LOW ABUNDANCE OF NATURALLY PROCESSED T-CELL DETERMINANTS FOLLOWING ANTIGEN PULSING OF ANTIGEN-SPECIFIC HUMAN B-CELL LINES

An attempt was made to recover naturally processed T-cell determinants following antigen pulsing of tetanus toxin-specific human B-cell clones with mu g/ml amounts of antigen. Class II major histocompatibility complex (MHC) molecules were isolated from cells pulsed under optimal conditions and the eluted peptides displayed by reverse-phase high-performance liquid chromatography (HPLC). Antigen-pulsed and control-unpulsed cells showed virtually identical optical density (OD)(215) profiles, although multiple peptides derived from the input antigen could be identified at the radiochemical level. At least four distinct HPLC fractions contained naturally processed versions of the determinant 830-844, detected using the specific T-cell clone Mix 111. Quantification of the most active fraction indicated that approximately 1 pmole of this determinant was recovered from similar to 5 x 10(9) antigen-pulsed cells. Based on the amount of antigen processed following uptake on membrane immunoglobulin, and quantification of the biologically active material recovered, it was estimated that the efficiency of determinant capture was no greater than 1-2%. Further method development and a considerable increase in the number of Cells used (>10(10)) would appear to be necessary before naturally processed determinants from exogenously pulsed antigens can be reliably and fully characterized. Finally, a theoretical analysis showed that accurate (+/-0.01%) mass information alone could identify a limited number of candidate peptides from known or putative antigens.