Structure-function analysis of a lupus anti-DNA autoantibody: central role of the heavy chain complementarity-determining region 3 Arg in binding of double- and single-stranded DNA.

To determine the contribution of the somatic point mutations and that of the complementarity-determining region (CDR)3 Arg to DNA binding, we engineered the germline V(H) and V(kappa) gene revertant and site-mutagenized the CDR3 Arg residues of the mutated and "antigen-selected" mAb 412.67. This anti-DNA autoantibody was derived from B-1 cells of a lupus patient and bore two H-CDR3 Arg, Arg105 and Arg107, encoded by N segment additions, and one kappa-CDR3 Arg, Arg97, resulting from a point mutation (Kasaian et al. 1994. J. Immunol. 152: 3137-3151; Kasaian et al. 1995. Ann. N.Y Acad. Sci. 764: 410-423). The germ-line revertant bound double-stranded (ds) DNA and single-stranded (ss) DNA as effectively as its wild-type counterpart (relative avidity: 6.4x10(-7) and 9.9x10(-9) vs. 6.7x10(-7) and 9.1 x10(-9) g/microl), raising the possibility that an antigen other than DNA was responsible for the selection of the mAb 412.67 V(H) and V(kappa) point mutations. H-CDR3 Arg105 and Arg107 were both required for dsDNA binding, but either Arg105 or Arg107 was sufficient for ssDNA binding. The central role of Arg105 and Arg107 in DNA binding reflected their solvent-exposed orientation at the apex of the H-CDR3 main loop. Consistent with its inward orientation afar from the antigen-binding surface, the kappa-CDR3 Arg97 played no role in either dsDNA or ssDNA binding.

Lack of intraclonal diversification in Ig heavy and light chain V region genes expressed by CD5+IgM+ chronic lymphocytic leukemia B cells: a multiple time point analysis.

To analyze the modalities of clonal expansion of chronic lymphocytic leukemia (CLL) cells, we sequenced at multiple time points the V(D)J genes expressed by CD5+IgM+CLL B cells in three patients. All three V(D)J gene sequences were found to be point mutated. The mutation frequency in the Ig VH (3.96 x 10(-2) and 2.41 x 10(-2) change/bp) and Vkappa and Vlambda (6.67 x 10(-2) and 1.74 x 10(-2) change/bp) genes of two CLLs (1.19 and 1.32, respectively) was similar, and higher than that in the corresponding gene segments of the third CLL (1.69; 3.4 x 10(-3) and 6.67 x 10(-3) change/bp). In all three CLLs, there was no preferential representation of nucleotide changes yielding amino acid replacement (R mutations), nor was there any preferential segregation of R mutations within the Ig V gene complementarity-determining regions. In all three CLLs, the somatic mutations were all identical in multiple Ig VHDJH transcripts at any given time point, and were all conserved at multiple time points throughout a 2-yr period. The lack of concentration of R mutations in the complementarity-determining regions and the lack of intraclonal heterogeneity suggest that Ag may no longer be able to play a significant role in the clonal expansion of these cells. This conclusion would be strengthened further by the germline configuration of the bcl-1 and bcl-2 proto-oncogenes that are translocated in neoplastic B cells that display significant traces of intraclonal diversification and Ag-dependent selection, such as B-prolymphocytic leukemia and low grade follicular non-Hodgkin lymphoma.

VHDJH gene sequences and antigen reactivity of monoclonal antibodies produced by human B-1 cells: evidence for somatic selection.

