CCL21 Expression in β-Cells Induces Antigen-Expressing Stromal Cell Networks in the Pancreas and Prevents Autoimmune Diabetes in Mice

Abstract

Tumors induce tolerance toward their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, nonobese diabetic (NOD) mice express CCL21 in pancreatic β-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes. Ins2-CCL21 mice develop TLOs by 4 weeks of age, consisting of naive CD4⁺ T cells compartmentalized within networks of CD45^-gp38⁺CD31^- fibroblastic reticular cell (FRC)-like cells. Importantly, 12-week-old Ins2-CCL21 TLOs contained FRC-like cells with higher contractility, regulatory, and anti-inflammatory properties and enhanced expression of β-cell autoantigens compared with nontransgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and a higher proportion of regulatory T cells in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for the regulation of autoimmunity, and although the effect is systemic, the induction is mediated locally likely by lymphocyte trafficking through TLOs. Overall, our findings suggest that CCL21 promotes TLOs that differ from inflammatory TLOs found in type 1 diabetic islets in that they resemble lymph nodes, contain FRC-like cells expressing β-cell autoantigens, and are able to induce systemic and antigen-specific tolerance leading to diabetes prevention.

Figure 1

CCL21 local secretion by β-cells prevents autoimmune diabetes in Ins2-CCL21 transgenic NOD mice. A: Percent of diabetes-free Ins2-CCL21 NOD (96%, red, n = 25) and nontransgenic littermate control (40%, black, n = 10) female mice. B: Confocal images of pancreatic sections of 4-week-old nontransgenic and Ins2-CCL21 NOD mice immunostained with insulin (red) and CCL21 (green) antibodies. Nuclei were counterstained with DAPI (gray). Scale bar, 50 μm. C: CCL21 concentrations in the supernatant of pancreatic islets isolated from 19- to 33-week-old nontransgenic (black) and Ins2-CCL21 NOD (red) mice stimulated with 2.2 mmol/L (LG) followed by 16.7 mmol/L (HG) glucose. ND, not detectable. ***P < 0.01. D: Normal (P = 0.83), fasting (P = 0.98), and postprandial (P = 0.23) secretion of CCL21 in the serum of 6- to 7-week-old nontransgenic (n = 3) compared with Ins2-CCL21 NOD (n = 3) mice. E: Confocal images of pancreatic sections of 21- and 52-week-old Ins2-CCL21 NOD mice stained with insulin (red) and Ki67 (green) antibodies. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. F: Quantification of islet size from pancreatic sections of 4- (n = 6), 8- (n = 5), and 12-week-old (n = 5) nontransgenic littermate control and age-matched Ins2-CCL21 NOD mice. G: Insulin secretion after stimulation of islets from 19- to 33-week-old nontransgenic (black, n = 3) and Ins2-CCL21 NOD (red, n = 3) mice with 2.2 mmol/L (LG) followed by 16.7 mmol/L (HG) glucose. Islets were isolated from a minimum of five mice per experiment and plated in n = 3 wells per condition. H: Percent of diabetes-free 10-week-old Ins2-CCL21 NOD (red, n = 6) or nontransgenic (black, n = 4) NOD mice treated daily with five low doses (50 mg/kg) of STZ. I: Percent survival of C57BL/6 fully allogeneic (left) or syngeneic NOD (right) skin grafts into Ins2-CCL21 mice (red, n = 5, median survival rate: 14 days) or control nontransgenic littermate NOD mice (black, n = 5). wks, weeks.

