In this project, transgenic mice will be produced on the non-obese diabetic (NOD) background expressing HLA DR3, DR4 (subtypes 0401, 0402, and 0405), DQ 2 and DQ 8. These mice will also be homozygous for a human CD 4 transgene and for a murine MHC Class II null targeted recombinant seventeenth chromosome (Abeta 0/0). These mice will be immunized with human pre pro-insulin, human glutamic acid decarboxylase 65 kD, and human IA-2 produced by recombinant DNA techniques in baculovirus in insect cell cultures. Mice of the above genotype will ensure optimal positive selection of CD 4 positive T cells on the HLA DR or DQ restricting element. At least two immunization and fusion experiments will be carried out for each protein and each HLA MHC Class II transgene. Immunizations will be carried out in incomplete Freund'S adjuvant, and if possible (in terms of numbers of responding T cells) by immunization with the protein antigen in aqueous solution injected intradermally in the hind footpads. Identified peptide epitopes will be provided to the collaborating centers at the Barbara Davis Diabetes Center, the University of Washington, and the Medical College of Wisconsin so that the presence or absence of T cell proliferative responses to these peptides can be verified in patients with recent onset diabetes or pre-diabetic individuals. In addition, through the use of peptide motifs for each HLA DR or DQ allele, and peptide binding studies (to be carried out by Dr. Gerald Nepom), peptide epitopes from each antigen which bind well to the DR or DQ allele under study but do not elicit a response in transgenic mice will be used to further test the accuracy of peptide epitope identification in transgenic mice. The functional characteristics of each peptide epitope will be tested analyzing the cytokine profile (IL-2, interferon gamma, TNF alpha, IL-4, and IL-10) elicited in T cells from the draining lymph nodes of immunized transgenic mice. These cytokine profiles can then be compared with similar cytokine profiles elicited in the peripheral blood T cells of pre-diabetic and recent onset diabetic patients. The results should provide a blueprint for the development of a rational, HLA genotype specific, preventative immunotherapy to prevent the development of type I diabetes in susceptible individuals.
|Freitag, Tobias L; Cham, Candace; Sung, Hsiang-Hsuan et al. (2010) Human risk allele HLA-DRB1*0405 predisposes class II transgenic Ab0 NOD mice to autoimmune pancreatitis. Gastroenterology 139:281-91|
|Durinovic-Bello, Ivana; Rosinger, Silke; Olson, Jennifer A et al. (2006) DRB1*0401-restricted human T cell clone specific for the major proinsulin73-90 epitope expresses a down-regulatory T helper 2 phenotype. Proc Natl Acad Sci U S A 103:11683-8|
|Gottlieb, Peter A; Hayward, Anthony R (2002) Cytokine and immunosuppressive therapies of type 1 diabetes mellitus. Endocrinol Metab Clin North Am 31:477-95|
|Gottlieb, Peter A; Eisenbarth, George S (2002) Insulin-specific tolerance in diabetes. Clin Immunol 102:2-11|