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. Hussman Institute for Human Genomics Center for Genome Technology. At least

10ng of total RNA verified by Agilent Bioanalyzer was used as input for the KAPA RNA

HyperPrep Kit with RiboErase (HMR), to create ribosomal RNA depleted sequencing

libraries, including sample indexing to allow for multiplexing. Cluster generation and

sequencing was carried out using the Illumina cBOT and HiSeq 3000, generating >32

million single-end 100 base reads per sample.

RNAseq Analysis. De-multiplexed FASTQ files were created with the Illumina supplied scripts in the BCL2FASTQ software (v2.17). The quality of the reads was determined with

FASTQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for per base sequence quality, duplication rates, and overrepresented k-mers. Illumina adapters Page 9 of 57 Diabetes

were trimmed using the Trim Galore! package

(http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and aligned to the

mouse reference genome (mm10) with the STAR aligner (v2.5.0a) (31) with default

alignment parameters. Gene count quantification for total RNA was performed using the

GeneCounts function within STAR against the GENCODE vM19 transcript file.

Differential expression analysis. Gene count data was input into edgeR for differential

expression analysis. Briefly, gene counts were normalized using the trimmed mean of M-

values (TMM) (32) method to account for compositional difference between the libraries.

Differential expression between groups was calculated with the exact test implemented

in edgeR.

Statistical Analysis

Prism 6 was used for all data analysis. Data are presented as mean ± standard deviation.

ANOVA with Tukey post-hoc test was performed on data sets with more than two

groups. A confidence level of 95% was considered significant. Actuarial survival curves

and log-rank test were used to compare diabetes reversal and graft survival amongst

experimental groups. Diabetes Page 10 of 57

Results

CCL21 local secretion by β-cells prevents autoimmune diabetes in Ins2-CCL21 transgenic NOD

mice

Ins2-CCL21 transgenic NOD mice do not develop diabetes (J. Bluestone, unpublished

communication, https://www.jax.org/strain/006254). In our colony, 96% of female transgenic

mice remained diabetes-free at 50 weeks of age compared to 40% littermate control female mice

(p<0.001, Fig. 1A).

CCL21 was expressed (Fig. 1B) and secreted (Fig. 1C) by pancreatic β-cells from

transgenic mice. CCL21 secretion from cultured transgenic islets was influenced by glucose

concentrations in vitro; the observation that serum levels of CCL21 did not differ between Ins2-

CCL21 NOD mice and control littermates suggests that CCL21 is consumed locally (Fig. 1D).

CCL21 expression in β-cells did not induce either β-cell proliferation (Fig. 1E) or islet

hypertrophy (Fig. 1F) or increased glucose-stimulated insulin release (GSIR) from isolated islets

(Fig. 1G). Additionally, β-cells from transgenic NOD islets were equally sensitive to STZ as β- cells from NOD islets (Fig. 1H). Overall, these data show that CCL21 expression in β-cells protects

from diabetes development independent of β-cell function, replication and resistance to toxicity.

We also transplanted Ins2-CCL21 mice with fully allogeneic B6 skin grafts and syngeneic

NOD grafts. Ins2-CCL21 NOD mice rejected B6 skin allografts at a similar rejection rate as NOD

mice, while syngeneic skin grafts were accepted in both mice strains (Fig. 1I). So, protection from

diabetes development of CCL21-expressing β-cells is not caused by impaired immune function. Page 11 of 57 Diabetes

CCL21 local secretion by β-cells does not prevent insulitis

At 4 weeks of age, most (86%) female NOD mice exhibited no insulitis and only 14% showed

grade 2-5 insulitis (Fig. 2A-B); in contrast, 83% of Ins2-CCL21 mice showed grade 2-5 insulitis

in 38% of islets. These results suggest that CCL21 expression did not prevent islet infiltration, yet

diabetes did not develop.

The relative proportions of naïve CD4+ T cells (Fig. 2C, p<0.001) and of CCR7+CD62L+

leukocytes (Fig. S1A, p<0.01) were increased in the pancreas of transgenic mice compared to

NOD mice. Additionally, the relative proportions (Fig. S1B and Fig. 2D, p<0.05) but not the total

number of circulating CD3+, CD4+, and CD45+CCR7+ cells and the total number of splenic CD3+,

+ + CD4 , Tregs and naïve CD4 T cells (Fig. S1C) were lower in transgenic compared to NOD mice

(p<0.001). These results suggest that CCL21 secretion by β-cells enriched the pancreas with naïve

T cells, likely via chemotactic recruitment through CCR7. Additionally, the relative proportion of

macrophages and immature DCs (imDC) was decreased in the pancreas of transgenic compared to

NOD mice (Fig. S1D).

Very few B and T cells were detected in the islets of NOD mice at 4wks of age (Fig. 2E).

However, B and T cells were observed in transgenic islets (Fig. 2E) with an organization that

resembled the LN paracortex (Fig. 2F). Therefore, transgenic CCL21 expression in β-cells

induced the formation of TLOs in the pancreas by 4 weeks of age.

By immunofluorescence Tregs were readily detected in 4 week-old transgenic islets but not

in age-matched NOD islets (Fig. 2E, 2G). Because the total number and the relative abundance

(Fig. S1A) of Tregs in the pancreas were unaffected, we conclude that CCL21 expression in β-cells

lead to redistribution of pancreatic Tregs and their enrichment in the islets, which may contribute to

explain the enhanced regulation. Diabetes Page 12 of 57

At 12 weeks of age, the total number of pancreatic lymphocyte populations were

comparable in both strains (Fig. 2H). Thus, CCL21 production from β-cells leads to alterations in

islet-infiltrating immune cells at early time points, but not at later time points.

Splenocytes from transgenic mice are not diabetogenic

While splenocytes from diabetic NOD mice transferred diabetes to 100% of NOD-scid recipients,

splenocytes from transgenic mice (Table 2) failed to transfer diabetes (Fig. 3A) suggesting that

they are non-diabetogenic and maintain this phenotype upon transfer to mice that do not express

CCL21 in the islets.

Through tetramer staining (Table S1) we found that the numbers of CD44+ IGRP-reactive

CD8+ T cells (CTLs) (Fig. 3B) and of CD44+ insulin-reactive CTLs (Fig. 3C) in pancreatic islets

and in blood of 12 week-old Ins2-CCL21 NOD mice were significantly decreased compared to

non-transgenic mice. These results suggest that CCL21 local expression by β-cells decreased the

number of autoreactive CTLs, which contributes to explain the lack of diabetogenic potential.

CCL21 local secretion by β-cells induces formation of TLOs containing FRC-like stromal cells

We hypothesized that protection from diabetes was associated with tolerogenic stromal cells

within TLOs, which may impact autoreactive T-cells. Indeed, the pancreatic islets of 4 week-old

transgenic NOD mice displayed a highly organized network of gp38+Lyve-1- FRC-like cells (Fig.

4A-B), closely resembling FRC reticular networks in LNs. This was not observed in non- transgenic littermates NOD mice until age 8 weeks (Fig. 4C). Thus, the transgenic expression of

CCL21 leads to the early appearance of highly organized TLOs in the pancreas. Page 13 of 57 Diabetes

By 16 weeks of age, when non-transgenic NOD mice started developing diabetes, FRC

networks started disappearing from the pancreas, as previously shown (14). In contrast, diabetes

resistance in transgenic mice was associated with persistence of both FRC-like networks

(ERTR7+Lyve-1-) and high endothelial venules (PNAd+CD31+) in the pancreas (Fig. 4D).

Further characterization of pancreatic and LN-derived stromal cells by flow cytometry

(Fig. S2A) revealed the presence of two populations of FRC-like cells: CD45-CD31-gp38hi and

CD45- CD31-gp38dim (Fig. 4E). Transgenic islets were enriched in CD45-CD31-gp38hi stromal

cells at 4 weeks (Fig. 4E-G) and 12 weeks (Fig.4H) compared with non-transgenic islets.

Proportions of FRCs in LNs were comparable between strains (Fig. S2 C, D).

Local CCL21 expression by β-cells is sufficient to induce FRC-like cell containing TLOs and

mediate systemic protection from diabetes

Next, we asked whether local formation of FRC like cell-containing TLOs in CCL21-expressing

islets were responsible for mediating systemic regulation of diabetogenic leukocytes. Adoptive

transfer of diabetogenic splenocytes from recently diabetic NOD mice into 10 week-old transgenic

NOD mice (Table 2) resulted in delayed diabetes development of 48 weeks compared to 18 weeks

in NOD recipients (p=0.007) (Fig. 5A), suggesting that formation of “regulatory” TLOs is crucial

to prevent diabetes in NOD mice.

However, transgenic islets were not protected against recurrence of autoimmunity after

transplantation in diabetic NOD mice (Fig. 5B) and from allo-rejection after transplantation into

fully MHC-mismatched diabetic Balb/C mice (Fig. S2E). To test whether lack of protection was

due to insufficient time for the formation of “regulatory” TLOs at the graft site, we transplanted Diabetes Page 14 of 57

Ins2-CCL21 or non-transgenic NOD islets (Table S2) under the kidney capsule of 7-week-old

NOD-scid mice and waited 4 weeks before adoptively transferring splenocytes from diabetic NOD

mice formation (Table 2), thus allowing time for TLO formation (Fig. 5C). Before adoptive

transfer, we could detect insulitis in recipient NOD-scid pancreas when either NOD or Ins2-

CCL21 islets were transplanted (Fig. 5D-F, Before AT) where lymphocytes originated from the

transplanted grafts as passenger leukocytes. Importantly, the formation of TLOs was observed in

the Ins2-CCL21 graft (Fig. 5E) but not in non-transgenic islet grafts (Fig. 5D). Diabetes

development after adoptive transfer of diabetogenic splenocytes was delayed in recipients of Ins2-

CCL21 islets compared to mice transplanted with NOD islets (Fig. 5C, p=0.0002). Importantly,

mice transplanted with Ins2-CCL21 NOD islets displayed lower grades of insulitis in the pancreas

(Fig. 5F, 5H, After AT; p=0.0491) than mice transplanted with NOD islets (Fig. 5G). Thus, CCL21

local expression in β-cell kidney grafts was sufficient to delay the onset of autoimmune diabetes

and provide systemic protection in the pancreas, if time was allowed for the formation of regulatory

TLOs at the graft site.

