a humanized mouse model to study type 1 diabetes · 7/5/2018 · key requirements in type 1...

A HUMANIZED MOUSE MODEL TO STUDY TYPE 1 DIABETES

LUCE Sandrine1,2, GUINOISEAU Sophie1,2, GADAULT Alexis1,2, LETOURNEUR Franck1,

BLONDEAU Bertrand3, NITSCHKE Patrick2, PASMANT Eric2,4, VIDAUD Michel4,

LEMONNIER François1,2, BOITARD Christian1,2

1INSERM U1016, Institut Cochin, Paris, France; 2 Université Paris Descartes, Faculté de Médecine René Descartes, Paris, France; 3 INSERM U938, Centre de recherche Saint-Antoine, Paris, France; 4Service de Biochimie et Génétique Moléculaire, Hôpital COCHIN, Paris, France;

2 Address correspondence to Pr. Christian BOITARD, INSERM U1016, Groupe Hospitalier

Cochin-Port-Royal, 123 bvd Port Royal, 75014 Paris, France. Tel: 33 (0) 1 58 41 24 40. Fax:

33 (0) 1 46 34 64 54; Email: [email protected]

of 54 Diabetes

Diabetes Publish Ahead of Print, published online July 6, 2018

Abstract

Key requirements in type 1 diabetes are in setting up new assays as diagnostic biomarkers that

will apply to prediabetes, likely T-lymphocyte assays, and in designing antigen-specific

therapies to prevent its development. New preclinical models of T1D will be required to help

advancing both aims. By crossing mouse strains that lack either murine major

histocompatibility complex class-I, class-II genes and insulin genes, we developed YES mice

that instead expresses human HLA-A*02:01, HLA-DQ8 and insulin genes as transgenes. The

metabolic and immune phenotype of YES mice is basically identical to that of the parental

strains. YES mice remain insulitis- and diabetes-free up to one year of follow up, maintain

normoglycemia to an intraperitoneal glucose challenge in the long-term range, have a normal

β-cell mass and show normal immune responses to conventional antigens. This new model

has been designed to evaluate adaptive immune responses to human insulin on a genetic

background that recapitulate human high susceptibility HLA-DQ8 genetic background.

Although insulitis-free, YES mice develop T1D when challenged with polyInosinic-

polyCytidylic acid. They allow characterizing preproinsulin epitopes recognized by CD8+ and

CD4+ T-lymphocytes upon immunization against human preproinsulin or along diabetes

development.

of 54Diabetes

INTRODUCTION

The non-obese diabetic (NOD) mouse has been instrumental in deciphering mechanisms

involved in type 1 diabetes (T1D). But it fails to cover clinical T1D heterogeneity, shows

phenotypic differences with human T1D and limitations when developing T-cell assays or

therapies to be applied to the human [1]. Immunosuppression has shown efficacy in

preserving β-cells in recent-onset T1D patients, but also side effects precluding its long-term

use [2]. Strategies to restore immune tolerance to β-cells should thus be prioritized [3] as

respecting responses to unrelated antigens. We lack rodent models to evaluate antigen-

specific immunotherapy that directly apply to the human as well as rodent models and to

explore environmental factors involved in triggering diabetes on conventional genetic

backgrounds.

We designed a mouse expressing both a human β cell autoantigen and a human antigen-

presenting module to characterize autoantigen-derived epitopes that translate to the human.

Based on evidence that insulin is a key autoantigen, we introgressed a human insulin (hINS)

transgene in our model. Indeed, insulin is targeted by autoreactive T-cells in T1D [4, 5]. Anti-

insulin autoantibodies are the first to be detected in children at risk and carry a high positive

predictive value for T1D in patient siblings [6]. The VNTR located 5’ of the hINS gene

predisposes to T1D. In the NOD, the lack of either the mouse insulin (mINS) 1 or 2 gene

markedly alters the diabetes phenotype [4, 7]. We selected HLA-A*02:01 and HLA-DQ

A1*0301/B1*03:02, i.e. DQ8, as human major histocompatibility complex (MHC) genes to

be expressed in addition to the hINS gene. HLA-DQ A1*0301/B1*03:02, i.e. DQ8, carries the

highest risk for T1D in man [8] and presumably present epitopes that drive the autoimmunity

to β-cells. Class-I HLA-A*02:01 also modify the risk for T1D and is the most common class-

I gene expressed in Caucasians [9].

of 54 Diabetes

We thus generated humanized mice that lack the expression of murine MHC class-I, class-II

and insulin genes, and express HLA-A*02:01, HLA-DQ8 and hINS transgenes, thereafter

called YES mice. YES mice develop T1D upon injection of polyInosinic-polyCytidylic acid

(pI:C).

of 54Diabetes

RESEARCH DESIGN AND METHODS:

Mice

YES mice were obtained by crossing mINS-/- H-2k mice that express a hINS transgene under

the control of the 353 bp human insulin gene promoter on a mix C57BL/6JxCBA background

[10] with double transgenic HLA-A*02:01/HLA-DQ8 mice carrying a dominant C57BL/6J

background. The HLA-A*02:01/HLA-DQ8 mouse was obtained by crossing transgenic H-

2Db mouse-ß2-microglobulin (mß2m) double KO HLA-A*02:01 [11] and IAb

IEb double KO

HLA-DQ8 mice [12, 13]. It expresses a chimeric HLA-A*02:01 HHD monochain containing

the HLA-A*02:01 α1 and α2 and H-2Db α3 domains linked to human β2m by a 15-mer

linker under the control of the HLA-A*02:01 promoter [11] and the HLA-DQ8 α and β chains

under the control of their promoter [13] (Supplemental Material-1). Mice were maintained

under specific pathogen-free conditions and experiments performed following Institutional

Animal Care and Use Guidelines accreditation CEEA34.CB.024.11 by the ethic committee.

Genotyping

Insulin PCRs were performed with Red Taq (Sigma-Aldrich) using primers listed in

Supplemental Material-2. Screening for mß2m and human HLA transgenes was performed

with GC2 polymerase Mix PCR (Clontech). Real time quantitative PCRs based on 3-point

dilutions using QuantiTect SybrGreen PCR Kit (Qiagen) on LC480 (Roche) allowed

discriminating mice carrying a single mINS1 and/or mINS2 allele for further crosses. Cell-

surface stainings were performed on blood cells for the loss of H-2Dk and IAkIEk using anti-

mouse TCR-APC-labeled and anti-H-2Dk-FITC-labelled antibodies (BDPharmingen) or with

anti-mouse CD19-APC-labelled and anti-H-2 IAk/IEk-FITC-labeled antibodies (BD

Pharmingen). Data were collected with a LSR-FORTESSA cytometer and analyzed using

FlowJo 9.2 software (FlowJo, Tree Star Inc).

Thymic epithelial cell (TEC) isolation

of 54 Diabetes

Thymi from 6 weeks-old-mice were digested with 0.125% Collagenase D with 0.1% DNase I

followed by two step digestions with 0.5 U/ml Liberase TM and 0.1% DNase I [14]. TEC

enrichments were then depleted using mouse CD45-MicroBeads from Miltenyi Biotec on LD

column according to manufacturer’s protocol.

Flow cytometry

Cell suspensions in RPMI 1640/FCS 10% (106 cells/ml) were stained with: anti-mouse

CD3ε-PE, CD3ε-APC, CD8α-PerCP, CD4-efluor450, CD44-FITC, CD45-APC, CD19-APC,

CD11c-APC, CD11b-efluor450 or CD11b-APC; anti-human β2m-FITC (clone TÜ99,

BDBiosciences), anti-HLA-DQ-Biot (Hybridoma SVPL3), Streptavidin-FITC, anti-H-2Dk-

FITC, anti-H-2IAk-FITC; anti-mouse NKp46-PE, CD25-PE, FoxP3-FITC, TCRβ-APC,

NK1.1-APC, LY6G-PE-labelled, TER119-FITC (homemade), CD45.2-PE or CD90.2-FITC,

Biotin-conjugated anti-insulin (DAKO), Biotin-conjugated anti-mouse CD62L and SAV-

PECy7 from ebiosciences if not specified. Data were collected with LSR-FORTESSA

cytometer and analyzed using FlowJo 9.2 software.

Medullary TEC (mTEC) and cortical TEC (cTEC) were sorted with a FACSAria II using an

anti-mouse CD45-PeCy7, an anti-H-2IAb-FITC or anti-H-2IAk-FITC or anti-HLA-DQ-FITC

depending on the mouse genetic background, an anti-mouse-Epcam-APC, an anti-mouse

BP1-PE, an anti-mouse UEA-I-biot and a Streptavidin-BrillantViolet-650 (ebiosciences

except for UEA-I-Biot (Ulex Europaeus Agglutinin I) (VECTOR Laboratories). CD45- MHC

Class-II+ Epcam+ cells define TEC. BP1 and UEA-I allowed sorting cTEC and mTEC; BP1+

UEA-I- and BP1- UEA-I+ respectively [14].

Gene expression

After RNA Later-stabilization, RNA extractions were performed on solid organs and inguinal

lymph nodes (iLN) with RNeasy Mini Prep and DNaseI treatment (Qiagen). Sorted cTEC and

mTEC were lysed in Qiazol lysis reagent and RNA extracted with miRNeasy Mini Prep and

of 54Diabetes

DNaseI treatment (Qiagen). Gene specific primers were used for reverse transcription on 10-

15ng RNA (Supplemental Material-3). First round PCRs were performed on 5µl of amplified

cDNA: for ßactin, Aire and Caseinß, 30 cycles/TM=54°C; for Insulin genes, 30

cycles/TM=58°C, Supplemental Material-4) and second round PCR on 2µl of the PCR1

product (for ßactin, Aire and Caseinß, 40 cycles/TM=54°C; for all Insulin genes, 40

cycles/TM=58°C, Supplemental Material-5) [15].

Immunizations

Mice were immunized against either 100µg HLA-A*02:01-restricted Influenza matrix protein

2 peptide GILGFVFTL (MatA258-66) in CFA or 100µg of hPPI peptides (Supplemental

Material-6) with 140µg HLA-DQ8-restricted helper Nef66-97 peptide; or 100µg hPPI

(Supplemental Material-7) in incomplete Freund’s adjuvant (IFA) subcutaneously at the base

of the tail, followed by 2 recall injections in IFA every two other weeks. Control

immunizations were realized injecting PBS1X or KLH in adjuvant.

Enzyme-linked Immunospot (ELISpot):

IFNγ-ELIspot assays were performed as previously reported [16]. Spots were counted using

Bioreader 5000 ProSF (BioSys GmbH). Data are mean of triplicate-wells and expressed as

spot-forming cells (SFC) per 106 cells, evaluating the background IFNγ responses in absence

of peptide. Positive controls were cells stimulated by 1µg/ml ConA (Sigma-Aldrich) and

negative controls by irrelevant Pyruvate Dehydrogenase (PDHase208-216) peptide. When

indicated, responses were inhibited by pre-incubating splenocytes for 20 min with 50µg/ml

anti-HLA-A*02:01 antibody (BB7.2). For CD4+ IFNγ-ELIspot assays, splenocytes were

depleted using mouse anti-CD8-microBeads (Miltenyi) on LD column according to

manufacturer’s protocol. When indicated, responses were inhibited by pre-incubating CD8+

T-cells-depleted splenocytes for 20 min with 50 µg/ml anti-HLA-DQ antibody (SVPL3).

