Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins that bind peptides derived from exogenous proteins and present them to CD4 T cells. MHC class II molecules associated with residual invariant chain fragments [class II-associated invariant chain peptides (CLIP)] are loaded with antigenic peptides by the catalytic action of the class II- like molecule, HLA-DM that also functions as a class II chaperone and peptide editor. HLA-DO (DO), yet another class II-like molecule that is expressed in B cells, dendritic cells and thymic epithelium, physically associates with DM both during and after transport to the MHC class II compartments (MIIC). The DM-DO association in the MIIC suggested that the functions of both molecules are linked. DO was shown to block DM function as purified DM-DO complexes could not catalyze CLIP exchange for peptide in vitro. Expression of DO in class II+ DO- cell lines caused the accumulation of class II-CLIP complexes at the cell surface, suggesting that DO blocked DM function in vivo. The inhibition of DM function results in down modulation of the class II processing pathway by decreasing the level of class II-peptide complexes on the cell surface. The biochemical, functional and immunological consequences of the observed DO-mediated inhibition of DM function remain unknown. Additionally, it is not known if DO has additional functions. The studies proposed in this application are designed to define the biological consequences of DO expression and to determine if there are alternative functions for DO in the class II processing pathway. This will be accomplished by three different biochemical approaches. First, bonafide antigen presenting cell lines that express the known components of the class II antigen processing pathway in the presence and absence of HLA-DO will be generated and analyzed using a biochemical approach. Second, HLA-DO molecules that can transport from the ER in the absence of DM but that retain their ability to associate with DM we be generated and examined to analyze DO structure and function. Third, it will be determined if internalization of the B cell receptor or if activation of B cells results in the release of Do from DM, thereby activating DM. The proposed work should answer important questions about the role of DO the class II processing pathway and has relevance in such areas as tumor surveillance and autoimmunity.
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