Immunologic memory to phosphocholine keyhole limpet hemocyanin: Recurrent mutations in the λ1 light chain increase affinity for antigen

Anti-phosphocholine (PC)-keyhole limpet hemacyanin hybridomas representative of a memory response that express the λ1 L chain isotype have a high reactivity to PC-protein. A common feature of these hybridomas possessing high affinity for PC-protein is the occurrence of somatic mutations resulting in replacement changes in three CDR2 positions of the λ1 L chain. The influence of each of these three positions on the Ag binding properties of these antibodies was examined by site-specific mutagenesis and expression of recombinant antibody molecules by transfected cells. Affinity measurements and fine specificity profile determinations demonstrated the importance of the three λ1 CDR2 positions in Ag binding. Compared to antibodies expressing germline λ1, including one with an additional junctional serine that is not encoded by V or J, those antibodies possessing critical changes in CDR2 would have a strong selective advantage based on affinity differences for Ag. Sequence analysis of a group of clonally related hybridomas expressing mutated λ1 genes allowed construction of a hypothetical genealogic tree that suggests selection based on changes in CDR2 of λ1 in the absence of H chain mutations. The results are consistent with stepwise acquisition of mutations and selection based on affinity constraints.