Cell proliferations:

of 54 Diabetes

105 spleen cells/well were incubated with 0.5µg antigen/well or 0.1µg peptide (Supplemental

Material-8)/well for 72h at 37°C in triplicate. Proliferation was evaluated with BrdU Cell

Proliferation Assay Kit (Cell Signaling) and expressed as proliferation index (PI).

Background and positive controls were evaluated in triplicate wells containing 105 cells/well

incubated without antigen or in the presence of 10µg/ml final concentration of anti-mouse

CD3ε antibody. When indicated, the response was inhibited adding 50µg/ml of anti-HLA-

DQ8 antibody (Hybridoma SVPL3).

Metabolic evaluations

Body weight was monitored weekly for 20 weeks. Intra-peritoneal glucose tolerance tests

(IGTT, 2g/kg body weight) were performed after 12h-16h fasting. Intra-peritoneal insulin

tolerance tests (IITT) (0.75U/kg body weight) were performed by injecting Actrapid

(Novonordisk) after 4h fasting. Blood glucose was measured using a glucometer (BG Star) at

0, 30, 60 and 120 min.

Immunostainings

Immunostainings were performed on formalin-fixed paraffin pancreas sections deparaffinized

in Xylene and dehydrated by Ethanol. After washing, antigen retrieval was realized by hot

incubation, followed by permeabilization (20 min in PBS1X/0,4% Triton-X100) and

saturation (20 min PBS 1X/1% Horse Serum) before immuno-staining with polyclonal

Guinea Pig anti-Insulin, polyclonal rabbit anti-glucagon (DAKO), anti-PDX1, anti-TLR3

(FITC, TLR3.7, Hycult Biotech) or rat anti-human CD3-Biot (ABDSerotec) antibodies

overnight. Slides were washed with PBS 1X/1% BSA/0.1% TritonX100 and stained with an

anti-rabbit Ig-FITC antibody, a goat anti-Guinea pig-CyA3 (Abcam) or SAV-Cy3 at RT.

Sections were mounted in Vectashield Mounting Medium for fluorescence with DAPI

(Vector Laboratory). Observations were made with fluorescence microscope Zeiss

AxioObserver Z1 coupled with MRm Axiocam Zeiss and pictures analyzed with the ImageJ

of 54Diabetes

software. Assessment of ß-cell mass was performed on scan stained-microscope slides with

ImageJ software using a guinea pig anti-hINS antibody (DAKO) as the ratio between ß-cell

surface (µm2)/pancreas surface (µm2) multiplied by pancreas weight (mg) [17].

TLR ligand treatment and additional controls

6-8 weeks old mice were daily injected (ip) with 100µg vac-pI:C (Invivogen), or 50µg CpG

(ODN 2395, Invivogen) or 10µg of LPS (Sigma) for 6 days, tested for glycosuria every 2-

days, and diagnosed diabetic on blood glucose levels >250mg/dl. YES mice were injected

with 6 daily dose of 2mg corticosterone/Kg as stress control or with 5 daily injection of 50mg

STZ/Kg as diabetes-induced control. To deplete in vivo in CD4+ and CD8+ T-cells, we

injected 500µg of GK1.5 and YTS169.4 48h prior the pI:C injection and we maintain CD4+

T-cells depletion during 3 days more[18, 19].

of 54 Diabetes

RESULTS

HLA-A*02:01/HLA-DQ8/hINS YES mice

YES mice lack the expression of mINS genes and express the hINS transgene in the pancreas

and the thymus (Fig.1B). The hINS transgene was expressed in thymic mTEC, co-expressed

with the AIRE gene, but not expressed in cortical cTEC (Fig.1C), a pattern similar to that of

hINS in parental mice and of mINS2 in C57BL/6 mice [15, 20]. We confirmed the loss of

mouse MHC class-I (H-2Dk and H-2Kk) (Fig.1D, upper panel) and class-II (IAk) (Fig.1D,

lower panel) and the presence of the transgenic HLA-A*02:01 molecule (Fig.1E, upper panel)

on T cells and transgenic HLA-DQ8 molecule (Fig.1E, lower panel) on B cells. We observed

the expression of HLA-A*02:01 on CD45-Insulin+ islet cells from YES mice as of murine H-

2Db in C57BL/6 mice (Fig.1F).

HLA-A*02:01/HHD expression was confirmed on immune cells in spleen, blood, and iLN by

flow cytometry (Supplemental Data-1). In all subsets analyzed, HLA-A*02:01 expression was

comparable in YES and in parental HLA-A*02:01/HLA-DQ8 double transgenic mice (not

shown). Expression of both HLA class-I and class-II transgenic molecules was lower than

expression of H-2Db (Supplemental Data-1A) and H-2 IAb (Supplemental Data-1B)

molecules in C57BL/6 mice.

As the YES genetic background is a mix of C57BL/6 and CBA strains, we characterized the

YES genome relative to C57BL/6 and NOD genomes (Supplemental Material-9). NOD and

C57BL/6 mice showed 80.4% identity. YES mice showed 79.3% identity with NOD but

90.9% identity with C57BL/6 mice (Table 1). We studied T1D-associated regions in the YES

genome. Mouse linkage regions associated with T1D were listed, and updated on the USCS

Genome Browser Home (https://genome.uscs.edu/index.html). SNPs that were identical in NOD

and C57BL/6 mice were not further considered. 0.21% of SNPs were different between YES

and C57BL/6 mice. SNPs were listed according to each chromosome and corresponding Idd

of 54Diabetes

loci when relevant. We identified SNPs for which at least one variation was identified. Using

Mouse Genome Informatics (MGI) web site, we found 86% of SNPs associated to a CBA

genetic background. 14% were not described or covered regions for which PCR probes do not

discriminate NOD, C57BL/6 and CBA Idd profiles or could not be defined in absence of

marker for the relevant region (Supplemental Data-2).

Distribution of immune cell subsets in YES mice

Absolute numbers of spleen, iLN and thymic cells were comparable in YES and in parental

HLA-A*02:01/HLA-DQ8 double transgenic mice (not shown). Distributions of most immune

cell subsets were comparable in YES and C57BL/6 mice, with few differences (Fig.2). Spleen

T-cell frequencies were lower in YES than in C57BL/6 mice (13.0% ± 2.4 vs 29.0% ± 2.1)

with an inverted CD3+CD4+/CD3+CD8+ T-cell ratio (0.7 and 1.35, respectively) (Fig.3A). A

significant fraction of cells (9.0% ± 3.9) were CD3- CD4- CD8- CD19- CD11b- CD11c- Nk1.1-

and not observed in parental HLA-A*02:01/HLA-DQ8 double transgenic mice (Supplemental

Data-3), but present in parental hINS transgenic mice (8.0% ± 2.9). Corresponding cells were

hematopoietic cells expressing CD45. In YES spleens, a majority of cells were analogous to

double negative DN3 (CD44-CD25+) pre-T cells (59.3%) while a minority was analogous to

DN1 (CD44+CD25-, 17.2%), DN2 (CD44+CD25+, 5.2%) pro-T cells and DN4 (CD44-CD25-,

16.3%) pre-T cells as compared to 3.4%, 32.2%, 3.1% and 61.2%, respectively, in hINS

transgenic mice [21]. In YES spleens, those cells were CD62L-, suggesting that they were

immature cells incapable of homing in peripheral lymph nodes. In iLN cells (Fig.2B), an

inverted CD3+CD4+/CD3+CD8+ T-cell ratio was also observed in YES as compared to

C57BL/6 mice (0.5 and 1.25, respectively) with a slight difference in T-cell frequency (47.0%

± 8.5 vs 53.45% ± 5.6). In peripheral blood, T-cells were under-represented in YES as

compared to C57BL/6 mice (12.0% ± 9.8 vs 48.0% ± 7.6, *p=0.0238 with Mann-Whitney

Test) with an inverted CD3+CD4+/CD3+CD8+ T cell ratio (0.69 and 1.3, respectively).

of 54 Diabetes

Dendritic cells (12.0% ± 1.83 vs 0.89% ± 1.1) and macrophages (30.0% ± 8.1 vs 8.83%± 3.4)

were overrepresented (Fig.2C). Thymus analyses showed no difference in YES as compared

to C57BL/6 mice. The dominant CD3+low T-cell subset, which is mainly constituted of

CD3+lowCD4+CD8+ double positive cells, was comparable in YES and C57BL/6 mice (90% ±

3.19 and 95% ± 3,09, respectively – Fig.2D). CD4+CD25+Foxp3+ regulatory T-cells were not

significantly different in YES and in C57BL/6 mice.

YES responses to conventional antigens

As differences were seen in immune cell distributions, we evaluated immune responses to

conventional antigens. After immunization against MatA258-66, YES splenocytes showed a

strong IFNγ-ELISpot response (Supplemental Data-4A) that was inhibited in the presence of

the anti-HLA-A*02:01BB7.2 antibody. CD8+ T-cell-depleted HLA-DQ8-restricted CD4+

spleen T-cells from YES mice immunized against KLH showed significant IFNγ responses

(Supplemental Data-4B) that were inhibited by an anti-HLA-DQ antibody (p≤0.03).

Altogether, these experiments indicate that YES mice respond to conventional antigens as

expected.

YES mice show conserved glucose homeostasis

As hINS binding to the mouse insulin receptor is in the normal range [22], YES mice are

expected to remain normoglycemic. They maintained a normal weight in the long-term range

(Fig.3A and B). Their overall islet morphology was comparable in YES and HLA-

A*02:01/HLA-DQ8 parental mice, as were insulin, glucagon and PDX1 staining (Fig.3C).

The β-cell mass was comparable in YES mice and parental hINS and HLA-A*02:01/HLA-

DQ8 mice at 15 weeks of age (Fig.3D). No significant difference was observed in YES

responses to 2mg/kg i.p. GTT at 6 months of age as compared to HLA-A*02:01/HLA-DQ8

parental mice (Fig.3E). Glycemic responses to 0.75 U/Kg i.p. insulin were similar in female

YES mice and parental HLA-A*02:01/HLA-DQ8 females, while a difference was observed in

of 54Diabetes

male mice (Fig.3F). In the long-term range, YES mice developed neither diabetes nor

insulitis.

