An idiotype D23-bearing polyspecific, murine anti-DNA monoclonal antibody forms glomerular immune deposits. Pathogenic role of natural autoantibodies?

Identification of the immunochemical and structural properties of pathogenic anti-DNA antibodies is a major goal for understanding their origins and the mechanisms whereby they induce tissue lesions. Herein, we report on the production of an IgG2a,k anti-DNA monoclonal antibody (4B1), derived from a 12-month-old (NZB x NZW)F1 lupus mouse, able to form glomerular immune deposits. mAb 4B1 is a polyspecific antibody able to bind to ssDNA, actin, tubulin, cardiolipin and to laminin as shown by solid phase ELISAs. Indirect immunofluorescence labeling of HEp-2 cells gave a cytoplasmic staining pattern similar to that obtained with anti-cytoskeleton antibodies. Western blot analysis demonstrated that mAb 4B1 bore idiotype D23, previously shown to be characteristic of natural antibodies derived from normal mice. After injecting the 4B1-secreting hybridoma intraperitoneally into normal (NZW x BALB/c)F1 mice, glomerular immune deposits were observed along the capillary wall. These deposits contained mainly IgM, IgG2a and mAb 4B1, as demonstrated by direct immunofluorescence using a biotinylated-rat anti-4B1 idiotype mAb and kidney eluate analysis. Nucleotide sequence analysis of the VH and VL genes showed that mAb 4B1 is encoded by VH Q52, DSP2.9 and JH2 genes with minimal mutations and by VK8 very similar to the canonic D23 light chain, and JK1 germline genes. No arginine residues were observed in the VH CDR and both chains lacked N-segment addition. Thus, no structural characteristics deduced from the primary structure of mAb 4B1 could explain its pathogenic potential. However, the immunochemical and structural properties suggest that autoantibodies closely related to natural autoantibodies may be pathogenic.

[Lupus diseases]. / Les maladies lupiques.

Systemic lupus erythematosus is a multifactorial, non organ-specific autoimmune disease. Studies of the disease in humans, of spontaneous murine models and of induced experimental models have made it possible to identify the various factors involved. These factors act on the immune system, the abnormalities of which include polyclonal B cell activation, selection of autoreactive B clones by autoantigen, and intervention of helper T cells. Other abnormalities, such as disturbance of the idiotypic network and alteration of suppressor T cell function, are not excluded.

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.

Structural characterization of an (NZB x NZW)F1 mouse-derived IgM monoclonal antibody that binds through V region-dependent interactions to murine IgG anti-DNA antibodies.

Among B/W-derived IgM mAb, 12.5H was selected because of its ability to recognize, in solid phase ELISAs, IgG mAb with DNA-binding activity. mAb 12.5H bound preferentially to IgG2a mAb and did not react with B/W- or BALB/c-derived IgG2a mAb with no DNA-binding activity, suggesting V region-mediated interactions. mAb 12.5H also bound to IgG2a isolated from the sera of old B/W mice but did not react with Ig present in the sera of young B/W (< 6 months) or BALB/c mice. Further analysis of IgM/IgG interaction showed that the binding of 12.5H to IgG polyclonal anti-DNA antibodies could be inhibited by DNA and that mAb 12.5H bound to the F(ab')2 fragments of B/W-derived IgG2a anti-DNA mAb, demonstrating that the interaction occurred through the variable regions of the molecules. When the antigen-binding capacity of mAb 12.5H was evaluated, it was demonstrated to bind to self-antigens such as myosin, actin, tubulin and histones, to bind poorly to ssDNA and not to react with dsDNA. mAb 12.5H gave a cytoplasmic staining pattern by indirect immunofluorescence on HEp-2 cells. Nucleotide sequence analysis of the H and L V genes showed features previously demonstrated to be recurrent in two categories of SLE autoantibodies, i.e. arginine residues in the VHCDR3 and at position 96 on the light chain, both considered to be characteristic of antibodies reacting with dsDNA and acidic amino-acid residues in VHCDR2 reported to be frequent on antihistone antibodies. Taken together, these results show that the B/W mouse repertoire contains IgM autoantibodies: (i) that react in an idiotypic manner with DNA binding antibodies and (ii) that, because of structural characteristics, may constitute the common precursor of different categories of SLE autoantibodies, and the prototype of the idiotypically connected SLE autoantibodies accounting for the production of autoantibodies upon immunization with cognate idiotype and the experimental model of cross-idiotype-induced lupus.

Obtaining a functional recombinant anti-rhesus (D) antibody using the baculovirus-insect cell expression system.

The cloning and production of a human anti-rhesus (Rh) D monoclonal antibody (mAb) using the baculovirus-insect cell expression system is described. This monoclonal recombinant antibody R.D7C2 derived from a human parental IgM lambda immunoglobulin was obtained after immortalization of lymphocytes by Epstein-Barr virus (EBV). The human heavy (VH) and light (VL) variable regions were cloned from the parental cell line and genetically fused to the human constant IgG1 heavy (H) and light (L) chain genes (gamma 1 and lambda, respectively). A recombinant baculovirus was constructed that directs the co-expression of genes encoding both genetically fused heavy and light chains under the control of two late and strong baculovirus promoters. After infecting the Spodoptera frugiperda (Sf9) insect cell line with this baculovector, a complete IgG1 mAb was secreted in the culture medium indicating that each immunoglobulin chain was correctly processed and assembled with a functional glycosylation into a tetrameric form. In vitro analysis showed that the functional properties of R.D7C2 using agglutination tests were efficient for the specific recognition of Rh-D-positive red blood cells (RBC). In addition, R.D7C2 showed effector functions of the gamma 1 heavy chain resulting in the lysis of Rh+ papain RBC by an antibody-directed cellular cytotoxicity mechanism. These results demonstrate that R.D7C2 can be produced in the baculovirus-insect cell expression system as a source for potential therapeutic application in the treatment of the haemolytic disease of the newborn.