Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient

Abstract

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.

Fig. 1

(A) Nucleotide sequence of the V_HIV and V_HIII genes utilized by the RF mAb. The top sequence in each cluster is used for germline comparison. Identities are indicated by dashes Asterisks indicate the boundaries of the CDR. The V58, VH4.21, V79, V71-2, and V_H4.18 genes are members and alleles of the V_HIV family (35,36). Parentheses in the V_H4.21 and V79 sequences denote deletions in these genes when compared with another member of the V_HIV family, V71-2 (35). Due to the unavailability of the leader sequences of V_H4.21 and V_H4.18, the leader sequences of their most similar published germline segments, [V58] and [V2-1], respectively, are provided. MLH4-1 is the sequence we obtained by targeted PCR amplification of the genomic DNA from the patient under study (boxes depict the sequence and the complementary sequence of the 5′ and 3′ primers, respectively, utilized in these experiments). Notice its perfect identity with the genomic V_H4.18 allele reported by Sanz et al. (36). Small letters denote the leader intron sequence of the V71-2 gene The V_H11 gene is a member of the V_HIII family (37). (B) Deduced amino acid sequences from the above nucleotide sequences. Identities are indicated by dashes. Blank spaces represent deletions. The new V_H nucleotide sequences presented here are available from EMBL/GenBank/DDBJ under the following accession numbers: mAb60, X54435; mAb61, X54437; mAb63, X54441; mAb65, X54443; mAb67, X54445; and MLH4-1, X54447.

Fig. 2

(A) Nucleotide sequences of the D segments utilized by the RF mAb. The top sequence in each cluster is used for germline comparison Identities are indicated by dashes (B) Deduced ammo acid sequences from the above nucleotide sequences Identities are indicated by dashes (C) Nucleotide sequences of the J_H segments utilized by the RF mAb The top sequence in each cluster is used for germline comparison. Identities are indicated by dashes. (D) Deduced amino acid sequences of the J_H segments Identities are indicated by dashes. The new nucleotide sequences presented here are available from the EMBL/GenBank/DDBJ under the accession numbers listed in the legend to Fig. 1.

Fig. 3

Sequences of the V_λ genes utilized by the RF mAb. The top sequence in each duster is used for comparison. Identities are indicated by dashes. Asterisks indicate the boundaries of the CDR and the 5′ untranslated regions (UT) regions. No term of comparison is given for the RF mAb 60, V_λIII gene. (B) Deduced amino acid sequences from the above nudeotide sequences. Identities are indicated by dashes. (C) Nudeotide sequences of the J_λ segments utilized by the RF mAb. The top sequence is each cluster is used for germline comparison Identities are indicated by dashes. (D) Deduced amino acid sequence of the J_λ segments. Identities are indicated by dashes. The new nudeotide sequences presented here are avaiable from the EMBL/GenBank/DDBJ under the following accession numbers: mAb60, X54436; mAb61, X54438; mAb63, X54442; mAb65, X54444; and mAb67, X54446.