YES immune responses to preproinsulin

We previously identified CD8+ T-cell responses hPPI peptides in human T1D [23]. After

immunization of YES mice against individual HLA-A*02:01-restricted hPPI-peptides, a

significant response was detected against hPPI2-11 (p≤0.03) and hPPI6-14 (p≤0.03) using an

IFNγ-ELIspot assay. A significant but weak response was detected against hPPI33-42 (p≤0.04)

and against hPPI101-109 in an individual mouse (Fig.4A).

YES mice were immunized against hPPI and tested for responses against a panel of hPPI

overlapping peptides. They remained insulitis-free upon immunization (not shown).

Significant IFNγ responses (Fig.4B) were detected against peptides overlapping the whole

hPPI sequence. When considering individual mouse responses, 93.75% mice showed IFNγ

responses against at least one hPPI peptide (Supplemental Data-5). Predominant individual

IFNγ responses were observed to hPPI20-35 (62.5% of mice, p≤0.008), hPPI25-40 (56.25% of

mice, p≤0.02), hPPI46-61 (56.25% of mice, p≤0.02), hPPI55-70 (68.75% of mice, p≤0.005),

hPPI61-76 (62.5% of mice, p≤0.008) and hPPI92-110 (68.75% of mice, p≤0.005) and hPPI

(81.25% of mice, p≤0.0005).

The number of epitopes that led to proliferative responses in vitro following hPPI

immunization was restricted when compared to IFNγ responses (Fig.4C). As for IFNγ

responses, proliferative responses were diversified. After determination of the threshold value

for each peptide in YES mice (Supplemental Data-6) using pairs of measurements and the

Bland and Altman test (Supplemental Data-7), responses were seen against both hPPI55-70

peptide (58.3% of mice, p≤0.006) and hPPI (66.6% of mice, p≤0.02) in a significant number

of mice. Proliferative responses were seen against at least one hPPI peptide in 91.67% mice

upon immunization against hPPI. These experiments show responses that preferentially

of 54 Diabetes

cluster around a region covering the leader sequence, B-chain and C-peptide sequence.

Induction of autoimmune diabetes in YES mice

We addressed whether YES mice are amenable to T1D induction. Following 6 daily 100µg

pI:C injections [24], diabetes developed within a week following the last injection (p≤0.0003)

but not after LPS or CpG injection (Fig.5A). As expected, diabetes occurred after multi STZ

low-dose stimulation (p≤0.0001) but not induced under cortisol stress conditions that were

tested as a control. Administration of anti-CD8 and CD4 mAbs prevented diabetes induced by

pI:C stimulation (p≤0.04, Fig.5B). An islet infiltration was observed in diabetic mice

(Fig.5C), with islets showing a loss of cells expressing insulin (Fig.5D) and the presence of

CD3+ T-cells (Fig.5E). No significant TLR3 expression was observed in control mice that

remained untreated (Fig.5F). TLR3 expression in diabetic pI:C-treated mice showed a

speckled distribution within the islet cell cytoplasm which mostly co-localized with insulin

(Fig.5G). A few glucagon-stained cells showed TLR3 co-localization. Spleen CD8+ T-cells

from diabetic pI:C-treated mice showed IFNγ responses to hPPI6-14 (p≤0.016) and hPPI15-24

(p≤0.02), although not to hPPI2-11 or hPPI33-42 as observed in hPPI-immunized YES mice

(Fig.6A). Individual responses were observed against at least one hPPI peptide in 80% mice.

No CD8+ T-cell responses were observed in non-diabetic pI:C-treated mice. We next

evaluated CD4+ T-cell responses in pI:C-treated and control YES mice (Fig.6B). Diabetic

pI:C-treated mice showed significant proliferative responses as compared to control YES

mice: hPPI16-30 (p≤0.048), hPPI18-30 (p≤0.008), hPPI33-47 (p≤0.05), hPPI40-55 (p≤0.016), hPPI46-

61 (p≤0.022), hPPI61-76 (p≤0.031), hPPI80-97 (p≤0.041) and hPPI92-110 (p≤0.005) and the hPPI

(p≤0.011) protein. As for CD4+ T-cell responses in hPPI-immunized YES mice, individual

responses were observed against at least one hPPI peptide in 86.7% mice (Supplemental

Data-7 and 8). Responses were against hPPI1-15 (53.3%, p≤0.02), hPPI33-47 (46.7%, p≤0.05),

hPPI46-61 (46.7%, p≤0.05), hPPI70-86 (53.3%, p≤0.02), hPPI92-110 (73.3%, p≤0.02) and hPPI

of 54Diabetes

(53.3%, p≤0.05) (Fig.6.C). Responses clustered in a region covering the leader sequence and

C-peptide sequence. A strong correlation were observed between the responses against hPPI

protein and most of all hPPI-peptides in diabetic pI:C-treated YES mice (Supplemental Data-

9). In contrast with CD8+ T-cells, we detected proliferative responses against hPPI peptides in

splenocytes from non-diabetic pI:C-treated YES mice (Fig.6B). All mice were also tested for

ZnT8 and GAD T-cell responses (Fig.6D). Significant proliferative responses were observed

against ZnT8166-179 and GAD536-550 peptides that are common to the human and to the mouse.

Stronger CD8+ T-cell (Fig.7.A) and CD4+ T-cell (Fig.7.B) responses were observed in

pancreatic infiltrates in diabetic as compared to the non-diabetic pI:C-treated YES mice.

Infiltrating-cells from non-diabetic pI:C-treated mice show an increased frequency of Treg

cells but a decreased frequency in CD11b+ and dendritic cells as compared to diabetic pI:C-

treated mice (Fig.7.C). In pancreatic LN, populations maintain similar frequencies with the

exception of DCs expressing the activator marker CD103 specifically in diabetic pI:C-treated

YES mice (Fig.7.D).

of 54 Diabetes

Discussion

The clinical management of T1D faces the lack of fully accurate biomarkers of autoimmunity

and the lack of efficient and specific immunotherapies. Major improvements in glucose

monitoring and insulin therapies have narrowed the mortality gap with the general population

and have shifted the safety line balancing risks and benefits towards the safest

immunotherapy approaches. Meanwhile, antigen-specific immunotherapy faces a long way to

go. The autoantigen dose, the delivery route, the stage of the disease process at which

immunotherapy is applied and the use of peptides rather than full-length autoantigens remain

open issues in the human [25]. Models that would allow direct translation of experimentally-

defined peptides in man are lacking. In addition, the search for environment factors that

trigger diabetes on a susceptible genetic background remains elusive. We almost exclusively

rely on the NOD and humanized-NOD mouse to address such issues [26, 27]. The NOD

mouse shows a unique phenotype that is unlikely to summarize the heterogeneity of T1D

mechanisms. It has been selected for spontaneous diabetes development, precluding its use in

strategies to explore the triggering of diabetes by environmental factors on conventional

genetic backgrounds.

The YES mouse is a new model that lacks mouse class-I, class-II and insulin genes and

expresses HLA-A*02:01, HLA-DQ8 and hINS. Parental mice carrying the hINS transgene

have normal insulin levels and glucose homeostasis [28]. Most differences seen between YES

and control mice were minor and likely represent the expected inter-strain variability. YES

mice remain normoglycemic up to one year of age, respond normally to an intra-peritoneal

glucose challenge and maintain a normal β-cell mass. The distribution of most immune cell

subsets in YES mice is comparable to that of a conventional mouse strain. However, T-cells

were underrepresented in YES mice. Given an increased percentage of CD3+CD4-CD8+ SP T-

cells in the thymus, shifts in peripheral T-cell representation in YES mice is likely to relate

of 54Diabetes

with shifted efficiency of human MHC molecules to select T-cells. The inverted CD4+/CD8+

T-cell ratio seen in YES and parental HLA-A*02:01/HLA-DQ8 mice suggests a lower

efficiency of class-II HLA-DQ8 to select CD4+-T cells than of class-I HLA-A*02:01 class I-

HLA-A*02:01 to select CD8+-T cells. As the expression of the insulin gene controls the

selection of insulin-specific T cells in the thymus [4, 29], we evaluated the expression of the

hINS transgene in thymic cells. YES mice exclusively express the hINS transgene in mTEC, a

pattern identical to expression patterns observed in C57BL/6 or NOD mice and in the human.

Immunization with hPPI induced neither insulitis nor diabetes in YES mice, indicating that

they are tolerant to the hPPI transgene.

The main bias towards T1D development in YES mice is the expression of DQ8. The YES

genetic background was closer to the C57BL/6 and CBA than to the NOD strain. YES mice

were tolerant to the islets, and remained insulitis-free in the long-term range, as do parental

hINS transgenic mice [10]. Upon immunization against hPPI or hPPI peptides, YES mice

developed CD8+ T-cell IFNγ responses to a diverse repertoire of HLA-A*02:01-restricted

peptides but remained insulitis-free. Based on evidence that enterovirus infections are

involved in T1D development [30], we addressed whether it was possible to induce diabetes

in YES mice. Upon pI:C injections, YES mice developed acute T1D. Diabetes-free mice were

refractory to ultimate T1D development upon a second set of pI:C injections or hPPI

immunization, suggesting an on/off mechanism in T1D triggering [31, 32]. Individual

responses were observed against a set of hPPI peptides that partially overlaps with peptides

that induced CD8+ T-cell responses upon hPPI immunization. Significant responses were seen

against leader sequence hPPI6-14 and hPPI15-24 peptides, against which we previously reported

significant expansions of CD8+ T-cells in recent-onset T1D patients, pointing to the relevance

of the YES model to human T1D [16, 23, 33].

In contrast with class-I-restricted epitopes, HLA-DQ8-restricted CD4+ T-cell responses

of 54 Diabetes

remain ill-defined in man. IFNγ CD4+ T-cell responses observed after immunization of YES

mice against hPPI covered a wide spectrum of peptides scattered along the hPPI sequence,

indicating the absence of a prevalent response. Proliferative CD4+ T- cell responses were

observed against a more restricted set of peptides overlapping the B-chain and C-peptide

sequences. CD4+ T-cells clones obtained from pancreatic infiltrates of a T1D patient have

been characterized as recognizing two HLA-DQ8-restricted C-peptide epitopes that are

overlapping with peptide hPPI61-76 in our model [34]. Upon induction of T1D by pI:C,

significant CD4+ T- cell proliferative responses were observed against hPPI61-76 and against

hPPI peptides that were only partially overlapping with responses induced by immunization of

YES mice against hPPI. The partial dissociation of IFNγ and proliferative responses on one

side, and of responses induced by immunization against hPPI and upon induction of diabetes

by pI:C on the other side, was not unexpected. Adjuvant-mediated immunization and pI:C

indeed involve different pathways in antigen-presenting cells and CD4+ T-cell activation [35-

37].

Mechanisms inducing T1D through the loss of immune tolerance to β-cells remain elusive.

