Page 1 of 57 Diabetes CCL21 Expression in -Cells Induces Antigen-Expressing Stromal Cell Networks in the Pancreas and Prevents Autoimmune Diabetes in Mice Running Title: Diabetes Prevention by CCL21-induced Pancreatic TLOs Freddy Gonzalez Badilloa,e, †, MS, Flavia Zisi Tegoua,e, †, MS, Maria M. Abreua, †, PhD, Riccardo Masinaa, BS, Divya Shaa, MS, Mejdi Najjara, BS, Shane Wrighta, BS, Allison L. Bayera,b, PhD, Éva Korposf, PhD, Alberto Pugliesea,b,c, MD, R. Damaris Molanoa, DVM, Alice A. Tomeia,d,e*, PhD aDiabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA bDepartment of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida, USA cDepartment of Medicine, Division of Diabetes, Endocrinology and Metabolism, University of Miami Miller School of Medicine, Miami, Florida, USA dDepartment of Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA eDepartment of Biomedical Engineering, University of Miami, Miami, FL, USA fInstitute of Physiological Chemistry and Pathobiochemistry and Cells in Motion (CiM), Cluster of Excellence, University of Muenster, Muenster, Germany *Corresponding author: Alice A. Tomei, 1450 NW 10th Avenue, Miami, FL-33136, USA; Phone: +1 305-243-3469; Email: [email protected] †These authors contributed equally to the work Diabetes Publish Ahead of Print, published online August 1, 2019 Diabetes Page 2 of 57 Abstract Tumors induce tolerance towards their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, non-obese 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 naïve 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 to non- transgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and higher proportion of Tregs in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for regulation of autoimmunity and while 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 T1D 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. Keywords Tertiary lymphoid organs, fibroblastic reticular cells, tolerance, non-obese diabetic Page 3 of 57 Diabetes Abbreviations BECs Blood Endothelial Cells DCs dendritic cells DNs Double Negative Lymphoid Stromal Cells EFP Epididymal Fat Pad FRCs Fibroblastic Reticular Cells GSIR Glucose-Stimulated Insulin Release KD Kidney Subcapsular Space LECs Lymphatic Endothelial Cells LN Lymph Node NOD Non-Obese Diabetic SCID Severe Combined Immunodeficiency T1D Type I Diabetes TLOs Tertiary Lymphoid Organs Tregs Regulatory T cells IL-1 Interleukin-1 TNF Tumor necrosis factor IL-6 Interleukin-6 IFNγ Interferon gamma LT Lymphotoxin MHC Major histocompatibility complex imDC immature DCs Diabetes Page 4 of 57 CTL Cytotoxic T lymphocyte Introduction Type 1 diabetes (T1D) is an autoimmune disease characterized by the progressive destruction of insulin-producing β-cells in pancreatic islets, resulting in hyperglycemia and insulin dependency (1; 2). Failure of central and peripheral immunological tolerance to islet cell autoantigens mediate T1D (3). Tumor cells are able to induce tolerance and promote their own survival (4). One mechanism utilized by tumor cells to induce tolerance is by secreting the secondary lymphoid chemokine CCL21 (5; 6). CCL21 is expressed by endothelial cells of high endothelial venules (HEV), fibroblastic reticular cells (FRCs) in the lymph node (LN) paracortex and by lymphatic endothelial cells (LECs). CCL21 promotes interactions that are crucial to the adaptive T cell immunity, by attracting various immune cell types expressing its receptor, CCR7, including dendritic cells, regulatory T cell (Treg) and naïve T cells (7-9). CCR7 signaling is critical for peripheral tolerance as it is required for Treg activation in the LN (10-12). Autologous secretion of CCL21 by melanoma cells is required for immune tolerance to melanoma antigens and is dependent on the induction of tolerogenic tertiary lymphoid organs (TLO) (6). TLO formation is reported in many organs during autoimmune diseases, chronic infections, inflammation, in allogeneic transplantation (13-20), and in the fetal pancreas (21); however, the role of TLOs in modulating immunity and self-tolerance remains unclear. In the pancreas of non- Page 5 of 57 Diabetes obese diabetic (NOD) mice, islet infiltration is associated with TLO formation. Characterized by compartmentalization of T and