Mechanisms of FRC-like cells-mediated immune regulation

Since FRCs promote tolerance by expressing self-antigens (25), we assessed β-cell antigens expression in pancreatic FRC-like cells by RNAseq and by immunostaining. RNAseq analysis revealed higher expression of β-cell antigens including Insulin (Ins1 and Ins2) autoantigen in pancreatic gp38dimCD31- FRC-like stromal cells from 12 week-old Ins2-CCL21

NOD compared with non-transgenic NOD (Fig. 6A; Table S3); we verified that pancreatic - cells did not contaminate our FRC populations because of (i) high sorting purity of Page 15 of 57 Diabetes

pancreatic CD45-gp38+CD31- FRC-like cells (Ins2-CCL21 NOD: 97±2%; non-transgenic

NOD: 96±3%); (ii) > 3-log reduction in Ins2 mRNA levels between FRC-like cells and islets

by qRT-PCR (non-transgenic fold-change: 8,287; transgenic fold-change: 4,958; data not

shown), which is consistent with what would be expected between Ins2 mRNA

expression in beta cells and ectopic expression in LN. Insulin protein (Fig. 6B, inset b) was

detected in conduits formed by islet-associated gp38+Pan-Laminin+ stromal cells of Ins2-CCL21

NOD mice at 4 weeks of age (Fig. 6B, inset a), reminiscent of FRC conduits found in LNs. These

data suggest that pancreatic gp38+ stromal cells induced by CCL21 local expression in Ins2-

CCL21 NOD mice express islet autoantigens, and this enhanced expression may be key for the

lack of diabetes development in CCL21-transgenic mice.

Next, we compared the RNAseq profiles of FRC-like cells from transgenic islets to

FRC-like cells from non-transgenic islets, to LN-derived FRCs and to islet-derived CD45+

leukocytes freshly sorted from both mouse strains. From principal component analysis,

LN-FRCs clustered independently from islet-derived FRC-like cells (gp38hi and gp38dim),

suggesting that they are distinct populations in both mouse strains (Fig. 6C). Similarly,

forward scattering of islet-derived FRC-like cells was higher than LN FRCs (Fig. S2 F, G).

Differential expression of genes previously associated with LN stromal cells (33) (Table

S4) and of genes associated with cytoskeletal organization and cell contractility (34) (Table S5)

was found in CCL21-induced islet FRC-like cells compared to FRCs from non-transgenic

mice. Cells from transgenic mice overexpressed genes that are implicated in cell shape regulation, Diabetes Page 16 of 57

rapid cytoskeletal reorganization and cell contractility and downregulated genes associated with actin polymerization.

Differential expression analysis of innate and adaptive immunity genes in CCL21- induced islet FRC-like cells compared to non-transgenic littermates (Fig. 6D and Table S6), suggest different immunological function. Genes implicated in prevention of terminal differentiation of B cells into antibody-forming cells and in BCR-induced B cell apoptosis were upregulated in transgenic compared to non-transgenic NOD-derived FRC-like cells; in contrast, genes involved in protecting pro-B cells from programmed cell death, promoting pre-B cell development, proliferation and activation were downregulated in transgenic FRC-like cells. Co- stimulatory molecules, pro-inflammatory genes implicated in IL-1, TNF, IL-6, IFNγ signaling, complement activation, Toll-like receptor signaling and in chemotaxis of activated T cells and other inflammatory cells were also down-regulated in transgenic compared to non-transgenic

NOD-derived FRC-like cells. Conversely, pro-tolerogenic genes involved in IgM/IgA endocytosis, in regulating T cell activation and maturation, Th17 T cell function, in antagonizing

IL-1 and TNF signaling and promoting macrophage polarization were upregulated in transgenic

FRC-like cells. Genes associated with lymphotoxin (LT) signaling were also upregulated in FRC- like cells from transgenic mice. Despite lower expression of β-cell antigens, FRC-like cells from non-transgenic islets expressed higher gene expression levels of proteins involved in antigen processing and presentation on MHC I and MHC class II. Accordingly, we found that Ins2-CCL21

NOD islet FRC-like cells aligned their immunology gene expression pattern more with LN FRCs

(Fig. 6E), which are well described to have tolerogenic properties (25) than with islet-derived

CD45+ cells though this hasn’t been quantified. Page 17 of 57 Diabetes

We conclude that CCL21-expression in β-cells is associated with highly organized TLOs

in the pancreas in which FRC-like cells express β-cell antigens, have enhanced contractility and

anti-inflammatory function, resulting in an immunological profile that is more similar to

tolerogenic LN-derived FRCs than pro-inflammatory FRC-like cells from the TLOs of inflamed

islets of non-transgenic NOD mice.

Discussion

Understanding the mechanisms employed in the immune tolerant tumor microenvironment

could advance approaches to re-establish self-tolerance in autoimmunity. Tumor tolerance can be

mediated by tumor-derived secretion of the CCL21 chemokine and TLO formation (6). Here, we

investigated whether local CCL21 secretion by β-cells could induce tolerogenic TLOs and whether

these could play a role in preventing autoimmune diabetes in NOD mice. Protection from

diabetes in the CCL21-transgenic NOD mice was associated with the formation of TLOs

containing stromal cells that we identified as FRC-like cells based on either gp38+Lyve-1-

or CD45-gp38+CD31- phenotype. We identified properties of FRC-like populations that

are unique to the CCL21 transgenic mice and changes in immune cell populations that

are associated with tolerance and can explain lack of diabetes development.

Transgenic islets developed immune infiltrates containing TLOs with gp38+Lyve-1- FRC-

like cells at earlier age (by 4 weeks) than non-transgenic mice, and unlike NOD mice in which

TLOs are lost over time (14; 35), these TLOs persisted in transgenic mice. We demonstrated the Diabetes Page 18 of 57

critical role of TLOs by using a transplant model, in which allowing time for the

formation of TLOs promoted protection of both the CCL21-transgenic islet grafts and of

the pancreas from diabetes transfer.

Local secretion of CCL21 by β-cells was predominantly associated with the recruitment

of naïve T cells to the pancreas, which are critical for the formation of TLOs (20; 22; 36-39).

Accordingly, LT pathways critical for TLO formation were upregulated in FRCs from transgenic islets. Since macrophages and imDCs are attracted by inflammation, their observed decrease in CCL21-transgenic islets suggest that the CCL21-induced TLOs are less inflammatory than TLOs found in inflamed islets of NOD mice. Additionally, we noted a re-distribution of

Tregs to CCL21-transgenic islets likely through expression of the CCL21 ligand CCR7, which could contribute to the regulatory phenotype we observe. We speculate that a different microenvironment created by local CCL21 secretion could impact the diabetogenic potential of autoreactive T cells. In agreement with this hypothesis, Ins2-

CCL21 NOD mice had far fewer β-cell autoreactive CTLs in pancreatic islets and blood.

Thus, CCL21 local expression by β-cells impacted autoreactive T cells at a systemic level,

similarly to CCL21-secreting tumors (6). However, CCL21-induced TLOs did not cause a globally suppressive environment as allogenic skin grafts were rejected suggesting that regulation was specific for β-cell antigen-specific T cells. We hypothesized that autoreactive CTLs could be impacted by tolerogenic expression of islet autoantigens by Page 19 of 57 Diabetes

FRCs that results in CTL deletion or functional inactivation in LNs (25; 40; 41) while interactions

with autoreactive CD4 T cells remain to be investigated.

Islet FRC-like cells from TLOs of CCL21 transgenic mice possessed higher expression of

T1D related autoantigen genes than pancreatic FRC-like cells from TLOs of inflamed islets of

non-transgenic mice. Downregulation of autoantigens in LN FRCs of 12 week-old NOD mice was

previously reported and is mediated by DEAF-1 downregulation (42). We did not observe

differences in DEAF-1 expression suggesting potentially different regulatory mechanisms.

Autoantigen downregulation in islet FRC-like cells from non-transgenic NOD mice was

not associated with downregulation of genes associated with antigen processing and presentation,

suggesting that reduced FRC antigen presentation was not a critical mechanism and emphasizing

the importance of autoantigen levels. CCL21-induced pancreatic FRC-like cells expressed lower

levels of pro-inflammatory genes than FRC-like cells found in TLOs of inflamed NOD islets.

Because inflammation leads to MHC class I upregulation in the pancreas, it is likely that reduced

inflammation in Ins2-CCL21 FRC-like cells might contribute to their observed downregulation of

H2-K and H2-A genes. Genes associated with maturation, activation and pro-survival of B

cells were downregulated in CCL21-transgenic FRC-like cells compared to non-

transgenic cells, suggesting that CCL21 may also regulate B lymphocytes, which also play

a role in T1D development (13).

TLOs of transgenic mice harbored higher proportions of gp38hi FRCs. Podoplanin

(gp38) regulates actomyosin contractility in FRCs and enables FRCs to re-organize their reticular

network in LNs (43). Ablating podoplanin in FRCs reduced FRC contractility and enhanced

immunity (43). Further, genes regulating contractility of islet gp38dim FRC-like cells were Diabetes Page 20 of 57

upregulated in transgenic compared to non-transgenic cells. Both increased proportions of gp38hi

FRC-like cells and increased contractility of gp38dim FRC-like cells observed in CCL21- transgenic islets may enhance immune regulation early in the natural history of islet autoimmunity.

