A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes

To circumvent the limitations of available preclinical models for the study of type 1 diabetes (T1D), we developed a new humanized model, the YES-RIP-hB7.1 mouse. This mouse is deficient of murine major histocompatibility complex class I and class II, the murine insulin genes, and expresses as transgenes the HLA-A*02:01 allele, the diabetes high-susceptibility HLA-DQ8A and B alleles, the human insulin gene, and the human co-stimulatory molecule B7.1 in insulin-secreting cells. It develops spontaneous T1D along with CD4+ and CD8+ T-cell responses to human preproinsulin epitopes. Most of the responses identified in these mice were validated in T1D patients. This model is amenable to characterization of hPPI-specific epitopes involved in T1D and to the identification of factors that may trigger autoimmune response to insulin-secreting cells in human T1D. It will allow evaluating peptide-based immunotherapy that may directly apply to T1D in human and complete preclinical model availability to address the issue of clinical heterogeneity of human disease.


INTRODUCTION
Type 1 diabetes (T1D) is a multifactorial autoimmune disease that remains a major health challenge (1). Its incidence increases by 3% to 4% yearly. There is presently no therapy to definitively revert or stop the autoimmune process responsible for the destruction of b cells (2). Upstream of therapies, immunological markers for the autoimmune response to b cells have shown limitations in predicting the development of type 1 diabetes in subjects with prediabetes, especially when a single autoantibody is detected (3). Following their use in preclinical models of T1D, antigen and peptide-specific immunotherapies have been proposed as strategies with a low risk/benefit ratio in human. Early attempts have shown minimal efficacy in human, as in using glutamic acid decarboxylase-65 (GAD65) (4). However, the use of an immunodominant proinsulin peptide has proven to be well-tolerated and to delay C-peptide decline in human (5). From both a diagnostic and a therapeutic standpoint, preclinical models of T1D have fallen short of translating into human. Current models do not allow testing peptides derived from human autoantigens that may directly apply to the human situation in vaccination strategies. A new preclinical model to study T1D in a humanized mouse model would be amenable to evaluate the relevance of T-cell assays or peptide immunotherapy that would directly apply to human diabetes.
Our aim in this study was to create a preclinical model that would develop spontaneous T1D and allow characterizing HLA-A class I and class II MHC-restricted peptides that directly apply to human. We chose human preproinsulin (hPPI) as a major T1D autoantigen (6)(7)(8), the most common class I MHC HLA-A*02:01 allele in the three major ethnic groups (50% in Caucasian and Asian and 30% in African) (9) and the high T1D susceptibility class II DQ8 A and B alleles (10). We previously generated the YES mouse that expresses the HLA-A*02:01, the HLA-DQ8, and the human insulin (hINS) genes and fails to develop spontaneous T1D, but develops T1D when challenged with polyinosonic-polycytidylic acid (pI:C) (11). As the expression of B7.1 in pancreatic b cells has been shown to trigger the development of T1D in conventional mice (12) and accelerate diabetes in the NOD mouse (13), we introduced the human costimulatory molecule B7.1 (hB7.1) under the control of the rat insulin promoter in pancreatic b cells onto the YES background to enforce the development of spontaneous T1D. The first objective was to identify autoreactive epitopes from hPPI involved in the T1D autoimmune response and evaluate their relevance in human. The second objective was to identify external factors that may accelerate the development of T1D within the frame of human MHC presenting molecules (14).
