B-1 cells are a distinct B cell subset in mice characterized by expression of CD19highCD43+IgMhighIgDlowCD5+/-, and by secretion of polyreactive IgM natural antibodies (NAb). Under steady state conditions, IgM NAb play a critical homeostatic function by enhancing efferocytosis and suppressing inflammation in response to apoptotic cells. Due to this innate immune function, these cells are also referred to as innate B cells. In contrast, there is still little consensus on how to define innate B cells i humans. In preliminary experiments, we generated human innate B cell clones secreting polyreactive NAb that react to apoptotic cells from human blood samples. Remarkably, we found that a fraction of these cells were memory B cells, which had undergone somatic hypermutation in vivo. Because their frequency in peripheral blood was relatively low (~1/20-100,000), we hypothesized that innate B cells reside primarily in tissue and do not often recirculate. Our proposed studies will make use of a collection of human tissue specimens collected from brain dead solid organ donors to investigate the distribution of innate B cells in the human body. We will also phenotype these cells in their anatomical niches. Our experiments will particularly examine the clonal composition of innate B cells through a deep sequencing based analysis of their IgH repertoire. Functionally, innate B cells participate in efferocytosis through the production of IgM Nab. We propose that upon activation, these cells undergo class-switch recombination resulting in the secretion of IgG and IgA Nab. Our studies will address compare the modulatory effect of different classes of Nabs on efferocytosis. Experiments will be carried out in 2 specific aims: Aim-1. To characterize human innate B cells and map their anatomical niches. Innate B cell clones reactive to apoptotic cells will be generated in vitro from a variety of human tissue specimens collected from brain dead solid organ donors. Their Ig heavy chain (IgH) rearranged CDR3 sequences will then be identified and used to assess their frequency in the various tissues using a deep sequencing strategy. The IgH sequences will also indicate whether these clones have accumulated different somatic mutations at different sites and have undergone class switch recombination (CSR) in situ. Lastly, we use flow cytometry to profile innate B cell surface markers in various tissues and blood. Aim-2. To determine the function of NAb produced by innate B cells in efferocytosis. We will first determine the classes and subclasses of Nabs produced by innate B cell clones derived from the various tissue specimens. In relation to their capacity to bind apoptotic cells, we will then examine the capacity of the different types classes (IgM, IgG and IgA) and subclasses (IgG1-4) of Nabs to 1) activate complement leading to the deposition complement opsonins on target apoptotic cells, 2) enhance or block efferocytosis by most common phagocytes and 3) trigger different cytokine secretion pattern by scavenger cells upon phagocytosis.