Increased contractility of self-antigen-expressing FRCs may promote their interactions with autoreactive T cells and enhance regulatory properties of FRCs.

In conclusion, we characterized molecular events induced by CCL21 expression in pancreatic β-cells that leads to T1D prevention in NOD mice. We demonstrate that local CCL21 secretion is associated with the formation of tolerogenic TLOs in the pancreas. FRC-like cells in the pancreatic TLOs of CCL21 transgenic mice are phenotypically and functionally different than FRC-like cells from TLOs of inflamed NOD islets. While inflammatory NOD TLOs stimulate polyclonal expansion of autoreactive T cells and autoantibody production (13),

CCL21-induced TLOs promote systemic and antigen-specific regulation of islet autoimmunity and this is mediated by FRC-like cells with enhanced expression of β-cell autoantigens, anti-inflammatory profiles, and higher contractility. Despite CCL21 local secretion having a systemic immunomodulatory effect, CCL21 was not detected systemically in CCL21- transgenic mice. This, together with the presence of HEVs, could imply that tolerance might be mediated locally by lymphocyte trafficking through TLOs. Overall, our findings suggest that

CCL21 promotes TLOs that differ from inflammatory TLOs associated with inflamed islets in

T1D 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. This provides a strong rationale of developing novel therapeutics based on inducing TLOs in confined sites to induce antigen-specific tolerance in autoimmune diseases by local CCL21 delivery through Page 21 of 57 Diabetes

biomaterials. Induction of TLO formation at other tissue sites will also allow comparing the TLO

characteristics in relation to their tissue localization and application of the approach to other

autoimmune diseases.

Acknowledgments

The authors are grateful to Dr. Vickie Zhang for technical assistance, the personnel of the DRI

Preclinical Cell Processing and Translational Models Core for their help with islet isolation,

transplantation and management of diabetic mice, the DRI Imaging Core for providing expertise

on confocal imaging, and the DRI Histology core headed by Kevin Johnson for his help with

histological processing of all samples. Additionally, we thank Dr. William Hulme and the Center

for Genome Technology of the John P. Hussman Institute for Human Genomics for their assistance

with RNA sequencing and data analysis.

Funding was provided by philanthropic funds from the Diabetes Research Institute

Foundation, a grant from the Juvenile Diabetes Research Foundation (grant # 2-SRA-

2016-316-S-B), a grant from the Iacocca Family Foundation, a grant from the National

Institute of Health (grant # DK109929), Dr. Tomei’s 2017 provost’s research award, Dr.

Tomei’s BME start-up package, and the European Foundation for the Study of Diabetes

(EFSD; ZUW80166).

Data and Resource Availability Diabetes Page 22 of 57

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

The RNAseq data were deposited in Gene Expression Omnibus (GEO; accession #

GSE130979).

Author Contributions F.G.B., F.Z.T., M.M.A., D.S., M.N., A.B., R.D.M., E.K., S.W. R.M., and A.A.T. generated data, reviewed/edited the manuscript and contributed to discussion. A.A.T., A.P., F.G.B. and M.M.A designed the research and A.A.T wrote the manuscript.

Disclosure

A.A.T. is co-inventor of intellectual property used in the study and may gain royalties from future commercialization of the technology.

Guarantor Statement

Freddy Gonzalez Badillo is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Tables

Table 1 Experimental details of experiments involving islet transplantation in mice.

Donors Dose Recipients Site n Figure Islets from Ins2-CCL21 NOD mice 1k IEQ Spontaneously diabetic NOD mice EFP 4 5B (red) Islets from non-transgenic NOD mice 1k IEQ Spontaneously diabetic NOD mice EFP 1 5C (black) Islets from CCL21 transgenic (CCL21+) 1k IEQ Chemically-diabetic BALB/c mice EFP 13 S2D (red) C57BL/6 mice Islets from CCL21- control C57BL/6 mice 1k IEQ Chemically-diabetic BALB/c mice EFP 4 S2D (black) Islets from Ins2-CCL21 NOD mice 0.5k IEQ 7 week-old NOD-scid mice Kidney capsule 7 5C (red) (KD) Islets from non-transgenic NOD mice 0.5k IEQ 7 week-old NOD-scid mice Kidney capsule 6 5C (black) (KD)

Table 2 Experimental details of experiments involving adoptive transfer of splenocytes

in mice.

Donors Dose Recipients site N Figure Splenocytes from 20-25 10 million 4-6 week-old female NOD-scid Mice IV 12 3A (red) week-old Ins2-CCL21 NOD Splenocytes from 20-25 10 million 4-6 week-old female NOD-scid Mice IV 3 3A (black) week-old diabetic NOD Splenocytes from 20-25 10 million 10 week-old female Ins2-CCL21 NOD IV 6 5A (red) week-old diabetic NOD Splenocytes from 20-25 10 million 10 week-old female NOD IV 5 5A (black) week-old diabetic NOD Splenocytes from 20-25 11 week-old NOD-scid (4 weeks after NOD islet 10 million IV 6 5C (black) week-old diabetic NOD transplantation in KD) Splenocytes from 20-25 11 week-old NOD-scid (4 weeks after Ins2-CCL21 islet 10 million IV 7 5C (red) week-old diabetic NOD transplantation in KD)

Figure Legends

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 Diabetes Page 30 of 57

non-transgenic littermate control (40%, black, n=10) female mice. (B) Confocal images of

pancreatic sections of 4 week-old non-transgenic and Ins2-CCL21 NOD mice

immunostained with insulin (red) and CCL21 (green) antibodies. Nuclei were

counterstained with DAPI (grey). Scale bar: 50µm. (C) CCL21 concentrations in the supernatant of pancreatic islets isolated from 19-33 week-old non-transgenic (black) and

Ins2-CCL21 NOD (red) mice stimulated with 2.2mM (LG) followed by 16.7mM (HG) glucose. ND: not detectable. ***: p<0.01 (D) Normal (p=0.83), fasting (p=0.98), and post-

prandial (p=0.23) secretion of CCL21 in the serum of 6-7 week-old non-transgenic (n=3)

compared to 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: 50µm. (F)

Quantification of islet size from pancreatic sections of 4 (n=6), 8 (n=5), and 12 (n=5) week- old non-transgenic littermate control and age-matched Ins2-CCL21 NOD mice. (G)

Insulin secretion after stimulation of islets from 19-33 week-old non-transgenic (black, n=3) and Ins2-CCL21 NOD (red, n=3) mice with 2.2mM (LG) followed by 16.7mM (HG) glucose. Islets were isolated from a minimum of 5 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 non-transgenic (black, n=4) NOD mice treated daily with five low doses (50mg/kg) of

streptozotocin. (I) Percent survival of C57BL/6 fully allogeneic (left) or syngeneic NOD Page 31 of 57 Diabetes

(right) skin grafts into Ins2-CCL21 mice (red, n=5, MSR: 14 days) or control non-transgenic

littermate NOD mice (black, n=5).

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 non-transgenic 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:

50µm. (B) Insulitis quantification (grade 1-5) of pancreatic sections of 4 (n=6), 8 (n=5), and

12 (n=5) week-old non-transgenic and age-matched Ins2-CCL21 NOD mice. 4week-old

non-transgenic NOD mice: 86%- grade 1, 2%- grade 2, 11%- grade 3, 1%- grade 4 and 1%-

grade 5. 4week-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 4week-old non-

transgenic (n=5) and Ins2-CCL21 NOD (n=5) mice by flow cytometry. Data are indicated

+ - - + as %CD4 live cells. Tn: naïve T cells (CD44 CD127 CD62L ) Transgenic: 38.05±0.08; non-

+ - transgenic: 19.45±4.62, p<0.001; Tef (effector T cells): CD44 CD127 ; Tm (memory T cells):

CD44+ CD127+. (D) Quantification of blood leukocytes of 4week-old non-transgenic (n=5)

and Ins2-CCL21 NOD (n=5) mice by flow cytometry. Data are indicated as % CD45+ live

cells. CCR7+: transgenic, 13±11; non-transgenic, 3±3, p<0.05) (E) Confocal images of

pancreatic sections of 4 week-old non-transgenic and Ins2-CCL21 NOD mice stained with

insulin (cyan), B cells (B220, red), and T cells (CD3, green) antibodies; insulin (red) and Diabetes Page 32 of 57

CD4 (green); insulin (red) and CD8 (green); insulin (cyan), T cells (CD3, green), and Tregs

(CD3+FoxP3+, red) antibodies. Scale bars: 50µm. (F) Confocal images of skin-draining

lymph node (LN) sections of 4 week-old Ins2-CCL21 NOD mice immunostained with

B220 (red) and CD3 (green) at two different magnifications. Scale bar: 50µm. (G)

Quantification of Tregs density in islets from pancreatic sections of 4 week-old non- transgenic (n=4) and age-matched Ins2-CCL21 (n=4) NOD mice. (H) Quantification of

pancreatic infiltrates of 12 week-old non-transgenic littermate (n=6) and Ins2-CCL21

NOD (n=6) mice by flow cytometry. Data are indicated as total number of live cells.

