The developmental pathway of fetal-derived B cells

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Abstract Stem cell theory states that all the blood cells are derived from hematopoietic stem cells (HSCs) and is a central concept of hematology and immunology. Bone marrow transplant therapy for blood disorders is rooted in the concept that donor HSCs can replace all blood cells in the recipient's body. However, recent research has challenged the stem cell theory by showing that some tissue-resident immune cells develop from endothelial cells (EC) of the embryo in an HSC-independent manner and these immune cells may not be replaced by HSCs. Furthermore, it has been reported that a significant percentage of conventional B cells (B-2 cells) also originate from fetal EC progenitors in the embryo and persist into old mice. These striking results highlight a huge knowledge gap in the hematology and immunology fields because HSC-independent B-2 cell development has yet to be recognized. Since B cells play important roles in protecting against infections and in the pathology of autoimmune and other diseases, it is critically important to understand the developmental pathways of HSC- independent B-cells and their functions compared to HSC-derived counterparts. However, it is challenging to clarify the origins of these B-cells since there are multiple waves of hematopoiesis from ECs in the embryo, in which HSCs and HSC-independent blood progenitors seem to be produced simultaneously. Our preliminary data and other studies suggest that there are at least three waves of B-cell development; 1) innate-immune B-1 cell development, 2) common progenitors for innate B-1 and conventional B-2 cells, and 3) B-2 dominant progenitors derived from HSCs. Our objective is to determine the functional differences of B-cells based on their origins and to establish a revised B-cell development map from embryo to adult, using combinations of various lineage tracing and in vivo barcoding mouse models. To pursue these goals, Aim 1 will utilize HSC-labeling mice (100% labeling of HSCs is achieved) and the HSC-derived lymphoid cell depletion model so that HSC-independent and dependent B-cell subsets will be separated. Then, these HSC-independent and dependent B-cell subsets will be sorted and examined for 1) antibody and cytokine secretion, class switching upon in vitro stimulation, and their IgM repertoire, 2) in vivo repsonse to S. pneumoniae infection, and 3) response to pneumococcal vaccination. In Aim 2, preliminary data identified the earliest innate-immune restricted B-progenitors in the early fetal liver. Also, scRNA-sequencing of the neonatal spleen revealed three separate B-progenitor clusters, supporting our hypothesis. Thus, the EC-lineage tracing mouse model that can mark HSC-independent blood progenitors will be combined with in vivo barcoding, which will enable visualization of the clonal relationships between HSCs and fetal- and HSC-derived B-progenitors. The results obtained from this proposal will revise the current paradigm of B-cell development and will be utilized to understand pathology not previously recognized in the absence of recognizing the existance of various fetal-derived B cell subsets.