Molecular and functional analysis of human B-1 cell subsets and natural IgM

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In humans, the existence of a separate B-1 cell lineage is debated. Our major findings from the first funding period revealed that mature CD5+ B cells represent the human B-1a cell counterpart, according to molecular, developmental and functional properties. We showed that human B-1a cells develop ontogenetically early from distinct hematopoietic precursors in the peritoneal cavity (PerC) of humanized mice and give rise to natural IgM titers in peripheral blood (PB). Moreover, human B-1a cells represent a major population in human PerC. The DNA methylome analysis of human B-1a cells revealed specific epigenetic marks resulting from divergent transcription factor activity and support their ontogenetical and functional distinctness. The IgH V gene repertoire of umbilical cord blood (UCB)-derived mature CD5+ B cells is biased, often lacking N-nucleotides at IGHD-IGHJ-joints. Germinal center-independent clonal expansions in UCB indicate a self-replenishing potential of these cells. Finally, functional analyses revealed an innate-like responsiveness and spontaneous Ig secretion potential of mature CD5+ B cells, the human B-1a lineage.In further experiments, we identified bona fide human B-1b cells in UCB, which differ from B-1a cells and conventional naïve B cells according to cell size and surface marker expression. Moreover, these cells show similar restrictions in their Ig rearrangement repertoire as B-1a cells.In this follow-up application, we plan to deepen our understanding of the functional role of human B-1a cells and plan to define and characterize their B-1b sister cells on a molecular and functional basis. For this, we aim at a comparative molecular characterization by Ig repertoire, RNA-sequencing and DNA-methylation analysis of both subsets. Subsequent functional analyses shall focus on migration, differentiation and secretion capacities of both subsets in comparison to conventional B cells. Furthermore, we plan to determine the BCR specificities of human B-1a, B-1b and human memory B cells by BCR cloning and protein microarray analysis to reveal an influence of auto-/ polyreactivity for the development and selection of B-1 cell subsets. Moreover, this strategy will reveal the specificity of human natural (B-1a-derived) versus specific (memory B cell-derived) IgM. Finally, we plan to perform a structural analysis of human IgM molecules ex vivo and generated in vivo by mass spectometry, and a functional characterization of the effector functions of human natural IgM.Taken together, this follow-up application aims at a refined definition and functional characterization of human B-1 cell subsets and natural IgM.