Figure 3. Splenocytes from transgenic mice are not diabetogenic. (A) Adoptive transfers of

splenocytes from 20-25 week-old Ins2-CCL21 NOD (100%, red, n=12) or from age-

matched diabetic non-transgenic (0%, black, n=3, median transfer time: 28 days) into 6 week-

old NOD-scid. At least three donors per group, two recipients per donor and 10 million

cells per adoptive transfer were used. ***: p<0.01. Experimental details are reported in

Table 2. (B) Quantification of CD44+ IGRP-reactive CD8+ T cells (CTL) in pancreatic islets

(p<0.05) and in blood (p<0.05) of 12 week-old Ins2-CCL21 NOD mice (n=5) compared to non- transgenic littermate mice (n=5) (Pancreatic islets: Ins2-CCL21 NOD, 0.11 ± 0.3 %; non-

transgenic NOD, 4.64 ± 2.31 %, p<0.05. Blood: Ins2-CCL21 NOD, 0.10 ± 0.10 %; non- transgenic NOD, 8.34 ± 9.68 %, p<0.05) (C) Quantification of CD44+ INS-reactive CD8+ T cells

(CTL) in pancreatic islets (p<0.05) and blood (p=0.08) of 12 week-old Ins2-CCL21 NOD mice Page 33 of 57 Diabetes

(n=5) compared to non-transgenic littermate NOD mice (n=5) (Pancreatic islets: Ins2-CCL21

NOD, 1.23 ± 0.17 %; non-transgenic NOD, 3.28 ± 1.04 %, p=0.01. Blood: Ins2-CCL21 NOD,

21.77 ± 10.66 %; non-transgenic NOD, 10.82 ± 3.60 %, p=0.08). Data are indicated as %

CD8+ cells.

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 non-

transgenic and Ins2-CCL21 NOD mice and of skin-draining LN, stained with insulin

(cyan), gp38 (stromal cell marker, green) and Lyve-1 (LEC marker, red) antibodies, and

nuclear counterstain (DAPI, white). FRC-like cells are identified by gp38+Lyve-1-. Scale

bars: 50µm. (B) Quantification of FRC density in pancreatic islets of 4 week-old non-

transgenic (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. **: p≤0.01 (C) Confocal

microscope images of pancreatic sections of 4-16 week-old non-transgenic mice stained

with insulin (red), glucagon (cyan), stromal cells (gp38, green) antibodies, and nuclear

counterstain (DAPI, grey). Scale bar: 50µm. (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 with HEVs antibodies (CD31: green; PNAd: red)

(right); nuclear counterstain: DAPI (blue). Scale bars: 50µm. (E-G) Gating strategy (E) and

relative quantification of CD45- CD31- gp38dim (F) and of CD45- CD31- gp38hi (G) FRCs Diabetes Page 34 of 57

from the skin-draining LNs and FRC-like cells from the pancreatic islets of 4 week-old

non-transgenic (n=4, black) and Ins2-CCL21 NOD mice (n=4, red) by flow cytometry. (H)

Relative proportions of CD45- CD31- gp38dim and of CD45- CD31- gp38hi FRC-like cells

from the pancreatic islets of 12 week-old non-transgenic (n=5, black) and Ins2-CCL21

NOD mice (n=5, red) by flow cytometry. ***: p≤0.001

Figure 5. Local CCL21 expression by β-cells is sufficient to induce FRC-like cell containing

TLOs and mediate systemic protection from diabetes. (A) Adoptive transfers (AT) of

splenocytes from diabetic non-transgenic NOD mice into 10 week-old Ins2-CCL21 NOD

(red, n=6, median survival time: 48 weeks, p=0.007) or into age-matched non-transgenic

(black, n=5, median survival time: 18 weeks). (B) Percent of diabetes free spontaneously

diabetic NOD mice transplanted (EFP) with 1k IEQ islets from Ins2-CCL21 NOD (red,

n=4) or non-transgenic (black, n=1) NOD mice. Experimental details are reported in Table

2. (C) AT of splenocytes from recently diabetic NOD mice into 7-11 week-old NOD-scid

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 non-transgenic (black, n=6, median survival time: 75 days) mice 4 weeks prior to

injection. (D, E) Confocal microscope images of kidney capsule islet graft sections (KD

graft) (left) and of pancreatic sections (right) of 7 week-old NOD-scid mice that received

either non-transgenic (D) or Ins2-CCL21 transgenic (E) NOD islets 4 weeks before. (F) Page 35 of 57 Diabetes

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 kidney capsule 4 weeks before the AT. (G, H) Confocal microscope

images of kidney capsule islet graft sections (KD graft) (left) and pancreatic sections

(right) of NOD-scid mice that received either non-transgenic (G) or Ins2-CCL21

transgenic (H) NOD islets followed by adoptive transfer 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 bars:

100µm

Figure 6. Mechanism of FRC like cells-mediated immune regulation. (A) Heatmap of T1D

autoantigens’ expression in gp38dim FRC-like cells freshly sorted from either Ins2-CCL21

NOD mice or non-transgenic 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),

Regenerating Family Member 3 Alpha (Reg3a). (B) Confocal microscope images of Diabetes Page 36 of 57

pancreatic sections of 4 week-old non-transgenic (top) and Ins2-CCL21 transgenic

(bottom, different magnifications) NOD mice immunostained with insulin (red), Pan-

Laminin (grey), stromal cells (gp38, green) antibodies, and nuclear counterstain (DAPI,

blue). Scale bars: 50µm. 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 non-transgenic NOD and n=3 for Ins2-CCL21 NOD), islet-derived gp38dim FRC-like cells (n=4 per mouse model), islet-derived gp38hi 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 littermate. (D) Heatmap 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

gp38dim FRC-like cells from Ins2-CCL21 NOD mice compared to non-transgenic

littermates. (E) Heatmap of inflammatory genes (Nanostring Immunology panel)

expressed by LN-derived FRCs, islet-derived gp38dim FRC-like cells, islet-derived

gp38high FRC-like cells and islet-derived CD45+ cells freshly sorted from 12 week-old from

Ins2-CCL21 NOD mice (right) or from non-transgenic littermates (left). Page 37 of 57 Diabetes

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 non-transgenic littermate control (40%, black, n=10) female mice. (B) Confocal images of pancreatic sections of 4 week-old non-transgenic and Ins2-CCL21 NOD mice immunostained with insulin (red) and CCL21 (green) antibodies. Nuclei were counterstained with DAPI (grey). Scale bar: 50µm. (C) CCL21 concentrations in the supernatant of pancreatic islets isolated from 19-33 week-old non-transgenic (black) and Ins2-CCL21 NOD (red) mice stimulated with 2.2mM (LG) followed by 16.7mM (HG) glucose. ND: not detectable. ***: p<0.01 (D) Normal (p=0.83), fasting (p=0.98), and post-prandial (p=0.23) secretion of CCL21 in the serum of 6-7 week-old non-transgenic (n=3) compared to 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: 50µm. (F) Quantification of islet size from pancreatic sections of 4 (n=6), 8 (n=5), and 12 (n=5) week-old non-transgenic littermate control and age-matched Ins2-CCL21 NOD mice. (G) Insulin secretion after stimulation of islets from 19-33 week-old non-transgenic (black, n=3) and Ins2-CCL21 NOD (red, n=3) mice with 2.2mM (LG) followed by 16.7mM (HG) glucose. Islets were isolated from a minimum of 5 mice per experiment and plated in n=3 wells per condition. (H) Diabetes Page 38 of 57

Percent of diabetes free 10 week-old Ins2-CCL21 NOD (red, n=6) or non-transgenic (black, n=4) NOD mice treated daily with five low doses (50mg/kg) of streptozotocin. (I) Percent survival of C57BL/6 fully allogeneic (left) or syngeneic NOD (right) skin grafts into Ins2-CCL21 mice (red, n=5, MSR: 14 days) or control non- transgenic littermate NOD mice (black, n=5).

175x202mm (300 x 300 DPI) Page 39 of 57 Diabetes

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 non-transgenic 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: 50µm. (B) Insulitis quantification (grade 1-5) of pancreatic sections of 4 (n=6), 8 (n=5), and 12 (n=5) week-old non-transgenic and age-matched Ins2-CCL21 NOD mice. 4week-old non-transgenic NOD mice: 86%- grade 1, 2%- grade 2, 11%- grade 3, 1%- grade 4 and 1%- grade 5. 4week-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 4week-old non-transgenic (n=5) and Ins2- CCL21 NOD (n=5) mice by flow cytometry. Data are indicated as %CD4+ live cells. Tn: naïve T cells (CD44- CD127- CD62L+) Transgenic: 38.05±0.08; non-transgenic: 19.45±4.62, p<0.001; Tef (effector T cells): CD44+ CD127-; Tm (memory T cells): CD44+ CD127+. (D) Quantification of blood leukocytes of 4week-old non-transgenic (n=5) and Ins2-CCL21 NOD (n=5) mice by flow cytometry. Data are indicated as % CD45+ live cells. CCR7+: transgenic, 13±11; non-transgenic, 3±3, p<0.05) (E) Confocal images of pancreatic sections of 4 week-old non-transgenic and Ins2-CCL21 NOD mice stained with insulin (cyan), B cells (B220, Diabetes Page 40 of 57

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 bars: 50µm. (F) Confocal images of skin-draining lymph node (LN) sections of 4 week-old Ins2-CCL21 NOD mice immunostained with B220 (red) and CD3 (green) at two different magnifications. Scale bar: 50µm. (G) Quantification of Tregs density in islets from pancreatic sections of 4 week-old non-transgenic (n=4) and age-matched Ins2-CCL21 (n=4) NOD mice. (H) Quantification of pancreatic infiltrates of 12 week-old non- transgenic littermate (n=6) and Ins2-CCL21 NOD (n=6) mice by flow cytometry. Data are indicated as total number of live cells.