Our data point to the importance of islet-environment interactions through signals carried by

Pattern Recognition Receptors (PPRs) [38, 39] and Toll Like Receptors (TLRs) in induction

of T1D via innate immune upregulation [40, 41]. This is remisniscent of data involving T1D

induction by Coxsackie B4 virus [32, 42]. The IFN induced with helicase C domain I

(IFIH1/MDA5) gene associates with T1D while mediating the early IFN response to viral

RNA [43]. Using a TLR3 agonist [44], we induced T1D in YES mice along with a T-cell

infiltrate. The ß-cells from pI:C-treated diabetic mice showed increased TLR3 expression.

Following data indicating TLR3 and MDA5 expression in the islets in vitro [24, 45], we

analysed TLR3 expression after pI:C treatment. pI:C itself had a direct effect on ß-cells in

boosting speckled TLR3 expression predominantly in ß-cells. Indeed, a key role of p-DCs has

of 54Diabetes

been reported in the NOD mouse [32, 46].

In conclusion, the YES mouse is a new model to characterize of HLA-A*02:01 and HLA-

DQ8-restricted hPPI epitopes involved in T1D and study mechanisms of T1D induction and

heterogeneity in the human. This model will provide a new avenue to evaluate immune

tolerance strategies that may directly apply to immunotherapy of T1D in the human.

of 54 Diabetes

Acknowledgments

This work was performed within the Département Hospitalo-Universitaire (DHU)

AUToimmune and HORmonal diseaseS. We acknowledge Sébastien Jacques and the

Genotyping platform at Cochin Institute. We acknowledge Raphaël Scharfmann for reading

manuscript, Latif Rachdi and Virginie Aiello-Lorenzo for technical assistance in evaluated ß-

cell mass and PDX1 staining.

Author Contributions:

L.S. performed experiments, involved in discussions and contributed to writing the

manuscript. G.S. performed metabolic experiments. G.A. performed DAB staining, P.E., L.F.

and V.M. were involved in Affymetrix genotyping array discussion and manuscript editing.

L.F. were involved in discussion and manuscript editing. B.B. addressed the question of

cortisol stress. N.P. performed big data analysis. B.C was designed experiments, chaired

discussions and wrote the manuscript.

Pr Christian BOITARD is the guarantor of this work and, as such, had full access to all data in

the study and takes responsibility for the integrity of the data and the accuracy of the data

analysis.

Conflict of interest statement:

The authors declare no duality of interest associated with this manuscript.

Funding

This work was supported by ANR grant R11189KK-RPV11189KKA, ANR2010-Biot-00801

and EFSD grant 1-2008-106.

Data avaibility

The data discussed in this publication have been deposited in NCBI's Gene Expression

Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number

GSE101551.

of 54Diabetes

(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101551).

of 54 Diabetes

FIGURE LEGENDS:

Figure 1: Insulin and MHC class-I and class-II expression in YES mice

(A) RT-PCR analysis of mIns expression in thymus and pancreas of YES mice compared to

the pancreas of C57BL/6 mice. Gene Ruler 1Kb+ DNA ladder to 500bp to 70bp. (B) Nested

RT-PCR analysis of hINS expression in different organs of YES mice. Gene Ruler 1Kb+

DNA ladder to 500bp to 70bp. (C) Nested RT-PCR analysis of hINS transgene and mIns

expression in thymocyte fractions, cTEC and mTEC of YES mice, parental hINS mice and

control C57BL/6 mice were compared to mAire and mCsn2 (casein2) genes expression. Gene

Ruler 1Kb+ DNA ladder to 500bp to 70bp. (D) Flow cytometry evaluation of the expression

of mouse class-I H-2Dk and class-II IAk, respectively on T (upper panel) and B-cells (lower

panel), in YES and parental hINS transgenic mice. (E) Flow cytometry evaluation of the

expression of HLA-A*02:01/HHD and HLA-DQ8, respectively on T (upper panel) and B-

cells (lower panel), in YES and parental hINS transgenic mice. (F) Flow cytometry evaluation

of the expression of HLA-A*02:01/HHD and H-2Db on islets cells (CD45- Ins+ cells) in YES

and C57BL/6 conventional mice.

Figure 2: Immune cell subsets in YES mice

Splenocytes (A), inguinal lymph node (iLN) cells (B), PBMCs (C) and thymocytes (D) were

stained using the following antibodies: anti-CD19 (B cells, blue), anti-CD11c (dendritic cells,

DC, red), anti-CD11b (macrophages, green), anti-NKp46 (Natural Killer, NK cells, pink) and

anti-CD3ε (T-cells; CD3low, light purple; CD3hight, bright purple). T-cells subsets were further

stained with anti-CD4 and anti-CD8α mAb (grey panels). A population of immature cells was

detected in YES mice (IC, gold and Supplemental Figure 2).

Figure 3: Metabolic characterization of YES mice

of 54Diabetes

(A) Body weight of male (�, n=6) and female (�, n=6) YES mice, male (�, n=6) and female

(�, n=6) HLA-A*02:01/HLA-DQ8 parental mice and male (�, n=6) and female (�, n=6)

hINS parental mice up to 20 weeks of age. (B) Insulin (red), Glucagon (green) and PDX1

(red) staining of YES and HLA-A*02:01/HLA-DQ8 pancreatic sections. (C) β-cell mass.

Male (�, n=6) and female (�, n=6) YES mice, male (�, n=3) and female (�, n=3) HLA-

A*02:01/HLA-DQ8 parental mice, male (�, n=3) and female (�, n=3) hINS parental mice. β-

cell mass is expressed in mg for the whole pancreas. (D) Intra-peritoneal GTT following

2g/kg glucose injection and Area Under the Curve (AUC) in 6 months old male (n=4, � and

black bars, respectively) and female (n=4, � and checkerboard black bars, respectively) YES

mice, male (n=4, � and grey bars, respectively) and female (n=4, � and checkerboard grey

bars, respectively) HLA-A*02:01/HLA-DQ8 transgenic mice. (E) Intra-peritoneal ITT curves

following 0.75U/kg insulin injection and AUCs in 15 weeks old male (n=4, � and black bars,

respectively) and female (n=6, � and checkerboard black bars, respectively) YES mice, male

(n=5, �and grey bars, respectively) and female (n=5, � and checkerboard grey bars,

respectively) HLA-A*02:01/HLA-DQ8 parental mice. * p ≤ 0.05; ns, non-significant; Mann-

Whitney test.

Figure 4: MHC Class-I and class-II restricted T-cell responses against hPPI in YES mice

(A) INFγ-responses against individual hPPI peptides restricted to MHC class-I. YES mice

were immunized against hPPI2-11, hPPI6-14, hPPI15-24, hPPI30-39, hPPI33-42, hPPI34-42, hPPI42-51,

hPPI101-109 peptides (100µg/mouse, CFA, HLA-DQ8-restricted helper Nef66-97 peptide) or PBS

and boosted (IFA, 2 times). INFγ-ELISpot assays were performed in the presence of the

immunizing peptide (�), PDHase208-216 as irrelevant peptide (�) or ConA as positive control

(�). Numbers of spot forming-cells were express for 106 cells. (B) INFγ-responses to hPPI.

Splenocytes from YES mice immunized against hPPI full length protein (�) or PBS (�) were

of 54 Diabetes

depleted in CD8+ T-cells and INFγ-ELISpot assays performed in the presence of individual

hPPI peptide, Nef66-97 as irrelevant peptide or ConA as positive control. Numbers of spot

forming-cells were expressed for 106 cells. (C) Proliferative responses to full-length hPPI and

hPPI peptides hPPI1-15, hPPI8-23, hPPI16-30, hPPI18-30, hPPI20-35, hPPI25-40, hPPI33-47, hPPI40-55,

hPPI46-61, hPPI55-70, hPPI61-76, hPPI70-86, hPPI80-97 and hPPI92-110. Splenocytes from YES mice

immunized against hPPI full length protein (�) or PBS (�) were stimulated in vitro for 3 days

and proliferation was evaluated by measuring BrdU incorporation.

Each dot represents individual mouse. *, p ≤ 0.05; **, p ≤ 0.01; Mann-Whitney test.

Figure 5: pI:C-induction diabetes in YES mice

(A) Diabetes incidence upon pI:C stimulation (6 daily injections (ip)/100µg pI:C, �) in YES

mice compared to the CpG-stimulated YES mice (6 daily injections (ip)/50µg ODN2395, �),

LPS-stimulated YES mice (6 daily injections (ip)/10µg LPS, �, or untreated-YES mice (�).

(B) Diabetes incidence upon pI:C stimulation (6 daily injections (ip)/100µg pI:C, �) in YES

mice compared to the STZ-stimulated YES mice (5 daily injections (ip)/50mg/Kg STZ, �),

corticosterone-stimulated YES mice (6 daily injections (ip)/2mg/Kg corticosterone, �), PBS-

treated YES mice (6 daily injection (ip)/100µl PBS1X, �) and pI:C-stimulated depleted YES

mice (2 daily injection (ip) of 500µg GK1.5 and 500µg YTS 169.4 following by 3 daily

injection (ip) of 100µg pI:C and 500µg GK1.5 then 3 daily injection (ip) of 100µg pI:C, �).

(C) Pancreas paraffin sections stained by hematoxylin-eosin in pI:C-induced diabetic YES

mice. (D) Glucagon (green), and insulin (red) immune-fluorescent staining of pancreatic

section from pI:C-induced diabetic YES mice and YES mice control. (E) Glucagon (green),

and CD3 (red) immune-fluorescent staining of pancreatic section from pI:C-induced diabetic

YES mice and YES mice control. Glucagon (grey), Insulin (red) and TLR 3 (green) staining

of pancreatic section from YES mice (F) and from pI:C-induced diabetic YES mice (G).

of 54Diabetes

Incidence curves comparison with Log-rank (Mantel-Cox) Test. *, p ≤ 0.05; ***, p ≤ 0.003.

Figure 6: T-cells responses against hPPI

(A) Analysis of CD8+ T-cells responses by INFγ-ELISpot assays performed in the presence of

individual hPPI peptide (hPPI2-11, hPPI6-14, hPPI15-24, hPPI30-39, hPPI33-42, hPPI34-42, hPPI42-51,

hPPI101-109 peptides), Nef66-97 as irrelevant peptide or ConA as positive control for splenocytes

from diabetic pI:C-treated YES mice (�), compared to the splenocytes from un-stimulated

YES mice (�) and non diabetic pI:C-treated YES mice (�). (B) Analysis of CD4+ T-cells

responses by proliferative assays performed in the presence of full-length hPPI and individual

hPPI peptides hPPI1-15, hPPI8-23, hPPI16-30, hPPI18-30, hPPI20-35, hPPI25-40, hPPI33-47, hPPI40-55,

hPPI46-61, hPPI55-70, hPPI61-76, hPPI70-86, hPPI80-97 and hPPI92-110. Splenocytes from diabetic

pI:C-treated YES mice (�) and non diabetic pI:C-treated YES mice (�) compared to the un-

treated YES mice (�) were stimulated in vitro for 3 days with individuals peptides and

proliferation was evaluated by measuring BrdU incorporation. (C) Graph radar representing

the percentage of frequencies recognition of hPPI peptides in diabetic pIC-treated YES mice

(in red), in hPPI-immunized YES mice (in black) and in YES mice control (in grey). (D)

Analysis of CD4+ T-cells responses by proliferative assays performed in the presence of full-

length hPPI and individual ZnT8166-179 peptide or GAD101-115, GAD126-135, GAD207-220, and

GAD536-550. Splenocytes from diabetic pI:C-treated YES mice (�) and non diabetic pI:C-

treated YES mice (�) compared to the un-treated YES mice (�) were stimulated in vitro for 3

days with individuals peptides and proliferation was evaluated by measuring BrdU

incorporation.


