VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)

Abstract

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of VH genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of VH3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged VH4 genes was also observed in these patients. These data suggest that although usage of individual VH genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.

Fig. 1

Somatic mutations among rearrangements of five V_H3 genes derived from peripheral blood B cells of four RA patients and four normal controls. Somatic hypermutation events were estimated by hybridizing V_H3 libraries with motif-specific oligonucleotide probes targeted at CDR1 or CDR2 sequences of each gene as described [33]. Filled symbols, RA patients; open symbols, normal controls. Paired samples are indicated by shape of symbol. (a) Percent of rearrangements with somatic mutations in CDR1. (b) Percent of rearrangements with somatic mutations in CDR2.

Fig. 2

Somatic mutations among rearrangements of three V_H4 genes in the blood samples of four RA patients and four healthy subjects. Somatic hypermutation events were estimated by hybridizing V_H4 libraries with motif-specific oligonucleotide probes targeted at CDR1 or CDR2 sequences of each gene as described [33]. Filled symbols, RA patients; open symbols, normal controls. Paired samples are indicated by shape of symbol. (a) Percent of rearrangements with somatic mutations in CDR1. (b) Percent of rearrangements with somatic mutations in CDR2.

Fig. 3

Somatic mutations in V_H3 versus V_H4 rearrangements among patients and controls. The mean incidence of somatic mutations was calculated from each subject from the data in Figs 1 and 2. Each mean value from each subject was treated as a single point in statistical analysis (pair-wise Student's t-test).

Fig. 4

The fraction of mutated rearrangements is larger in RA patients than controls. Rearrangements that had 0–2 substitutions (unmutated and system background) are compared with rearrangements that had three or more substitutions (mutated). ▪, RA patients; □, normal controls.

Fig. 5

Nucleotide substitutions in FRs and CDRs of V3–23 rearrangements from RA patients and healthy subjects. A total of 59 V3–23 rearrangements from four RA patients and 62 V3–23 rearrangements from four healthy subjects were randomly selected for DNA sequence analysis. Filled symbols, RA patients; open symbols, normal controls. Paired samples are indicated by shape of symbol. Data are presented as number of nucleotide substitutions per 100 nucleotides among the respective regions. FR and CDR are defined according to Kabat et al. [62].

Fig. 6

D segment usage in RA patients and in controls. Individual D segments were identified as described [24]. Total number of each D segment from four RA patients or from four controls is presented.

Fig. 7

Somatic mutations in B cell subsets. V_H3 rearrangement libraries were made from unsorted PBL and from sorted IgD⁺/CD19⁺ B cells. Purity of the sorted IgD⁺ CD19⁺ B cells was 95%. V3–23⁺ clones were identified by hybridization with at least one of the V3–23-specific probes, M18, M8, or E87. Somatic hypermutation events in CDR1 and CDR2 were estimated by loss of concordant hybridization to M8 or E87 probes, respectively.