166x227mm (300 x 300 DPI) Page 41 of 57 Diabetes

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

178x130mm (300 x 300 DPI) Diabetes Page 42 of 57

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 non-transgenic and Ins2-CCL21 NOD mice and of skin-draining LN, stained with insulin (cyan), gp38 (stromal cell marker, green) and Lyve-1 (LEC marker, red) antibodies, and nuclear counterstain (DAPI, white). FRC-like cells are identified by gp38+Lyve- 1-. Scale bars: 50µm. (B) Quantification of FRC density in pancreatic islets of 4 week-old non-transgenic (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. **: p≤0.01 (C) Confocal microscope images of pancreatic sections of 4-16 week-old non-transgenic mice stained with insulin (red), glucagon (cyan), stromal cells (gp38, green) antibodies, and nuclear counterstain (DAPI, grey). Scale bar: 50µm. (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 with HEVs antibodies (CD31: green; PNAd: red) (right); nuclear counterstain: DAPI (blue). Scale bars: 50µm. (E-G) Gating strategy (E) and relative quantification of CD45- CD31- gp38dim (F) and of CD45- CD31- gp38hi (G) FRCs from the skin-draining LNs and FRC-like cells from the pancreatic islets of 4 week-old non-transgenic (n=4, black) and Ins2-CCL21 NOD mice (n=4, red) by flow cytometry. (H) Relative proportions of CD45- CD31- gp38dim and of CD45- CD31- gp38hi FRC-like cells from the pancreatic islets of 12 week-old non-transgenic (n=5, black) and Ins2-CCL21 NOD mice (n=5, red) by flow cytometry. ***: p≤0.001

200x172mm (300 x 300 DPI) Page 43 of 57 Diabetes

Figure 5. Local CCL21 expression by β-cells is sufficient to induce FRC-like cell containing TLOs and mediate systemic protection from diabetes. (A) Adoptive transfers (AT) of splenocytes from diabetic non-transgenic NOD mice into 10 week-old Ins2-CCL21 NOD (red, n=6, median survival time: 48 weeks, p=0.007) or into age-matched non-transgenic (black, n=5, median survival time: 18 weeks). (B) Percent of diabetes free spontaneously diabetic NOD mice transplanted (EFP) with 1k IEQ islets from Ins2-CCL21 NOD (red, n=4) or non-transgenic (black, n=1) NOD mice. Experimental details are reported in Table 2. (C) AT of splenocytes from recently diabetic NOD mice into 7-11 week-old NOD-scid 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 non-transgenic (black, n=6, median survival time: 75 days) mice 4 weeks prior to injection. (D, E) Confocal microscope images of kidney capsule islet graft sections (KD graft) (left) and of pancreatic sections (right) of 7 week-old NOD-scid mice that received either non-transgenic (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 kidney capsule 4 weeks before the AT. (G, H) Confocal microscope images of kidney capsule islet graft sections (KD graft) (left) and pancreatic sections (right) of NOD-scid mice that received either non-transgenic (G) or Ins2-CCL21 transgenic (H) NOD islets followed by adoptive transfer of Diabetes Page 44 of 57

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 bars: 100µm

199x222mm (300 x 300 DPI) Page 45 of 57 Diabetes

Figure 6. Mechanism of FRC like cells-mediated immune regulation. (A) Heatmap of T1D autoantigens’ expression in gp38dim FRC-like cells freshly sorted from either Ins2-CCL21 NOD mice or non-transgenic 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), Regenerating Family Member 3 Alpha (Reg3a). (B) Confocal microscope images of pancreatic sections of 4 week-old non-transgenic (top) and Ins2-CCL21 transgenic (bottom, different magnifications) NOD mice immunostained with insulin (red), Pan-Laminin (grey), stromal cells (gp38, green) antibodies, and nuclear counterstain (DAPI, blue). Scale bars: 50µm. 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 non-transgenic NOD and n=3 for Ins2-CCL21 NOD), islet-derived gp38dim FRC-like cells (n=4 per mouse model), islet-derived gp38hi 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 littermate. (D) Diabetes Page 46 of 57

Heatmap 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 gp38dim FRC-like cells from Ins2-CCL21 NOD mice compared to non-transgenic littermates. (E) Heatmap of inflammatory genes (Nanostring Immunology panel) expressed by LN-derived FRCs, islet-derived gp38dim FRC-like cells, islet- derived gp38high FRC-like cells and islet-derived CD45+ cells freshly sorted from 12 week-old from Ins2- CCL21 NOD mice (right) or from non-transgenic littermates (left).

199x252mm (300 x 300 DPI) Page 47 of 57 Diabetes

Supplemental Tables

Table S1 Tetramer sequences

Tetramers were provided by the NIH tetramer core.

Tetramer target MHC molecule Sequence

Mouse/human Insulin B 15-23 H-2K(d) LYLVCGERG

Mouse IGRP 206-214 H-2K(d) VYLKTNVFL

Table S2 Islet donors for kidney capsule transplants

Number of donors per age and gender for kidney capsule transplants

Strain # of donors Age Gender NOD 1 29 weeks Female 1 22 weeks Female 2 14 weeks Female 2 12 weeks Female 1 10 weeks Female 1 9 weeks Female 3 9 weeks Male 1 7 weeks Female 1 7 weeks Male 3 5 weeks Male NOD Ins2-CCL21 2 14 weeks Female 3 12 weeks Female 1 10 weeks Female 4 9 weeks Female 2 9 weeks Male 4 7 weeks Male 3 5 weeks Female 1 5 weeks Male

Table S3 Islet autoantigen gene expression in pancreatic FRC-like cells. Diabetes Page 48 of 57

Differential expression of T1D autoantigen genes between gp38dim FRC-like cells freshly sorted from either Ins2-CCL21 NOD mice or non-transgenic NOD littermates and processed for RNAseq calculated with the exact test implemented in edgeR. Linear

Fold Change (linearFC) compares cells isolated from Ins2-CCL21 transgenic NOD mice over non-transgenic NOD littermates.

Upregulated Gene Gene Linea Symbol type name location r FC Pvalue FDR Protein ENSMUSG00000037852.8 coding Cpe chr8:64592542-64693054 3.44 5.63E-11 1.51E-08 Protein ENSMUSG00000029644.7 coding Pdx1 chr5:147269959-147275848 3.51 5.02E-06 2.68E-04 Protein ENSMUSG00000062995.12 coding Ica1 chr6:8630527-8778488 3.83 2.14E-12 9.55E-10 Protein ENSMUSG00000021194.6 coding Chga chr12:102554969-102565028 6.00 8.96E-12 3.21E-09 Protein ENSMUSG00000017316.13 coding Ppy chr11:102099930-102101319 6.22 7.33E-07 5.31E-05 Protein ENSMUSG00000041681.2 coding Iapp chr6:142298423-142303961 9.21 1.38E-15 1.28E-12 Protein ENSMUSG00000022315.3 coding Slc30a8 chr15:52295553-52335798 10.45 3.19E-07 2.63E-05 Protein ENSMUSG00000000215.11 coding Ins2 chr7:142678656-142743381 10.54 3.80E-05 1.46E-03 Protein ENSMUSG00000056553.14 coding Ptprn2 chr12:116485720-117276849 12.13 2.92E-25 4.98E-22 Protein ENSMUSG00000026204.15 coding Ptprn chr1:75247027-75264502 14.50 1.70E-13 1.08E-10 Protein ENSMUSG00000000394.15 coding Gcg chr2:62474530-62483650 16.02 1.44E-25 2.61E-22 Protein ENSMUSG00000035804.4 coding Ins1 chr19:52264323-52265456 16.20 2.61E-08 3.16E-06 Protein ENSMUSG00000079516.2 coding Reg3a chr6:78380709-78383827 173.57 3.27E-08 3.82E-06

Table S4 FRC genes Page 49 of 57 Diabetes

Differential expression of FRC genes between gp38dim FRC-like cells freshly sorted from

either Ins2-CCL21 NOD mice or non-transgenic NOD littermates and processed for

RNAseq calculated with the exact test implemented in edgeR. Linear Fold Change

(linearFC) compares cells isolated from Ins2-CCL21 transgenic NOD mice over non-

transgenic NOD littermates.

Upregulated Gene Linear Symbol Gene type name location FC Pvalue FDR Protein ENSMUSG00000056978.8 coding Hamp2 chr7:30922372-30924681 31.89 6.89E-04 1.61E-02 Protein ENSMUSG00000019874.11 coding Fabp7 chr10:57784881-57788450 10.46 1.61E-04 4.88E-03 Protein ENSMUSG00000024331.11 coding Dsc2 chr18:20030633-20059554 3.99 1.30E-07 1.24E-05 Protein ENSMUSG00000026321.7 coding Tnfrsf11a chr1:105780718-105847981 3.28 1.85E-05 7.90E-04 Protein ENSMUSG00000037605.16 coding Adgrl3 chr5:81020138-81825133 3.26 1.81E-06 1.13E-04 Protein ENSMUSG00000000567.5 coding Sox9 chr11:112782224-112787760 3.21 2.21E-08 2.75E-06 Protein ENSMUSG00000024621.15 coding Csf1r chr18:61105572-61132149 2.79 6.65E-10 1.28E-07 Protein ENSMUSG00000032135.15 coding Mcam chr9:44134469-44142727 2.46 4.91E-06 2.63E-04 Protein ENSMUSG00000003545.3 coding Fosb chr7:19302696-19310051 2.27 7.14E-06 3.55E-04 Protein ENSMUSG00000041559.7 coding Fmod chr1:134037254-134048277 1.77 1.36E-05 6.14E-04