This new humanized mouse expressing HLA-A*02:01, HLA-DQ8, hINS, and the RIP-hB7.1 transgenes, thereafter called YES-RIP-hB7.1 mice, develops spontaneous T1D and shares immunological features with human T1D. This new model of spontaneous diabetes completes the previously reported YES model. According to the clinical complexity of human T1D that is likely to be a heterogeneous set of diseases, these new models altogether provide a larger set of preclinical tools to study human T1D.
Mice YES-RIP-hB7.1 mice were obtained by lentiviral transgenesis of YES mice (H-2 D b , mouse b2 microglobulin, IAa b , b b , IEb b quintuple KO mice expressing a chimeric-a3 H-2 D b domain, human b2 microglobulin-HLA-A*02:01 monochain molecule named HHD-and the HLA-DQB1*03.02 and HLA-DQA1*03.01 genes) (15,16). The RIP-hB7.1 transgene was inserted in a HIV-derived recombinant lentiviral vector (LV-RIP-hB7.1; insert of 4,640 bp) as previously reported into YES mice (17,18). Retroviral pseudotypes were injected into fertilized eggs obtained from super-ovulated female YES mice mated with male YES mice (16). Fertile and transduced eggs were reimplanted into pseudo-pregnant C57BL/6xCBA F1 mice. All mice were maintained under specific pathogen-free conditions, and experimental studies were performed in accordance with the Institutional Animal Care and Use Guidelines and accredited by the Ethics Committee n°34 of Paris Descartes under number CEEA34.CB.024.11. Mice were monitored three times a week for glycosuria. When glycosuria was detected, diabetes was diagnosed when two successive glycemic values >250 mg/dl were detected at 24 h interval. Diabetes incidences curves correspond to the percentage of mice diagnosed as diabetic referring to the aforementioned criteria.
Molecular Characterization of LV-RIP-hB7.1 Transgene YES mice submitted to lentiviral transgenesis were genotyped using the following primers: 5′end (AGGGAACATCAC CATCCAAG) and 3′end (TGCCAGTAGATGCGAGTTTG), annealing temperature: 62°C, amplicon: 181 bp. A positive RIP-hB7.1 mouse was selected to perform the molecular characterization of the LV-RIP-hB7.1 transgene. To characterize insertion sites in genomic DNA of the founder, we realized a sequence capture design using the SeqCap EZ system (Roche NimbleGen) targeting 97.6% of the transgene. After nebulization, fragmented genomic DNA was end-repaired and ligated with adapters. A double capture was realized to obtain a GS Junior library ready for emulsion PCR (emPCR). Fragments were then annealed to capture beads and clonally amplified by emPCR (emPCR Amplification Method Manual LibL; GS Junior Titanium Series, Roche). Beads with the cloned amplicons were then enriched, loaded on a 454 picotiter plate, and sequenced on the Roche GS Junior Sequencer according to the protocol of the manufacturer (Sequencing Method Manual, GS Junior Titanium Series, Roche). Image analysis and base calling of the raw sequencing data were performed using the default "shotgun" Roche GS Junior data analysis pipeline. To obtain the position of transgene insertions in genomic DNA, sequence reads were aligned to the 50 bp of each end of the transgene primary sequence To compare the genetic background of YES-RIP-hB7.1 and YES mice, we performed a GenScan SNP Affymetrix. Briefly, high-quality genomic DNAs (250 ng) were digested with NspI and StyI enzymes. NspI and StyI adaptors were then ligated to restricted fragments followed by PCR using the universal primer PCR002. Each amplicon was purified, pooled, and used for fragmentation and end-labeling with biotin using terminal In Vivo T-Cell Depletion Treatment and pI:C Stimulation

Cytotoxic Assay
T-cell cytotoxicity assay was performed on HHD-transfected P815 cells prepulsed with 10 mg peptide for 2 h at 37°C using the LDH Cytotoxicity Detection Kit PLUS (Roche) or hPPI/HHD-doubled transfected P815 cells. High control of lysis corresponded to cell killing with a lysis solution (Tween-20), which provide the maximum LDH release. Low control of lysis, which provide the spontaneous cytotoxicity, was evaluated on cells that were not submitted to additional treatment. Specific lysis was calculated as optical density of (targeted condition − spontaneous lysis)/ (maximum LDH release − spontaneous lysis) × 100.