Figure 7: Pancreatic infiltrates analysis

of 54 Diabetes

(A) Analysis of CD8+ T-cells responses by INFγ-ELISpot assays performed in the presence of

pool of hPPI peptide (hPPI2-11, hPPI6-14, hPPI15-24, hPPI30-39, hPPI33-42, hPPI34-42, hPPI42-51,

hPPI101-109 peptides) and Nef66-97 as irrelevant peptide for pancreatic infiltrating cells from

diabetic pI:C-treated YES mice (�, n=6) compared to the non-diabetic pI:C-treated YES mice

(�, n=3). Each dot represents individual mouse. (B) Analysis of CD4+ T-cells responses by

proliferative assays performed in the presence of full-length hPPI and irrelevant peptide Nef66-

97. Pancreatic infiltrating cells from diabetic pI:C-treated YES mice (�, n=12) and non

diabetic pI:C-treated YES mice (�, n=3) were stimulated in vitro for 3 days with hPPI portein

and proliferation was evaluated by measuring BrdU incorporation. Each dot represents

individual mouse. Each dot represents individual mouse. (C) Immunophenotypage of

pancreatic infiltrating cells from diabetic pI:C-treated YES mice (�, n=3) and non diabetic

pI:C-treated YES mice (�, n=3) for the frequency of B-cells, T-cells, CD8+ T-cells, CD4+ T-

cells, Treg defined as CD4+CD25+Foxp3+ T-cells, CD11b+ cells and DC defined as CD11C+

and CD11c+CD11b+ cells. (D) Immunophenotypage of pancreatic lymphe node cells from

diabetic pI:C-treated YES mice (�, n=3), non diabetic pI:C-treated YES mice (�, n=3) and

un-treated YES mice (�, n=3) for the frequency of B-cells, T-cells, CD8+ T-cells, CD4+ T-

cells, Treg defined as CD4+CD25+Foxp3+ T-cells, CD11b+ cells, DC defined as CD11c+ and

CD11c+CD11b+ cells and for the CD103+ DCs.

of 54Diabetes

Table 1: Similarity of genetic backgrounds of YES, NOD and C57BL/6 mice

Identity % NOD C57BL/6 YES

NOD Nd 80.4 % 79.3 %

C57BL/6 80.4 % nd 90.9 %

YES 79.3 % 90.9 % 99.9 %

nd: not determined

of 54 Diabetes

REFERENCES:

1. Donath MY, H.C., Palmer E., What is the role of autoimmunity in type 1 diabetes? A

clinical perspective. Diabetologia, 2014. 57(4): p. 653-5. 2. Bluestone JA, H.K., Eisenbarth G., Genetics, pathogenesis and clinical interventions

in type 1 diabetes. Nature, 2010. 464(7293): p. 1293-300. 3. Mahil SK, C.M., Di Meglio P, Dand N, Ahlfors H, Carr IM, Smith CH, Trembath RC,

Peakman M, Wright J, Ciccarelli FD, Barker JN, Capon F., An analysis of IL-36

signature genes and individuals with IL1RL2 knockout mutations validates IL-36 as a

psoriasis therapeutic target. Sci Transl Med, 2017. 9(411). 4. Thébault-Baumont K, D.-L.D., Krief P, Briand JP, Halbout P, Vallon-Geoffroy K,

Morin J, Laloux V, Lehuen A, Carel JC, Jami J, Muller S, Boitard C., Acceleration of

type 1 diabetes mellitus in proinsulin 2-deficient NOD mice. Journal of Clinical Investigation, 2003. 111(6): p. 851-7.

5. Nakayama M, A.N., Moriyama H, Babaya N, Liu E, Miao D, Yu L, Wegmann DR, Hutton JC, Elliott JF, Eisenbarth GS., Prime role for an insulin epitope in the

development of type 1 diabetes in NOD mice. Nature, 2005. 435(7039): p. 220-3. 6. Ziegler AG, N.G., Prediction and pathogenesis in type 1 diabetes. Immunity, 2010.

32(4): p. 468-78. 7. Moriyama H, A.N., Paronen J, Sikora K, Liu E, Miao D, Devendra D, Beilke J,

Gianani R, Gill RG, Eisenbarth GS., Evidence for a primary islet autoantigen

(preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse. Proceedings of National Academy of Sciences of the United States of America, 2003. 100(18): p. 10376-81.

8. Jones EY, F.L., Strominger JL, Siebold C., MHC class II proteins and disease: a

structural perspective. Nature reviews. Immunology, 2006. 6(4): p. 271-82. 9. Noble JA, V.A., Varney MD, Carlson JA, Moonsamy P, Fear AL, Lane JA, Lavant E,

Rappner R, Louey A, Concannon P, Mychaleckyj JC, Erlich HA, HLA class I and

genetic susceptibility to type 1 diabetes: results from the Type 1 Diabetes Genetics

Consortium. Diabetes, 2010. 59(11): p. 2972-9. 10. Bucchini D, R.M., Stinnakre MG, Desbois P, Lorès P, Monthioux E, Absil J, Lepesant

JA, Pictet R, Jami J., Pancreatic expression of human insulin gene in transgenic mice. Proc Natl Acad Sci U S A., 1986. 83(8): p. 2511-5.

11. Pascolo S, B.N., Ure JM, Smith AG, Lemonnier FA, Pérarnau B., HLA-A2.1-

restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2

microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double

knockout mice. Journal of Experimental Medicine, 1997. 185(12): p. 2043-51. 12. Madsen L, L.N., Engberg J, Dierich A, Svejgaard A, Benoist C, Mathis D, Fugger L.,

Mice lacking all conventional MHC class II genes. Proceedings of the National Academy of Sciences of the United States of America, 1999. 96(18): p. 10338-43.

13. Nabozny GH, B.J., Cheng S, Cosgrove D, Griffiths MM, Luthra HS, David CS., HLA-

DQ8 transgenic mice are highly susceptible to collagen-induced arthritis: a novel

model for human polyarthritis. Journal of Experimental Medicine, 1996. 183(1): p. 27-37.

14. Jain R, G.D., Isolation of thymic epithelial cells and analysis by flow cytometry. Current Protocols in Immunology, 2014. 107: p. 3.26.1-3.26.15.

15. Villaseñor J, B.W., Benoist C, Mathis D., Ectopic expression of peripheral-tissue

antigens in the thymic epithelium: probabilistic, monoallelic, misinitiated. Proceedings of the National Academy of Sciences of the United State of America, 2008. 105(41): p. 15854-9.

of 54Diabetes

16. Toma A, H.S., Briand JP, Camoin L, Gahery H, Connan F, Dubois-Laforgue D, Caillat-Zucman S, Guillet JG, Carel JC, Muller S, Choppin J, Boitard C., Recognition

of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic

patients. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(30): p. 10581-6.

17. Rachdi L, A.V., Duvillié B, Scharfmann R., L-leucine alters pancreatic β-cell

differentiation and function via the mTor signaling pathway. Diabetes, 2012. 61(2): p. 409-17.

18. Kurasawa K, S.A., Maeda T, Sumida T, Ito I, Tomioka H, Yoshida S, Koike T., Short-

term administration of anti-L3T4 MoAb prevents diabetes in NOD mice. Clin Exp Immunol, 1993. 91(3): p. 376-80.

19. Grcević D, L.S., Marusić A, Lorenzo JA., Depletion of CD4 and CD8 T lymphocytes

in mice in vivo enhances 1,25-dihydroxyvitamin D3-stimulated osteoclast-like cell

formation in vitro by a mechanism that is dependent on prostaglandin synthesis. J Immunol, 2000. 165(8): p. 4231-8.

20. Fan Y, R.W., Grupillo M, He J, Sisino G, Trucco M., Thymus-specific deletion of

insulin induces autoimmune diabetes. EMBO Journal, 2009. 28(18): p. 2812-24. 21. Godfrey DI, K.J., Suda T, Zlotnik A., A developmental pathway involving four

phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative

adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol, 1993. 150(10): p. 4244-52.

22. Muggeo M, G.B., Roth J, Neville DM Jr, De Meyts P, Kahn CR., The insulin receptor

in vertebrates is functionally more conserved during evolution than insulin itself. Endocrinology, 1979. 104(5): p. 1393-402.

23. Luce S, L.F., Briand JP, Coste J, Lahlou N, Muller S, Larger E, Rocha B, Mallone R, Boitard C., Single insulin-specific CD8+ T cells show characteristic gene expression

profiles in human type 1 diabetes. Diabetes, 2011. 60(12): p. 3289-99. 24. Wen L, P.J., Li Z, Wong FS., The effect of innate immunity on autoimmune diabetes

and the expression of Toll-like receptors on pancreatic islets. J Immunol, 2004. 172(5): p. 3173-80.

25. Harbige J, E.M., Peakman M., New insights into non-conventional epitopes as T cell

targets: The missing link for breaking immune tolerance in autoimmune disease? J Autoimmun, 2017. 84: p. 12-20.

26. Marron MP, G.R., Chapman HD, Serreze DV., Functional evidence for the mediation

of diabetogenic T cell responses by HLA-A2.1 MHC class I molecules through

transgenic expression in NOD mice. Proc nati Acad Sci U S A, 2002. 99(21): p. 13753-8.

27. Racine JJ, S.I., Ratiu J, Christianson G, Lowell E, Helm K, Allocco J, Maser RS, Chen YG, Lutz CM, Roopenian D, Schloss J, DiLorenzo TP, Serreze DV., Improved

Murine MHC-Deficient HLA Transgenic NOD Mouse Models for Type 1 Diabetes

Therapy Development. Diabetes, 2018. 67(5): p. 923-35. 28. Lamothe B, B.A., Desbois P, Lamotte L, Bucchini D, De Meyts P, Joshi RL., Genetic

engineering in mice: impact on insulin signalling and action. Biochem J, 1998. 335(Pt2): p. 193-204.

29. Pugliese A, B.D., Garza D, Murchison D, Zeller M, Redondo MJ, Diez J, Eisenbarth GS, Patel DD, Ricordi C., Self-antigen-presenting cells expressing diabetes-associated

autoantigens exist in both thymus and peripheral lymphoid organs. Journal of Clinical Investigation, 2001. 107(5): p. 555-64.

