Regulation of Natural Antibody-Producing B-1 Cells - Summary The selective absence of IgM in humans, a rare genetic disorder, is linked to enhanced autoimmunity, as well as increased risk of infections, consistent with studies in mice. In mice, most spontaneously generated “natural” IgM is produced by broadly autoreactive B-1 cells, cells that develop in multiple waves during the fetal and early neonatal period, through a developmental path that is controlled in part by epigenetically regulators of fetal hematopoiesis (Lin28/Let7). Their distinct developmental processes select for a unique and self-reactive Ig- repertoire that characterizes these B-1 cells, which in turn defines their transcriptional profile. Our recent studies have identified two distinct subsets of nIgM-secreting B-1 cells in bone marrow and spleen: B-1 plasma cells (B- 1PC) and non-terminally differentiated B-1 cells (B-1sec). The distinct pathways these cells take to becoming nIgM-producing cells, and thus the mechanisms controlling these autoreactive, yet functionally critical nIgM- secreting B cell subsets, is largely unknown. Filling this gap in knowledge is important as it may identify novel control mechanisms of B cell-mediated autoimmunity, guide the identification of human B-1 orthologues, and eventually their manipulation for therapeutic or prophylactic uses. We have been involved in a large-scale phenotypic reverse genetics screening effort of 14 weeks-old C57BL/6 mice with single, known gene deficiencies (“knockouts”), generated by an NIH-supported consortium. We used two multicolor flow cytometric panels to screen the spleens of 4,390 mice (3F/3M mice per genotype) and 617 distinct genotypes for changes in leukocyte populations. Using an unbiased data analysis approach, we recently identified 18 knockout mice with significant and selective changes in splenic B-1 cells. 17 of those have not previously been shown to affect B cell development or differentiation. One, Rac-2 was previously shown to affect B-1 and marginal zone B cells demonstrated the validity of our approach. Aim 1 of this application is to explore the functional impact of 7 of these novel genes on the development of B cell subsets, especially the nIgM-secreting B-1PC and B-1sec in spleen and bone marrow and their B cell extrinsic and intrinsic expression. In Aim 2 we propose to study the functional impact of the genes, by measuring antibody production from birth – 14 weeks of age by ELISA and ELISpot and assessing the repertoire via auto-antigen array. Neonatal allotype-chimera approaches will determine to which extent validated genes regulates B-1 cells and their differentiated nIgM secreters in a B cell- intrinsic versus extrinsic manner. Expected results would identify novel genes and/or regulatory pathways selectively responsible for the regulation of natural Ig production. This would enable future follow-up studies to define the molecular mechanisms by which each of the identified genes regulate these cells and might help to develop gene-signatures that could aid the identification of B-1 orthologues in humans or other model species. It may also reveal pathways that more broadly control antibody production by autoreactive B cells.
