EXCEED THE SPACE PROVIDED. MHC class ll-restricted antigen processing by Antigen Presenting Cells (APCs) is essential for the development of CD4-positive T cell responses. In this proposal we will further investigate the involvement in this process of the enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT). GILT is constitutively present in the lysosome-like MHC class II compartment of APCs, and a GILT-deficient mouse strain that we created is defective in making CD4-positive T cell responses to protein antigens containing disulfide bonds. These mice also exhibit defects in B cell development, with reduced CD23 expression and displaced marginal zone B cells. We will identify substrate proteins and peptides and accessory proteins that interact with GILT, and use molecular and mutational approaches to investigate the B cell developmental anomaly. We will also investigate the phenomenon of 'reverse cross-presentation', in which peptides derived from cytoplasmic proteins associate with MHC class II molecules. Such peptides are normally found in association with MHC class I molecules, while peptides from extracytoplasmic proteins associate with MHC class II molecules. Initial experiments show that peptides from cytoplasmic proteins predominate in MHC class II molecules from a human dendritic-like cell line. Peptides from cytoskeletal proteins and those that associate with the cytoplasmic face of cellular membranes are particularly well- represented. We suggest that dendritic cells are specially equipped to use this pathway, and propose to determine the molecular mechanisms which govern it. These studies will unravel novel features of MHC class ll-restricted antigen processing which may prove of critical importance in immunological diseases, particularly autoimmunity.
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