30. Ghazarian L, D.J., Simoni Y, Beaudoin L, Lehuen A., Prevention or acceleration of

type 1 diabetes by viruses. Cell Mol Life Sci, 2013. 70(2): p. 257.

of 54 Diabetes

31. Serreze DV, P.C., Chapman HD, Johnson EA, Lu B, Rothman PB., Interferon-gamma

receptor signaling is dispensable in the development of autoimmune type 1 diabetes in

NOD mice. Diabetes, 2000. 49(12): p. 2007-11. 32. Ghazarian L, D.J., Beaudoin L, Larsson PG, Puri RK, van Rooijen N, Flodström-

Tullberg M, Lehuen A., Protection against type 1 diabetes upon Coxsackievirus B4

infection and iNKT-cell stimulation: role of suppressive macrophages. Diabetes, 2013. 62(11): p. 3785-96.

33. Toma A, L.T., Haddouk S, Luce S, Briand JP, Camoin L, Connan F, Lambert M, Caillat-Zucman S, Carel JC, Muller S, Choppin J, Lemonnier F, Boitard C., Recognition of human proinsulin leader sequence by class I-restricted T-cells in HLA-

A*0201 transgenic mice and in human type 1 diabetes. Diabetes, 2009. 58(2): p. 394-402.

34. Pathiraja V, K.J., Campbell PD, Krishnamurthy B, Loudovaris T, Coates PT, Brodnicki TC, O'Connell PJ, Kedzierska K, Rodda C, Bergman P, Hill E, Purcell AW, Dudek NL, Thomas HE, Kay TW, Mannering SI., Proinsulin specific, HLA-DQ8 and

HLA-DQ8 transdimer restricted, CD4+ T cells infiltrate the islets in type 1 diabetes. Diabetes, 2015. 64(1): p. 172-82.

35. Nakamura T, M.R., Kogure K, Harashima H., Incorporation of polyinosine-

polycytidylic acid enhances cytotoxic T cell activity and antitumor effects by

octaarginine-modified liposomes encapsulating antigen, but not by octaarginine-

modified antigen complex. Int J Pharm, 2013. 441(1-2): p. 476-81. 36. Francica JR, Z.D., Linde C, Siena E, Johnson C, Juraska M, Yates NL, Gunn B, De

Gregorio E, Flynn BJ, Valiante NM, Malyala P, Barnett SW, Sarkar P, Singh M, Jain S, Ackerman M, Alam M, Ferrari G, Tomaras GD, O'Hagan DT, Aderem A, Alter G, Seder RA., Innate transcriptional effects by adjuvants on the magnitude, quality, and

durability of HIV envelope responses in NHPs. Blood Adv, 2017. 1(25): p. 2329-2342. 37. Speth MT, R.U., Müller E, Spanier J, Kalinke U, Corthay A, Griffiths G., Poly(I:C)-

Encapsulating Nanoparticles Enhance Innate Immune Responses to the Tuberculosis

Vaccine Bacille Calmette-Guérin (BCG) via Synergistic Activation of Innate Immune

Receptors. Mol Pharm, 2017. 14(11): p. 4098-4112. 38. Hutton MJ, S.G., Johnson JD, Verchere CB., Role of the TLR signaling molecule TRIF

in β-cell function and glucose homeostasis. Islets, 2010. 2(2): p. 104-11. 39. Giarratana N, P.G., Amuchastegui S, Mariani R, Daniel KC, Adorini L., A vitamin D

analog down-regulates proinflammatory chemokine production by pancreatic islets

inhibiting T cell recruitment and type 1 diabetes development. J Immunol, 2004. 173(3): p. 2280-7.

40. Alkanani AK, H.N., Lien E, Ir D, Kotter CV, Robertson CE, Wagner BD, Frank DN, Zipris D., Induction of diabetes in the RIP-B7.1 mouse model is critically dependent

on TLR3 and MyD88 pathways and is associated with alterations in the intestinal

microbiome. Diabetes, 2014. 63(2): p. 619-31. 41. Swiecki M, M.S., Wang Y, Colonna M., TLR7/9 versus TLR3/MDA5 signaling during

virus infections and diabetes. J Leukoc Biol, 2011. 90(4): p. 691-701. 42. McCall KD1, T.J., Courreges MC, Benencia F, James CB, Malgor R, Kantake N,

Mudd W, Denlinger N, Nolan B, Wen L, Schwartz FL., Toll-like receptor 3 is critical

for coxsackievirus B4-induced type 1 diabetes in female NOD mice. Endocrinology, 2015. 156(2): p. 453-61.

43. Nejentsev S, W.N., Riches D, Egholm M, Todd JA., Rare variants of IFIH1, a gene

implicated in antiviral responses, protect against type 1 diabetes. Science, 2009. 324(5925): p. 387-9.

44. Moriyama H, W.L., Abiru N, Liu E, Yu L, Miao D, Gianani R, Wong FS, Eisenbarth

of 54Diabetes

GS., Induction and acceleration of insulitis/diabetes in mice with a viral mimic

(polyinosinic-polycytidylic acid) and an insulin self-peptide. Proc Nati Acad Sci USA, 2002. 99(8): p. 5539-44.

45. Lincez PJ, S.I., Horwitz MS., Reduced expression of the MDA5 Gene IFIH1 prevents

autoimmune diabetes. Diabetes, 2015. 64(6): p. 2184-93. 46. Diana J, B.V., Beaudoin L, Dalod M, Mellor A, Tafuri A, von Herrath M, Boitard C,

Mallone R, Lehuen A., Viral infection prevents diabetes by inducing regulatory T

cells through NKT cell-plasmacytoid dendritic cell interplay. J Exp Med, 2011. 208(4): p. 729-45.

of 54 Diabetes

Fig.1

of 54Diabetes

CD

3+C

D4+

48

%

CD

3+C

D8+

12

%

CD

3+C

D4+

CD

8+

40%

C

D3+

CD

4+

51%

C

D3+

CD

8+

28%

CD

3+C

D4+

C

D8+

21

%

!"#$%&!"

'(&

#)&

!"#$%&!"

*(&

+)&

!"#$%&!"

'(&

!"*(&

,-)&

Thym

us T

-cel

ls, Y

ES m

ice !

!"#$%&!"

'(&

#)&

!"#$%&!"

*(&

+)&

!"#$%&!"

'(&

!"*(&

**)&

Thym

us T

-cel

ls, C

57BL

/6 m

ice!

CD

8+!

55%!

CD

4+!

40%!

CD

3+C

D4+

C

D8+!

1%!

CD

4+C

D25

+ Fo

xP3+!

4%!

Bloo

d T-

cells

, YES

mic

e !

CD

8+!

54%!

CD

4+!

40%!

CD

3+C

D4+

C

D8+!

2%!

CD

4+C

D25

+ Fo

xP3+!

4%!

Sple

en T

-cel

ls, Y

ES m

ice !

CD

8+!

40%!

CD

4+!

54%!

CD

4+C

D25

+ Fo

xP3+!

6%!

Sple

en T

-cel

ls, C

57BL

/6 m

ice!

CD

8+!

40%!

CD

4+!

52%!

CD

3+C

D4+

C

D8+!

1%!

CD

4+C

D25

+ Fo

xP3+!

7%!

Bloo

d T-

cells

, C57

BL/6

mic

e!

B-c

ells

38

%

DC

s 1%

Mac

roph

ages

3%

T-ce

lls

53%

iLN

C57

BL/

6 m

ice

B-c

ells

65

%

DC

s 4%

Mac

roph

ages

5%

T-ce

lls

13%

NK

-cel

ls

4%

IC

9%

othe

r cel

ls

13%

Spl

enoc

ytes

YE

S m

ice

B-c

ells

38

%

DC

s 12

%

Mac

roph

ages

30

%

T-ce

lls

12%

NK

-cel

ls

5%

IC

3%

othe

r cel

ls

8%

PB

MC

s Y

ES

mic

e

B-c

ells

42

%

DC

s 2%

Mac

roph

ages

2%

T-ce

lls

47%

NK

-cel

ls

2%

IC

5%

othe

r cel

ls

7%

iLN

YE

S m

ice

B-c

ells

4%

DC

s 1%

M

acro

phag

es

1%

CD

3 lo

ce

lls

90%

NK

-cel

ls

1%

CD

3+ c

ells

3%

othe

r cel

ls

4%

Thym

ocyt

es Y

ES

mic

e

B-c

ells

60

%

DC

s 5%

Mac

roph

ages

5%

T-

cells

29

%

NK

-ce

lls

1%

othe

r cel

ls

1%

Spl

enoc

ytes

C57

BL/

6 m

ice

B-c

ells

39

%

DC

s 1%

Mac

roph

ages

8%

T-ce

lls

48%

NK

-cel

ls

4%

Oth

er c

ells

4%

PB

MC

s C

57B

L/6

mic

e

A B D

!"#$%

&'(%

!")$%

*+(%

!"*$!"

)$%

!"#$%

)(%

!")$!"

,&$%

-./0*$%

1(%

iLN

T-c

ells

, YES

mic

e !

CD

8+!

39%!

CD

4+!

49%!

CD

3+C

D4+

C

D8+!

2%!

CD

4+C

D25

+ Fo

xP3+!

10%!

iLN

T-c

ells

, C57

BL/6

mic

e!

B-c

ells

1%

D

Cs

1%

CD

3 lo

ce

lls

95%

CD

3+ c

ells

3%

othe

r cel

ls

3%

Thym

ocyt

es C

57B

L/6

mic

e

Fig.2

of 54 Diabetes

Fig.3

of 54Diabetes

Fig.4

of 54 Diabetes

Fig.5

of 54Diabetes

Fig.6

of 54 Diabetes

Fig.7

of 54Diabetes

Supplementary Material-1: YES mouse crosses.

of 54 Diabetes

Supplementary Material-1: YES mouse crosses.

To obtain YES mice, F1 crosses of HLA-A*02:01/HLA-DQ8 and hINS transgenic mice

were backcrossed with hINS transgenic mice. Female breeders were progressively

selected for full loss of mINS1 and mINS2 alleles, expression of at least one allele of the

hINS transgene and one allele of HLA-A2*02:01 and HLA-DQ8 transgenes. Additional

crosses allowed to obtain homozygous YES mice with a mINS1-/- mINS2-/- hINS+/+ mβ2m-

/- H-2D-/- IA-/- IE-/- A2.1+/+ DQ8+/+ genotype. Twenty brother-sister crosses led to the

inbred YES mouse

of 54Diabetes

Supplementary Material-2: Nucleotidic sequence of primers used for genotyping.

