B Lymphocytes Emerge De Novo Independently in the YS and PSP Before E10, but Lack B-2 Potential.

Abstract 1501

Poster Board I-524

In the developing mouse embryo, hematopoietic stem cells that can reconstitute all hematopoietic lineages in irradiated adult murine recipients emerge at E10.5 in the AGM region. However, lymphoid potential has been reported in the YS and PSP region prior E10.5. Since the embryonic heartbeat starts at E8.25, the presence of circulation makes it difficult to identify the site of origin of lymphoid progenitors. Indeed, YS lymphoid progenitors have been thought to be derived from the PSP and seeded via circulation. The Ncx1 gene encodes a sodium-calcium channel and is essential for heart function. Ncx1 null embryos lack circulation due to failure to initiate a heartbeat and survive until day 11 of gestation as previously reported. Therefore, Ncx1 null embryos represent a unique model to determine the origin of lymphoid progenitors in a circulation free environment. B-1 cells are innate effectors that spontaneously secrete 'natural' antibodies independent of T cell help. These B-1 cells comprise a high proportion of the B cells in the pleural and peritoneal cavities and can be identified by their sIgM^hi sIgD^lo CD11b⁺ CD5^+/− phenotype. It has been known that mid-gestation fetal liver (FL) can efficiently reconstitute B-1 cells in murine recipients, and recent studies have localized this potential to a lineage negative (Lin⁻) CD45R^lo-neg CD19⁺ AA4.1⁺ B-1 cell specified progenitor present in that tissue. Thus, B-1 progenitors are enriched in FL, but whether these progenitors emerge elsewhere is not fully known. The earliest B-1 cell origin has been reported in the early PSP region, and not in the YS by transplantation assay. Here we demonstrated de novo B cell emergence in the E9.0-9.5 YS as well as the PSP in the NCX1 mouse model using in vitro OP9 stromal cell co-culture. These B cells were derived from VE-cadherin⁺ hemogenic endothelial cells and were confirmed to show Ig rearrangement and anti-phosphorylcholine IgM secretion upon challenge. Cells directly isolated from Ncx1 null embryos did not engraft in the peritoneal cavities of recipient NOD/SCID/IL2gc^null neonate, whereas WT YS and PSP cells became B-1 cells in vivo. However, cultured B cells derived from Ncx1 null YS (>5×10⁶ B cell produced/1YS, n=7 out of 42 null YS tested) and PSP (>5×10⁶ B cell produced/1PSP, n=13 out of 39 null PSP tested) were transplantable and displayed a prominent B-1 phenotype in the host peritoneal cavity (85.2±5.8% compared to 48.8% control non-transplanted C57BL/6 B-1 cells), became marginal zone B cells (39.6%±18.3 of donor IgM+ cells compared to 3.6% control MZ B cells) and displayed a B-1 cell phenotype (10.4±4.6% of donor IgM+ cells compared to 4±2.3% control B-1 cells) in the spleen of transplanted mice, and failed to display a B-2 phenotype (4.6±5.2% of donor IgM+ cells compared to 31.9±4.1% control follicular B cells). Thus, we have redefined de novo B cell potential in the YS and PSP and reported that this B cells potential generates transplantable B-1 cells that may play a role in innate immunity after birth.