Human rheumatoid B-1a (CD5⁺ B) cells make somatically hypermutated high affinity IgM rheumatoid factors

To analyze the structure and formally ascertain the B-1a cellular origin of IgM rheumatoid factor (RF) autoantibodies, we generated 4 IgM RF mAb-producing cell lines using sorted (surface CD5⁺) B-1a cells from a patient with active rheumatoid arthritis. The RF mAb111, mAb112, mAb113, and mAb114 were monoreactive and displayed a relatively high affinity for human IgG Fc fragment (K_d, 3.1 × 10^-7 to 6.8 × 10^-7 M). The B-1a origin of the lymphocytes that gave rise to the IgM RF was confirmed by the expression of surface CD5 and specific CD5 mRNA by all mAb-producing cell lines. Analysis of the genes encoding the RF mAb V_H and V_L regions revealed that members of the V_HI and V_HIII families were utilized in conjunction with VκIIIa, VκIIIb, or VλI genes. J_H3 and J_H4 genes were each utilized twice. The H chain CDR3 sequences were divergent and variable in length. The RF mAb V_H genes were identical or closely related to those expressed in the "restricted" fetal B cell repertoire and/or were JH-proximal. For instance, mAb111 V_H gene likely constituted a mutated variant of the expressed fetal 20P3 which is the second most J_H-proximal gene (125 kb from J_H). In addition, the expressed V_H and V_L genes were among those that have been found to encode other RF, different autoantibodies, high affinity antibodies induced by exogenous Ag, and natural autoantibodies in the adult and neonatal B cell repertoires. When compared with those of known germline genes, the expressed V gene sequences displayed a number of differences. By cloning and sequencing DNA from PMN of the same patient whose B lymphocytes were used for the mAb generation, we showed that such differences resulted from somatic hypermutation in the RF mAb112 V_H gene. The germline gene (112GL) that presumably gave rise to the RF mAb112 V_H segment was identical to the expressed fetal 51P1 gene. The distribution and the high replacement to silent mutation ratio of the nucleotide mutations in RF mAb112 VH segment were highly consistent with their selection by Ag. RF mAb113 was clonally related to RF mAb112, as shown by the utilization of the same sets of V_HI-D-J_H4 and VκIIIb-Jκ4 genes, displaying identical junctional sequences, and the presence of two identical replacement and one silent mutations. The presence of a high number of unique amino acid changes, a total of 14 and 9 in the two RF mAb V_H and V_L segments, respectively, suggested that RF mAb112-and mAb113-producing cells diverged relatively early in their expansion from the common progenitor clone. These studies show that high affinity RF can arise through a hypermutation process from antibodies expressed by B-1a cells. They also suggest that the affinity maturation of a human autoimmune response can apply to IgM and is independent of Ig class switch.