Enzyme-Linked Immunospot Assay
IFNg-ELISpot assays were achieved as previously reported (21). Spots were counted using Bioreader 5000 Pro Sf (BioSys GmbH). Splenocytes from mice were stimulated overnight with peptides and Il-2 (5 U/ml final). The enzyme-linked immunospot (ELISpot) assay was performed using mouse g-ELISpot antibody pair, from U-CyTech biosciences. Data are the mean of triplicate wells. The background of IFNg responses was evaluated in the absence of peptide. Specific IFNg responses were expressed as spot-forming cells (SFC) per 10 6 cells after normalization of the background. Positive controls were triplicates of cells stimulated by 1 mg/ml ConA (Sigma-Aldrich) and negative controls by pyruvate dehydrogenase (PDHase 208-216 ) irrelevant peptide.

Human Recombinant-PPI Protein Production
The cDNA sequence of preproinsulin was mutated to convert the Ala codon in position 3 of the leader DNA sequence to Asp by site-directed mutagenesis (NEB) to abolish the signal-sequence site cleavage of PPI (22). Mutated PPI was cloned in pFastBac vector to generate the PPI-recombinant bacmid, then PPIrecombinant Baculovirus to produce human PPI protein into Bac-to-Bac Baculovirus Expression System (Invitrogen) according to the instruction of the manufacturer. Purification was performed on pelleted infected insect cells, following preparation and extraction procedures for insoluble proteins (inclusion body) from Escherichia coli (23). Purity was confirmed on SDS/PAGE gel and quantification was performed using the BCA Protein assay kit (Pierce).

Cell Proliferation Assay
Spleen cells (10 5 /well) were incubated with 0.5 mg antigen/well or 0.1 µg peptide/well for 72 h 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 10 5 cells/well incubated without antigen or in the presence of 10 mg/ml final concentration of anti-mouse CD3ϵ antibody, respectively.

Immunohistochemistry and Fluorescence
Immunofluorescence stainings were performed on formalin-fixed paraffin pancreas sections that were deparaffinized in xylene and dehydrated by ethanol. After washing, antigen retrieval was realized by hot incubation in citrate buffer, followed by permeabilization (20 min in PBS 1×/0.4% Triton X-100) and saturation (30 min PBS 1×/1% horse serum) before immunostaining with biotinylated rat anti-human CD3e(AbD Serotec) and polyclonal rabbit anti-glucagon (DAKO) antibodies overnight. Slides were washed with PBS 1×/1% BSA/0.1% Triton X-100 and stained with an anti-rabbit Ig-FITC antibody (Abcam) and SAV-Cy3 (Abcam) at RT. Sections were mounted in Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories). Observations were made with a spinning disk confocal apparatus at the Cochin Institute Imaging Platform and pictures analyzed with the ImageJ software. Stable immunohistochemical stainings were performed on paraffin-embedded pancreas sections and stained with polyclonal guinea-pig anti-human insulin (DAKO) or rat antimouse CD4 biotin (eBioscience), followed by incubation with peroxidase-labeled antibodies. All reactions were revealed with diaminobenzidine (DAB, Genemed). Sections were counterstained with hematoxylin and mounted. Observations were made with Zeiss AxioObserver Z1 microscope coupled with MRm Axiocam Zeiss and pictures analyzed with the ImageJ software.
Assessment of total b-cell mass was performed on scanstained microscope slides with Inform software using a guinea pig anti-human insulin antibody (DAKO) as the ratio between bcell surface (µm 2 )/pancreas surface (µm 2 ) multiplied by pancreas weight (mg) (24). Assessment of total a-cell mass was performed on scan-stained microscope slides with Inform software using a rabbit anti-glucagon antibody (DAKO) as the ratio between acell surface (µm 2 )/pancreas surface (µm 2 ) multiplied by pancreas weight (mg). Quantification of insulin-positive or glucagonpositive areas was performed from the entire pancreas on serially cut 8-µm-thick sections. Five to 10 pancreatic sections were processed for immunostaining. Cellular mass obtained for each group was compared using the non-parametric Mann-Whitney statistical test.