Figure 2

CCL21 local secretion by β-cells does not prevent insulitis. A: Light and confocal microscope images of pancreatic sections of 4-week-old nontransgenic and Ins2-CCL21 NOD mice stained with hematoxylin and eosin (left) or with insulin (red) and glucagon (green) (right) antibodies. Nuclei were counterstained with DAPI (white). Scale bar, 50 μm. B: Insulitis quantification (grade 1–5) of pancreatic sections of 4- (n = 6), 8- (n = 5), and 12-week-old (n = 5) nontransgenic and age-matched Ins2-CCL21 NOD mice. Four-week-old nontransgenic NOD mice: 86% grade 1, 2% grade 2, 11% grade 3, 1% grade 4, and 1% grade 5. Four-week-old Ins2-CCL21 NOD mice: 62% grade 1, 9% grade 2, 22% grade 3, 6% grade 4, and 1% grade 5. C: Quantification of pancreatic infiltrates of 4-week-old nontransgenic (n = 5) and Ins2-CCL21 NOD (n = 5) mice by flow cytometry. Data are indicated as % CD4⁺ live cells. T_n: naive T cells (CD44⁻CD127⁻CD62L⁺) transgenic: 38.05 ± 0.08, nontransgenic: 19.45 ± 4.62, P < 0.001; T_ef (effector T cells): CD44⁺CD127⁻; T_m (memory T cells): CD44⁺CD127⁺. D: Quantification of blood leukocytes of 4-week-old nontransgenic (n = 5) and Ins2-CCL21 NOD (n = 5) mice by flow cytometry. Data are indicated as % CD45⁺ live cells. CCR7⁺: transgenic, 13 ± 11; nontransgenic, 3 ± 3 (P < 0.05). E: Confocal images of pancreatic sections of 4-week-old nontransgenic and Ins2-CCL21 NOD mice stained with insulin (cyan), B cells (B220, red), and T cells (CD3, green) antibodies; insulin (red) and CD4 (green); insulin (red) and CD8 (green); insulin (cyan), T cells (CD3, green), and Tregs (CD3⁺FoxP3⁺, red) antibodies. Scale bar, 50 μm. F: Confocal images of skin-draining LN sections of 4-week-old Ins2-CCL21 NOD mice immunostained with B220 (red) and CD3 (green) at two different magnifications. Scale bar, 50 μm. G: Quantification of Treg density in islets from pancreatic sections of 4-week-old nontransgenic (n = 4) and age-matched Ins2-CCL21 (n = 4) NOD mice. H: Quantification of pancreatic infiltrates of 12-week-old nontransgenic littermate (n = 6) and Ins2-CCL21 NOD (n = 6) mice by flow cytometry. Data are indicated as total number of live cells. wks, weeks.

Figure 3

Splenocytes from transgenic mice are not diabetogenic. A: ATs of splenocytes from 20- to 25-week-old Ins2-CCL21 NOD mice (100%, red, n = 12) or from age-matched diabetic nontransgenic mice (0%, black, n = 3, median transfer time 28 days) into 6-week-old NOD-scid mice. At least three donors per group, two recipients per donor, and 10 million cells per AT were used. ***P < 0.01. Experimental details are reported in Table 2. B: Quantification of CD44⁺ IGRP-reactive CTLs in pancreatic islets (P < 0.05) and in blood (P < 0.05) of 12-week-old Ins2-CCL21 NOD mice (n = 5) compared with nontransgenic littermate mice (n = 5). Pancreatic islets: Ins2-CCL21 NOD, 0.11 ± 0.3%; nontransgenic NOD, 4.64 ± 2.31%, P < 0.05. Blood: Ins2-CCL21 NOD, 0.10 ± 0.10%; nontransgenic NOD, 8.34 ± 9.68%, P < 0.05. C: Quantification of CD44⁺ INS-reactive CTLs in pancreatic islets (P < 0.05) and blood (P = 0.08) of 12-week-old Ins2-CCL21 NOD mice (n = 5) compared with nontransgenic littermate NOD mice (n = 5). Pancreatic islets: Ins2-CCL21 NOD, 1.23 ± 0.17%; nontransgenic NOD, 3.28 ± 1.04%, P = 0.01. Blood: Ins2-CCL21 NOD, 21.77 ± 10.66%; nontransgenic NOD, 10.82 ± 3.60%, P = 0.08. Data are indicated as % CD8⁺ cells. wks, weeks.

Figure 4

CCL21 local secretion by β-cells induces formation of TLOs containing FRC-like stromal cells. A: Confocal microscope images of pancreatic sections of 4-week-old nontransgenic and Ins2-CCL21 NOD mice and of skin-draining LN, stained with insulin (cyan), gp38 (stromal cell marker, green), Lyve-1 (lymphatic endothelial cell marker, red) antibodies, and nuclear counterstain (DAPI, white). FRC-like cells are identified by gp38⁺Lyve-1⁻. Scale bar, 50 μm. B: Quantification of FRC density in pancreatic islets of 4-week-old nontransgenic (n = 24 islets) and Ins2-CCL21 NOD (n = 36 islets) mice by image analysis of pancreatic sections. At least n = 3 mice per group were analyzed. C: Confocal microscope images of pancreatic sections of 4- to 16-week-old nontransgenic mice stained with insulin (red), glucagon (cyan), stromal cell (gp38, green) antibodies, and nuclear counterstain (DAPI, gray). Scale bar, 50 μm. D: Confocal microscope images of pancreatic sections of 52-week-old transgenic mice stained with insulin (red), Lyve-1 (cyan), FRC (ER-TR7, green) antibodies (left), and HEV antibodies (CD31: green; PNAd: red) (right); nuclear counterstain: DAPI (blue). Scale bar, 50 μm. Gating strategy (E) and relative quantification of CD45⁻CD31⁻gp38^dim (F) and of CD45⁻CD31⁻gp38^hi (G) FRCs from the skin-draining LNs and FRC-like cells from the pancreatic islets of 4-week-old nontransgenic (n = 4, black) and Ins2-CCL21 NOD mice (n = 4, red) by flow cytometry. H: Relative proportions of CD45⁻CD31⁻gp38^dim and of CD45⁻CD31⁻gp38^hi FRC-like cells from the pancreatic islets of 12-week-old nontransgenic (n = 5, black) and Ins2-CCL21 NOD mice (n = 5, red) by flow cytometry. **P ≤ 0.01, ***P ≤ 0.001. wks, weeks.

