The immune response to phosphocholine (PC)–protein is characterized by a shift in antibody repertoire as the response progresses. This change in expressed gene combinations is accompanied by a shift in fine specificity toward the carrier, resulting in high affinity to PC–protein. The somatically mutated memory hybridoma, M3C65, possesses high affinity for PC–protein and the phenyl-hapten analogue, p -nitrophenyl phosphocholine (NPPC). Affinity measurements using related PC–phenyl analogues, including peptides of varying lengths, demonstrate that carrier determinants contribute to binding affinity and that somatic mutations alter this recognition. The crystal structure of an M3C65–NPPC complex at 2.35-Å resolution allows evaluation of the three light chain mutations that confer high-affinity binding to NPPC. Only one of the mutations involves a contact residue, whereas the other two have indirect effects on the shape of the combining site. Comparison of the M3C65 structure to that of T15, an antibody dominating the primary response, provides clear structural evidence for the role of carrier determinants in promoting repertoire shift. These two antibodies express unrelated variable region heavy and light chain genes and represent a classic example of the effect of repertoire shift on maturation of the immune response.

Decreasing antigen in vivo may preferentially stimulate cells with the potential for synthesis of high-affinity antibody through activation of surface receptors with similarly high affinity. This selection should result in cells with increased sensitivity to lower antigen concentrations, cells with greater avidity for antigen. We have followed the in vivo changes in anti-TNP memory-cell sensitivity by initiating the secondary anti-hapten response in vitro. This response was determined by anti-TNP plaque assay. The results indicate that cell populations with increased sensitivity for antigen continue to emerge with time after priming and that this sensitivity may increase 1000-fold in a 4 month period. Increased sensitivity to stimulation by antigen is concomitant with suppression by higher, previously stimulatory doses as in high zone immune tolerance. The data support the hypothesis that memory cells of high avidity result from the selective pressure of diminishing in vivo antigen concentration.

Anamnestic antihapten responses were obtained to trinitrophenyl (TNP) when rabbits sensitized to trinitrophenyl-hemocyanin (TNP-KLH) were challenged with TNP-heterologous protein conjugates. Hapten-heterologous carrier conjugates elicited antihapten titers similar in magnitude to those elicited by the homologous carrier conjugate. Hapten-heterologous carrier recall of antihapten was successful as early as 37 days and as late as 11 months after sensitization. There was no correlation between anti-TNP-precipitating antibody titer after sensitization and the ability to respond to challenge by hapten-heterologous carrier. The results are discussed in terms of immunogenicity of sensitization, suppressive effects of persisting postsensitization antibody, and submolecular haptenic environment as factors possibly affecting the heterologous recall process.