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Islet Cell Autoimmunity in NOD Mice Transgenic to HLA-DQ8 and Lacking I-Ag71
Islet Cell Autoimmunity in NOD Mice Transgenic to HLA-DQ8 and Lacking I-Ag7 R. Raju, S.R. Munn, C. Majoribanks, and C.S. David
T
YPE I diabetes is an autoimmune disease with a strong genetic predisposition as seen by the presence of specific HLA haplotypes like HLA DR3, DR4, DQ8, and/or DQ2.1 However, the role of any particular HLA allele in the development of the disease is still unclear due to the presence of multiple class I and class II molecules on the cell surface. The genetics of autoimmune diabetes is further complicated due to the role of several non-MHC genes also in the initiation of the disease.2 We addressed this problem by generating HLA-DQ8 transgenic mice lacking endogenous mouse class II molecules and backcrossing them on to the NOD background. MATERIALS AND METHODS The generation of DQ8.Ab0 mice was previously described.3,4 The DQ8.Ab0 mice were crossed to the NOD and the DQ81 Ab0/g7 F1 mice were selectively backcrossed further to the NOD to generate N2 generation NOD/DQ8 mice. The DQ81 Ab0/g7 mice of N2 generation were intercrossed to obtain mice of different class II phenotypes: DQ81 Ab0/g7, DQ82Ab0/g7, DQ81Abg7/g7, DQ82Abg7/g7, and DQ81Ab0/0 mice. The neo gene (Ab0) was detected by a PCR specific for the neomycin resistant gene incorporated in Ab gene for the gene disruption. The surface expression of DQ8 and Ag7 molecules were detected using FACS analysis using monoclonal antibodies IVD12 and K24-199, respectively. Microsatellite analysis for idd3 and idd10 were performed by a polymerase chain reaction (PCR) method. D3Mit5 and D3Nds7 primer pairs were used for the specific amplification of idd3 and idd10 microsatellite repeat units. Cyclophosphamide (200 mg/kg body weight) was administered to 5 to 6 month old mice at day 0 and day 14, blood glucose was tested every week for four weeks and sacrificed on day 28.
RESULTS AND DISCUSSION
The HLA DQ8.Ab0 mice we generated did not develop diabetes or spontaneous inflammation in the islet. Multiple immunizations of these mice with insulin peptides or insulin
0041-1345/98/$19.00 PII S0041-1345(97)01362-6 474
also did not result in diabetes or inflammation of the pancreas/islet. So we crossed DQ8.Ab0 mice to the NOD background. There were a total of 35 mice of NOD/ DQ81Ab0/0 phenotypes of N2 generation. None of these mice developed spontaneous overt diabetes by 5 to 6 months of age. Sixteen of the 35 mice were administered cyclophosphamide. The administration of cyclophosphamide also did not induce diabetes in any of these mice. The histology of the pancreas did not show any signs of insulitis, though there was extensive periinsulitis and perivasculitis in about 70% of the mice treated or untreated with cyclophosphamide. Among the mice that did not receive cyclophosphamide, six mice were homozygous at both idd3 and idd10 loci and there were five mice homozygous at these loci in the group that received cyclophosphamide. The number of mice in other class II phenotypes were small, however cyclophosphamide could induce diabetes in DQ81Ab0/g7, DQ81Abg7/g7 and DQ82Ab0/g7 mice. This result shows that though idd3 and idd10 together with Ag7 molecule contribute significantly to diabetes, these non-H2 susceptibility genes in the presence of DQ8 do not initiate insulitis or diabetes unless at least one copy of the Ag7 molecule is present. REFERENCES 1. Lernmark A: Diabetologia 37:S73, 1994 2. Vyse TJ, Todd JA: Cell 85:311, 1996 3. Cosgrove D, Gray D, Dierich A, et al: Cell 66:1051, 1991 4. Nabozny GH, Baisch JM, Cheng S, et al: J Exp Med 183:27, 1996 From the Department of Immunology and Transplantation, Mayo Clinic, Rochester, Minnesota. Support provided by Juvenile Diabetes Foundation International and NIH grant, AI 14764. Address reprint requests to C.S. David, Department of Immunology and Transplantation, Mayo Clinic, Rochester, MN 55905.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 474 (1998)
