Relationship of variable region genes expressed by a human B cell lymphoma secreting pathologic anti-Pr2 erythrocyte autoantibodies

Abstract

To study the biology of cold agglutinin disease we previously established EBV-transformed B cell clones isolated from a patient with splenic lymphoma of an early plasmacytic cell type and immune hemolysis due to an anti-Pr2 cold agglutinin. These clones had an aberrant chromosomal marker identical to the patient's B cell lymphoma and each secreted IgM(k) anti-Pr2 similar to the pathologic autoantibody in the serum of the patient. In this study, we have further investigated the Pr2-specific autoimmune response through nucleotide sequencing of V(H) and V(L) region genes. We have shown that the seven clones share the same VDJ/VJ gene segments and junctional elements confirming their clonal origin. The V(H) sequences were 88% homologous to a V(HI) germline gene while the V(L) sequences were 97% homologous to a V(kIII) germline gene. Only 4 somatic mutations (3 silent and 1 conservative) were found in >5,000 bp sequenced, suggesting that a low mutation rate existed. Based on a tumor mass of 1012 cells and a minimum of 40 divisions, we estimated the somatic mutation rate to be 4.45 x 10-5 m/bp/d. This somatic mutation rate is similar to those estimated for acute lymphocytic leukemia (pre-B cell) and chronic lymphocytic leukemia (intermediate B cell), but significantly lower than the mutation frequency in follicular lymphomas (activated B cell). We propose that the difference in somatic mutation frequency of a B cell tumor may be related to the stage of B cell differentiation. In addition, the low mutation frequency observed in the Pr2-specific B cell tumor may also reflect, in part, selection by autoantigen to conserve sIg structure and specificity.