Molecular mechanisms resulting in pathogenic anti-mouse erythrocyte antibodies in New Zealand black mice

Abstract

SUMMARYThe New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. In order to investigate the molecular mechanisms which lead to anti-mouse erythrocyte autoantibody production we have generated eight anti-mouse erythrocyte MoAbs producing hybridomas from splenocytes of 9-and 12-month-old NZB with spontaneous autoimmune anaemia. IgG2a was the predominant isotype, while IgM, IgGl and IgG2b were each produced by one hybridoma cell line. All anti-mouse erythrocyte MoAbs were characterized for their antigen specificities. None of the MoAbs cross-reacted with ss-or dsDNA or with other species' erythrocytes, with the exception of one MoAb which cross-reacted with rat erythrocytes. None ofthe eight hybridomas was demonstrated to express surface or cytoplasmic CD5, suggesting that they derived from CD5" B lymphocytes. All hybridomas when implanted intraperitoneally into BALB/c mice caused anaemia. In order to define the genetic basis and investigate the molecular mechanisms resulting in pathogenic anti-mouse erythrocyte autoantibody production, the pattern of immunoglobulin variable region gene use has been studied. Five ofthe eight MoAbs whose IgVH genes were sequenced all have functionally rearranged genes from the VH J558 gene family. There is evidence for somatic point mutations in the complementarity-determining regions (CDR) ofthe IgVH genes in all of these five MoAbs when compared with the closest known germline gene. We suggest that these nucleotide sequence changes are likely to reflect selection by an antigen-driven mechanism. Furthermore, the data indicate that pathogenic anti-mouse erythrocytes are not derived from 'natural' autoantibodies. Keywords autoantibodies complementarity-determining region monoclonal antibody H genes B lymphocytes tNTRODUCTtONAntibodies have diverse antigen specificities resulting from the structure ofthe assembled V, D and J heavy chain and V and J light chain gene products. Antibodies generated during the rearrangement of germline V(D)J gene segments without the presence of foreign antigen represent the pre-immune repertoire. Due to the almost unlimited potential for V region diversity caused by the combinatorial events during VH-D-JH and VL-JL gene rearrangements and subsequent somatic mutations, antibodies with specificity for autoantigen can occur [1]. The immunoglobulin coding region is organized into largely non-overlapping Vn-gene family clusters, the most proximal to the D and J region loci being the 7183 and Q52 VH-gene families, and J558 and 3609 VH-gene families being most distal.

Regulatory T cells essential to prevent the loss of self-tolerance in murine models of erythrocyte-specific autoantibody responses

“…Early studies by Linder and Edgington [61] demonstrated that the target of the NZB MRBC-specific autoantibodies was a glycoprotein of high molecular weight that they referred to antigen 'X'. As indicated above, panels of NZB-derived monoclonal anti-MRBC hybridomas demonstrated heterogeneous V region gene usage [29][30][31], suggesting diversity also in target binding. Autoantibodies extracted from Coombs' positive erythrocytes from actively autoimmune NZB mice, like many (but not all) of the MRBC-specific pathogenic IgG mAbs produced from these mice, immunoprecipitate exclusively Band 3 from MRBC membranes [62][63][64].…”

“…Analyses of VH and VL regions of NZB-derived pathogenic MRBC-specific hybridomas in three different laboratories have demonstrated that the genes encoding these monoclonal autoantibodies exhibit no obvious restriction of the V gene families utilized. Extensive somatic mutation seen within the V region genes [29][30][31] is indicative of an antigen-driven response. Furthermore, these MRBC-specific mAbs use the same general genetic repertoire used in responses to exogenous antigens, supporting the conclusion that the anti-MRBC, as well as exogenous antigen responses, are antigen driven and not due to natural antibodies [29][30][31].…”

“…Extensive somatic mutation seen within the V region genes [29][30][31] is indicative of an antigen-driven response. Furthermore, these MRBC-specific mAbs use the same general genetic repertoire used in responses to exogenous antigens, supporting the conclusion that the anti-MRBC, as well as exogenous antigen responses, are antigen driven and not due to natural antibodies [29][30][31]. These findings, in addition to the CD5-negative phenotypes of a panel of NZB hybridomas producing pathogenic anti-MRBC antibodies, argue for CD5-negative B cells being the predominant B-cell type contributing to the NZB anti-MRBC response [32].…”

Lessons learned from mouse models of hemolytic transfusion reactions

“…For example, the New Zealand black mouse strain is predisposed to develop AIHA at approximately 6 months of age, which is caused by the production of anti-RBC autoantibodies [45]. The antigen specificities of multiple monoclonal antibodies derived from these mice have been determined [46], and, when injected into other strains of mice, these purified monoclonal antibodies induce autoimmune hemolysis [39].…”

Involvement of Fcα/μ Receptor in IgM Anti-Platelet, but Not Anti–Red Blood Cell Autoantibody Pathogenicity in Mice

“…These IgM autoantibodies induced in vivo thrombocytopenia through uptake of opsonized platelets (10). In contrast, IgM anti-erythrocyte autoantibodies generated from NZB mice (11) cause anemia through spleen accumulation of agglutinated erythrocytes rather than through erythrophagocytosis (12,13).…”