Chronic expression of membrane-bound FLT3 ligand by t-lymphocytes in severe aplastic anemia

The fit3 ligand (FL) is a proliferative factor for primitive hematopoietic cells. Biologically active FL exists in a soluble (sFL) and a membrane-bound (nvbFL) form. Serum levels of sFL increase dramatically in diseases characterized by deficiency of primitive hematopoietic progenitors, such as severe aplastic anemia (SAA). In this work, we analysed expression of m-bFL in peripheral blood and bone marrow cells of patients with SAA. Using FLspecific antibodies in flowcytometry (FACS), we detected high levels of FL expressed on the surface of CD3+, but not CD3". cell population. In agreement with serum concentrations of sFL, expression of m-bFL was the highest in patients at diagnosis (p<0.005, as compared to normal), decreased after immunosuppressive therapy, but remained significantly elevated (p<0.01) for up to 10 years after treatment. Relapse of SAA was more frequent in patients with elevated, than in patients with m-bFL rapidly decreasing after the therapy. Levels of m-bFL inversely correlated with the hematopoietic function in SAA, as measured by the in vitro colony assay (R=-07), granulocyte (R=0.8) and platelet (R=-0.8) count. Expression levels of m-bFL in peripheral blood and bone marrow T lymphocytes were similar (R=0.9). These results suggest a diagnostic usefulness of FACS analysis of m-bFL in patients' peripheral blood as a rapid tool to assess the severity and persistence of SAA. To study the mechanism of FL upregulation in SAA, we analysed FL mRNA and protein expression, using RT-PCR and Western analysis. The results indicate that regulation of FL expression occurs at posttranslational rather than transcriptional level. FL mRNA and protein were constitutively expressed in normal peripheral blood cells and their levels increased only about 2-fold in patients with SAA at diagnosis. Instead, as demonstrated by FACS analysis of intracellular FL in saponin-permeabilized cells and by confocal microscopy, FL is prestored intracellularly in normal cells and translocates to the surface of T lymphocytes in SAA. Chronic, T lymphocytes-specific expression, points to the involvement of m-bFL in cell-cell interactions during a compensatory growth factor response to overcome the bone marrow failure in, S A A.