Objective. To determine the immunologic effects of anti-CD40 ligand (anti-CD40L) therapy in 5 patients with systemic lupus erythematosus nephritis who participated in an open-label study of a humanized anti-CD40L monoclonal antibody.Methods. Serum and peripheral blood mononuclear cells were obtained before, during, and after treatment, and the frequency of Ig and anti-DNA antibody-secreting B cells was analyzed by enzymelinked immunospot assay and by analysis of EpsteinBarr virus (EBV)-transformed B cell lines. To determine the effect of treatment on somatic mutation of Ig genes, reverse transcriptase-polymerase chain reaction was performed on messenger RNA from 4 patients, using primers specific for the DP-47 heavy chain gene and for IgG. Finally, B cell phenotype was investigated using flow cytometry.Results. Even a brief period of treatment with anti-CD40L markedly reduced the frequency of IgG and IgG anti-DNA antibody-producing B cells, and these changes persisted for several months after cessation of treatment. To confirm these findings, EBV-transformed B cell lines were screened from each of 3 patients, and a 10-fold decrease in anti-DNA antibody-secreting cell lines was found after treatment in all 3 patients. Few differences in mutation patterns were observed before and after treatment; however, the frequency of germlineencoded DP-47 sequences was significantly increased before treatment and normalized following treatment. Flow cytometric analysis of B cells revealed expansion of a CD27؊/IgD؊ B cell subset in some of the patients, which did not change with treatment.Conclusion. These are the first mechanistic studies of the effect of anti-CD40L therapy in human autoimmune disease. The results suggest that further studies of CD40L blockade are warranted.

Receptor editing or secondary Ig gene rearrangement occurs in immature, autoreactive B cells to maintain self-tolerance. Here we show that nonspontaneously autoimmune mice immunized with a peptide mimetope of DNA develop peptide-and DNA-reactive antibodies. Antigen-specific B cells display a follicular B cell phenotype. As these cells move into the memory compartment, many express RAG protein and acquire expression of both and light chains. Thus, this study provides evidence for receptor editing occurring in a mature, antigen-activated B cell population. Because the receptor editing observed here occurred in an autoreactive response to antigen, it may function to maintain peripheral tolerance.autoimmunity ͉ B cell selection ͉ autoantibodies A utoreactive B cells routinely arise during the immune response to foreign antigen. Although it has been demonstrated that the processes of apoptosis, anergy, and receptor editing maintain tolerance in immature B cells, it is clear that autoreactivity can also arise in mature B cells in a germinal center response (1-4). Mechanisms that limit autoreactivity in this population are less well characterized. Studies investigating tolerance induction of antigen-specific B cells within a native repertoire have been limited by the rarity the population. In murine models of lupus, DNA-reactive B cells represent Ͻ0.1% of the splenic cell population (5). Thus, studies examining the fate of antigen-specific B cells have relied on transgenic mouse models, in which B cells expressing particular B cell receptors dominate the repertoire. Although analyses of these mice have been very informative, it is clear that the behavior of a B cell may differ when it competes for a particular niche within a lymphoid organ with other B cells (6-11). Thus, it remains important to study antigen-specific B cells within a complete B cell repertoire.We have reported a peptide sequence (DWEYS-peptide) that behaves as a dsDNA mimetope (12). Immunization of nonspontaneously autoimmune BALB͞c mice with an octameric form of this peptide (DWEYS-MAP) results in T cell-dependent production of pathogenic IgG anti-dsDNA antibodies (13,14). To identify the dsDNA-reactive B cells participating in this response, we developed a specific staining methodology using a fluorochrome-labeled tetrameric form of the DWEYS peptide (DWEYS-tetramer) (15). We can use this technique to examine the development of a small autoreactive B cell population in the context of a normal repertoire.We now demonstrate that dsDNA-reactive B cells arise in the follicular B cell population with little contribution from B1 or marginal zone B cells. As the autoreactive B cells mature, they undergo heavy chain class switching and develop phenotypic features of memory B cells. Furthermore, they express RAG and many acquire coexpression of and light chains.

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