Target gene Primer name Primer sequences Amplicon size

human insulin hINS 5'-CGC AGC CTT TGT GAA CCA AC-3’ 100

5'-GCG GGT CTT GGG TGT GTA GAA-3’

mouse insulin 1 mINS1 5'-CCA GCC AAG ACT CCA GCG ACT TTA-3’ 234

5'-CCC ACA CAC CAG GTA GAG AGC CTC T-3’

mouse insulin 2 mINS2 5'-GGT GAG TTC TGC CAC TGA ATT C-3’

5'-GGC ATC AGC AGC ACA GAA GCA A-3’

436

mouse ß2m mB2m 5'-GTC AGA TAT GTC CTT CAG CAA G-3’

5'-GAT GCT GAT CAC ATG TCT CG-3’

mB2m KO: 1800

mB2m WT: 657

HLA-A*02:01 HLA-A2 5'-GAC GCC GCG AGC CAG AGG AT-3’

5'-TGC AGC GTC TCC TTC CCG TT-3’

671

HLA-DQ8 HLA-DQ 5'-GAA GAC ATT GTG GCT GAC CAT GTT GCC-3’

5'-AGC ACA GCG ATG TTT GTC AGT GCA AAT TGC GG-3’

250

Supplementary Material-3: Gene-specific primers for reverse transcription

Target gene Primer name Primer sequences TM (°C)

human Insulin

hINS

5'-CCA CCT GCC CCA CCT GCA GG-3’

58

mouse Insulin mINS 5'-TAG TGG TGG GTC TAG TTG CAG-3’ 58

mouse Aire mAire 5'-TCA TCT CTA CCA GGT ATA GTG AC-3’ 55

mouse Caseinß mCsn2 5'-TGG TGG CTT TAG CTT TAA GG-3’ 55

mouse ß-actin mActin 5'-ACG CTC GGT CAG GAT CTT C-3’ 55

of 54 Diabetes

Supplementary Material-4: Nucleotidic sequence of primers used for first round PCR

Supplementary Material-5: Nucleotidic sequence of primers used for second round PCR


human Insulin

hINS

5' CGC AGC CTT TGT GAA CCA AC-3’

58

5'-CCA CCT GCC CCA CCT GCA GG-3’

mouse Insulin mINS 5'-GCC TAT CTT CCA GGT TAT TGT TTC A-3’ 58

5'-AGG TTT TAT TCA TTG CAG AGG GGT A-3’

mouse Aire mAire 5’-CTC TTG GAA ACG GAA TTC AGA C-3’

5'-TCA TCT CTA CCA GGT ATA GTG AC-3’

55

mouse Caseinß

mCsn2 5'-TCC TCT GAG ACT GAT AGT ATT TCC-3’

5'-TGG TGG CTT TAG CTT TAA GG-3’

55

mouse ß-actin mActin 5’-GTG AAA AGA TGA CCC AGA TCA TGT-3’

5'-ACG CTC GGT CAG GAT CTT C-3’

55


human Insulin

hINS

5' CGC AGC CTT TGT GAA CCA AC-3’

58

5'-GCG GGT CTT GGG TGT GTA GAA-3’

mouse Insulin mINS 5'-GCC TAT CTT CCA GGT TAT TGT TTC A-3’ 58

5'-GTG GGT CTA GTT GCA GTA GTT CTC C-3’

mouse Aire

mouse Caseinß

mouse ß-actin

mAire

mCsn2

mActin

5’-CTC TTG GAA ACG GAA TTC AGA C-3’

5'-GCC TTG TTC TTC AAA TTG CC-3’

5'-TCC TCT GAG ACT GAT AGT ATT TCC-3’

5'-AGC TTT AAG GAA GGA TTC CAG-3’

5’-GTG AAA AGA TGA CCC AGA TCA TGT-3’

5'-GGA GAG CAT AGC CCT CGT AG-3’

55

55

55

of 54Diabetes

Supplementary Material-6: Sequences of peptides restricted to HLA-A2*02:01

Name Sequence

hPPI 2-11 RLLPLLALL

hPPI 6-14 ALWGPDPAAA

hPPI 15-24 LLWGPDPAAA

hPPI 30-39 LCGSHLVEAL

hPPI 33-42 SHLVEALYLV

hPPI 34-42 HLVEALYLV

hPPI 42-51 VCGERGFFYT

hPPI 101-109 SLYQLENYC

MatA2 GILGFVFTL

Supplementary Material-7: Human recombinant-PPI protein production

cDNA sequence of prepoinsulin were mutated to convert the Ala codon in position 3 to Asp

by Site-directed mutagenesis (NEB) and abolish the signal-sequence site cleavage of PPI [16].

Mutated PPI was cloned in pFastBac vector to generate the PPI-recombinant bacmid and PPI-

recombinant Baculovirus to produce human PPI protein into Bac-toBac Baculovirus

Expression System (Invitrogen) according to the manufacturing instruction. Purification was

performed on infected-insect cells pellet, following preparation and extraction procedures for

insoluble proteins (Inclusion body) from E. Coli [17]. Purity was confirmed on SDS/PAGE

gel and quantification was performed using the BCA Protein assay kit (Pierce).

of 54 Diabetes

Supplementary Material-8: Sequences of peptides restricted to HLA-DQ8

Name Sequence

hPPI 1-15 MALWMRLLPLLALLA

hPPI 8-23 LPLLALLALWGPDPAA

hPPI 16-30 WGPDPAAAFVNQHL

hPPI 18-30 PDPAAAFVNQHL

hPPI 20-35 DPAAAFVNQHLCGSHL

hPPI 25-40 FVNQHLCGSHLVEALY

hPPI 33-47 SHLVEALYLVCGERG

hPPI 40-55 YLVCGERGFFYTPKTR

hPPI 46-61 RGFFYTPKTRREAEDL

hPPI 55-70 RREAEDLQVGQVELGG

hPPI 61-76 LQVGQVELGGGPGAGS

hPPI 70-86 GGPGAGSLQPLALEGSL

hPPI 80-97 LALEGSLQKRGIVEQCCT

hPPI 92-110 VEQCCTSICSLYQLENYCN

Nef 66-97 VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL

of 54Diabetes

Supplementary Material-9: Affymetrix mouse diversity genotyping array

High quality genomic DNAs (250ng) were restricted with NspI and StyI. NspI and StyI

adaptors were then ligated to restricted fragments followed by PCR using universal primer

PCR002. All amplicons were purified, pooled and used for fragmentation and end labelling

with biotin using Terminal Transferase. Labelled targets were then hybridized overnight to

Genechip® Mouse Diversity Genotyping Array (Affymetrix, ref: 901615) at 49°C. Chips

were washed on the fluidic station FS450 following specific protocols (Affymetrix) and

scanned using the GCS3000 7G. The image is then analyzed with GCOS software to obtain

raw data (cel files). Genotypes were called by the Affymetrix Genotyping Console tool using

Dynamic Model (DM) and Bayesian Robust Linear Model with Mahalanobis (BRLMM)

mapping algorithms. Genotypes were then extracted for each SNP of the chip. Big data were

exploited in R console Software.

of 54 Diabetes

B-cells

DCs

Macrophages

T-cells

Splenocytes YES mice

B-cells

DCs

Macrophages

T-cells

Splenocytes C57BL/6 mice

**

B-cells

DCs

Macrophages

T-cells

PBMCs YES mice

B-cells

DCs

Macrophages

T-cells

PBMCs C57BL/6 mice

B-cells

DCs

Macrophages

T-cells

iLN YES mice

B-cells

DCs

Macrophages

T-cells

iLN C57BL/6 mice

Cells expressing class II MHC (%)

*

100

50

0

B-cells

DCs

Macrophages

T-cells

B-cells

DCs

Macrophages

T-cells

B-cells

DCs

Macrophages

T-cells B-

cells

DCs

Macrophages

T-cells

B-cells

DCs

Macrophages

T-cells

B-cells

DCs

Macrophages

T-cells

100

50

0

100

50

0

100

50

0

100

50

0

100

50

0

BA

100

50

0

100

50

0

100

50

0

100

50

0

100

50

0

100

50

0

B-cells

DCs

Macrophages

T-cells

CD8+

T-cells

CD4+

T-cells

PBMCs YES mice

B-cells

DCs

Macrophages

T-cells

CD8+

T-cells

CD4+

T-cells

Splenocytes YES mice

B-cells

DCs

Macrophages

T-cells

CD8+

T-cells

CD4+

T-cells

Splenocytes C57BL/6 mice

*

B-cells

DCs

Macrophages

T-cells

CD8+

T-cells

CD4+

T-cells

PBMCs C57BL/6 mice

B-cells

DCs

Macrophages

T-cells

CD8+

T-cells

CD4+

T-cells

iLN YES mice

B-cells

DCs

Macrophages

T-cells

CD8+

T-cells

CD4+

T-cells

iLN C57BL/6 mice

Cells expressing class I MHC (%)

*

*

*

B-cells

DCs

Macrophages

T-cells

CD8+ T-cells

CD4+ T-cells

B-cells

DCs

Macrophages

T-cells

CD8+ T-cells

CD4+ T-cells

B-cells

DCs

Macrophages

T-cells

CD8+ T-cells

CD4+ T-cells

B-cells

DCs

Macrophages

T-cells

CD8+ T-cells

CD4+ T-cells

B-cells

DCs

Macrophages

T-cells

CD8+ T-cells

CD4+ T-cells

B-cells

DCs

Macrophages

T-cells

CD8+ T-cells

CD4+ T-cells

Supplementary Data-1: HLA-A*02:01 and HLA-DQ8 expression on immune cells

(A) Expression of HLA-A*02:01 in YES mice as compared to H-2Db in C57BL/6 mice. B

(CD19+) cells, T (CD3ε

+,

CD4+

or CD8+) cells, dendritic cells (DCs, CD11c

+) and

macrophages (CD11b+) from spleen, blood (PBMCs) and inguinal lymph node (iLN) were

stained with an anti-human β2m (Tü99) mAb or an anti-H-2Db class-I mAb and analyzed by

flow cytometry. Results are percentages of cells expressing either HLA-A*02:01 (YES mice)

or H-2Db (C57BL/6 mice) at a basal state. (B) Expression of HLA-DQ8 in YES mice as

compared to IAb in C57BL/6 mice. B (CD19

+), T (CD3ε

+) cells, dendritic cells (DCs,

CD11c+) and macrophages (CD11b

+) from spleen, blood (PBMCs), and inguinal lymph node

(iLN) were stained with an anti-HLA-DQ (SVPL3 hybridoma) for YES mice and an anti-H-

2IAb mAb for C57BL/6 mice. Results are percentages of cells expressing MHC class-II

molecules at a basal state. *, p ≤ 0.05; **, p ≤ 0.01; Mann-Whitney test.

of 54Diabetes

Supplementary Data-2: Idd localization and CBA associated polymorphism in YES

mice.

