SEQUENCE-ANALYSIS OF V4-34-ENCODED ANTIBODIES FROM SINGLE B-CELLS OF 2 PATIENTS WITH SYSTEMIC LUPUS-ERYTHEMATOSUS (SLE)

SLE is an autoimmune disease characterized by the presence of autoanti bodies against double-stranded (ds)DNA. A large proportion (approx. 40 %) of patients with lupus also have increased levels of serum immunogl obulin encoded by the V4-34 heavy chain gene, which often fluctuate wi th disease activity, and this gene is utilized by a subset of anti-dsD NA antibodies. In order to probe the nature of the V4-34- encoded immu noglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gen e product. Following cell picking, single-cell polymerase chain reacti on (PCR) amplification of cDNA was used to investigate bath 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 e xpected, and the isotype-switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, VL (12 kappa and 3 lambda) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated VH sequences were associated with unmuta ted VL sequences. Analysis of the distribution of mutations revealed o nly minor clustering in complementarity-determining regions (CDRs) cha racteristic of antigen selection. The CDR3 lengths of VH ranged from f ive to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed rep ertoire. Basic amino acids were also found at the V-L-J(L) junctions i n 4/15. These findings provide insight into the V4-34-V-L gene combina tions used by B cells in patients with SLE which might have clinical r elevance.