Hilary J. McKenna

Molecular cloning of a ligand for the flt3 flk-2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Polyethylene Glycol-Modified GM-CSF Expands CD11b high CD11c high But Not CD11b low CD11c high Murine Dendritic Cells In Vivo: A Comparative Analysis with Flt3 Ligand

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11bhighCD11chigh or CD11blowCD11chigh. Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11bhighCD11chigh but not CD11blowCD11chigh DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11bhighCD11chigh DC were CD8α−, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11blowCD11chigh DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11bhighCD11chigh DC that share phenotypic and functional characteristics with FL-generated CD11bhighCD11chigh DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.

Flt3 ligand promotes engraftment of allogeneic hematopoietic stem cells without significant graft-versus-host disease

Background. Graft-versus-host (GVH) reactions contribute to stable engraftment of allogeneic hematopoietic stem cell transplants. It was hypothesized that the in vivo expansion of recipient dendritic cells (DC) with the administration of ligand for Flt3 (FL) could promote allogeneic engraftment after reduced-intensity conditioning by enhancing the GVH effect. Methods. FL was first administered to three nonirradiated healthy dogs for 13 days at a dosage of 100 &mgr;g/kg/day. Next, nine dogs received 4.5 Gy total-body irradiation (TBI) and unmodified marrow grafts from dog leukocyte antigen (DLA)-identical littermates without posttransplant immunosuppression. FL was administered to the recipients at a dosage of 100 &mgr;g/kg/day from day −7 until day +5. Results. In normal dogs, FL produced significant increases in monocytes (CD14+) and neutrophils in the peripheral blood, a marked increase in CD1c+ cells with DC-type morphology in lymph nodes, and increased alloreactivity of third-party responders to peripheral blood mononuclear cells in mixed lymphocyte reactions (P <0.001). Sustained engraftment was observed in eight of nine (89%) FL-treated dogs compared with 14 of 37 (38%) controls (P =0.02, logistic regression). All engrafted FL-treated dogs became stable complete (n=2) or mixed (n=6) hematopoietic chimeras without significant graft-versus-host disease (GVHD). Recipient chimeric dogs (n=4) were tolerant to skin transplants from their marrow donors but rejected skin grafts from unrelated dogs within 7 to 9 days (median, 8 days). Conclusions. In this study, the authors showed that FL administered to recipients promotes stable engraftment of allogeneic marrow from DLA-identical littermates after 4.5 Gy TBI without significant GVHD.

CHAPTER 42 – Flt3 ligand

This chapter discusses the interleukins, and the colony-stimulating factors (CSFs), which play a large role in regulating the survival, growth, and differentiation of hematopoietic cells. In the case of two CSFs, CSF-1, and Steel factor, this regulation was shown to result from the interaction of these proteins with specific tyrosine kinase receptors on the surface of these cells. The flt3 (fms like tyrosine kinase 3) tyrosine kinase receptor was isolated independently by two groups using different strategies. One group used low stringency hybridization with a DNA probe from the kinase domain of the CSF-1 receptor (c-fms) to isolate a partial cDNA clone of a related DNA sequence. The gene fragment was then used to isolate a full-length receptor clone, which was named flt3. A second group used a PCR-based strategy to isolate a novel receptor fragment from purified mouse fetal liver stem cells. This fragment was used to isolate a full-length receptor clone that was named flk-2 (fetal liver kinase 2). A comparison of the mouse flt3 and flk-2 receptor sequences revealed that these sequences differ by only two amino acids in their extracellular domain. The flt3 receptor has also been referred to as Stk-1 (stem cell kinase-1).