Figure 5

Local CCL21 expression by β-cells is sufficient to induce FRC-like cell–containing TLOs and mediate systemic protection from diabetes. A: ATs of splenocytes from diabetic nontransgenic NOD mice into 10-week-old Ins2-CCL21 NOD mice (red, n = 6, median survival time 48 weeks, P = 0.007) or into age-matched nontransgenic mice (black, n = 5, median survival time 18 weeks). B: Percent of diabetes-free spontaneously diabetic NOD mice transplanted (epididymal fat pad) with 1,000 islet equivalents (IEQ) islets from Ins2-CCL21 NOD (red, n = 4) or nontransgenic (black, n = 1) NOD mice. Experimental details are reported in Table 2. C: AT of splenocytes from recently diabetic NOD mice into 7- to 11-week-old NOD-scid mice transplanted (TX) in the kidney capsule (KD) with a marginal dose (500 IEQ/mouse) of islets from either Ins2-CCL21 NOD (red, n = 7, median survival time 22.5 days, P = 0.0002) or nontransgenic (black, n = 6, median survival time 75 days) mice 4 weeks prior to injection. Confocal microscope images of KD islet graft sections (KD graft) (left) and of pancreatic sections (right) of 7-week-old NOD-scid mice that received either nontransgenic (D) or Ins2-CCL21 transgenic (E) NOD islets 4 weeks before. F: Insulitis quantification (grades 1–5) of pancreatic sections of NOD-scid mice before (left) and after (right) AT of diabetic splenocytes. Mice had received a transplant of either NOD (n = 3 for both timepoints) or Ins2-CCL21 NOD (n = 3 after transplant and n = 4 after AT) islets under the KD 4 weeks before the AT. G and H: Confocal microscope images of KD islet graft sections (KD graft) (left) and pancreatic sections (right) of NOD-scid mice that received either nontransgenic (G) or Ins2-CCL21 transgenic (H) NOD islets followed by AT of splenocytes from recently diabetic NOD mice. Sections were stained for insulin (red), stromal cells (gp38, green), and glucagon (cyan) (top panels) or for T cells (CD3, red), insulin (green), and B cells (B220, cyan) (bottom panels). Nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. wks, weeks.

Figure 6

Mechanism of FRC-like cell–mediated immune regulation. A: Heat map of T1D autoantigen expression in gp38^dim FRC-like cells freshly sorted from either Ins2-CCL21 NOD mice or nontransgenic NOD littermates analyzed byRNAseq. T1D autoantigens: carboxypeptidase E (Cpe), pancreatic and duodenal homeobox 1 (Pdx1), islet cell autoantigen 1 (Ica1), chromogranin A (Chga), pancreatic polypeptide (Ppy), islet amyloid polypeptide (Iapp), solute carrier family 30 member 8 (Slc30a8), insulin 2 (Ins2), protein tyrosine phosphatase, receptor type N (Ptprn), glucagon (Gcg), insulin (Ins1), and regenerating family member 3 α (Reg3a). B: Confocal microscope images of pancreatic sections of 4-week-old nontransgenic (top) and Ins2-CCL21 transgenic (bottom, different magnifications) NOD mice immunostained with insulin (red), pan-laminin (gray), stromal cell (gp38, green) antibodies, and nuclear counterstain (DAPI, blue). Scale bar, 50 μm. Bottom panels are higher magnifications of the areas indicated by the white boxes. C: Principal component analysis (PCA) of gene expression in LN-derived FRCs (n = 4 for nontransgenic NOD and n = 3 for Ins2-CCL21 NOD), islet-derived gp38^dim FRC-like cells (n = 4 per mouse model), islet-derived gp38^hi FRC-like cells (n = 2 per mouse model), and islet-derived CD45⁺ cells (n = 4 per mouse model) freshly sorted from 12-week-old Ins2-CCL21 NOD mice and from control NOD littermates. D: Heat map of significantly relevant (false discovery rate <0.05) inflammatory genes (list of genes taken from the Nanostring Immunology panel available on their website) in islet-derived gp38^dim FRC-like cells from Ins2-CCL21 NOD mice compared with nontransgenic littermates. E: Heat map of inflammatory genes (Nanostring Immunology panel) expressed by LN-derived FRCs, islet-derived gp38^dim FRC-like cells, islet-derived gp38^hi FRC-like cells, and islet-derived CD45⁺ cells freshly sorted from 12-week-old Ins2-CCL21 NOD mice (right) or nontransgenic littermates (left). wks, weeks.