Analysis of T cell reactivities to phosphorylcholine-conjugated hen egg lysozyme in C57BL/6 mice: hapten-conjugate specificity reflects an altered expression of a major carrier epitope

Abstract

We have selected several different T cell hybridoma clones reactive to hen egg lysozyme (HEL) conjugated to phosphorylcholine (PC) after fusion of PC-HEL-primed C57BL/6 lymphocytes with BW5147 parent cells. These hybridoma clones preferentially recognize PC-HEL over unconjugated HEL, but not other carrier molecules conjugated with the same hapten. All the PC-HEL-reactive clones are similarly responsive to not only p-azobenzenearsonate (ABA)-conjugated HEL (ABA-HEL) but also to a variety of other diazotized hapten-HEL conjugates. However, these clones are not stimulated by fluoresceinated or dinitrophenylated HEL beyond the level of HEL carrier alone. Therefore, the type of hapten linkage (diazonium) to the carrier molecule appears to affect T cell recognition. The hybridoma clones apparently recognize the carrier molecule alone, although the level of stimulation is relatively low compared to that induced by either PC-HEL or ABA-HEL. Interestingly, HEL unfolded by S-carboxymethylation is capable of stimulating the hybridomas to a level comparable to that obtained with PC-HEL. T cell recognition of the unfolded HEL is independent of antigen processing, which is different from that of PC-HEL. The peptide sequence corresponding to the amino acids 81-93 of HEL appears to contain the epitope region for the hybridoma clones based on testing stimulation activity with synthetic peptides. Previously, the peptides including this region (81-96) have been reported as the determinant recognized by T cells derived from C57BL/6 mice after immunization with an HEL peptide (HEL 13-105) but not with native HEL. These results suggest that a hapten conjugation via diazonium linkage modifies antigen presentation and consequently the presentation of the major T cell epitopes similar to that of the HEL fragment.