Flt3 ligand is prestored in t-lymphocytes and is released in response to chemotherapy-induced stem cell depletion

FU3 ligand (flt3L) is a hematopoietic cytokine implicated in the regulation of early hematopoietic progenitor/stem cells. The level of fH3L in normal human serum is low (14±39 pg/ml) but increases to high values (2500 pg/ml) as a result of bone marrow failure in patients with congenital and acquired aplastic anemia and patients with leukemia under chemotherapy treatment (Lyman et al. Blood 86:4091, 1995; Wodnar-Filipowicz et al. Blood 88:4493, 1996). We have analysed mechanisms regulating expression of flt3L mRNA and protein in vivo, in peripheral blood and bone marrow mononuclear cells from normal donors and from patients undergoing chemoradiotherapy for leukemia and lymphoma. Flt3L mRNA was constitutively expressed in normal cells and its levels increased 4-6 fold during treatment. However, this increase was less pronounced and delayed compared with the increase of soluble flt3L in serum. Total cellular flt3L, measured by immuno-precipitation and Western analysis, was readily detectable in normal peripheral blood and bone marrow cells, likely arising from the constitutively expressed fU3L mRNA. The presence of intracellular flt3L was confirmed by flow cytometry in saponin-perforated cells, and by visualisation of the ligand in cytoplasmic structures by confocal microscopy. The level of cellular flt3L remained stable during chemotherapy. However, treatment-induced pancytopenia was paralleled by the appearance of membrane-bound fltSL on the cell surface, as measured by flow cytometry. Double staining of peripheral blood and bone marrow mononuclear cells with lineage specific surface antigens identified CD3+ cells as the major source of fU3L. Our results suggest the existence of an intracellular reservoir of flt3L. We speculate that it is prestored rather than de novo sythesized flt3L which is released into the circulation in response to an as yet unknown trigger activated upon stem cell depletion. Considering an early hematopoietic function of flt3L, prestorage of this cytokine may be a physiological mechanism assuring protection or replenishment of the stem cell pool.