Downregulated Gene Linear Symbol Gene type name location FC Pvalue FDR Protein ENSMUSG00000029417.9 coding Cxcl9 chr5:92321347-92328079 -5.56 1.77E-24 2.75E-21 Protein ENSMUSG00000036594.14 coding H2-Aa chr17:34282744-34287827 -3.92 1.59E-08 2.08E-06 Protein ENSMUSG00000079363.7 coding Gbp4 chr5:105115767-105139586 -3.71 9.10E-06 4.36E-04 Protein ENSMUSG00000003477.5 coding Inmt chr6:55170626-55175043 -3.13 1.27E-06 8.37E-05 Protein ENSMUSG00000073421.5 coding H2-Ab1 chr17:34263209-34269418 -3.05 1.36E-07 1.28E-05 Diabetes Page 50 of 57

Protein ENSMUSG00000060586.10 coding H2-Eb1 chr17:34305877-34316199 -2.63 9.90E-07 6.87E-05 Protein ENSMUSG00000023046.6 coding Igfbp6 chr15:102144362-102149511 -2.48 7.24E-05 2.54E-03 Protein ENSMUSG00000020427.11 coding Igfbp3 chr11:7206086-7213923 -2.30 3.22E-09 4.99E-07 Protein ENSMUSG00000105504.4 coding Gbp5 chr3:142493978-142522344 -2.20 3.56E-05 1.38E-03 Protein ENSMUSG00000030116.14 coding Mfap5 chr6:122505845-122529290 -2.04 1.26E-04 3.95E-03 Protein ENSMUSG00000020101.14 coding Vsir chr10:60346851-60372684 -2.01 1.23E-05 5.59E-04 Protein ENSMUSG00000031750.15 coding Il34 chr8:110741829-110805924 -1.97 1.59E-05 6.96E-04 Protein ENSMUSG00000024620.11 coding Pdgfrb chr18:61045150-61085061 -1.91 1.01E-06 6.97E-05 Protein ENSMUSG00000035678.8 coding Tnfsf9 chr17:57105325-57107757 -1.91 2.65E-05 1.08E-03 Protein ENSMUSG00000031497.9 coding Tnfsf13b chr8:10006467-10039072 -1.88 8.50E-08 8.75E-06 Protein ENSMUSG00000016496.7 coding Cd274 chr19:29367455-29388095 -1.82 8.90E-05 2.99E-03 Protein ENSMUSG00000028583.14 coding Pdpn chr4:143267431-143299564 -1.80 8.95E-05 3.00E-03 Protein ENSMUSG00000017493.12 coding Igfbp4 chr11:99041244-99054392 -1.72 6.87E-04 1.60E-02 Protein ENSMUSG00000042333.16 coding Tnfrsf14 chr4:154921933-154928563 -1.71 1.45E-05 6.45E-04 Protein ENSMUSG00000029231.15 coding Pdgfra chr5:75152292-75198215 -1.66 4.15E-05 1.57E-03 Protein ENSMUSG00000016494.9 coding Cd34 chr1:194938819-194961279 -1.65 8.25E-04 1.86E-02 Protein ENSMUSG00000024610.14 coding Cd74 chr18:60803848-60812646 -1.62 1.41E-03 2.84E-02 Protein ENSMUSG00000022425.16 coding Enpp2 chr15:54838901-54952892 -1.58 1.99E-04 5.79E-03 Protein ENSMUSG00000054675.5 coding Tmem119 chr5:113793729-113800516 -1.55 1.70E-03 3.28E-02 Protein ENSMUSG00000019899.16 coding Lama2 chr10:26980036-27619758 -1.49 2.24E-03 4.07E-02

Table S5. Contractome genes

Differential expression of genes involved in controlling cell contractility and cytoskeleton structure and dynamic between gp38dim FRC-like cells freshly sorted from either Ins2-CCL21 NOD mice or non-transgenic NOD littermates and processed for

RNAseq calculated with the exact test implemented in edgeR. Linear Fold Change Page 51 of 57 Diabetes

(linearFC) compares cells isolated from Ins2-CCL21 transgenic NOD mice over non-

transgenic NOD littermates.

Upregulated Gene Gene Linear Symbol type name location FC Pvalue FDR Protein ENSMUSG00000038167.6 coding Plekhg6 chr6:125362660-125380793 4.93 4.50E-06 2.43E-04 Protein ENSMUSG00000074923.10 coding Pak6 chr2:118663303-118698020 4.21 7.36E-09 1.04E-06 Protein ENSMUSG00000021108.18 coding Prkch chr12:73584796-73778185 4.17 1.56E-11 5.17E-09 Protein ENSMUSG00000031284.16 coding Pak3 chrX:143518591-143797796 4.04 2.11E-08 2.64E-06 Protein ENSMUSG00000047281.3 coding Sfn chr4:133600556-133602168 3.97 7.89E-09 1.10E-06 Protein ENSMUSG00000030739.18 coding Myh14 chr7:44605803-44670843 3.66 1.42E-09 2.46E-07 Protein ENSMUSG00000020782.18 coding Llgl2 chr11:115824049-115855780 3.55 9.21E-11 2.31E-08 Protein ENSMUSG00000029381.16 coding Shroom3 chr5:92683435-92965318 3.37 2.61E-07 2.24E-05 Protein ENSMUSG00000045180.13 coding Shroom2 chrX:152609509-152769465 3.34 2.86E-08 3.43E-06 Protein ENSMUSG00000044641.7 coding Pard6b chr2:168081004-168101203 3.11 4.58E-07 3.55E-05 Protein ENSMUSG00000035873.8 coding Pawr chr10:108332121-108414240 3.00 3.87E-07 3.09E-05 Protein ENSMUSG00000018387.12 coding Shroom1 chr11:53457205-53467766 2.77 3.52E-06 1.98E-04 Protein ENSMUSG00000029053.16 coding Prkcz chr4:155260129-155361361 2.31 1.85E-06 1.15E-04 Protein ENSMUSG00000028518.8 coding Prkaa2 chr4:105029874-105109890 2.13 2.91E-04 7.86E-03 Protein ENSMUSG00000035133.9 coding Arhgap5 chr12:52503972-52571975 2.02 1.42E-03 2.87E-02 Protein ENSMUSG00000024769.7 coding Cdc42bpg chr19:6306456-6325652 1.90 7.90E-05 2.72E-03 Protein ENSMUSG00000006494.11 coding Pdk1 chr2:71873224-71903858 1.89 2.75E-07 2.33E-05 Protein ENSMUSG00000039031.16 coding Arhgap18 chr10:26753421-26918648 1.85 1.76E-04 5.26E-03 Protein ENSMUSG00000003534.17 coding Ddr1 chr17:35681567-35704621 1.70 4.75E-06 2.55E-04 Protein ENSMUSG00000030774.13 coding Pak1 chr7:97788541-97912381 1.61 1.05E-04 3.42E-03 Protein ENSMUSG00000015143.15 coding Actn1 chr12:80167547-80260371 1.51 2.10E-03 3.86E-02 Protein ENSMUSG00000030602.13 coding Pak4 chr7:28558819-28598185 1.45 8.15E-04 1.84E-02 Diabetes Page 52 of 57

Downregulated Gene Gene linear Symbol type name location FC Pvalue FDR Protein ENSMUSG00000006457.3 coding Actn3 chr19:4861223-4877909 -2.80 2.66E-07 2.27E-05 Protein ENSMUSG00000056367.14 coding Actr3b chr5:25759997-25850688 -1.87 1.69E-03 3.27E-02 Protein ENSMUSG00000002233.13 coding Rhoc chr3:104788375-104794459 -1.87 1.16E-04 3.70E-03 Protein ENSMUSG00000029581.14 coding Fscn1 chr5:142960343-142973185 -1.79 3.86E-05 1.48E-03 Protein ENSMUSG00000029674.13 coding Limk1 chr5:134656039-134688598 -1.76 1.10E-04 3.55E-03 Protein ENSMUSG00000030409.15 coding Dmpk chr7:19083849-19093821 -1.71 2.18E-05 9.10E-04 Protein ENSMUSG00000023972.10 coding Ptk7 chr17:46564471-46629504 -1.68 2.38E-04 6.67E-03 Protein ENSMUSG00000019370.10 coding Calm3 chr7:16915379-16924114 -1.54 2.52E-03 4.45E-02 Protein ENSMUSG00000029465.14 coding Arpc3 chr5:122391878-122414184 -1.51 2.62E-03 4.59E-02 Protein ENSMUSG00000005469.10 coding Prkaca chr8:83972993-83996443 -1.51 1.98E-03 3.70E-02 Protein ENSMUSG00000004665.10 coding Cnn2 chr10:79988584-79996062 -1.48 2.27E-03 4.12E-02 Protein ENSMUSG00000051391.9 coding Ywhag chr5:135908409-135934616 -1.46 2.22E-03 4.05E-02 Protein ENSMUSG00000029621.14 coding Arpc1a chr5:145083830-145108761 -1.45 2.16E-03 3.96E-02 Protein ENSMUSG00000029622.16 coding Arpc1b chr5:145114215-145130705 -1.44 2.52E-03 4.46E-02

Table S6. Immunology genes

Differential expression of immunoregulatory genes (NS_IMMUNOLOGY_MM_C2269 panel takn from the Nanostring website) between gp38dim FRC-like cells freshly sorted from either Ins2-CCL21 NOD mice or non-transgenic NOD littermates and processed for RNAseq calculated with the exact test implemented in edgeR. Linear Fold Change

(linearFC) compares cells isolated from Ins2-CCL21 transgenic NOD mice over non- transgenic NOD littermates. Page 53 of 57 Diabetes