B cell infiltrates as well as appearance of high endothelial venules (HEVs), TLOs are considered sites of antigen presentation and activation of the immune response (22; 23). In a C57BL/6 mouse model, Luther et al. showed that TLO formation in the endocrine pancreas is induced by ectopic expression of CCL21 by the pancreatic islets without any signs of diabetes development (24). The presence and function of FRCs, which induce peripheral tolerance in LNs (25), remain to be elucidated in pancreatic TLOs in T1D. Here, we investigated CCL21 as a novel regulator of immune tolerance to self- molecules implicated in the development of T1D. Local secretion of CCL21 in the pancreas of NOD mice was associated with the formation of TLOs containing β-cell autoantigen-expressing FRC-like cells, which induced systemic regulation of diabetogenic splenocytes. Diabetes Page 6 of 57 Research Design and Methods Mice All animal procedures were approved by the Institutional Animal Care and Use Committee of University of Miami. Female NOD.Cg-Tg (Ins2-Ccl21b)2Cys/JbsJ (herein referred to as Ins2- CCL21) mice, NOD.CB17-Prkdcscid/J (NOD-scid), NOD/ShiLtJ, BALB/cJ, and C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). Histological Evaluation, Immunofluorescence and Insulitis Grading Sections from formalin-fixed paraffin-embedded or OCT frozen blocks were stained and imaged as reported (26). Islet size, Treg and FRCs density within pancreatic islets and islet size were quantified with ImageJ (NIH). Insulitis was graded depending on the percent of lymphocyte infiltration in the islet; 0%=grade 1, 1-10%=grade 2, 10-25%=grade 3, 25-50%=grade 4, and >50%=grade 5. Islet isolation, culture, in vitro functionality and CCL21 assays. Isolation of murine pancreatic islets were performed at the DRI Preclinical Cell Processing and Translational Models Core as described (27). Glucose stimulated insulin release (GSIR) was performed as described (27). CCL21 levels were measured by ELISA (R&D Systems, Minneapolis, MN). Diabetes Induction, Blood Glucose Monitoring, Islet and Skin Transplantation, Adoptive Transfer of Splenocytes Page 7 of 57 Diabetes Diabetes was chemically-induced with a single intravenous injection of 200mg/kg streptozotocin (STZ) (27) or with 50 mg/kg of STZ on five consecutive days. Diabetes was determined by three, consecutive readings of non-fasting glycemic values above 250mg/dL. Transplants in the renal subcapsular space (KD) were performed as described (26-28). Islet transplant experiments and donor age/gender can be found in in Table 1 and Table S2. Skin grafting was performed as described (29). Adoptive transfer experiments by intravenous (IV) injection are summarized in Table 2. Immune cell isolation from pancreas, spleens, LNs and blood LNs and spleens were processed by manual disruption. Collagenase D (Sigma) was used for the pancreatic distention and cell isolation. Single cell suspensions were stained for live/dead (Invitrogen) and using the following anti-mouse antibodies: CD3, CD8, CD44, CD62L, CD25, CD127, Ki-67, B220 (BD Bioscience), CD4, FoxP3 (eBioscience), CD45 (Biolegend) and acquired on a CytoFLEX or a BD LSRII. Tetramer staining for insulin and IGRP (NIH Tetramer Core) is detailed in Table S1. FRC isolation from islets and LNs FRCs were isolated from skin draining LNs (axillary, brachial and inguinal) and pancreatic islets by adapting published protocols (30). Briefly, harvested tissues were digested with Dispase II, DNAse I and Collagenase P for 1 hour maximum. Every 15 Diabetes Page 8 of 57 minutes, released cells were collected on ice, while fresh enzyme solution was added to the undigested tissue. Single cell suspensions were stained with the following antibodies: gp38-PE (eBioscience), CD31-APC (Biolegend) and CD45-PeCy7 (Tonbo Biosciences) and sorted using a Beckman Coulter MoFlo Astrios EQ. LN FRCs and islet-derived FRC-like cells were gated as CD45- CD31- gp38+ cells. Characterization of FRC-like cells by RNA sequencing RNAseq Sample Preparation and Sequencing. Total RNA was extracted from FRCs freshly sorted from LNs or from pancreatic islets using TRIzol reagent (Invitrogen) and the RNeasy microKit (Qiagen). Preparation and sequencing of RNA libraries was carried out in the John P.