The characterization of anti -dsDNA transgenic mice
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Part I. Systemic Lupus Erythematosus (SLE) is characterized by the production of anti-dsDNA antibodies. The anti-dsDNA B cells can arise from bone marrow where they produce IgM antibodies or from periphery through somatic mutation, which happens concomitantly with class switching, resulting in the production of IgG anti-dsDNA Abs. The differences between IgM and IgG antibody constant regions may lead to different regulation of anti-dsDNA producing B cells. The regulation of anti-dsDNA B cells was studied previously in an R4A-gamma2b transgenic mouse model. In order to compare the regulation of IgG and IgM DNA binding B cells, the R4A-Cmu transgenic mice were generated. In this study the characterization of R4A-Cmu transgenic mice was done to compare with its R4A-gamma2b counterpart.;Like R4A-gamma2b transgenic mice, a high affinity, anergized population exists in R4A-Cmu transgenic mice, indicated by an LPS responsive anti-DNA population present in the primary spleen cells. However in stimulation with a surrogate for T cell help, CD40/IL4, only spleen cells from R4A-Cmu, not R4A-gamma2b Tg mice, showed an increase of anti-DNA Ab production. Also unlike the situation in R4A-gamma2b Tg mice, a population of high affinity DNA-binding hybridomas was obtained without LPS stimulation prior to fusion. Most hybridomas exhibit no somatic mutations, suggesting survival of a population that is deleted in R4A-gamma2b Tg mice. These studies demonstrated that IgG dsDNA binding B cells are more stringently regulated than IgM dsDNA binding B cells.;Part II. It was demonstrated previously that in R4A-gamma2b Tg mice the existence of high affinity dsDNA binding B cells was tightly correlated with the presence of endogenous IgM heavy chain. To test the hypothesis that the presence of second heavy chain permits the high affinity autoreactive B cells to escape deletion, the R4A-gamma2b mice was crossed onto membrane mu knockout mice, referred as R4AmuKO below.;The serum titer of anti-DNA in R4AmuKO mice is comparable to that in R4A-gamma2b mice, both maintain tolerance to dsDNA. There was a decrease in splenocytes in R4AmuKO mice compared to R4A-gamma2b mice. Unlike what was found in R4A-gamma2b mice, LPS stimulation was not able to activate a DNA binding population in R4AmuKO mice, as assayed by ELISA and ELISpot. Sequence analysis showed a similarity of light chain usage to a previously defined indifferent population that escapes negative selection. The disappearance of the LPS responding population demonstrates the need of a second heavy chain for the survival of anergic B cells.
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