Characterization of antibody-defined epitopes on HLA-DRB1*04 molecules

Spurrell, David R. (2004) Characterization of antibody-defined epitopes on HLA-DRB1*04 molecules. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

We have previously identified antibody-defined epitopes on the HLA-DRB1*04 molecules that are associated with development of rheumatoid arthritis (RA). The expression of a NFLD.D11-defined epitope (D11⁺0401) on B cell lines (BCL) is dependent on HLA-DM. NFLD.D13 recognizes DRB1*0404, but not DRB1*0401 in DM⁺ BCL (D13⁺0404). However, in DM⁻ BCL, NFLD.D13 recognized an epitope on HLA-DRB1*0401 molecules (D13⁺0401). It was speculated that these epitopes arise either through acquisition of peptides from differentiation antigens of through differential processing and acquisition of a peptide from a common protein. Initially, we show that these epitopes were not detected on the surface of synovial fibroblasts from RA patients, despite the up-regulation of DR, DM molecules and compartments necessary for class II antigen processing and presentation. Further investigation showed that the expression of the D11⁺0401 and D13⁺0404 epitopes was restricted to professional antigen presenting cells, with the exception of the melanoma cell MDA MD 435.0401, which expressed D11⁺0401. To Analyze the intracellular mechanisms by the D11⁺0401 and D13⁺0404 are generated, we treated normal and DM⁻ BCL with various inhibitors, including brefeldin A and protease inhibitors, and employed confocal and immunoelectron microscopy to dissect the compartments within the endocytic pathway where these epitopes form. We found that both the D11⁺0401 and D13⁺0404 epitopes from within CD63⁺CD82⁺ lysosomal compartments in normal BCL. The D11⁺0401 epitope requires cytoplasmic and endosomal cysteine proteases, whereas D13⁺0404 epitope expression is protease independent. Both epitopes appear to require tetraspan protein-enriched microdomains for proper surface expression. Interestingly, the D13⁺0401 epitope also required cysteine proteases for its expression, suggesting that a unique set of peptides are responsible for the formation of these DRB1*04 epitopes.

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