We propose to examine the peptide-binding properties of the class II MHC molecule (I-Ag7) from NOD mice. Our proposal is based on the following findings; i) Ag7 molecules isolated from a variety of Antigen Presenting Cells are unstable when examined by SDS. PAGE, ii) this unstability does not seem to be caused by a lack of association with Invariant chain or by a lack of processing and assembly of intracellular Ag7, iii) all peptides so far examined bind very weakly to Ag7 either from purified molecules or in APC, iv) the rate of dissociation of all peptides tested is very fast and t his increased off-rate is dramatically evident in T cell assays. Indeed T cells fail to respond if the peptide is not in the culture all of the time, v) preliminary studies indicate that the beta cell diabetogenic antigen behaves the same. These results led us to speculate that the weak binding of peptides by Ag7 is, contrary to expectations, a feature that does not allow for tolerance of autoreactive T cells. The proposal called for, first, an extensive biochemical characterization of Ag7 and its interaction with peptides, including the use of photoaffinity labeled peptides. Second, we will examine how APC bind peptides, their immunogenicity and their time of persistence. For both these issues we select foreign peptides, known autoreactive peptides, and peptides that are isolated from Ag7 molecules. A third goal examines the immune response to peptides bound weakly to Ag7. We propose two experiments, one is an analysis of a peptide processed by APC from Ealpha chain and which interacts in dramatically different ways when binding to Ag7 or Ab. We will test the immune response in (NOD X B6) F1 mice expressing the E chain (and, in principle, tolerant to it). Another experiment attempts to engineer the Aalpha chain of NOD so as to produce a stable, high affinity binding Ag7. If successful we will compare the reaction of T cells to it, both in culture and in in vivo experiment. Finally, we plan to examine these phenomena on human IDDM susceptible class II molecules DQ2 and DQ8.
Showing the most recent 10 out of 14 publications
