STRUCTURE OF THE V-H AND V-L SEGMENTS OF MONOREACTIVE AND POLYREACTIVE IGA AUTOANTIBODIES TO DNA IN PATIENTS WITH SYSTEMIC LUPUS-ERYTHEMATOSUS

Anti-DNA IgA autoantibodies play an important immunopathologic role in SLE patients. To analyze the cellular origin and the V-H and V-L stru cture of anti-DNA IgA autoantibodies, we generated five IgA1 mAbs to D NA using B lymphocytes from three SLE patients. Two mAbs bound to ssDN A only and one to both ssDNA and dsDNA (monoreactive antibodies). The remaining two mAbs bound to DNA (one to ssDNA and the other to both ss DNA and dsDNA) and to other self and foreign Ag (polyreactive antibodi es). The IgA mAb relative avidity for DNA ranged from 7.5 x 10(-8) to 8.0 X 10(-10) g/mu l. The anti-DNA IgA mAb used V-H segments of the VH I (VI-3b), VHII (V(H)2-MC2), VHIII (WHG16G and V(H)26c), and VHIV (V71 -2) families in conjunction with V kappa I, V kappa IIIb, or V lambda I segments. All IgA mAb V-H segments were juxtaposed with J(H)4b segme nts. The heavy chain CDR3 sequences were divergent in composition and length. When compared with those of the closest reported germ line gen es, the IgA mAb V-H and V-L gene sequences displayed a number of diffe rences. That these differences represented somatic point mutations was formally proved in both the monoreactive IgA mAb 412.67.F1.3 and the polyreactive IgA mAb 412.66.F1 V-H segments by differential PCR amplif ication and cloning and sequencing of genomic DNA from the mAb-produci ng cell lines and autologous polymer-phonuclear cells. The sequences o f the germ line genes that putatively gave rise to the mAb 412.67.F1.3 and mAb 412.66.F1 V-H segments were identical with those of the WHG16 G and V(H)26c genes, respectively. In not only the monoreactive mAb 41 2.67.F1.3 but also the polyreactive mAb 412.66.F1 and mAb 448.9G.F1 V- H segments, the higher concentration of replacement (R) mutations and the higher R:S (silent) mutation ratios in the complementarity-determi ning region (infinity; 19:0) than in the framework region (1.0) (p = 0 .00001, chi(2) test) were highly consistent with selection by Ag. In t he five IgA mAb V-H and V-L segments, the putative and verified somati c point mutations yielded 68 amino acid replacements, of which 38 were nonconserved. Twenty of these yielded positively charged or polar res idues that play a major role in DNA binding, including seven Arg, five Lys, three Tyr, two Gln, two His, and a Thr. The conserved amino acid changes included seven Asn. These findings suggest that anti-DNA IgA autoantibodies use a broad selection of V-H and V-L genes and enhance their fit for Ag by undergoing somatic hypermutation and Ag selection. Such a hypermutation and Ag selection process would apply to original ly polyreactive, in addition to monoreactive natural DNA binding IgA a utoantibodies.