FLT-3 ligand and marrow stroma-derived factors promote CD3γ, CD3δ, CD3ξ, and RAG-2 gene expression in primary human CD34+LIN-DR- marrow progenitors

We hypothesize that early lymphoid commitment from primitive hematopoietic marrow progenitors is governed by signals from the marrow microenvironment leading to sequential induction of lineage specific genes. Using expression of lymphoid genes as markers of differentiation, we characterize a highly purified population (>99.8% by double sorting) of primary human CD34+Lin(-)DR(-) progenitors. This population was then used to evaluate the effects of supplemental cytokines (interleukin-2[IL-2], IL-3, IL-7, c-kit ligand), FLT-3 ligand (FL), and stroma-derived factors on lymphoid differentiation in vitro. CD3 epsilon, RAG-1, Ikaros, CD10, and TdT transcripts were detected in the starting CD34(+)Lin(-)DR(-) population. By contrast, CD3 gamma, CD3 delta, CD3 zeta, and RAG-2 transcripts were not present in any samples tested. The presence of supplemental cytokines alone at culture initiation permitted stimulation of the expression of CD3 zeta, but not of CD3 gamma or CD3 delta. However, when FL and stroma derived factors were added to cytokines, CD3 gene expression was induced in all samples. The predominant CD3 transcripts induced by optimal culture conditions were alternatively spliced isoforms lacking transmembrane sequences (CD3 delta and CD3 gamma) and portions of the intracellular and extracellular domains (CD3 gamma). The combination of cytokines, FL, and stromal factors also provided a potent stimulus for RAG-2 gene expression. These findings show that FL in combination with stroma-derived factors provide important signals to promote early events required for lymphoid differentiation. (C) 1998 by The American Society of Hemetology.