To understand whether the distinct VHDJH gene utilization by natural polyreactive Abs reflects the developmentally restricted Ig VHDJH rearrangements putatively expressed by B-1 cells, we generated 11 (8 IgM, 1 IgG3, 2 IgA1), 7 (6 IgM, 1 IgG1), and 7 (2 IgM, 3 IgG1, 2 IgG3) mAb-producing lines using B-1a (surface CD5+, CD45RAlow), B-1b (surface CD5-, CD45RAlow, CD5 mRNA+), and B-2 (surface CD5-, CD45RAhigh, CD5 mRNA-) cells, respectively, sorted from adult human peripheral blood. Most B-1a and B-1b, but no B-2, cell-derived mAbs were polyreactive; i.e., they bound different self and foreign Ags with different affinities. B-1a and B-2 mAbs preferentially utilized VH4 (p = 0.003) and VH3 (p = 0.010) genes, respectively. All three mAb populations utilized DXP, DLR, DN DH genes, and JH6, but no mAb utilized DHQ52. There were fewer unencoded nucleotide (N) additions in the VHDJH junctions of B-1b (3.00 +/- 2.52, mean +/- SD) than of B-1a (12.45 +/- 3.93, p = 1.23 x 10(-5)) or B-2 (8.29 +/- 4.75, p = 0.020) mAbs. Partly due to the fewer N additions and a paucity of D-D fusions, the B-1b mAb CDR3s were significantly shorter than the B-1a mAb CDR3s (p = 0.013), which contained a nonrandom Tyr distribution (p = 0.003). Finally, all but two B-1 cell-derived mAbs were mutated, in a fashion similar to that of the Ag-selected B-2 mAbs. Thus, in the human adult, B-1 cells that make natural polyreactive Abs may not be representative of the predominantly B-1 developmental waves of colonization of the fetal and neonatal B cell repertoires, and are somatically selected.

Structure and function of natural antibodies.

Natural antibodies arise independently of known antigenic stimulation, are mostly IGM, polyreactive, and are generally encoded by V genes in germline configuration. Polyreactive IgM natural antibodies are produced by mainly B-1 cells which account for most of the B cell repertoire in the fetus and neonate, and possibly play a major role in the development and physiology of the human B cell repertoire. Although endowed with self-reactivity, natural antibodies also bind exogenous antigens [73, 74]. Exposure to environmental antigens is not necessary for the emergence of natural antibody-producing cell precursor clones to exogenous antigens, as suggested by the significant population of B cells capable of producing antibodies to a variety of bacterial antigens in germ-free animals. Because of their ability to bind a variety of exogenous antigens, including those on bacteria and viruses, natural antibodies play a major role in the primary line of defense against infections. A central issue related to the understanding of the physiopathologic roles of natural antibodies is whether precursors of cells producing natural antibodies, B-1a and B-1b lymphocytes, are capable of undergoing an antigen-driven clonal selection process, thereby producing autoantibodies with a high affinity for the selecting antigen. In this respect, we have clearly established that B-1 cells can express a hypermutation mechanism similar to that of conventional (B-2) cells. Furthermore, we have shown by gene shuffling, site-directed mutagenesis, and in vitro human Ig gene expression, that the main structural correlate for antibody polyreactivity is provided by the somatically generated H chain CDR3. We have also shown that this Ig V region provides the main structural correlate for antigen-binding in monoreactive antigen-induced autoantibodies. These findings in the human are at the basis of our proposed structure-function model in which the antigen binding features of the germline template antibody are dictated by the somatically generated H chain CDR3, and perhaps, but at a lower degree, L chain CDR3; the point-mutation changes underlying the antigen-driven affinity maturation process would impact mainly the Ig V gene encoded segments. This structure-function model is being tested in our laboratory by analyzing the antigen binding activity of somatically mutated polyreactive autoantibodies that have been structurally reverted to their original putative unmutated configuration. Precise identification of the Ig gene and/or somatic recombination products mediating recruitment of unmutated B cell clonotypes, as well as those that are preferentially modified by an antigen-dependent selection process, should further our understanding of the mechanisms that shape the B cell repertoire in development and disease.

Cellular origin, antigen reactivity, and VH segment structure of IgM mAbs from AIDS lymphomas.

