Plasmacytoid Dendritic Cells Derived from Chronic Myeloid Leukemia Induced by Low Dose Cytosine Arabinoside Combined with Flt-3L and TPO

Objective Plasmacytoid dendritic cells(pDC) as a subtype of dendritic cells, play an important immunological effects in the body.It is a focus in resent research. After stimulated by viruses or CpG ODNs, pDC produce large amounts of IFN-α rapidly, result in an strong non-specific immunological effect, and then become differentiation and maturation, and attained certain antigen presenting function in adaptive immune response, act as a bridge connecting innate immunity with adaptive immunity. In vitro, FLT-3L combination with TPO can successfully develop pDC from hematopoietic stem cells. Chronic myeloid leukemia(CML) is a hematopoietic stem cell malignant proliferation of the disease. There are series of immune abnormalities in patients with CML. It is well known that Interferon - α treatment of CML is effective, patients can be gained part of cytogenetic and molecular biology mitigation, and effective in patients with interferon therapy, the long-term prognosis is superior to other methods of treatment. Clinical experience also found that low dose cytosine arabinoside(LD-Ara-C) in combination with interferon is superior therapeutic effect of interferon treatment alone. We assumed that whether the LD-Ara-C in patients has an improvement in the immune dysfunction? Whether it will affect on CML-derived pDC differentiation, maturation and function? Therefore, we used LD-Ara-C joint FLT-3L, TPO cultivante CML-derived hematopoietic stem cells that make it differentiate into CML derived pDC to study LD-Ara-C for the treatment of CML may be immune mechanism for the clinical treatment theory.

Method Bone marrow mononuclear cells (BMMNCs) were isolated from CML patients in chronic phase at diagnosis by density gradient centrifugation. BMMNCs were incubate with a cocktail of Flt-3 and TPO, Ara-C were added at the same time of 5ng/ml (A1), 10ng/ml (A2), 25ng/ml (A3), 50ng/ml (A4) and zero as the control, respectively. After 30 days of culture, the morphologic features were observed and CD4,CD11c, CD123, BDCA-2 were analyzed by flow cytometry, IFN-α concentration in supernate were detected by ELISA kits after added influenza vaccine.

Results after 25d of culture, cells clustered with increased size and widespread cytoplasmic projection. Wright-Giemsa-stained cytospin preparation the pDC displays an eccentric kidney-shaped nucleus.The immunophenotype expression of CD4,CD123 and BDCA-2 on pDCs of group A1 and A2 were obviously higher than control group(p<0.05),and group A1 were higher than A2(p<0.05). The majority cells of group A3 and all cells of A4 were died. The group A1 had the highest level of the secretion of IFN-α than A2 and than control group(p<0.05)

Conclusion LD-Ara-C in combination with Flt-3 and TPO can induce CML cells into pDCs which express the typical immunophenotype, Increase the production of IFN-α on stimulated by influenza vaccine. This study indicates that LD-Ara-C increase the quantity of pDC and IFN-α production, and this maybe explain why the therapy with LD-Ara-C in CML patients have better outcome.