Cell Surface Modifications with Trifluoromethyl Dinitrophenyl-Soluble Protein Conjugates: Immunogenic Role of Noncovalently Bound Hapten

Abstract

Bovine serum albumin (BSA) was derivatized, under mild conditions, with trifluoromethyl-dinitrophenyl (CF3-DNP), a haptenic group cross-reacting with trinitrophenyl (TNP). High-field nuclear magnetic resonance of fluorine (¹⁹F-NMR) permitted to calculate the number of covalently and noncovalently bound haptenic groups per BSA molecule. Further dialysis against paratoluene sulfonic acid permitted to obtain CF₃-DNP-BSA conjugates from which noncovalently bound hapten had been removed. Soluble conjugates containing 4 covalenty bound plus 1 noncovalently bound hapten groups, or only 4 covalently bound groups, were added to splenocytes in culture. These splenocytes, after such treatments, were added to effector lymphocytes in a 5-day culture aimed at the generation of cytotoxic T lymphocytes (CTL) against the CF₃-DNP-induced cell surface modification antigens. It was found that only the BSA conjugates that contained noncovalently bound haptens were able to generate CTL against target cells whose surface had been directly modified with CF₃-DNP, whereas BSA bearing only covalently bound hapten groups were not. Replacing BSA by human serum albumin, or CF₃-DNP by TNP, gave comparable results. Thus, under the conditions used, haptenic groups covalently bound to their soluble carrier protein did not generate hapten-dependent CTL, but noncovalently bound or free haptenic groups at very low concentration were able to do so.