Important Role for flt3 Ligand In the Generation of Early B and T Cell Precursors From Human Primitive Hematopoietic Progenitors, Revealed by Telomerized Human Stromal Cells.

Abstract 2582

The regulatory mechanism of human early T and B lymphopoiesis has not been well defined. It has been mostly studied, using mouse stromal cell lines, but either T or B lineage cells were developed. We first tested if human telomerase catalytic subunit (hTERT)-transfected telomerized human stromal cells, which have been shown to efficiently support myelopoiesis in serum-containing and serum-free cultures, are able to support the generation of T and B lineage cells from human primitive hematopoietic progenitors.

When we examined the relationship of the expression of lymphoid markers CD7, CD19, CD10, and cytoplasmic CD79a (cyCD79a) with CD38 on cord blood CD34⁺ cells, these antigens were mainly expressed on CD38^high population. The CD19⁺ or CD10⁺ cell population mostly overlapped with the cyCD79a⁺ population. The CD19⁺ or CD10⁺ population was largely mutually exclusive with the CD7⁺ population. We therefore isolated CD34⁺CD38^low/-CD7⁻CD19⁻CD10⁻ cells to obtain immature hematopoietic progenitors and incubated the cells with the hTERT-stromal cells in the presence of 20%FCSaMEM. After three weeks, CD19⁻CD10⁻CD20⁻VpreB⁻cyCD79a⁺ early B, CD19⁺CD10⁺CD20^+/−VpreB⁻cyCD79a⁺ proB, and CD7⁺CD56⁻CD2^+/−CD1a⁻cyCD3⁻ proT stage of cells were detected, in addition to myeloid cells.

We then examined the effect of cytokines on the generation of the proB and proT cells. Addition of flt3 ligand (flt3L) to the culture led to an increase in the numbers of the early B, proB, and proT cells by 8-, 13- and 12-fold, respectively, while flt3L did not significantly affect their differentiation. On the other hand, SCF or TPO alone did not significantly affect the generation of early B, proB, and proT cells, but, in combination with flt3L, enhanced the generation of these cells. No effects were seen with IL-7. In serum-free cultures without cytokines, considerable number of CD7⁺ cells was detected, but few or no B lineage cells including cyCD79a⁺CD19⁻ early B cells were not observed. Nevertheless, flt3L not only enhanced the generation of CD7⁺ cells but also induced the development of cyCD79a⁺CD19⁻ early B cells.

Our data show the usefulness of telomerized human stromal cells to assess human lymphopoiesis in vitro and suggest an important role for flt3L in human early B and T lymphopoiesis in the human bone marrow environment.