Upregulated Gene Linear Symbol Gene type name location FC Pvalue FDR Protein ENSMUSG00000029656.13 coding C8b chr4:104766317-104804548 30.77 6.21E-04 1.48E-02 Protein ENSMUSG00000026415.11 coding Fcamr chr1:130800902-130814740 28.59 7.16E-04 1.66E-02 Protein ENSMUSG00000074272.10 coding Ceacam1 chr7:25461707-25477603 7.01 6.89E-05 2.43E-03 Protein ENSMUSG00000000489.7 coding Pdgfb chr15:79995874-80014977 5.22 3.44E-08 3.96E-06 Protein ENSMUSG00000027111.15 coding Itga6 chr2:71745616-71858416 4.42 8.09E-09 1.13E-06 Protein ENSMUSG00000061132.13 coding Blnk chr19:40928927-40994535 3.72 2.67E-10 5.77E-08 Protein ENSMUSG00000042784.9 coding Muc1 chr3:89229057-89233381 3.67 4.20E-10 8.54E-08 Protein ENSMUSG00000005672.12 coding Kit chr5:75574916-75656722 3.67 3.65E-08 4.16E-06 Protein ENSMUSG00000000409.14 coding Lck chr4:129548344-129573641 3.63 2.84E-05 1.14E-03 Protein ENSMUSG00000047139.9 coding Cd24a chr10:43578284-43584265 3.59 1.29E-10 3.09E-08 Protein ENSMUSG00000015966.17 coding Il17rb chr14:29996135-30008896 3.58 2.98E-04 8.00E-03 Protein ENSMUSG00000043088.16 coding Il17re chr6:113458484-113470758 3.48 3.11E-12 1.32E-09 Protein ENSMUSG00000049176.14 coding Frmpd4 chrX:167471309-168577231 3.36 1.66E-03 3.22E-02 Protein ENSMUSG00000027544.16 coding Nfatc2 chr2:168476410-168601657 3.35 8.77E-05 2.95E-03 Protein ENSMUSG00000026070.15 coding Il18r1 chr1:40465552-40500854 3.31 1.14E-05 5.28E-04 Protein Tnfrsf11 ENSMUSG00000026321.7 coding a chr1:105780718-105847981 3.28 1.85E-05 7.90E-04 Protein ENSMUSG00000026981.15 coding Il1rn chr2:24336853-24351494 3.24 1.75E-06 1.10E-04 Protein ENSMUSG00000022037.15 coding Clu chr14:65968483-65981547 3.22 2.55E-11 7.66E-09 Protein ENSMUSG00000025494.8 coding Sigirr chr7:141091175-141100572 3.16 2.47E-12 1.09E-09 Protein ENSMUSG00000024401.14 coding Tnf chr17:35199381-35202007 3.13 2.25E-07 1.96E-05 Protein ENSMUSG00000028150.14 coding Rorc chr3:94372794-94398276 3.11 8.25E-05 2.83E-03 Protein ENSMUSG00000022797.16 coding Tfrc chr16:32608920-32632794 2.89 4.09E-06 2.25E-04 Protein ENSMUSG00000024621.15 coding Csf1r chr18:61105572-61132149 2.79 6.65E-10 1.28E-07 Protein ENSMUSG00000004266.15 coding Ptpn6 chr6:124720707-124738714 2.77 1.85E-07 1.67E-05 Protein ENSMUSG00000058952.12 coding Cfi chr3:129835884-129875332 2.76 1.64E-07 1.50E-05 Protein ENSMUSG00000026417.13 coding Pigr chr1:130826684-130852249 2.71 4.10E-05 1.56E-03 Protein ENSMUSG00000024399.5 coding Ltb chr17:35194439-35196320 2.60 9.30E-05 3.09E-03 Diabetes Page 54 of 57

Protein ENSMUSG00000040584.8 coding Abcb1a chr5:8660077-8748575 2.57 1.66E-05 7.20E-04 Protein ENSMUSG00000051439.6 coding Cd14 chr18:36725074-36726736 2.49 1.39E-12 6.61E-10 Protein ENSMUSG00000062939.11 coding Stat4 chr1:51987148-52107189 2.46 2.48E-05 1.01E-03 Protein ENSMUSG00000079164.8 coding Tlr5 chr1:182954788-182976044 2.44 5.52E-06 2.90E-04 Protein ENSMUSG00000041607.16 coding Mbp chr18:82475146-82585637 2.40 1.83E-11 5.83E-09 Protein ENSMUSG00000079105.4 coding C7 chr15:4988762-5063740 2.10 1.20E-06 7.99E-05 Protein ENSMUSG00000027820.12 coding Mme chr3:63241537-63386030 2.01 4.45E-04 1.12E-02 Protein ENSMUSG00000030560.17 coding Ctsc chr7:88278085-88310888 1.98 4.77E-05 1.77E-03 Protein ENSMUSG00000024948.14 coding Map4k2 chr19:6341135-6355615 1.86 8.99E-05 3.01E-03 Protein ENSMUSG00000026923.15 coding Notch1 chr2:26457903-26516663 1.72 5.59E-05 2.03E-03 Protein ENSMUSG00000019850.11 coding Tnfaip3 chr10:19000910-19015657 1.69 1.52E-05 6.72E-04 Protein ENSMUSG00000026029.14 coding Casp8 chr1:58795374-58847503 1.54 1.38E-03 2.80E-02 Protein ENSMUSG00000060477.14 coding Irak2 chr6:113638467-113695026 1.53 3.69E-04 9.56E-03 Protein ENSMUSG00000046223.10 coding Plaur chr7:24462484-24475968 1.49 2.48E-03 4.40E-02 Protein ENSMUSG00000006932.17 coding Ctnnb1 chr9:120929216-120960507 1.43 2.52E-03 4.45E-02

Downregulated Gene Linear Symbol Gene type name location FC Pvalue FDR Protein ENSMUSG00000029417.9 coding Cxcl9 chr5:92321347-92328079 -5.56 1.77E-24 2.75E-21 Protein ENSMUSG00000026073.13 coding Il1r2 chr1:40074079-40125231 -4.08 1.33E-07 1.26E-05 Protein ENSMUSG00000036594.14 coding H2-Aa chr17:34282744-34287827 -3.92 1.59E-08 2.08E-06 Protein ENSMUSG00000096727.2 coding Psmb9 chr17:34181988-34187764 -3.57 2.41E-10 5.31E-08 Protein ENSMUSG00000009185.2 coding Ccl8 chr11:82115185-82116799 -3.42 1.88E-04 5.53E-03 Protein ENSMUSG00000073421.5 coding H2-Ab1 chr17:34263209-34269418 -3.05 1.36E-07 1.28E-05 Protein ENSMUSG00000038037.5 coding Socs1 chr16:10782240-10785536 -3.03 1.70E-13 1.08E-10 Protein ENSMUSG00000061232.15 coding H2-K1 chr17:33996017-34000333 -2.73 1.59E-12 7.41E-10 Protein ENSMUSG00000067235.14 coding H2-Q10 chr17:35470089-35474563 -2.68 3.94E-10 8.19E-08 Protein ENSMUSG00000060586.10 coding H2-Eb1 chr17:34305877-34316199 -2.63 9.90E-07 6.87E-05 Protein ENSMUSG00000037321.17 coding Tap1 chr17:34187553-34197225 -2.62 2.23E-09 3.61E-07 Protein ENSMUSG00000031897.9 coding Psmb10 chr8:105935735-105938444 -2.45 7.19E-08 7.60E-06 Protein ENSMUSG00000021356.10 coding Irf4 chr13:30749226-30766976 -2.44 6.13E-06 3.17E-04 Page 55 of 57 Diabetes

Protein ENSMUSG00000021822.3 coding Plau chr14:20836660-20843385 -2.41 1.19E-09 2.12E-07 Protein ENSMUSG00000024164.15 coding C3 chr17:57203970-57228136 -2.36 2.97E-08 3.52E-06 Protein ENSMUSG00000025888.5 coding Casp1 chr9:5298517-5307265 -2.30 3.13E-05 1.24E-03 Protein ENSMUSG00000056749.7 coding Nfil3 chr13:52967209-52981073 -2.28 9.93E-08 9.81E-06 Protein ENSMUSG00000046718.8 coding Bst2 chr8:71534255-71537456 -2.27 6.15E-08 6.59E-06 Protein ENSMUSG00000026770.5 coding Il2ra chr2:11642807-11693193 -2.25 3.15E-06 1.79E-04 Protein ENSMUSG00000056501.3 coding Cebpb chr2:167688915-167690418 -2.23 2.65E-09 4.19E-07 Protein ENSMUSG00000025498.15 coding Irf7 chr7:141262706-141266481 -2.22 1.54E-07 1.43E-05 Protein ENSMUSG00000046879.7 coding Irgm1 chr11:48861968-48871683 -2.19 3.59E-10 7.48E-08 Protein ENSMUSG00000025491.14 coding Ifitm1 chr7:140967221-140969825 -2.19 5.13E-06 2.73E-04 Protein ENSMUSG00000003420.8 coding Fcgrt chr7:45092990-45103851 -2.16 1.30E-05 5.90E-04 Protein ENSMUSG00000060802.8 coding B2m chr2:122147686-122153083 -2.11 5.89E-09 8.46E-07 Protein ENSMUSG00000053113.3 coding Socs3 chr11:117966079-117970047 -2.03 1.43E-05 6.38E-04 Protein ENSMUSG00000006611.15 coding Hfe chr13:23702034-23710854 -1.99 7.07E-06 3.53E-04 Protein ENSMUSG00000055172.10 coding C1ra chr6:124512405-124523443 -1.94 1.67E-06 1.06E-04 Protein ENSMUSG00000024620.11 coding Pdgfrb chr18:61045150-61085061 -1.91 1.01E-06 6.97E-05 Protein ENSMUSG00000028599.10 coding Tnfrsf1b chr4:145213463-145246870 -1.89 9.51E-08 9.53E-06 Protein ENSMUSG00000026104.14 coding Stat1 chr1:52119440-52161865 -1.88 8.84E-08 9.01E-06 Protein ENSMUSG00000031497.9 coding Tnfsf13b chr8:10006467-10039072 -1.88 8.50E-08 8.75E-06 Protein ENSMUSG00000016496.7 coding Cd274 chr19:29367455-29388095 -1.82 8.90E-05 2.99E-03 Protein ENSMUSG00000023067.14 coding Cdkn1a chr17:29090976-29100727 -1.81 5.06E-06 2.69E-04 Protein ENSMUSG00000097328.7 coding Tnfsf12 chr11:69686250-69695849 -1.81 1.04E-04 3.39E-03 Protein ENSMUSG00000023224.12 coding Serping1 chr2:84765387-84775444 -1.81 1.24E-04 3.91E-03 Protein ENSMUSG00000033467.12 coding Crlf2 chr5:109554709-109558993 -1.80 6.11E-06 3.16E-04 Protein ENSMUSG00000026193.15 coding Fn1 chr1:71585520-71653200 -1.80 2.25E-04 6.36E-03 Protein ENSMUSG00000048120.16 coding Entpd1 chr19:40612366-40741602 -1.79 8.41E-05 2.87E-03 Protein ENSMUSG00000042190.12 coding Cmklr1 chr5:113612354-113650426 -1.78 2.79E-06 1.61E-04 Protein ENSMUSG00000027947.11 coding Il6ra chr3:89864059-89913196 -1.77 5.14E-06 2.73E-04 Protein ENSMUSG00000024371.14 coding C2 chr17:34862604-34898265 -1.77 8.91E-05 2.99E-03 Protein ENSMUSG00000019843.14 coding Fyn chr10:39368855-39565381 -1.76 3.70E-05 1.42E-03 Diabetes Page 56 of 57

