These expressed human antibodies can be tested for specificity against self and non-self antigens. configuration of a peptide mimetope of DNA, we recognized peptide-reactive B cells in peripheral blood. Antibodies derived from these B cells bound to peptide and were largely cross-reactive to dsDNA. This methodology enables us to track the development of autoreactive B cells, which identify peptide and dsDNA, in individual patients with SLE and permits the isolation of autoreactive B cells for further characterization. strong class=”kwd-title” Keywords: lupus, STAT6 B cells, immunoglobulins, peptide Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of a wide variety of autotantibodies, many of which react with nuclear antigens. In particular, autoantibodies against dsDNA are essentially diagnostic of the disease, displaying changes in titer that correlate with disease activity and contributing to lupus nephritis (Vlahakos et al., 1992; Ehrenstein et al., 1995). However, given the rather low frequency of autoreactive B cells it has been hard to isolate and investigate their functional and molecular properties. Numerous transgenic mouse models have been generated to obtain an enhanced quantity of transgenic autoreactive B cells. In combination with antibodies targetting a specific idiotype, it has been possible to study the effects of different parameters, such as genetic background, hormones and antigen exposure, around the devolpment of autoreactive B cells. However, it has been demonstrated that this fate of the self-reactive repertoire is usually affected by competition with non-self reactive cells and the maturation of self-reactive B cells is usually altered in the absence of competition in these transgenic mice (Cyster et al., 1994). In addition, it remains to be elucidated if experiences obtained from autoimmune mouse models are also relevant in patients with SLE. Human hybridomas and Epstein-Barr computer virus transformed human B cells have been previously used to generate human antibodies. These techniques are low efficiency SHP099 hydrochloride and involve a selection bias; therefore, they are unable to permit an analysis of the frequency of autoreactive B cells in individual B cell subsets, although they have permitted the molecular characterization of particular autoantibodies. In recent years, a new strategy has been developed to sample large numbers of antibodies from human peripherl blood B cells. Immunoglobulin (Ig) heavy and light chain genes derived from individual SHP099 hydrochloride B cells of specific B cell populations are amplified by single cell RT-PCR and expressed in vitro. These expressed human antibodies can be tested for specificity against self and non-self antigens. Based on this new technique, an assessment of the percentage of self- or poly-reactive B cells in early B cell populations has revealed two tolerance checkpoints. In a study of a small number of lupus patients, a breach in the tolerance checkpoint at the transitional to na?ve B cell junction could be demonstrated. Although this method allows us to directly analyze the frequency of autoreactive B cells, it would be useful to establish a more convenient and economical method which could be used to enumerate autoreactive SHP099 hydrochloride B cells in specific B cell subsets in the routine analysis of large numbers of patients. Our laboratory has previously reported that immunization of BALB/c mice with an octameric form of the peptide DWEYSVWLSN (MAP-peptide) in which DWEYS is usually a mimetope of dsDNA, results in the production of pathogenic IgG anti-dsDNA antibodies, glomerular immunoglobulin deposition (Putterman and Diamond, 1998) and excitotoxic neuronal loss following a breach in the blood-brain barrier (Huerta et al., 2006; Kowal et al., 2006). To track the peptide-reactive B cell populace in this immune response, we generated a fluorochomeClabeled tetrameric DWEYS peptide (DWEYS-tetramer), which has higher avidity than monomer for B cells with a peptide-reactive B cell receptor (BCR). Using this reagent, we identified peptide-reactive and dsDNA-cross-reactive B cells in immunized mice (Newman et al., 2003). Our goal in this study was to test whether we could identify autoreactive B cells reactive to peptide and dsDNA in lupus patients by virtue of their binding to fluorochome-tagged tetramer. For this purpose, human monoclonal antibodies derived from isolated tetramer positive and negative B cells were expressed and analyzed for antigenic specificity using the above in vitro antibody expression methodology. Materials and Methods Tetramer generation DWEYSVWLSN-streptavidin-allophycocyanin tetramers were generated by combining 30l biotinylated peptide (650M) (AnaSpec, San Jose, CA) with 90l allophycocyanin-labeled streptavidin (6.1M) (Molecular Probes, Eugene, OR). Each mixture was incubated at 4C overnight. Subsequently, peptideCAPC complexes were separated from free peptide by gel filtration using a Bio-Gel P-30 spin column.