Autoantibodies, ly‐1, and immunoglobulin v gene expression in hybridomas obtained from young and from old new zealand black mice

Abstract

We obtained a large number of hybridomas from l-month-old and 16-month-old New Zealand black mice to study the fine specificities of the autoantibodies produced, the expression of Ly-1, and the expression of the immunoglobulin V gene families in this autoimmune strain. Analysis of the autoantibody specificities yielded 2 major classifications: those specific for a single autoantigen and those that exhibited multispecific binding. Among the multispecific antibodies, 2 categories were found: an antigen-inhibitable group and an antigennoninhibitable group. A large proportion of V,J558 and V,7183 gene families was observed in hybridomas obtained from l-month-old mice, and in hybridomas obtained from 16-month-old mice, there was a large proportion of V,J558 and V,36-60 gene families. Among the autoantibody K chains secreted by the hybridomas, there was a higher frequency of the VJ, VJ3, and V Q gene families. Autoantibodies were produced by both the Ly-l+ and the Ly-l-B cell subsets.The New Zealand black (NZB) strain of mice is prone to the development of a lupus-like syndrome and autoimmune hemolytic anemia. This strain is therefore an excellent animal model for studying the immuno- chemical and molecular characteristics of both organspecific and non-organ-specific autoantibodies. Whereas Coombs anti-erythrocyte autoantibodies represent typical organ-specific pathogenic autoantibodies (1 ), anti-DNA, anti-Sm, antihistone, and anti-rheumatoid factor (RF) autoantibodies represent non-organ-specific autoantibodies that are associated with various systemic autoimmune diseases (2). There is evidence that a discrete subset of B cells bearing the CDS antigen (Ly-1 in mice and Leu-1 in humans) is the major contributor to the production of certain autoantibodies (3,4). It has been shown that the Ly-I B cell subset is larger in the NZB strain of mice (3.5) and that aged NZB mice show an unusual clonal expansion of Ly-1 B cells exhibiting premalignant properties, such as autonomous proliferation resistant to immunoregulatory signals (6,7). NZB mice, then, are a good model for studying the cellular origin of autoantibodies as well.In the past. the autoreactivity of antibodies was defined by the ability of a heterogenous population of immunoglobulin molecules to bind autoantigens. These Ig were isolated from the sera of patients with autoimmune diseases or from animals prone to developing autoimmune diseases. The development of hybridoma technology and sensitive cellular immunologic techniques has permitted a detailed analysis of the self-reactivity of homogenous populations of antibodies. Using these techniques, investigators have shown that an important proportion of antibodies is multispecific (8.9) and that these antibodies have a moderate affinity to g d i t e r ) for self antigens and for foreign antigens (10).Studies of the immunochemical and molecular properties of monoclonal antibodies can provide important information on the origin of autoantibodies. In particular, we can now examine the properties of the

Human CD5‐positive B Cells in lymphoid malignancy and connective tissue diseases

“…A high proportion of clones specific for DNA were obtained from normal mice [88], but multi-reactive hybridomas were found to be equally frequent in NZB mice and in mice not prone to auto-immunity [89]. In one report [90], monospecific anti-double stranded (ds)DNA antibodies were claimed to be relatively frequent in young and old auto-immune mice and, furthermore, clones that displayed specificities devoid of detectable self-reactivity, found to be of the CD5+ and conventional B cell phenotypes. Taken together, these data raise the important question as to whether autoantibodies produced by CD5+ B cells from both patients or auto-immune strains of mice, differ from auto-antibodies secreted by CD5+ B cells from normal individuals or mice not prone to autoimmunity.…”

Stochastic pairing of heavy-chain and kappa light-chain variable gene families occurs in polyclonally activated B cells.

“…The method of Manser and Gefter (15) was used to identify BALB/c-derived hybridomas expressing the VHX-24 gene family and NZB-derived hybridomas expressing the VK1 gene family, as described (16,17). All positive BALB/c lines and 18 randomly selected NZB lines were cloned by limiting dilution and rescreened under high-stringency conditions.…”

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

“…This was demonstrated by immunofiuorescence staining of the hybridomas for cytoplasmic CD5. Fidanza et al [21] have recently reported that hybridomas derived from CD5 B lymphocytes, which normally lose expression of surface CD5 during the fusion with the myeloma cell line, retain cytoplasmic expression of the CD5 antigen. By detecting the expression or non-expression of cytoplasmic CD5 in our eight anti-mouse erythrocyte MoAbs we have been able to identify the cellular origin of these hybridomas (whether derived from CD5+ or CD5" B lymphocytes) (Sadigh et al, manuscript submitted).…”