Immunologic memory to phosphorylcholine (PC) VIII. Expression of the V_H−12 gene product in the response to PC‐keyhole limpet hernocyanin

Three murine anti‐phosphorylcholine (PC) hybridomas with group II‐like fine specificity patterns isolated during a memory response to PC‐keyhole limpet hemocyanin (KLH) are examined at the molecular level to determine the origins of the V_H and V_L used by these antibodies. Southern blots of Hind III cut DNA were hybridized with a probe specific for the V1 gene of the T15 V_H family. The V1 germ‐line configuration is retained in these hybridomas indicating that this gene which encodes the V_H gene product expressed by most group I anti‐PC hybridomas is not used for antibody production. Southern blots of Eco RI cut DNA hybridized to a probe specific for J_H1‐J_H4 indicated that all three hybridomas PCG1‐2, PCG1‐3 and PCM‐23 share a 5.2‐kb rearranged J_H band, suggesting utilization of a common V_H gene segment. N‐terminal amino acid sequence analysis of the heavy chains of two of the hybridoma proteins PCG1‐2 and PCG1‐3 indicates that they belong to mouse heavy chain subgroup II and are closest in sequence to a V_H−12 isotype anti‐PC hybridoma protein, HPC‐104, derived from BALB/c mice suppressed for the T15 idiotype; PCG1‐2 and PCG1‐3 each differed from HPC‐104 at only 1/20 residues. In addition, these proteins have in common a lysine at position 1 which has not been found previously in 203 other heavy chain sequences reported. N‐terminal sequences of the light chains of PCG1‐2 and PCG1‐3 are each shown to differ at only 1/22 residues from V_x24, and PCM‐23 had previously been shown to use V_x8; both of these light chains have been associated previously with heavy chains derived from the V1 gene in anti‐PC antibodies. These results indicate that the V_H−12 isotype can be used during a normal antibody response to PC and thus that heavy chains derived from both subgroup II and subgroup III (the T15 heavy chain) contribute to the molecular heterogeneity observed in memory responses to PC‐KLH.