Statistics
The biostatistic method used to compare diabetes incidences between different groups of mice was the log-rank Mantel-Cox test. Comparison of distribution scores between the different mouse strains used the non-parametric Mann-Whitney test. T-cell reactivity was compared using non-parametric Mann-Whitney test and non-parametric Kruskal-Wallis test. ns, non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. For T-cell proliferation assays, we used the Bland and Altman test to determine the threshold value for a global reliability statistic for significant proliferation response.

Accession Numbers
We submitted the data generated by the Affymetrix SNP Array detection to a public repository (http://www.ncbi.nlm.nih.gov/ geo) and the GEO Series accession numbers were GSE101551 and GSE151644.

Characterization of the YES-RIP-hB7.1 Mice
In order to enforce the development of T1D in YES mice, we introduced the hB7.1 gene under the control of the rat insulin gene promoter (RIP) using the LV-RIP-hB7.1 vector (17). We selected three mice that were positive for hB7.1 for backcrossing onto the YES background. Throughout the crosses, one founder progeny was selected. To further stabilize its lineage, we realized a NimbleGen Sequence Capture of the RIP-hB7.1 transgene of the founder and one of its offspring. Inserted hB7.1 sequences were identified as indicated in Table  S2, matching with regions located on chr. 11, 14, 16, and 19 in the founder mouse. Nevertheless, the only region located in chr.19 remained detectable in the offspring, a chromosome with no Idd known to associate with TID. We did not evidence hot spots of enhancer transcripts close to the LV-RIP-hB7.1 insertions using the mouse ENCODE database from UCSC Genome Bioinformatics (GSE101551 and GSE151644 GEO data accession numbers). We screened 128 mice from the progeny for the selected insertion sites in order to stabilize the lineage at a homozygote status using a classical progeny test.