In the present studies we analyzed the Ag specificity, VH gene structure, and cellular origin of three IgM mAb-producing cell lines established in vitro from bioptic specimens of three AIDS patients with BL. We found that (i) both HBL-2 and HBL-3 IgM mAbs were cold agglutinins highly specific for the i blood group determinant, a self Ag the expression of which is dominant in the early stages of life, and both mAbs used somatically point-mutated VH 4-21 segments; (ii) HBL-1 IgM mAb, the Ag-specificity of which has not been determined, used a putatively mutated member of the VHIII family; and (iii) both HBL-1 and HBL-2, but not HBL-3, cells expressed CD5 mRNA, suggesting a B-1 cell origin. The utilization of VH4-21 by the HBL-2 and HBL-3 cold agglutinins is consistent with the usage of this gene segment by all the reported pathogenic except the naturally occurring cold agglutinins. This restricted VH gene usage may reflect an inherent affinity of the germline VH4-21 gene product for the i/I carbohydrate structure, and, perhaps, an overrepresentation of VH4-21 in the human early and late B-cell repertoire. Consistent with both an early and late developmental expression of the VH4-21 gene is the B-1 and B-2 cellular origin of the two VH4-21+ cold agglutinins reported here. Thus, the two cold agglutinin autoantibodies possibly emerged at different stages of the natural history of the B-cell repertoires of these patients and might display a different temporal relationship, as discussed below, to the time of emergence of the respective tumoral cells. The somatically mutated status of the HBL-2 and HBL-3 mAb VH segments was suggested by the monomorphism of the human VH4-21 gene, the extension of the nucleotide differences to the, in general, highly conserved JH segment; and it was formally proved in HBL-3 mAb. Positive selection by Ag of the R mutations in the HBL-2 and HBL-3 mAb VH segments was suggested by the differential R:S mutation ratios in the CDRs and FRs (HBL-2 mAb, 5.0 and 1.1, respectively; HBL-3 mAb, 2.2 and 0.3, respectively) but not substantiated by appropriate statistical analysis according to the binomial distribution model.(ABSTRACT TRUNCATED AT 400 WORDS)

Two acquired immunodeficiency syndrome-associated Burkitt's lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments.

We analyzed the reactivity and the structure of the VH and VL segments of two IgM monoclonal antibodies (MoAbs) produced by spontaneously in vitro outgrowing cell lines, HBL-2 and HBL-3, established from two acquired immunodeficiency syndrome (AIDS) patients with Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL). These B-cell clones were representative of the respective neoplastic parental clones, as determined by immunophenotypic and molecular genetic analysis. The IgM MoAbs were highly specific for the i determinant on red blood cells (cold agglutinins), but bound none of the other eight self and nine foreign antigens (Ags) tested, including those most commonly recognized by natural antibodies or autoantibodies. Structural analysis showed that the IgM MoAb VH segment sequences were 93.5% and 84.2% identical with that of the germline VH4-21 gene, which encodes the vast majority of cold agglutinins that are specific for the i/l carbohydrate Ag and are produced under chronic lymphoproliferative conditions. The HBL-2 MoAb VH4-21 gene segment was juxtaposed with 20P3 and JH6 genes and paired with a V lambda 1 segment, the sequence of which was 95.5% identical to that of the germline Humlv117 gene; the HBL-3 MoAb VH4-21 gene segment was juxtaposed with DXP'1 and JH5 genes and paired with a V lambda 1 segment, the sequence of which was 86.7% identical to that of the germline Humlv1L1 gene. The high degree of conservation of the VH4-21 gene in the human population, the nature of the nucleotide differences in the expressed VH4-21 segments, and the presence of nucleotide substitutions in the HBL-2 and HBL-3 IgM MoAb JH and/or J lambda segments suggested that the MoAb V segments underwent a process of somatic hypermutation. This was formally shown in the HBL-3 MoAb VH segment, by differentially targeted polymerase chain reaction amplification of the HBL-3 MoAb-producing cell genomic DNA. In addition, cloning and sequencing of the genomic DNA from fibroblasts of the same patient whose neoplastic B cells gave rise to the HBL-3 cell line yielded a germline copy of the VH4-21 gene. Thus, the expression of VH4-21 gene products may be involved in a self Ag-driven process of clonal B-cell expansion and selection associated with BL in these AIDS patients.

VH and V kappa segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus. Evidence for somatic selection.

