Jul 14, 1993 - A97, E-, Db) were kindly provided by E. Leiter (The Jackson. Laboratory). ..... and Donna Louie for transgenic mouse production. S.M.S. was.
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9566-9570, October 1993 Immunology
An Abd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells (autoimmunity/major histocompatibility complex/class I)
STEVEN M. SINGER*, ROLAND TISCH*, XIAO-DONG YANG*, AND HUGH 0. MCDEVITT*tt Departments of *Microbiology and Immunology and tMedicine, Stanford University Medical Center, Stanford, CA 94305
Contributed by Hugh 0. McDevitt, July 14, 1993
ABSTRACT Susceptibility to the human autoimmune disease insulin-dependent diabetes meilitus is strongly associated with particular haplotypes of the maijor histocompatibility complex (MHC). Similarly, in a spontaneous animal model of this disease, the nonobese diabetic (NOD) mouse, the genes of the MHC play an important role in the development of diabetes. We have produced transgenic NOD mice that express the class II MHC molecule I-Ad in addition to the endogenous I-A97 molecules in order to study the role of these molecules in the disease process. Although the inflammatory lesions within the islets of Langerhans in the pancreas appear similar in transgenic and nontransgenic animals, transgenic mice develop diabetes with greatly diminished frequency compared to their nontransgenic littermates (10% of transgenic females by 30 weeks of age compared to 45% of nontransgenic females). Furthermore, adoptive transfer experiments show that T cells present in the transgenic mice are able to interfere with the diabetogenic process caused by T cells from nontransgenic mice. Thus, the mechanism by which I-Ad molecules protect mice from diabetes includes selecting in the thymus and/or inducing in the periphery T cells capable of inhibiting diabetes development.
of diabetes in NOD mice. Introduction of the I-Ak genes (11, 12) or of a site-specific mutant of the AbW7 allele (His-56 to Pro-56) (13) was also able to prevent diabetes in transgenic NOD mice. Class II MHC genes are normally expressed on thymic epithelial cells and on certain bone marrow-derived cells, including B cells, macrophages, and dendritic cells, and any of these cell types could express the defect(s) leading to diabetes. Using bone marrow chimeras, bone marrowderived cells but not thymic epithelial cells from NOD mice have been implicated as the cells expressing the MHC-linked genetic defect responsible for development of diabetes (14). These experiments, however, do not define the mature cell types responsible for diabetes. Several lines of experimentation show that antigen presenting cells and T lymphocytes are both intimately involved in the pathogenesis of diabetes. Diabetes can be prevented by treating NOD mice with reagents that inhibit either macrophage function or T-cell function (15-19). Furthermore, diabetes can be induced in syngeneic recipients by the adoptive transfer of T lymphocytes from diabetic donors (20, 21). However, while murine T cells do not express class II MHC molecules, T-cell development and activation are regulated by these molecules. It therefore appears that the MHC-linked genetic defect in NOD mice is expressed by bone marrow-derived antigen presenting cells and that this influences T-cell development so as to induce diabetes. We have produced transgenic NOD mice by using the Abd gene in order to understand how the interaction of I-A molecules with T lymphocytes affects diabetogenesis. NOD.Abd transgenic mice express functional I-Ad molecules in both the thymus and the periphery. These mice develop diabetes at a much reduced rate compared to nontransgenic littermates. Furthermore, T lymphocytes from nondiabetic transgenic mice are able to inhibit the adoptive transfer of diabetes by spleen cells from diabetic nontransgenic mice. We therefore propose that the protective effect of certain I-A molecules is due to their ability to select and induce regulatory T-lymphocyte populations in transgenic mice.
Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the insulin-producing ,8 cells in the islets of Langerhans in the pancreas. Susceptibility to this disease is determined by multiple genetic and environmental factors (1). The strongest genetic component found thus far is encoded by particular haplotypes of the human leukocyte antigen complex, but due to linkage disequilibrium within this region it has been difficult to identify the actual diseaserelated locus. Sequence analysis of DQB alleles on susceptible and protective haplotypes, however, suggests that the class II molecule DQ plays an important role in the pathogenesis of insulin-dependent diabetes mellitus (2). The nonobese diabetic (NOD) mouse is a spontaneous model of insulin-dependent diabetes mellitus (3, 4). It shares many features with the human disease including similar pathologic findings and genetic predispositions. Early studies of the genetic basis for diabetes found that the major histocompatibility complex (MHC) region of NOD mice contained at least one recessive locus responsible for this susceptibility (5, 6). The MHC class II region in NOD mice encodes a unique I-A molecule (I-A97) and a nonfunctional I-E molecule (5, 7). The gene encoding the A, chain in NOD mice is identical to the Aad allele, and it is the AbW7 allele that is unique. Recently, transgenic mice have been used to show that both the lack of I-E expression and the unique I-A molecule in NOD mice contribute to their susceptibility to diabetes (8-13). Restoring I-E expression by introducing a functional I-Ea allele (8-10, 13) prevented the development
MATERIALS AND METHODS Mice. Original breeding pairs of NOD/Lt mice (H-2; Kd, A97, E-, Db) were kindly provided by E. Leiter (The Jackson Laboratory). Our colony (NOD/Mcd) has been maintained by strict brother-sister mating for 24 generations, using the offspring of diabetic mothers for further breeding. Typically, 70% of females in our colony develop diabetes. B10.GD mice (H-2; Kd, Ad, E-, Db) and (NOD x BALB/c)F1 mice (H-2; Kd, Ad/g7, Ed/g7, Dbld) were bred and maintained in our facility.
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Abbreviations: MHC, major histocompatibility complex; NOD, nonobese diabetic; GAD, glutamic acid decarboxylase. tTo whom reprint requests should be addressed.
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Immunology: Singer et al. Production and Screening of Transgenic Mice. The cosmid 41.1 containing the Abd locus was kindly provided by M. Steinmetz (22). Cosmid DNA was partially digested with Xho I and the 10.5-kb fragment was purified from agarose gels with Geneclean (Bio 101). This DNA was microinjected into NOD embryos, and embryos were implanted in pseudopregnant (BALB/c x 129)F1 females. Tail DNA of offspring was digested with BamHI, resolved on agarose gels, transferred to nylon membranes (Nytran; Schleicher & Schull), and hybridized to a 32P-labeled Abk cDNA as described (23). Transgenic mice were backcrossed to NOD mice so that all analyses were performed with mice heterozygous for the transgene. Antibodies and Conjugates. Monoclonal anti-I-Ad (MKD.6; ref. 24) antibody was purified from ascites fluid by ammonium sulfate precipitation and protein A chromatography. Antibodies were conjugated to N-hydroxysuccinimide biotin (Pierce) as described (25). For cell depletion, anti-I-Ag7 (10-2.16; ref. 26) antibody was prepared as tissue culture supernatant. Fluorescein isothiocyanate-conjugated sheep anti-mouse IgD (Nordic), biotin-conjugated Thyl.2 (Becton Dickinson Immunocytometry Systems), avidin horseradish peroxidase (Sigma), and avidin Texas Red (Vector Laboratories) were obtained commercially. Flow Cytometry. Single cell suspensions of spleens were prepared and erythrocytes were lysed in 140 mM NH4Cl/17 mM Tris HCl, pH 7.5. Cells were stained with appropriate concentrations of the antibodies described. Data were collected on a FACS IV (Becton Dickinson Immunocytometry Systems) and analyzed with FACS-Desk software (Stanford University Shared FACS Facility). Cells that stained with propidium iodide were excluded from analysis. Diabetes Monitoring. Animals were checked for glycosuria every 2 weeks using Chemstrip UG (Boehringer Mannheim). When animals were positive in two consecutive urine tests, blood glucose was measured using Chemstrip BG and an Accutest IIm meter (Boehringer Mannheim). Mice were considered diabetic if they had glycemia levels >200 mg/dl. Inhibition of Diabetes Transfer. Disease was induced in 8to 12-week old, irradiated (600 rads from an x-ray source; 1 rad = 0.01 Gy) NOD males as described (20). Briefly, 107 erythrocyte-depleted splenocytes from recently diabetic NOD females were injected into recipients intravenously in 0.2 ml of saline. For cell mixing experiments, T cells were purified from the spleens of nondiabetic mice by nylon wool chromatography followed by two rounds of depletion with anti-I-Ag7 antibody and complement (LowTox-M; Cedarlane Laboratories). The resulting populations were analyzed for purity by flow cytometry and routinely contained >95% Thyl.2+ cells and
