Structure of the VH and VL segments of monoreactive and polyreactive IgA autoantibodies to DNA in patients with systemic lupus erythematosus

Abstract

Anti-DNA IgA autoantibodies play an important immunopathologic role in SLE patients. To analyze the cellular origin and the VH and VL structure of anti-DNA IgA autoantibodies, we generated five IgA1 mAbs to DNA using B lymphocytes from three SLE patients. Two mAbs bound to ssDNA 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 ssDNA and dsDNA) and to other self and foreign Ag (polyreactive antibodies). The IgA mAb relative avidity for DNA ranged from 7.5 x 10(-8) to 8.0 x 10(-10) g/microliters. The anti-DNA IgA mAb used VH segments of the VHI(VI-3b), VHII (VH2-MC2), VHIII (WHG16G and VH26c), and VHIV (V71-2) families in conjunction with V kappa I, V kappa IIIb, or V lambda I segments. All IgA mAb VH segments were juxtaposed with JH4b segments. The heavy chain CDR3 sequences were divergent in composition and length. When compared with those of the closest reported germ line genes, the IgA mAb VH and VL gene sequences displayed a number of differences. 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 VH segments by differential PCR amplification and cloning and sequencing of genomic DNA from the mAb-producing cell lines and autologous polymorphonuclear cells. The sequences of the germ line genes that putatively gave rise to the mAb 412.67.F1.3 and mAb 412.66.F1 VH segments were identical with those of the WHG16G and VH26c genes, respectively. In not only the monoreactive mAb 412.67.F1.3 but also the polyreactive mAb 412.66.F1 and mAb 448.9G.F1 VH segments, the higher concentration of replacement (R) mutations and the higher R:S (silent) mutation ratios in the complementarity-determining 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 the five IgA mAb VH and VL segments, the putative and verified somatic point mutations yielded 68 amino acid replacements, of which 38 were nonconserved. Twenty of these yielded positively charged or polar residues 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 VH and VL genes and enhance their fit for Ag by undergoing somatic hypermutation and Ag selection.(ABSTRACT TRUNCATED AT 400 WORDS)

FIGURE 1

Nucleotide (A) and deduced amino acid (B) sequences of the V_H genes used by the anti-DNA IgA mAb. In each cluster, the top sequence represents the germ-line V_H gene sequence displaying the highest degree of identity to the expressed V_H gene sequences of the cluster. The VI-3b, V_H2-MC2, V_H26c, WHG16G, and V71-2 genes belong to the V_HI, V_HII, V_HIII, V_HIII, and V_HIV families, respectively. Dashes indicate identities. Solid lines on the top of each cluster depict CDR. Small letters denote intron sequences. VCE-1 is a V_HII genomic sequence. M60 is a V_H gene expressed in fetal liver. Oligonucleotide (sense) primer sequences or reverse complementary (antisense) primer sequences are underlined. 412.67gl.5a is the portion of the germ-line V_H gene sequence derived from PMN genomic DNA using the combination of the Hl-3 and 67c primers; 412.67gl.6 is the sequence derived from PMN genomic DNA using the combination of 67b and 67a primers (see Materials and Methods and Results). The 67b′/67a is the sequence derived from mAb 412.67.F1.3-producing B cell genomic DNA using the combination of 67b′ and 67a primers. The 412.66gl is the germ-line V_H gene sequence amplified from PMN genomic DNA using the combination of the V_H26cl and 66a primers. The 66b′/66a is the sequence derived from mAb 412.66.F1-producing B cell genomic DNA using the combination of the 66b′ and 66a primers. These sequences are available from the EMBL/GenBank/DDBJ under accession numbers L28049, L28050, L28051, L28052, and L28053.

