2.5mo), or KRas^G12D-PDEC orthotopic lesions (2 weeks), as indicated. After gating on CD19 and CD1d populations, cells were analyzed for the presence of CD5 marker (n=8 p48^Cre, n=8 Kras^G12D-PDEC, n=8 KC). (C) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRas^G12D-PDEC implantation into WT or μMT mice were stained with anti-GFP antibody. Where indicated, μMT mice were reconstituted with WT CD19⁺CD1d^highCD5⁺ or with CD19⁺CD1d^lowCD5^- 2 days prior to orthotopic implantation. Representative images are shown. Scale bars, 100μm. (D) Graph depicts quantification of the data from (C) indicating the average fraction of GFP⁺ area per FOV of the implant (10 FOV per animal; n=12 WT, n=11 μMT, n=9 μMT+ CD1d^lowCD5^-, n=9 μMT+ CD1d^highCD5⁺, animals). (E) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRas^G12D-PDEC implantation into WT or μMT mice were stained with H&E or anti-GFP antibody. Where indicated, μMT mice were reconstituted with WT B cells or with IL10-/- B cells 2 days prior to orthotopic implantation. Representative images are shown. Scale bars, 100μm. (F) Graph depicts quantification of the data from (E) indicating the average fraction of GFP⁺ area per FOV of the implant (10 FOV per animal; n=14 WT, n=12 μMT, n=12 μMT+WT B cell, n=12 μMT+IL10-/- B cell animals). Error bars indicate SD; P values were determined by Student's t-test (unpaired, two-tailed); p value: *<0.05; **<0.01; ***<0.001; NS – not significant.

Figure 3. Expression of IL-35 by B cells is functionally important for sustaining growth of Kras^G12D-PDEC in vivo

(A) Levels of IL12a mRNA in immune cells from spleen or pancreata of p48^Cre (control) or KC mice were assessed by quantitative RT-PCR (n = 9 p48^Cre, n=9 KC). (B) Levels of IL12a mRNA in CD19⁺CD1d^highCD5⁺ and CD19⁺CD1d^lowCD5^- sub-populations of B cells sorted from pancreata of KC mice were assessed by quantitative RT-PCR (n=9 KC). (C) Levels of Ebi3 mRNA in B cells and non-B cells from pancreata of KC mice were assessed by quantitative RT-PCR (n=9 KC). (D) Levels of Ebi3 mRNA in CD19⁺CD1d^highCD5⁺ and CD19⁺CD1d^lowCD5^- sub-populations of B cells sorted from pancreata of KC mice were assessed by quantitative RT-PCR (n=9 KC). (E) Immunofluorescence staining for p35 and CD20 in samples of human pancreatic cancer containing PanIN lesions. Scale bars, 10μm (top) and 20μm (bottom). Two independent fields of view are shown. (F) Immunofluorescence staining for p35 and B220 in samples of KC pancreata. Scale bars, 20μm. Two independent fields of view are shown. (G) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRas^G12D-PDEC implantation into WT or μMT mice were stained with H&E or anti-GFP antibody. Where indicated, μMT mice were reconstituted with WT B cells or with IL12a-/- B cells 2 days prior to orthotopic implantation. Representative images are shown. Scale bars, 100μm. (H) Graph depicts quantification of the data from (G) indicating the average fraction of GFP⁺ area per FOV of the implant (10 FOV per animal; n = 9 WT, n=9 μMT, n= 9 μMT+WT B cell, n=9 μMT+IL12a-/- B cell animals). (I) Immunohistochemical staining for phospho-Histone H3 of GFP-KRas^G12D-PDEC implanted into mice as described in (G) above. Representative images are shown. Scale bars, 50μm. (J) Graph depicts quantification of the data in (I) and indicates the fraction of phospho-Histone H3⁺ signal in epithelial cells (10 FOV per animal; n=6 WT, n=6 μMT, n=6 μMT+WT B cell, n=6 μMT+IL12a-/- B cell animals). Error bars indicate SEM in A, SD in B-D, H, J; P values were determined by Student's t-test (unpaired, two-tailed); p value: *<0.05; **<0.01; ***<0.001.