Evaluation of Role for flt3L in Human Early B and T/NK Lymphopoiesis in a Novel Stromal Cell Culture System

Abstract 2385

The regulatory mechanism of human early lymphopoiesis remains less defined. A major limitation of conventional in vitro assay is that B and T lymphopoiesis cannot be assessed simultaneously. We exploded a novel culture assay and found that the hTERT-transduced telomerized human stromal cells support the generation of CD19⁺CD34^lo/-CD10⁺cyCD79a⁺CD20^+/−VpreB⁻ pro B- and CD7⁺CD34⁺CD45RA⁺CD56⁻cyCD3⁻ early T-cell precursors from human hematopoietic progenitors without cytokines, which was enhanced by flt3L (2010, ASH). We further characterized the generated lymphoid precursors, and verified that lineage-specific transcription factors for B (Pax-5 and EBF) and T/NK-cell precursors (GATA-3, HEB, Id2) were expressed by the CD19⁺ and CD7⁺CD56⁻ cells, respectively. The CD7⁺CD56⁻ cells showed the differentiation potential to T- and NK-lineage cells, by replating with OP9-Delta1 in the presence of flt3L+IL-7, and with the telomerized-stromal cells in the presence of flt3L+IL-7+IL-15, respectively. In serum-free culture condition, B-cell differentiation was minimally supported by the stromal cells, while low number of CD7⁺ cells was observed. Nonetheless, a significant number of CD7⁺, CD19-cyCD79a⁺, and CD19⁺ cells developed in the presence of flt3L, suggesting an important role for flt3L in the generation of early T/NK- and, particularly, B-lineage cell precursors on the stromal cells. The cellular interaction between Notch and Notch ligand Delta-1 or -4 in the presence of appropriate cytokines is considered to be crucial in T-lineage cell development in mice. We therefore examined whether Notch pathway is involved in the T/NK-cell development under this culture condition. While the gene expression of Delta-1 or -4 was detected in the telomerized stromal cells, the protein expression of these ligands was not detected with Western-blotting analysis. After co-culture of hematopoietic progenitor cells with the telomerized stromal cells, the gene expression of Notch target gene, HES1, was not increased. These data suggest that Notch signaling is not involved in the generation of early T/NK cell precursors on the stromal cells. This novel in vitro culture system suggests that the development of early B- and T/NK- cell precursors from hematopoietic precursors take places on the stromal cells in a Notch-independent manner and that flt3L plays a principal role in the stimulation of the early B and T/NK lymphopoiesis.