Maturation of the immune response to (4-hydroxy-3-nitrophenyl)-acetyl (NP) haptens in C57BL/6 mice

Changes with time in specificity and affinity of anti-NP antibodies in C57BL/6 mice after immunization with NP22-chicken gamma-globulin (CGG) were studied by comparing the abilities of the antibodies to bind to NP3-bovine serum albumin (NP3-BSA) at pH 5 and 8. Early anti-NP antibodies (on day 14 after immunization) bound to NP3-BSA at pH 8, but not pH 5. This pH-dependence of binding was explained in terms of the low affinity of the antibody to the phenolic form of NP on the basis of results of fluorescence quenching titration of a monoclonal anti-NP antibody that showed similar specificity to that of the early anti-NP antibodies. Since NP on the CGG molecule ionized with an apparent pK of about 7.4, more than half the NP should be in the unionized (phenolic) form under the immunization conditions. However, early anti-NP antibodies bound preferentially to the ionized (phenolate) form of NP, which was a minor form at neutral pH, whereas later anti-NP antibodies showed ability to bind to both the phenolate and phenolic forms of NP. This change in specificity with time was observed on immunization with T cell-dependent (TD) antigens such as NP-CGG and NP keyhole limpet hemocyanin (KLH), but not with a T cell-independent (TI) antigen such as NP-Ficoll. The heavy (H) chains from the two monoclonal antibodies 3G6 and 3C6, which bound to the phenolate form and both the phenolate and phenolic forms, respectively, were recombined with lambda 1 chains (L3G6 and L3C6) from these antibodies as well as a lambda 1 chain (LHOPC-1) with the amino acid sequence of the germline. Ability to bind to the phenolate form of NP was recovered in all the reconstituted IgGs, while ability to bind to both the phenolate and phenolic form of NP was observed only with IgG reconstituted from H3C6 and L3C6. These results suggest that the specificity corresponding to early anti-NP antibodies were generated even by lambda 1 chains of a germline sequence, but that of late anti-NP antibodies was expressed only by an appropriate pair of H and L chains. The contribution of amino acid substitution by somatic point mutation to the change of specificity with time was discussed.