T
YPE I diabetes is an autoimmune disease with a strong genetic predisposition as seen by the presence of specific HLA haplotypes like HLA DR3, DR4, DQ8, and/or DQ2.1 However, the role of any particular HLA allele in the development of the disease is still unclear due to the presence of multiple class I and class II molecules on the cell surface. The genetics of autoimmune diabetes is further complicated due to the role of several non-MHC genes also in the initiation of the disease.2 We addressed this problem by generating HLA-DQ8 transgenic mice lacking endogenous mouse class II molecules and backcrossing them on to the NOD background. MATERIALS AND METHODS The generation of DQ8.Ab0 mice was previously described.3,4 The DQ8.Ab0 mice were crossed to the NOD and the DQ81 Ab0/g7 F1 mice were selectively backcrossed further to the NOD to generate N2 generation NOD/DQ8 mice. The DQ81 Ab0/g7 mice of N2 generation were intercrossed to obtain mice of different class II phenotypes: DQ81 Ab0/g7, DQ82Ab0/g7, DQ81Abg7/g7, DQ82Abg7/g7, and DQ81Ab0/0 mice. The neo gene (Ab0) was detected by a PCR specific for the neomycin resistant gene incorporated in Ab gene for the gene disruption. The surface expression of DQ8 and Ag7 molecules were detected using FACS analysis using monoclonal antibodies IVD12 and K24-199, respectively. Microsatellite analysis for idd3 and idd10 were performed by a polymerase chain reaction (PCR) method. D3Mit5 and D3Nds7 primer pairs were used for the specific amplification of idd3 and idd10 microsatellite repeat units. Cyclophosphamide (200 mg/kg body weight) was administered to 5 to 6 month old mice at day 0 and day 14, blood glucose was tested every week for four weeks and sacrificed on day 28.
RESULTS AND DISCUSSION
The HLA DQ8.Ab0 mice we generated did not develop diabetes or spontaneous inflammation in the islet. Multiple immunizations of these mice with insulin peptides or insulin
0041-1345/98/$19.00 PII S0041-1345(97)01362-6 474
also did not result in diabetes or inflammation of the pancreas/islet. So we crossed DQ8.Ab0 mice to the NOD background. There were a total of 35 mice of NOD/ DQ81Ab0/0 phenotypes of N2 generation. None of these mice developed spontaneous overt diabetes by 5 to 6 months of age. Sixteen of the 35 mice were administered cyclophosphamide. The administration of cyclophosphamide also did not induce diabetes in any of these mice. The histology of the pancreas did not show any signs of insulitis, though there was extensive periinsulitis and perivasculitis in about 70% of the mice treated or untreated with cyclophosphamide. Among the mice that did not receive cyclophosphamide, six mice were homozygous at both idd3 and idd10 loci and there were five mice homozygous at these loci in the group that received cyclophosphamide. The number of mice in other class II phenotypes were small, however cyclophosphamide could induce diabetes in DQ81Ab0/g7, DQ81Abg7/g7 and DQ82Ab0/g7 mice. This result shows that though idd3 and idd10 together with Ag7 molecule contribute significantly to diabetes, these non-H2 susceptibility genes in the presence of DQ8 do not initiate insulitis or diabetes unless at least one copy of the Ag7 molecule is present. REFERENCES 1. Lernmark A: Diabetologia 37:S73, 1994 2. Vyse TJ, Todd JA: Cell 85:311, 1996 3. Cosgrove D, Gray D, Dierich A, et al: Cell 66:1051, 1991 4. Nabozny GH, Baisch JM, Cheng S, et al: J Exp Med 183:27, 1996 From the Department of Immunology and Transplantation, Mayo Clinic, Rochester, Minnesota. Support provided by Juvenile Diabetes Foundation International and NIH grant, AI 14764. Address reprint requests to C.S. David, Department of Immunology and Transplantation, Mayo Clinic, Rochester, MN 55905.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 474 (1998)