No difference was observed in dsDNA binding between variants 4 and 5, suggesting the mutation P46F does not affect dsDNA reactivity. activation occurs in response to self and non-self antigens, while selection after activation may be mediated by self antigen in SLE. Moreover, ineffective tolerance checkpoints may exist before and after antigen activation in SLE. == INTRODUCTION == Antibodies to a wide variety of autoantigens are a hallmark of systemic lupus erythematosus (SLE) (1). In particular, anti-double-stranded DNA (anti-dsDNA) antibodies are associated with lupus nephritis and disease activity (25). Two unique models have been proposed to explain the origin of pathogenic antibodies in SLE. One model suggests that pathogenic anti-dsDNA autoantibodies arise from nave autoreactive B cells through polyclonal B cell activation, which is usually antigen-independent; the alternative model proposes that anti-dsDNA antibodies acquire autoreactivity by somatic mutation and the anti-dsDNA response in SLE is usually antigen driven. Analysis of various murine anti-dsDNA antibodies has exhibited that somatic mutation, often with the introduction of basic amino acids, can result in dsDNA binding (6). Although this observation is usually consistent with antigen-dependent affinity maturation, the nature of the triggering and selecting antigens remains to be elucidated. Whether nucleosomes, DNA, or phospholipid antigens released by apoptotic cells, or alternatively, foreign antigens trigger the response is usually unclear. Back mutation of four human IgG TC-S 7010 (Aurora A Inhibitor I) anti-DNA antibodies derived from lupus patients demonstrates that in their germline configuration these antibodies may fail to bind DNA (79). This observation would suggest that polyclonal activation is not the mechanism for the generation of pathogenic autoantibodies and that self-DNA is usually a critical eliciting antigen. These antibodies are the only anti-dsDNA antibodies from lupus patients that have been analyzed for antigenic specificity in their germline configuration. It has been reported recently that patients with SLE have a defect in early B cell tolerance checkpoints, leading to the accumulation of many autoreactive B cells in the mature nave B cell compartment (10). This observation again raises the question whether pathogenic anti-dsDNA antibodies might originate from these nave autoreactive B cells. Nave B cells can become IgM memory B cells through antigen-dependent mechanisms TC-S 7010 (Aurora A Inhibitor I) and IgG-expressing cells through antigen-independent pathways (11). IgM memory B cells are diminished in lupus patients (12), probably due to increased Ig-class switching of IgM B cells which may be caused by elevated BAFF-levels, overexpression of costimulatory molecules, and certain cytokines, such as IL-10 and IL-21 (1315). This subset, therefore, may be a major precursor populace for pathogenic antibodies in SLE. We, therefore, chose to examine this subset to determine the origins of IgM anti-dsDNA antibodies and their antigenic cross-reactivity, because of the possibility that they might undergo class switch recombination even in the absence of cognate T-cell conversation. So far, only a few mutated IgM anti-dsDNA antibodies have been isolated from lupus patients (16), and none have been back-mutated to assess germline-encoded antigenic specificity. Therefore, we isolated IgM anti-dsDNA antibodies from peripheral blood B cells of a patient with SLE. Three somatically mutated IgM anti-DNA antibodies with pathogenic potential, as reflected in their ability to bind to isolated glomeruli, were reverted to their germline configuration. Reactivity against dsDNA and other antigens was decided in both somatically mutated and reverted antibodies in order to understand the impact of somatic mutation around the generation of dsDNA-reactive IgM memory B cells in SLE, and to determine whether pathogenic human anti-DNA autoantibodies are derived from DNA- or non-DNAreactive B cells. == MATERIALS AND METHODS == == Production of Human Anti-dsDNA Monoclonal Antibodies from Peripheral Blood of Lupus Patients == G3, A9, and B5 are human monoclonal antibodies derived from peripheral blood B cells of a lupus patient, M55, who met the revised ACR criteria for SLE (17). Patient Rabbit Polyclonal to BRS3 M55 was 37 years old and offered no indicators of active disease at the time of the blood draw. The patient exhibited elevated serum titers of anti-dsDNA antibodies and was being treated with hydroxychloroquine and low dose TC-S 7010 (Aurora A Inhibitor I) prednisone. In brief, individual B cells, recognized by reactivity with a fluorochrome-tagged peptide mimetope of dsDNA (18), were sorted into 96-well PCR plates and IgH ( only) and IgL chain gene rearrangements were amplified in two rounds of PCR (50 cycles each) before being cloned into human Ig 1 and expression vectors (gift of MC Nussenzweig, Rockefeller University or college, New York, NY, USA). Human embryonic kidney fibroblast 293T cells were cotransfected with IgH- and IgL-encoding plasmid DNA by calcium phosphate precipitation as explained previously (10,19)..