Protein ENSMUSG00000027995.10 coding Tlr2 chr3:83836272-83841767 -1.75 1.12E-04 3.61E-03 Protein ENSMUSG00000053475.5 coding Tnfaip6 chr2:52038009-52056686 -1.75 1.81E-04 5.37E-03 Protein ENSMUSG00000032508.8 coding Myd88 chr9:119335934-119341411 -1.74 3.09E-05 1.23E-03 Protein ENSMUSG00000042333.16 coding Tnfrsf14 chr4:154921933-154928563 -1.71 1.45E-05 6.45E-04 Protein ENSMUSG00000036427.5 coding Gpi1 chr7:34201330-34230336 -1.70 1.72E-03 3.31E-02 Protein ENSMUSG00000024308.14 coding Tapbp chr17:33915899-33929288 -1.70 7.86E-05 2.71E-03 Protein ENSMUSG00000032011.5 coding Thy1 chr9:44043384-44048579 -1.68 4.72E-04 1.17E-02 Protein ENSMUSG00000026637.13 coding Traf5 chr1:191997205-192092559 -1.67 8.71E-05 2.95E-03 Protein ENSMUSG00000016494.9 coding Cd34 chr1:194938819-194961279 -1.65 8.25E-04 1.86E-02 Protein ENSMUSG00000041515.10 coding Irf8 chr8:120736358-120756694 -1.64 1.60E-03 3.13E-02 Protein ENSMUSG00000000555.8 coding Itga5 chr15:103344286-103366763 -1.64 3.50E-04 9.14E-03 Protein ENSMUSG00000017344.4 coding Vtn chr11:78499091-78502324 -1.63 1.86E-03 3.52E-02 Protein ENSMUSG00000035000.8 coding Dpp4 chr2:62330073-62412231 -1.63 9.83E-04 2.14E-02 Protein ENSMUSG00000014599.10 coding Csf1 chr3:107741048-107760469 -1.63 8.74E-05 2.95E-03 Protein ENSMUSG00000024610.14 coding Cd74 chr18:60803848-60812646 -1.62 1.41E-03 2.84E-02 Protein ENSMUSG00000040033.16 coding Stat2 chr10:128270559-128292849 -1.62 8.52E-05 2.90E-03 Protein ENSMUSG00000039377.7 coding Hlx chr1:184727140-184732619 -1.61 1.81E-03 3.45E-02 Protein ENSMUSG00000020227.10 coding Irak3 chr10:120141648-120202130 -1.61 9.37E-05 3.11E-03 Protein ENSMUSG00000073889.10 coding Il11ra1 chr4:41699989-41769474 -1.61 1.49E-03 2.97E-02 Protein ENSMUSG00000020484.18 coding Xbp1 chr11:5520659-5525893 -1.60 7.24E-04 1.68E-02 Protein ENSMUSG00000002603.15 coding Tgfb1 chr7:25687002-25705077 -1.57 2.78E-03 4.81E-02 Protein ENSMUSG00000055435.6 coding Maf chr8:115682942-115707794 -1.57 2.64E-03 4.61E-02 Protein ENSMUSG00000020919.11 coding Stat5b chr11:100780731-100850724 -1.57 2.59E-04 7.12E-03 Protein ENSMUSG00000003873.11 coding Bax chr7:45461697-45466898 -1.55 1.13E-03 2.38E-02 Protein ENSMUSG00000053175.17 coding Bcl3 chr7:19808462-19822770 -1.54 2.92E-04 7.89E-03 Protein ENSMUSG00000022971.18 coding Ifnar2 chr16:91372783-91405589 -1.51 4.36E-04 1.10E-02 Protein ENSMUSG00000070942.8 coding Il1rl2 chr1:40324610-40367562 -1.51 2.74E-03 4.76E-02 Protein ENSMUSG00000073489.6 coding Ifi204 chr1:173747293-173766943 -1.48 1.64E-03 3.19E-02 Protein ENSMUSG00000022965.8 coding Ifngr2 chr16:91547072-91565623 -1.46 2.51E-03 4.43E-02 Page 57 of 57 Diabetes Diabetes Page 58 of 57

Supplemental Figures

Figure S1. (A) Quantification of T cell phenotypes in pancreatic infiltrates of 4 week-old non-transgenic littermate (n=5) and Ins2-CCL21 NOD (n=5) mice by flow cytometry. Data Page 59 of 57 Diabetes

are indicated as % CD45+ live cells. (B) Quantification of T cell phenotypes in blood of

4week-old non-transgenic (n=5) and Ins2-CCL21 NOD (n=5) mice by flow cytometry.

Data are indicated as % CD45+ live cells. (C) Quantification of T cell phenotypes in spleen

of 4 week-old non-transgenic (n=5) and Ins2-CCL21 NOD (n=5) mice by flow cytometry.

Data are indicated as total numbers of live cells. (D) Quantification of innate and antigen

presenting cell phenotypes in pancreatic infiltrates of 4week-old non-transgenic (n=5)

and Ins2-CCL21 NOD (n=5) mice by flow cytometry. Data are indicated as % CD45+ live

cells. ***, p<0.01. Statistically significant populations are underlined and highlighted by

boxes.

+ + + hi - T (T cells): CD3 ; Tregs (regulatory T cells): CD4 FoxP3 CD25 ; Tn (naïve T cells): CD44

- + + - + CD127 CD62L ; Tef (effector T cells): CD44 CD127 , Tcm (central memory T cells): CD44

+ + + + - CD127 CD62L ; Tefm (effector memory T cells): CD44 CD127 CD62L ; Macs (macrophages):

CD11b+ F4/80+ B220-; Neutr (neutrophils): CD11b+ GR-1+; Eos (eosinophils): CD11b+ GR-1+

F4/80+; B cells: CD11b- F4/80- B220+; mmDCs (mature myeloid dendritic cells): CD11b+ CD11c+

MHCII+; imDCs (immature myeloid dendritic cells): CD11b+ CD11c+ MHCII-; ilDCs (immature

lymphoid dendritic cells): CD11b- CD11c+ I MHCII-; mlDCs (mature lymphoid dendritic cells):

CD11b- CD11c+ I MHCII+. Diabetes Page 60 of 57

Figure S2. (A) Quantification of FRC (CD45- gp38+ CD31-) total number in lymph nodes

(LNs) and of FRC-like cells in pancreatic islets of 4 week-old non-transgenic (n=4) and Page 61 of 57 Diabetes

Ins2-CCL21 NOD mice (n=4) by flow cytometry. (B) Quantification of gp38 mean

fluorescence intensity (MFI) of gp38dim CD31-, gp38hi CD31-, and gp38+ CD31- FRCs-like

cells from LNs and from islets of CCL21-trnasgnic NOD mice (red) and from non-

transgenic NOD littermates. Also shown are quantification of gp38 mean fluorescence

intensity of all gp38+ CD31- FRCs-like cells from LNs and from islets of CCL21-trnasgnic

NOD mice (red) and from non-transgenic NOD littermates and MFI gp38 values of

CCL21-transgnic NOD normalized to non-transgenic littermates. (C, D) Quantification of

relative proportions of CD45- lymphoid stromal cells (FRC: gp38+ CD31-; LECs: gp38+

CD31+; BECs: gp38- CD31+; DN: gp38- CD31-) from LNs of 4 week-old (C) and 12 week-

old (D) non-transgenic (n=4) and Ins2-CCL21 NOD mice (n=4) by flow cytometry. (E)

Percent of diabetes free chemically-diabetic BALB/c mice transplanted (EFP) with islets

from fully MHC-mismatched CCL21+ (red, n=13) transgenic or CCL21- (black, n=4)

control B6 mice; ***, p<0.01; *, p<0.05. (F, G) Comparison of morphology quantified by

forward scatter (FSC-A) of CD45- gp38+ CD31- FRC isolated from the LNs or of FRC-like

cells from the pancreatic islets of 4 week-old (F) or from 12 week-old (G) non-transgenic

(n=4) and Ins2-CCL21 NOD mice (n=4) by flow cytometry.