YES-RIP-hB7.1 Mice Develop Spontaneous Type 1 Diabetes
Insulitis and spontaneous diabetes developed in founder YES-RIP-hB7.1 offspring that were submitted to 20 brother-sister mattings. In the founder progeny, 46 out of 128 YES-RIP-hB7.1 mice developed diabetes, while diabetes was not observed in nontransgenic YES mice. Diabetes incidence of stabilized YES-RIP-hB7.1 mice is shown in Figure 1A. Age at onset varied from one mouse to another, spanning from 9 to 51 weeks of age. The overall prevalence of diabetes was similar in female (53.8%) and male (50.0%) YES-RIP-hB7.1 mice. The difference in diabetic incidence was not statistically different between males and females. The average glycemia in YES-RIP-hB7.1 mice when diagnosed as diabetic was 520 ± 115 and 532 ± 99 mg/dl in female and male, respectively. The average glycemia in nondiabetic YES-RIP-hB7.1 mice at the end of the experiment (40 weeks) was 143 ± 35 and 125 ± 18 mg/dl in female and male, respectively. The average glycemia in YES mice was 107 ± 13 and 102 ± 12 mg/dl in female and male, respectively. In order to address whether diabetes was immune-related, we analyzed hematoxylin-eosin-stained pancreas paraffin sections from diabetic and non-diabetic YES-RIP-hB7.1 mice. As shown in Figure 1B, insulitis was detected by immunofluorescence staining using an anti-CD3ϵ antibody and a rabbit antiglucagon antibody to locate remnant islets, showing the infiltration of islets by CD3 + T lymphocytes ( Figure 1C). Glucagon-positive cells were dispersed in remnant islets that showed a dislocated architecture in YES-RIP-hB7.1 diabetic mice ( Figure 1D) compared with the YES mice control ( Figure 1E). Stable immunohistochemistry staining with an anti-CD4 antibody confirmed the detection of CD4 + T cells within the infiltrate ( Figure 1F). As shown in Figure 2, the number of islets expressing insulin was decreased by 70.9% in diabetic YES-RIP-hB7.1 mice as compared with YES mice or non-diabetic YES-RIP-hB7.1 mice. Islet size ( Figure 2B) was decreased by over 60% in diabetic YES-RIP-hB7.1 mice as compared with YES controls. A dramatic decrease of the b-cell mass was observed in diabetics YES-RIP-hB7.1 mice as compared with age-matched YES mice (0.60 ± 0.49 versus 3.93 ± 0.65, respectively, p ≤ 0.02) and non-diabetic YES-RIP-hB7.1 mice (7.01 ± 2.06) ( Figure 2C). The a-cell mass ( Figure 2D) also showed a significant decrease in diabetic YES-RIP-hB7.1 mice as compared with non-diabetic YES-RIP-hB7.1 mice and YES controls (0.60 ± 0.49 versus 3.93 ± 0.65, respectively, p ≤ 0.02). In non-diabetic YES-RIP-hB7.1 mice, the a-cell mass was heterogeneous (7.01 ± 2.06). Infiltrates from three diabetic mice were recovered and pooled to be analyzed ( Figure S1). They were composed of 52% T cells, among which 81% were CD8 + T cells, 8% were CD4 + T cells, 21% were b cells, 0.8% were dendritic cells, and 0.6% were macrophages. Among CD4 + T cells, 7% were CD4 + CD25 + FoxP3 + T cells. Infiltrates recovered from three non-diabetic YES-RIP-hB7.1 mice ( Figure S1) were composed of 56% T cells, among which 56% were CD8 + T cells, 34% were CD4 + T cells, 34% were b cells, and 2.3% were dendritic cells or macrophages. Among CD4 + T cells, 1% were CD25 + FoxP3 + . These data demonstrate the presence of an immune response along the development of diabetes in YES-RIP-hB7.1 mice, a dramatic decrease in b cells and a reduced a-cell mass. As the extent of infiltration was milder than the infiltrate seen in the NOD mouse model, we addressed whether autoimmune development could be delayed by a transient treatment with anti-CD4 and anti-CD8 monoclonal antibodies. As shown in Figure 3A, in vivo depletion of T cells by anti-CD4 and anti-CD8 antibodies from either 2 to 5 or 8 to 11 weeks of age significantly delayed T1D development in YES-RIP-hB7.1 mice. There was no difference in the protection observed in early-treated and late-treated mice. As previously described in the YES mouse model (16), we addressed whether diabetes development could be triggered in YES-RIP-hB7.1 mice by poly(I:C). As shown in Figure 3B, diabetes was induced within 6 to 17 days following the first poly(I:C) injection.

