Debora R. Sekiguchi

Anti-dsDNA Abs are specific diagnostic markers of systemic lupus erythematosus, and are also implicated in kidney pathology. Anti-dsDNA B cells have been shown to be tolerized in nonautoimmune mice. The immunodysregulation that causes these cells to break tolerance is presumably part of the fundamental defects in systemic lupus erythematosus. To explore these mechanisms, we used the chronic graft-versus-host model mediated by MHC class II differences. Induction of chronic graft-vs-host in anti-DNA H chain knockin (3H9.KI) transgenic mice on a nonautoimmune background resulted in specific activation of anti-dsDNA B cells, as evidenced by high titers of soluble Ab in sera and a high frequency (70%) of anti-dsDNA B cell clones recovered as hybridomas. In addition, the lambda(+)-anti-dsDNA B cells developed increased expression of cell surface activation markers, and concentrated in the T cell area of the follicle with an Ab-forming cell-compatible phenotype. Genetic analysis of the hybridoma clones showed strong evidence of secondary rearrangements of the L chain associated with anti-dsDNA reactivity. Thus, our study indicates that alloreactive T cell help can break tolerance in a complex manner, involving several events.

The chronic graft-versus-host (cGVH) reaction results in a syndrome that closely resembles systemic lupus erythematosus (SLE). It is induced in nonautoimmune mice by the transfer of alloreactive T cells. The availability of anti-DNA transgenes allows us to study the genetic origins of autoantibodies in this model. We induced cGVH in two anti-DNA H chain site-directed transgenic mouse strains. This resulted in clonal expansion and selection of specific mutations in the anti-double-stranded (ds) DNA B cell population. These data, together with a high frequency of anti-dsDNA B cell clones recovered as hybridomas, suggested that anti-dsDNAs are the product of an antigen-driven immune response. Genetic analysis associated this response with the generation of anti-dsDNA B cells through secondary rearrangements that replaced the site-directed transgene (sd-tg) with endogenous VH genes.

T cell responses to different protein Ags have been shown to focus on a few ("immunodominant") determinants. We have addressed three major, interrelated questions regarding immunodominance. First, can each area within hen eggwhite lysozyme (HEL) serve as an immunodominant focus in different inbred mouse strains or are there structural constraints that limit the utilization of certain segments of the molecule? Second, in MHC-congenic mice with identical non-MHC genes, is response to HEL restricted to one or more members of a set of HEL determinants owing to processing constraints imposed by the background genes? Third, does a truncated TCR repertoire influence the immunodominance of certain determinants of HEL? Our results in 19 strains of mice, representing 11 different MHC haplotypes, demonstrate that the immunodominant determinants within HEL are distributed all over the molecule, suggesting that there is no inherent structural constraint imposed on certain regions to be always immunorecessive. However, in different mouse strains, the emergence of identical regions of HEL as immunodominant sites strictly correlates with the identity of their MHC haplotypes but not genetic background (non-MHC) genes. We attribute this relationship to "MHC-guided processing" of native Ag. Finally, our results demonstrate that a truncated TCR repertoire can result not only in the loss of response to certain immunodominant determinants, but can also result in a gain. These results should contribute significantly to further understanding of the mechanism of immunodominance.

Tolerance to dsDNA is broken in mice with a high-affinity anti-DNA H chain transgene, 56R, on the C57BL/6 background (B6.56R). B6.56R produce more anti-dsDNA Abs than BALBc.56R. To investigate how anti-DNA Abs are regulated on the B6 background, phenotypic and genetic studies were performed. B6.56R have reduced numbers of B cells and phenotypically altered B cell subsets, including relative increases in the proportions of IgM-negative bone marrow B cells, cells with a marginal zone phenotype, and cells with a transitional T3 phenotype. The peripheral B cell repertoire in B6.56R is restricted: most B cells express the 56R H chain and use a similar, limited subset of editor L chains. DNA binding is more common in B6.56R because the repertoire is shifted toward L chains that are more permissive for DNA binding. H chain editing is also observed and is increased in spontaneous as compared with LPS hybridomas. A subset of spontaneous hybridomas appears to lack H chain expression.

Although autoantibodies have been used for decades as diagnostic and prognostic markers in type 1 diabetes (T1D), further analysis of developmental abnormalities in B cells could reveal tolerance checkpoint defects that could improve individualized therapy. To evaluate B cell developmental progression in T1D, immunophenotyping was used to classify circulating B cells into transitional, mature naïve, mature activated, and resting memory subsets. Then each subset was analyzed for the expression of additional maturation-associated markers. While the frequencies of B cell subsets did not differ significantly between patients and controls, some T1D subjects exhibited reduced proportions of B cells that expressed transmembrane activator and CAML interactor (TACI) and Fas receptor (FasR). Furthermore, some T1D subjects had B cell subsets with lower frequencies of class switching. These results suggest circulating B cells exhibit variable maturation phenotypes in T1D. These phenotypic variations may correlate with differences in B cell selection in individual T1D patients.