Sequence analysis of V4–34-encoded antibodies from single B cells of two patients with systemic lupus erythematosus (SLE)

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Abstract
SLE is an autoimmune disease characterized by the presence of autoantibodies against double-stranded (ds)DNA. A large proportion (approx. 40%) of patients with lupus also have increased levels of serum immunoglobulin encoded by the V4–34 heavy chain gene, which often fluctuate with disease activity, and this gene is utilized by a subset of anti-dsDNA antibodies. In order to probe the nature of the V4–34-encoded immunoglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gene product. Following cell picking, single-cell polymerase chain reaction (PCR) amplification of cDNA was used to investigate both VH and VL genes. Sequences were obtained from B cells synthesizing IgM (n = 10), IgG (n = 4) and IgA (n = 1). For VH, all were derived from V4–34 as expected, and the isotype-switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, VL (12 κ and 3 λ) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated VH sequences were associated with unmutated VL sequences. Analysis of the distribution of mutations revealed only minor clustering in complementarity-determining regions (CDRs) characteristic of antigen selection. The CDR3 lengths of VH ranged from five to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed repertoire. Basic amino acids were also found at the VL–JL junctions in 4/15. These findings provide insight into the V4–34–VL gene combinations used by B cells in patients with SLE which might have clinical relevance.