Biochemical characterization of a beta cell membrane fraction antigenic for autoreactive T cell clones

The two NOD-derived T cell clones, BDC-2.5 and BDC-6.9, are CD4+, V beta 4, islet-specific, and diabetogenic. These two T cell clones show different response patterns to whole islet cell antigen, but were found to respond to the same fraction isolated from beta granule membranes. The clones were us ed to follow the antigenic activity in the biochemical purification of a be ta cell membrane detergent lysate subjected to HPLC anion exchange (IEX) an d size exclusion chromatography (SEC). Antigenic activity could be retained after lysis in only one detergent (octyl-beta-glucoside) among several tes ted. In order to detect solubilized antigen, beta membrane proteins were co valently linked to microlatex beads prior to being added to T cell prolifer ation assays, a technique that eliminated detergent toxicity and resulted i n increased assay sensitivity. To purify the antigen, membrane proteins wer e absorbed onto an anion exchange column and after elution using a st gradi ent, activity for the clones was found in a fraction containing 0.15-0.2M N aCl. Subsequent analysis of this material by size exclusion chromatography provided an apparent molecular weight of the antigen to be between 50 and 8 0 kDa. Further attempts to purify the protein by SDS-PAGE resulted in loss of antigenic activity. It is possible that the elusive nature of this prote in is a clue to its importance as an autoantigen. (C) 2000 Academic Press.