Background/Purpose: Mapping studies in the lupus-prone New Zealand Black (NZB) mouse strain identified an interval from 170.8-181 Mb on chromosome 1 sufficient to induce B cell-intrinsic increases in B cell activation, germinal center formation and anti-ssDNA autoantibody production when introgressed onto a non-autoimmune C57BL/6 background. It was previously shown that B cells from these mice (denoted c1) expressing a transgene for hen egg lysozyme (HEL)-specific immunoglobulin in the presence of soluble HEL were able to breach anergy. The purpose of the current study was to determine if the autoimmune phenotype in c1 mice similarly resulted from a breach of anergy in DNA-reactive B cells.

Methods: To generate mice with a homogeneous, anergic B cell repertoire, genes for heavy (3H9) and light (Vκ8) chains specific for ssDNA were backcrossed onto the c1 strain (c1.Vκ8/3H9). Mice with both knock-in genes were aged to 8 months, at which time autoantibody production and B cell localization, activation and differentiation were assessed by ELISA and flow cytometry, respectively. Adoptive transfers were conducted by staining 10 million negatively isolated splenic B cells from 8-10 week old B6.Vκ8/3H9 or c1.Vκ8/3H9 mice with carboxyfluorescein succinimidyl ester (CFSE) and injecting them via tail vein into B6 or c1 mice. Splenic B cell subsets were analyzed by flow cytometry 7 days post-injection to assess survival, activation, plasma cell differentiation and germinal center recruitment. Germinal centers and plasma cells were further quantified by immunofluorescence microscopy.

Results: Surprisingly, analysis of autoantibody production and splenic B cell subsets in c1.Vκ8/3H9 mice revealed a reduced breach of tolerance to ssDNA as compared to c1 mice and a failure to recapitulate the cellular defects observed in the HEL model. To examine whether the preponderance of anergic B cells in the repertoire of c1.Vκ8/3H9 mice was suppressing the breach of tolerance that would otherwise occur, adoptive transfers of B6.Vκ8/3H9 and c1.Vκ8/3H9 B cells into recipients lacking the knock-in genes were performed. Supporting the previous observations of a breach of B cell tolerance in the c1 HEL model, transferred c1.Vκ8/3H9 B cells showed significantly increased activation compared to their B6 counterparts. Furthermore, increased marginal zone localization and a corresponding decrease in the size of the mature follicular subset for transferred c1.Vκ8/3H9 B cells were observed. Despite this, preliminary data did not reveal a difference between transferred B6.Vκ8/3H9 B cells and transferred c1.Vκ8/3H9 B cells with respect to recruitment to germinal centers in either B6 or c1 recipients, suggesting that the activation of transferred c1.Vκ8/3H9 B cells may be extra-follicular in nature.

Conclusion: The results reaffirm previous findings that c1 mice breach tolerance to nuclear self-antigen through an intrinsic B cell defect. There is also indication of an active role for anergic B cells in maintaining tolerance through immune suppression.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/breach-of-b-cell-tolerance-in-new-zealand-black-chromosome-1-congenic-mice/