FIGURE 1

Nucleotide (A) and deduced amino acid (B) sequences of the V_H genes used by the anti-DNA IgA mAb. In each cluster, the top sequence represents the germ-line V_H gene sequence displaying the highest degree of identity to the expressed V_H gene sequences of the cluster. The VI-3b, V_H2-MC2, V_H26c, WHG16G, and V71-2 genes belong to the V_HI, V_HII, V_HIII, V_HIII, and V_HIV families, respectively. Dashes indicate identities. Solid lines on the top of each cluster depict CDR. Small letters denote intron sequences. VCE-1 is a V_HII genomic sequence. M60 is a V_H gene expressed in fetal liver. Oligonucleotide (sense) primer sequences or reverse complementary (antisense) primer sequences are underlined. 412.67gl.5a is the portion of the germ-line V_H gene sequence derived from PMN genomic DNA using the combination of the Hl-3 and 67c primers; 412.67gl.6 is the sequence derived from PMN genomic DNA using the combination of 67b and 67a primers (see Materials and Methods and Results). The 67b′/67a is the sequence derived from mAb 412.67.F1.3-producing B cell genomic DNA using the combination of 67b′ and 67a primers. The 412.66gl is the germ-line V_H gene sequence amplified from PMN genomic DNA using the combination of the V_H26cl and 66a primers. The 66b′/66a is the sequence derived from mAb 412.66.F1-producing B cell genomic DNA using the combination of the 66b′ and 66a primers. These sequences are available from the EMBL/GenBank/DDBJ under accession numbers L28049, L28050, L28051, L28052, and L28053.

FIGURE 2

Nucleotide (A) and deduced amino acid (B) junctional V_H-D-J_H sequences of the anti-DNA IgA mAb heavy chain genes. Germ-line D genes are given for comparison. Dashes indicated identities. The V_H, D, J_H gene and unencoded (N) origin of the individual nucleotides are indicated. The deduced amino acid sequences are depicted as segregated into CDR3 and FR4. These data are available from the EMBL/GenBank/DDBJ under accession numbers L28049, L28050, L28051, L28052, and L28053.

FIGURE 3

Nucleotide (A) and deduced amino acid (B) sequences of the V_L genes used by the anti-DNA autoantibodies. In each cluster, the top sequence represents the germ line V_L gene sequence displaying the highest degree of identity to the expressed V_L gene sequences of the cluster. The HumKv325 and kalc6 genes belong to the VκIII and VκI subgroups, respectively. The YM-1 gene is most likely a member of a novel Vλ. subgroup. The kalc6 and YM-1 are an expressed RF gene and a bone marrow-derived gene. Dashes indicate identities. Solid lines on the top of each cluster depict CDR. Oligonucleotide primer sequences (sense) or reverse complementary primer sequences (antisense) are underlined. These sequences are available from the EMBL/GenBankDDBJ under accession numbers L28043, L28045, L28046, L28047, and L28048.

FIGURE 4

Nucleotide (A) and deduced amino acid (B) J_L sequences of the anti-DNA IgA mAb. Germ line JL genes are given for comparison. Dashes indicated identities. The deduced amino acid sequences are depicted as segregated into CDR3 and FR4. These data are available from the EMBL/GenBank/DDBJ under accession numbers L28043, L28045, L28046, L28047, and L28048.

FIGURE 5

Evidence for somatic mutations in the IgA mAb 412.67.F1.3 (left panels) and IgA mAb 412.66.F1 (right panels) V_H genes. Lanes A depict ethidium bromide staining of DNA PCR amplified from hybridomas and fractionated in agarose gel electrophoresis (right lanes). A 124-bp PCR product was obtained from the genomic DNA of the B cell hybridoma producing mAb 412.67.F1.3 using the sense CDR2 (67b′) and the antisense FR3 (67a) oligonucleotide primers, and a 134-bp PCR product was obtained from the genomic DNA of the B cell hybridoma producing mAb 412.66.F1 using the sense CDR2 (66b′) and antisense FR3 (66a) oligonucleotide primers (see Materials and Methods). The same combinations of primers failed to amplify PMN DNA from the same patient whose B cells were used for the generation of mAb 412.67.F1.3 and mAb 412.66.F1 (left lanes). Lanes B depict ethidium bromide staining of DNA amplified from PMN and hybridoma genomic DNA and fractionated in agarose gel electrophoresis. The sense CDR2 (67b) and the antisense FR3 (67a) oligonucleotide primers whose sequences are shared by the mAb 412.67.F1.3 and WHG16G genes yielded a 143-bp PCR product from both PMN (left lane) and mAb 412.67.F1.3-producing cell lines (right lane). Accordingly, the sense V_H26cl and antisense 66a oligonucleotide primers whose sequences are shared by the mAb 412.66.F1 and V_H26c genes yielded amplification products of 422 bp from both autologous PMN (left lane) and mAb 412.66.F1-producing cell DNA (right lane). Lanes C depict Southern hybridization of the PCR products shown in lanes B with the appropriate [γ-³²P]-labeled 67b′ or 66b′ oligonucleotide used as a probe. In both cases, a strong positive hybridization signal was detected with DNA amplified from the hybridoma cell line but not with that amplified from autologous PMN genomic DNA (right lanes).