CD5+ B1 B cells in mice constitute a pool of natural autoreactive B cells that carry out many important functions, including clearance of apoptotic cells, making rapid responses to pathogens, and regulating inflammation by secreting IL-10. However, their self-reactive BCRs predispose them to clonal expansion and progression to lymphoma and leukemia in aged animals. Hence, it is important to elucidate the regulatory mechanisms that give rise to these cells both from the point of view of normal and pathological immune responses. An interesting feature of these cells is their predominant origin from a distinctive fetal/neonatal developmental pathway, distinguishing it from most B cell development in bone marrow of adult mice. Recently the role of the Lin28b/Let-7 axis in regulating the switch from fetal to adult development in mice has become apparent, making it possible to generate CD5+ B1 B cells from mouse bone marrow precursors by retroviral transduction of Lin28b. While natural autoreactive B cells also exist in humans, identifying them and elucidating their developmental origins is much more challenging, as CD5 expression in human B cells is more transient and may reflect recent activation, and so is not a useful marker for the fetal B cell lineage. Hence, we have compared Lin28b expression in human fetal and adult hematopoietic progenitors and found that its expression is the same as in mice, present in cord blood progenitors and absent from adult progenitors. These results suggest that the Lin28b/Let-7 axis also functions in human B cell development as a regulatory switch. We will test this hypothesis by characterizing changes in cell surface phenotype, gene expression, and autoreactivity induced in adult B cells when we alter Lin28b/Let-7 expression in human B cell progenitors. Specifically, we will explore: 1) the molecular basis by which Lin28b and Let-7 miR control fate choices in human B cell development; and 2) how adoption of the fetal fate influences the specificity of the B cell antigen receptor.
In Aim 1, we will express Lin28b and deplete Let-7 miRNA by Lentiviral transduction of adult human progenitors, differentiating the cells in vitro, then performing flow cytometry to characterize surface protein expression and RNA-Seq to determine genes altered in progenitors and newly-formed B cells.
In Aim 2, we will transfer similarly transduced progenitors into immunodeficient NOD/SCID/IL2R?c- mice to facilitate the differentiation of mature B cells. We will compare changes in gene expression in the mature B cell progeny of adult development reprogrammed to resemble fetal type, again by RNA-Seq. We will also test for differences in autoreactivity of the B cell antigen receptors expressed on B cels generated in these mice by normal and Lin28b reprogrammed development. This work will form the basis for further investigation of human B1 B cells, shedding light on the distinctive mechanism of their development, their selection, and their contribution to immunity, autoimmunity, and leukemia. Moreover, gaining insights into the phenotype of human B1 B cells will enable their monitoring during aging, particularly their role in the origin of chronic lymphocytic leukemia.
This project addresses issues relating to the regulation of development of human B cell precursors and the generation of a pool of autoreactive mature B cells by a distinctive fetal pathway. Self-reactivity, selected for during this type of development generates natural autoreactive B cells that appear particularly susceptible to genetic changes, resulting in clonal expansion and uncontrolled growth with aging, leading to the most common leukemia of individuals over 65, chronic lymphocytic leukemia. Information gained during the course of this project will increase our understanding of the origins and growth of these cells, potentially aiding in the design of rational therapies for this disease of the aged.
