Title: A Humanized Mouse Model of Autoimmune Insulitis

Many mechanisms of and treatments for Type 1 diabetes studied in the NOD mouse model have failed to replicate in human disease. Thus, the field of diabetes research remains hindered by lack of an in vivo system in which to study the development and onset of autoimmune diabetes. To this end, we have characterized a system using human CD4+ T cells pulsed with autoantigen-derived peptides. Six weeks after injection of as few as 0.5x10 antigenpulsed cells into the NOD-Scid Il2rg-/mouse expressing the human HLA-DR4 transgene, infiltration of mouse islets by human T cells was seen. While islet infiltration occurred with both healthy and diabetic donor antigen-pulsed CD4+ T cells, diabetic donor injections yielded significantly greater levels of insulitis. Additionally, significantly reduced insulin staining was observed in mice injected with CD4+ T cell lines from diabetic donors. Increased levels of demethylated β cell-derived DNA in the bloodstream accompanied this loss of insulin staining. Together, these data show that injection of small numbers of autoantigen-reactive CD4+ T cells can cause a targeted, destructive infiltration of pancreatic β cells. This model may be valuable for understanding mechanisms of induction of human diabetes. Introduction The development of Type 1 diabetes involves a combination of genetic and environmental factors governing susceptibility to and/or protection from disease (1). NOD mice, the most widely studied model of human Type 1 diabetes, share a number of disease characteristics including autoantigens, the chronicity of the autoimmunity, and MHC homology, but significant differences between the two still remain (e.g., the time of progression from insulitis to clinical diabetes and the sex bias of disease incidence) (2). Because of these differences and others, many mechanisms and treatments that have been verified in NOD mice have failed to translate into successful treatments in humans (3,4). It therefore is imperative to develop model systems in which human cells that are involved in diabetes can be directly studied. The antigens that are involved in Type 1 diabetes have largely been identified through autoantibodies that are found in individuals at risk for and with the disease. They include preproinsulin (PPI), glutamic acid decarboxylase 65 (GAD65), and islet-specific glucose-6phosphatase catalytic subunit-related protein (IGRP), as well as other antigens recognized by polyclonal antibodies (islet cell antibodies, ICA) (5). T cells that are directed against these antigens are believed to cause β cell destruction, but there is little direct evidence that this is the case. The technical problems in studying the functions of autoreactive T cells include difficulties in growing and maintaining autoantigen-reactive lines and the lack of a suitable model system in which they can be studied. Previous studies have analyzed histopathology (6–8) and T cell tetramer staining (9) of pancreata from cadaveric diabetic donors. In these studies, CD8+ T cells that are reactive with IGRP were detected by immunohistochemical staining. However, staining of pre-diabetic insulitic lesions in humans is still conspicuously missing from the literature. Better visualization and understanding of these earliest events is of great significance, as it is unknown how the cellular composition of these lesions may have changed up to the point of clinical Type 1 diabetes diagnosis, let alone over a lifetime of disease in an individual. Knowledge of these very early events could allow for design of therapeutics aimed at prevention, as well as treatment of Type 1 diabetes. Herein, we tested whether CD4+ T cells, derived from HLA-matched diabetic and healthy donors and expanded on diabetes antigens, could cause insulitis and β cell destruction in NOD mice devoid of endogenous T cells, B cells and NK cells (NOD-Scid Il2rγ-/mice, referred to here as NSG mice) and also transgenic for human HLA-DR4 (10,11) (referred to as NSG.DR4). Parallel injections of PBMC from diabetic or healthy control individuals were also performed, allowing for direct comparisons of both the extent of insulitis and non-specific organ involvement of the two systems. We show that injections of antigen-pulsed expanded CD4+ T cells from patients with Type 1 diabetes resulted in varying degrees of islet infiltration, from peri-insulitis to severe insulitis. In these mice, there was a significant loss of insulin and increased levels of demethylated Ins1 DNA and Caspase-3 staining compared to control mice, reflecting β cell death. Interestingly, we isolated increased numbers of mouse CD45+ cells from the pancreata of mice injected with diabetic donor CD4+ T cells, suggesting that in this model cells from diabetic patients are able to establish an inflammatory environment in which murine leukocytes collaborate. These studies are the first to show β cell destruction mediated by human cells in a hybrid humanized mouse system. This model will be useful for studies of early insulitis and β cell destruction mediated by human immune cells. Research Design and Methods HLA Haplotype Determination PBMC were collected from patients with Type 1 diabetes and non-diabetic donors via leukopheresis or whole blood collection. Lymphocytes were isolated via Ficoll gradient. DNA was isolated from each prospective donor (Qiagen DNeasy Blood & Tissue Kit) and MHC haplotype was determined using the DRDQ 2 Test SSP UniTray Kit (Invitrogen). Only donors determined to be DRB1*0401 were used for further analysis. HLA-DR typing and disease status of donors is shown in Table 1. Written informed consent was obtained from all donors. The use of human cells was approved by the Yale Institutional Review Board. MACS-based cell sorting To isolate CD4+ T cells, PBMC were incubated with a depleting “antibody cocktail”, and processed according to manufacturer’s instructions (CD4+ T Cell Isolation Kit II – human, Miltenyi Biotech). The effluent population was considered CD4+ T cell-enriched and the population remaining on the column to be CD4+ T cell-depleted; this second population was used as donor-autologous APCs. Purity of the isolated CD4+ T cell population was routinely