DISCUSSION
We developed a new preclinical model of spontaneous T1D in YES mice engineered to express the human co-activation hB7.1 gene in b cells by injecting a HIV-derived recombinant lentiviral vector in which a RIP-hB7.1 transgene has been inserted as previously reported (17), in addition to expression of human MHC and insulin genes instead of the corresponding mouse genes (16). We obtained a founder in which four insertions were detected and one stabilized as homozygous in the progeny. This stable insertion was located at a distance from any known Idd loci. The YES genetic background on which the C57BL/6 background is dominant was previously described (16). This likely indicates that the main genetic constraint that favors diabetes development, beyond expression of hB7.1, is the expression of the human class II HLA-DQ8 and, to a lower extent, class I HLA-A*02:01 alleles. While less than 2% conventional RIP-hB7.1 transgenic mice developed diabetes by 8 months of age (30), spontaneous diabetes was commonly observed in transgenic mice that co-expressed RIP-hB7.1 and the human insulin gene b cells (12,31). Co-expression of RIP-hB7.1 in addition to HLA-DQ8 has been shown to allow the development of diabetes on a C57BL/6 genetic background (32)(33)(34)(35). Along with the development of diabetes and islet infiltration by CD4 + and CD8 + T cells, a dramatic decrease of b-cell mass and a decrease in a-cell mass were observed in diabetic YES-RIP-hB7.1 mice, as previously been reported in NOD mice (36). In addition, RNA sequencing of human islet cells obtained from T1D patients showed a decrease in the expression of glucagon and other a-cell genes (37,38), which is confirmed by a recent study on T1D patients based on the network for Pancreatic Organ Donors repository (39). In diabetic YES-RIP-hB7.1 mice in which the islet infiltrate was recovered from the pancreas, the islet infiltrate was predominantly composed of lymphocytes as observed in human T1D insulitis (40). CD8 + T cells were largely predominant, suggesting that they were a driving force in the diabetes process. We previously reported an increased percentage of single positive CD8 + T cells in the YES mouse, likely related to a lower efficiency of class II HLA-DQ8 to select CD4 + T cells than of class I HLA-A*02:01 to select CD8 + T cells (10). However, CD4 + T cells were recovered from non-diabetic YES-RIP-hB7.1 infiltrates in addition to CD8 + T cells. Spontaneous diabetes was significantly delayed in YES-RIP-hB7.1 mice following transient treatment with depleting anti-CD4 and anti-CD8 monoclonal antibodies, leaving open the issue of the predominant role of either CD4 + or CD8 + T cells in our model. In most T1D preclinical models, CD4 + T cells have been reported as dominant although a major role of CD8 + T cells has been reported in some models (41). In addition, an acute form of diabetes was induced in 8-week-old, prediabetic, YES-RIP-hB7.1 mice by seven daily poly(I:C) injections, as reported in the YES funders (16), indicating that different triggering events may concur to autoimmune T1D, as is probably the case in human disease. These data are reminiscent of data involving T1D induction by Coxsackie B4 virus (42)(43)(44), pointing to isletenvironment interactions through signals carried by pattern recognition receptors (PPRs) (45,46) in induction of T1D (47,48).
Mouse models have been developed that express T1D susceptibility HLA class I (65,66) or class II genes (31,67). They allow defining epitopes on murine autoantigens that possibly correspond to epitopes recognized on human autoantigen along human T1D (37,54). Sequence differences between murine and human MHC presenting molecules cannot exclude, however, that sets of epitopes defined on murine autoantigen differ from those recognized in human T1D. Models have further been reported that express T1D susceptibility HLA class I A*02:01 and/or the high-susceptibility DQ8 class II gene along with either the human preproinsulin or GAD genes (68,69). These models are likely to allow characterizing autoantigen epitopes that may directly apply to human T1D (11,70). Among these models, the YES-RIP-hB7.1 mouse is expected to allow the characterization of hPPI-specific CD8 + and CD4 + hPPI epitopes on a major autoantigen targeted in T1D, including new epitopes, such as spliced or modified epitopes, in this proinflammatory context (70,71). Beyond allowing the identification of HLA-A02*01 and DQ8-restricted epitopes, it allows exemplifying different mechanisms of induction of T1D in the context of human disease that is likely heterogeneous (55,56). Such models may prove valuable in developing T-cell assays in T1D and evaluating strategies to induce immune tolerance in T1D patients using peptides targeted by the autoimmune response to b cells.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. These data can be found here: http://www.ncbi.nlm. nih.gov/geo, GSE101551 and GSE151644.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Research Ministry Authorization MESR under number DC-2015-2536/IDRCB number 2015-A01875-44. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee n°34 of Paris Descartes under number CEEA34.CB.024.11.

AUTHOR CONTRIBUTIONS
SL performed the experiments, was involved in the discussion, and contributed to the writing of the manuscript. SG was in charge of the transgenic animals. AG performed DAB staining. FLet and MV were involved in Affymetrix genotyping array discussion and RIP-hB7.1 capture strategy. PN and MB performed big data analysis. PC participated in the production of lentiviral particles for the lentiviral transgenesis. EL was responsible for patient recruitment and follow-up. MC and DE were implicated and collaborated in the alternative spliced hPPI section. FLem was involved in the discussion and manuscript editing. CB designed the experiments, chaired discussions, and wrote the manuscript. All authors contributed to the article and approved the submitted version.