In some patients with insulin-dependent (type I) diabetes mellitus (IDDM), autoantibodies to insulin are present at diagnosis. After initiation of the treatment with not only animal but also human insulin, anti-insulin, mainly IgG, autoantibodies become a major component of the autoimmune response in virtually all IDDM patients. Their structure, however, is still relatively unknown. We analyzed the structure of the VH and V kappa segments of three human IgG mAb derived from three IDDM patients. The sequences of VH genes of two IgG, mAb13 and mAb48, were 98.3 and 96.6% identical with those of the H11 and 1.9III genes (VHIII family), respectively. The sequence of the VH gene of the third IgG, mAb49, was 98.6% identical with that of the 51p1 gene (VHI family). All three IgG mAb used V kappa III segments. The V kappa III gene sequences of mAb13 and mAb49 were 97.9 and 98.9% identical, respectively, to that of the kv3g gene; the mAb48 V kappa gene sequence was 96.5% identical to that of the kv328 gene. The VH and/or V kappa segments of these anti-insulin IgG mAb are similar to Ig V genes expressed in the fetal, and adult normal and autoimmune B cell repertoires. The nucleotide differences displayed by the three anti-insulin IgG mAb VH gene sequences, when compared with those of the closest reported germ-line genes, were concentrated in the CDR (6.2 x 10(-2) and 0.8 x 10(-2) difference/base in CDR and FR, respectively; p < 0.01, chi 2 test), and yielded a significantly higher putative replacement (R) to silent (S) mutation ratio in the CDR (12.0) than in the framework (0.2). The concentration of nucleotide differences in the CDR and their high R:S putative mutation ratios were consistent with the hypothesis that these expressed VH genes underwent a process of somatic mutation and Ag-driven clonal selection. That such differences constituted somatic point-mutations was formally proved in IgG mAb13, by differentially targeted PCR amplification and Southern blot hybridization of the mAb13-producing cell line DNA. The putative germ-line gene that gave rise to the expressed VH segment was cloned using genomic DNA from PMN of the same patient whose B cells were used for the generation of this mAb. Overall, in the anti-insulin IgG mAb VH and V kappa III genes, the (putative and verified) somatic point-mutations yielded 27 amino acid replacements, of which 14 nonconserved. Four of these resulted in positively charged residues, three Arg and one His.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural analysis of the VH-D-JH segments of human polyreactive IgG mAb. Evidence for somatic selection.

Polyreactive (natural) antibodies are primarily IgM and account for a major proportion of circulating Ig in humans. They use various V gene segments, in general, in germ line (unmutated) configuration. To analyze the VH regions of polyreactive antibodies, with particular attention at their somatically mutated status, we generated five IgG (three IgG1 and two IgG3) mAb (using B cells from a healthy subject, a patient with insulin-dependent diabetes mellitus and a patient with SLE), which bound with various efficiencies a number of different self and foreign Ag. Gene cloning experiments showed that the VH region sequences were unique to each IgG mAb. The H chain complementary determining region (CDR3) of two IgG (mAb10 and mAb426.4.2F20) displayed an identical stretch of five amino acids (RFLEW), but the other three IgG mAb CDR3 were divergent in both length and composition. The VH gene sequences of two IgG, mAb426.4.2F20 and mAb410.7.F91, were 99% identical to those of the germ line VH4.11 and VH4.21 genes, respectively. Those of the remaining three IgG mAb displayed a number of differences (93.6 to 95.9% identity) when compared with the germ line VH4.18, VH4.11, and hv1263 gene sequences. These and the VH4.21 gene have been found to encode polyreactive IgM and IgA and, in mutated configuration, monoreactive high affinity autoantibodies and antibodies induced by foreign Ag. When compared with the respective framework region, the CDR of three IgG mAb VH segment sequences displayed a significantly higher: 1) frequency of total nucleotide differences (6.1 x 10(-2) vs 4.5 x 10(-2) difference/base); 2) frequency of putative nucleotide changes yielding amino acid replacements (5.6 x 10(-2) vs 1.4 x 10(-2) replacement change/base); and 3) ratio of overall putative replacement to silent (R:S) mutations (11.0 vs 0.4). Thus, the distribution and nature of the nucleotide differences were consistent with a process of somatic mutation and Ag-dependent clonal selection. This was formally proved in IgG mAb426.12.3F1.4 and IgG mAb10 by differentially targeted polymerase chain reaction amplification and cloning and sequencing of the germ line genes that gave rise to the expressed VH segments, using DNA from polymorphonuclear cells of the same subjects whose B cells were used for the generation of these IgG mAb. Somatic mutations might have been responsible for bringing about polyreactivity in originally monoreactive antibodies or, more likely, they accumulated in originally polyreactive antibodies, which after undergoing a process of Ag selection, retained polyreactivity and may have or may have not acquired a higher affinity for the selecting Ag.