The main objective of this project is to analyze the molecular and functional structure of the DQ3.2 beta molecule, a putative susceptibility gene product in Insulin-Dependent Diabetes Mellitus (IDDM). Results of these studies will lead to further understanding of the role of class II molecules, both in normal immunity and potentially in the pathogenesis of IDDM. These studies will also lead to understanding of the cellular regulatory mechanism for expression of class II genes. The approach will be to use retroviral vectors to transfer and express class II cDNA in human B-LCLs.
The specific aims are: 1) To determine the precise structural determinants of DQ3.2 beta molecules which are important for MoAb and T cell recognition. 2) To assay for the presence of haplotype-mismatched and mixed isotype DQ3.2 beta molecules and to identify amino acid residues which influence class II alpha and beta chain interactions. 3) To manipulate intracellular levels of DQ mRNA as a means to study anomalous class II pairing and competition between alpha and beta molecules. 4) To probe the nature of class II expression defects in DQ defective cell lines. In these experiments, amphotropic retroviral vectors with HLA cDNA or in vitro mutagenized cDNA under the regulatory control of either the CMV promoter or an endogenous class II promoter will be used. Another series of vectors which link the HLA cDNA in either the sense or antisense orientation to dhfr cDNA wiLL aLso be used to co-amplify the linked HLA cDNA. Viruses produced from packaging cells with the appropriate constructs will be used to infect human B-LCLs and DQ defective lymphoid cells. The introduction of these various constructs will be used to generate quantitative and qualitative changes in intra-cellular levels of class II molecules. Infected cells will be analyzed for mRNA, intra-cellular and cell surface class II expression, and ability to stimulate alloreactive T-cells, to address the aims listed above. In this manner, a comprehensive molecular picture will emerge of the structural characteristics important for expression of a particular DQ beta allele, the DQ beta 3.2 gene. As this gene is potentially implicated in HLA-associated genetic susceptibility to IDDM, these studies will identify important variables contributing to the role of individual class II molecules in autoimmunity.
