This application addresses the mechanism responsible for variations in susceptibility to diabetes based on HLA genotypes. Specifically, the investigator proposes to test the hypothesis that a diabetogenic peptide binds to DQ0302 with weak affinity, that this leads to a predominantly Th1 response, and that this response mediates damage to islet cells. In contrast, he proposes that high affinity binding of these same peptides to DQ0602 causes a Th2 response which protects islet cells from destruction by either direct T-cell mediated attack or general inflammation. The different phenotypes, as reflected by these two different alleles, can also be a consequence of the intrinsic physical properties of these two different DQ molecules. The theses of this application have not been previously examined due to difficulties in obtaining DQ restricted T-cells. However, recent findings indicated that Herpes Simplex Virus (HSV-2) VP16 protein is highly immunogenic, and DQ restricted T-cells directed toward its carboxyl region can be detected from the skin lesions of HSV-2 infected individuals. DQ transgenic animals also have become available, and they will also be used to develop an experimental system for testing the current hypothesis for the mediation of susceptibility and resistance by specific HLA DQ alleles. There are 3 specific aims. The first is to contrast the structural difference between susceptible and protective alleles. These studies will involve a continuation of the investigator's previous characterizations of the binding specificity of these HLA DQ alleles. The major goal of this section will be to continue to characterize the binding patterns of DQ0302 and DQ0602 for specific peptides and determine whether they consistently demonstrate a """"""""reverse"""""""" binding pattern with respect to binding affinity for specific peptides. In this specific aim, they will also assess the stability of these alleles with peptides using the SDS gel dimer stabilization assay. They will also attempt to characterize the kinetics and affinity of peptide binding by these alleles using two new technologies, namely surface plasmon resonance and/or calorimetry analysis. Collaborators with the appropriate instrumentation and expertise are available.
Specific aim 2 is to assess the influence of variable avidity of DQ/peptide for TcR on T-cell differentiation. The goal of this aim will be to use the HSV-2 VP16 peptide system as a model system to assess the influence of peptide binding affinity and molecular stability on the nature of the T-cell immune response.
This specific aim contains experiments in which peptides from VP16 which show variations in their affinity for DQ0302 and DQ0602 will be used to determine whether the Th1 versus Th2 phenotypes in HLA DQ restricted human T-cells are potentiated on the basis of the MHC class-peptide stability of the stimulating epitope. A specific T-cell clone as well as populations of T-cells derived from herpetic lesions will be assessed for their cytokine profiles subsequent to stimulation with various phenotypes. The expectation will be that high affinity and stability will potentiate Th2 versus Th1 cytokine profiles.
This specific aim also contains experiments using transgenic mice expressing HLA DQ0302 or HLA DQ0602 molecules. These mice, which will be provided by Dr. Chella David, uniquely express only these MHC class II molecules and will be used to test the Th1 versus Th2 effect of immunization with high and low affinity peptides.
Specific Aim 3 is to examine the immune response of VP16/DQ0302 and VP16/DQ0602 transgenic mice. Experiments in this specific aim will test the ability of VP16 transgenic mice expressing various VP16 gene fragments under the regulation of the RIP promoter to tolerize T-cells responding to various peptides. The goal of these experiments will be to determine whether variations in affinity will have effects on the nature of tolerance induction to self antigens.
| Nepom, Gerald T; Buckner, Jane H; Novak, Erik J et al. (2002) HLA class II tetramers: tools for direct analysis of antigen-specific CD4+ T cells. Arthritis Rheum 46:5-12 |
| Koelle, D M; Chen, H B; Gavin, M A et al. (2001) CD8 CTL from genital herpes simplex lesions: recognition of viral tegument and immediate early proteins and lysis of infected cutaneous cells. J Immunol 166:4049-58 |
| Novak, E J; Masewicz, S A; Liu, A W et al. (2001) Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset. Int Immunol 13:799-806 |
| Ettinger, R A; Liu, A W; Nepom, G T et al. (1998) Exceptional stability of the HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus. J Immunol 161:6439-45 |