Chromosome Idd region genes number /Idd target

genes

CBA associated

polymorphism

1 Idd-5.2 62 Speg 8/8

3

Idd-17 81 Ni 240

Idd-18 16

Cam 1/1

Igfs3 2/2

Atp1a1 1/1

4 Idd-9.1 78

Phc2 2/2

A3galt2 1/1

Zfp362 1/1

Tmem39b 2/2

Khdrbs1 2/2

Ptp4a 1/2

6 Idd-20 185

Gm16039 14/28

Glcci1 8/8

Ica1 4/6

7 Idd-27 763 Ni 361

11 Idd-4.3 223

Hormad2 1/1

Nf2 3/3

Ap1b1 4/4

Ewsr1 2/2

Rhbdd3 1/1

Amid1 1/1

17 Idd-24 192

Esp8 2/2

Esp6 0/1

Esp4 2/2

Esp3 2/2

Esp1 1/1

Crisp2 9/9

Rhag 1/6

Cenpq 3/3

Mut 5/5

18

Idd-21.3 148

Zfp521 12/13

Psma8 7/7

Taf4b 7/7

Kctd1 3/3

Aqp4 1/1

Chst9 1/1

Garem 1/1

Idd-21.2 108 Ni 197

Idd-21.2 182 Ni 403

ni : not identified

of 54 Diabetes

Supplementary Data-3: Immature YES spleen cells.

(A) Frequency of spleen cells identified as immature cells (i.e. negative for conventional

lymphocyte and APC markers, IC cells). Yes mice (closed bars), hINS transgenic mice (grey

bars) and HLA-A*02:01/HLA-DQ8 mice (open bars). Results are expressed in % of IC cells

(B) Characterization of IC cells. Flow cytometric analysis of IC cells using developmental

lymphocyte markers: anti-CD45, anti-CD62L, anti-CD44 and anti-CD25 mAb. YES mice

(left bar), hINS-transgenic mice (right bar), pro-T cells DN1 (CD44+CD25

-, closed bars); pro-

T cells DN2 (CD44+CD25

+, grey bars), pre-T cells DN3 (CD44

-CD25

+, checkerboard bars)

and pre-T cells DN4 (CD44-CD25

-, open bars). Results are expressed in % IC cells.

of 54Diabetes

Supplementary Data-4: MHC Class-I and class-II restricted T-cell responses against

conventional antigens in YES mice

(A) INFγ-responses against Mat-A2 stimulation. YES mice immunized against MatA258-66

(closed symbols) or PBS (open circles) were individually re-stimulated overnight with the

MatA258-66 (20 µg/ml final concentration) in the absence (�) or presence (�) of the BB7.2.

Numbers of spot forming-cells were expressed for 106 cells. (B) INFγ-responses against KLH

stimulation. CD8+ T-cells depleted-splenocytes from experimental YES mice immunized and

boosted with KLH (n=6, �) and from control YES mice immunized and boosted with PBS

(n=6, �) were individually re-stimulated overnight with KLH (10µg/ml final concentration)

in the absence or in the presence of SVPL3 anti-HLA-DQ mAb (50 µg/ml final

concentration). Numbers of spot forming-cells were expressed for 106 cells.


of 54 Diabetes

Supplementary Data-5: IFNγγγγ-ELISpot responses to hPPI peptides in YES mice

immunized against hPPI or PBS.

‡ mean (range) of spot number

# response against the whole hPPI protein

Peptide

Response ‡ Frequency of recognition (±3SD)

YES mice

immunized against

hPPI (n=16)

YES mice

immunized

against PBS

(n=7)

pvalue YES mice immunized

against hPPI

YES mice immunized

against PBS

pvalue

hPPI 1-15 97.44 (0-617) 9.22 (0-83) ≤0.007 5/16 0/6

hPPI 8-23 151.6 (0-842) 0 (0) ≤0.002 8/16 0/6

hPPI 16-30 115.1 (0-283) 0 (0) ≤0.02 7/16 0/6

hPPI 18-30 98.56 (0-467) 26.14 (0-183) 6/16 0/6

hPPI 20-35 178.1 (0-758) 42.86 (0-283) ≤0.03 10/16 0/6 ≤0.008

hPPI 25-40 222.4 (0-700) 9.37 (0-50) ≤0.002 9/16 0/6 ≤0.02

hPPI 33-47 156.6 (0-725) 0 (0) ≤0.005 7/16 0/6

hPPI 40-55 55.25 (0-233) 6 (0-42) 6/16 0/6

hPPI 46-61 159.8 (0-508) 34.57 (0-242) ≤0.02 9/16 0/6 ≤0.02

hPPI 55-70 208.4 (0-783) 29.71 (0-142) ≤0.005 11/16 0/6 ≤0.005

hPPI 61-76 167.2 (0-575) 0 (0) ≤0.006 10/16 0/6 ≤0.008

hPPI 70-86 87 (0-350) 33.43 (0-192) 7/16 0/6

hPPI 80-97 152.1 (00-500) 38 (0-250) ≤0.01 8/16 0/6

hPPI 92-110 215.6 (0-775) 11.86 (0-58) ≤0.008 11/16 0/6 ≤0.005

hPPI # 323.4 (92-1700) 0 (0) ≤0.0002 13/16 0/6 ≤0.0005

of 54Diabetes

Supplementary Data-6: Anti-hPPI proliferative responses in YES mice immunized against

hPPI or PBS.


Peptide

Response (Proliferation Index) Frequency of recognition (±3SD)

YES mice

immunized against

hPPI (n=12)

YES mice

immunized against

PBS (n=9)

pvalue YES mice immunized

against hPPI

YES mice immunized

agaisnt PBS

pvalue

hPPI 1-15 1.06 (0.4-2.1) 0.89 (0.7-1.24) 4/12 2/9

hPPI 8-23 1.42 (0.33-4.6) 0.81 (0.36-1.2) 4/12 0/9

hPPI 16-30 1.08 (0.1-4) 0.65 (0.09-1.5) 5/12 2/9

hPPI 18-30 0.91 (0.19-1.87) 0.84 (0.3-1.21) 5/12 3/9

hPPI 20-35 1.42 (0.6-4.7) 0.99 (0.8-1.5) 5/12 1/9

hPPI 25-40 1.32 (0.55-4.1) 0.86 (0.33-1.6) 2/12 1/9

hPPI 33-47 0.96 (0.2-2.8) 0.65 (0.11-1.4) 1/12 0/9

hPPI 40-55 1.01 (0.2-2.47) 0.89 (0.62-1.24) 4/12 3/9

hPPI 46-61 1.37 (0.74-2.6) 0.90 (0.62-1,24) ≤0.009 3/12 0/9

hPPI 55-70 1.59 (0.42-5) 0.71 (0.26-1.1) ≤0.005 7/12 0/9 ≤0.006

hPPI 61-76 1.12 (0.23-4.1) 0.52 (0.13-1.2) ≤0.04 2/12 0/9

hPPI 70-86 0.94 (0.3-2.18) 0.89 (0.4-1.6) 2/12 1/9

hPPI 80-97 1.31 (0.82-2.8) 1.0 (0.79-1.28) 4/12 0/9

hPPI 92-110 1.29 (0.52-4) 0.84 (0.37-1.5) 5/12 2/9

hPPI # 1.57 (0.41-4.6) 0.62 (0.14-1.5) ≤0.01 8/12 1/9 ≤0.02

of 54 Diabetes

Supplementary Data-7: Treshold value determination of BrdU proliferation assay


Peptide YES mice (n=20) SD of biais Treshold (mean±3SD)

hPPI 1-15 0.62 (0.3-0.95) 0.161 1.11

hPPI 8-23 0.87 (0.33-1.3) 0.183 1.42

hPPI 16-30 0.71 (0.1-1.2) 0.151 1.17

hPPI 18-30 0.55 (0.17-1.3) 0.156 1.02

hPPI 20-35 0.8 (0.49-1.25) 0.176 1.33

hPPI 25-40 0.96 (0.36-1.4) 0.205 1.57

hPPI 33-47 0.83 (0.18-1.5) 0.25 1.58

hPPI 40-55 0.61 (0.1-1.26) 0.151 1.06

hPPI 46-61 0.85 (0.4-1.4) 0.221 1.513

hPPI 55-70 1.01 (0.41-2.3) 0.108 1.33

hPPI 61-76 0.86 (0.16-2.2) 0.249 1.61

hPPI 70-86 0.59 (0.2-1.28) 0.205 1.20

hPPI 80-97 0.79 (0.4-1.6) 0.226 1.47

hPPI 92-110 0.98 (0.24-1.8) 0.094 1.26

hPPI # 0.77 (0.15-1.5) 0.160 1.25

of 54Diabetes

Supplementary Data-8: Anti-hPPI proliferative responses in diabetic pI:C-treated YES

mice compared to the YES mice control.


Peptide

Response (Proliferation Index) Frequency of recognition (±3SD)

Diabetic pI:C-treated

YES mice (n=15)

Control YES mice

(n=7) pvalue Diabetic pI:C-treated

YES mice (n=15)

Control YES mice

(n=7)

pvalue

hPPI 1-15 2.54 (0.27-6.75) 0.68 (0.54-1) 8/15 0/7 ≤0.02

hPPI 8-23 1.81 (0.21-6.93) 0.76 (0.21-1.39) 5/15 0/7

hPPI 16-30 1.86 (0.22-6.81) 1.13 (0.11-4.4) ≤0.048 9/15 1/7

hPPI 18-30 1.35 (0.4-5.77) 0.43 (0.19-0.64) ≤0.008 5/15 0/7

hPPI 20-35 0.9 (0.32-1.77) 0.58 (0.28-0.89) 3/15 0/7

hPPI 25-40 2.39 (0.24-7.84) 0.88 (0.36-1.81) 6/15 1/7

hPPI 33-47 2.66 (0.31-9.63) 0.65 (0.18-1.42) ≤0.05 7/15 0/7 ≤0.05

hPPI 40-55 2.64 (0.46-8.99) 0.7 (0.52-1.08) ≤0.016 9/15 1/7

hPPI 46-61 2.13 (0.54-7.92) 0.79 (0.34-1,22) ≤0.022 7/15 0/7 ≤0.05

hPPI 55-70 1.67 (0.31-6.24) 0.75 (0.23-1.35) 7/15 1/7

hPPI 61-76 0.95 (0.15-2.15) 0.45 (0.16-0.74) ≤0.031 3/15 0/7

hPPI 70-86 2.75 (0.14-7.41) 0.72 (0.46-1.12) 8/15 0/7 ≤0.02

hPPI 80-97 3.4 (0.26-11.78) 0.89 (0.42-1.55) ≤0.041 8/15 1/7

hPPI 92-110 3.49 (0.68-14.43) 0.77 (0.24-1.39) ≤0.005 11/15 1/7 ≤0.02

hPPI # 2.66 (0.28-8.63) 0.47 (0.15-0.92) ≤0.011 8/15 0/7 ≤0.05

of 54 Diabetes

Supplementary Data-9: Correlation between hPPI protein and hPPI-peptides

proliferative response in diabetic pI:C-treated YES mice:

Proliferation index of hPPI protein were compared to index proliferation oh each individually

hPPI-peptides by Spearman r correlation analysis.

of 54Diabetes

a humanized mouse model to study type 1 diabetes · 7/5/2018 · key requirements in type 1...

Documents