Leukemia (2002) 16, 1484–1489  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Interferon- induces dendritic cell differentiation of CML mononuclear cells in vitro and in vivo RL Paquette 1 , N Hsu 1 , J Said 2 , M Mohammed 2 , NP Rao 2 , G Shih 1 , G Schiller 1 , C Sawyers 1 , and JA Glaspy 1 1 UCLA Department of Medicine, Division of Hematology/Oncology, Los Angeles, CA, USA; and 2 UCLA Department of Pathology, Los Angeles, CA, USA The ability of interferon- (IFN-) to induce dendritic cell (DC) differentiation in chronic myeloid leukemia (CML) was evalu- ated. Peripheral blood mononuclear cells from CML patients cultured with IFN- and granulocyte–macrophage colony-sti- mulating factor (GM-CSF) developed a dendritic morphology. Fluorescence in situ hybridization demonstrated that the DCs harbored the bcr/abl translocation. The DCs prepared with IFN- /GM-CSF expressed significantly higher levels of class I and II HLA than those grown in interleukin-4 (IL-4) and GM-CSF. The DCs prepared from newly diagnosed CML patients using IFN- /GM-CSF expressed immunoregulatory proteins at levels comparable to normal DCs. In contrast, DCs cultured from CML patients who did not achieve a cytogenetic response to IFN- expressed significantly lower levels of class I HLA, CD40, CD54, CD80 and CD86 than normal DCs. The expression of CD86 by CML DCs was enhanced when they were cultured with IFN-/IL-4/GM-CSF, or when IFN-/GM-CSF-treated cells were induced to mature by CD40 ligand. The DCs from IFN- failures were less stimulatory than normal DCs in the allogeneic mixed leukocyte reaction. CML patients who had a cytogenetic response to IFN- initially had low numbers of bone marrow DCs that increased significantly with treatment, while nonre- sponders had more prevalent DCs at baseline that showed no consistent change with treatment. Therefore, IFN- can induce DC differentiation from CML progenitor cells both in vitro and in vivo. The therapeutic activity of IFN- in CML may be due to its ability to stimulate the generation of DCs that can present CML-specific antigens. Resistance to IFN- may result when DC differentiation becomes impaired. Leukemia (2002) 16, 1484–1489. doi:10.1038/sj.leu.2402602 Keywords: dendritic cell; chronic myeloid leukemia; interferon alpha; granulocyte–macrophage colony-stimulating factor; fascin; interleukin-4 Introduction Chronic myeloid leukemia (CML) is a clonal malignancy of the hematopoietic stem cell characterized by massive over- production of granulocytic cells. 1 CML cells harbor a 9;22 translocation, which fuses the abl proto-oncogene to the bcr gene. The fusion gene encodes a novel 210 kDa protein with constitutive tyrosine kinase activity that is necessary and suf- ficient for cell transformation. The principal pharmacological therapy for CML is interferon- (IFN-), which is capable of inducing major cytogenetic remissions in 10–20% of CML patients. 1–3 The discovery of IFN- as an effective CML treat- ment was serendipitous and its mechanism of action is unknown. Dendritic cells (DCs) are potent antigen-presenting cells derived from hematopoietic progenitors. 4,5 Immature DCs uptake and process antigen in the peripheral tissues, then migrate to lymphoid organs where they mature and present Correspondence: RL Paquette, UCLA Division of Hematology/ Oncology, 42-121 Center for the Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095-1678, USA; Fax: 01-310-206-5511 Received 12 April 2001; accepted 21 February 2002 processed antigen to T lymphocytes. The transition of DCs from antigen-processing cells to mature antigen-presenting cells is accompanied by upregulation of class I and class II major histocompatibility complex proteins, and B7 co-stimu- latory molecules (CD80, CD86) and various adhesion mol- ecules that enhance their ability to present antigen and acti- vate T lymphocytes. 6 DCs occur rarely in the blood but they can be derived from peripheral blood monocytes by culturing them in interleukin-4 (IL-4) and granulocyte–macrophage col- ony-stimulating factor (GM-CSF). 7–9 This culture method, and others, have been used to prepare DCs from peripheral blood or bone marrow cells of CML patients. 10–15 The DCs have been shown to be derived from the leukemic clone and there- fore, they should be capable of presenting CML-specific anti- gens to cytotoxic T lymphocytes. As a result, preliminary clini- cal trials using ex vivo generated CML DCs as immunotherapy for CML have been conducted. 16–18 We previously reported that peripheral blood monocytes cultured in media containing IFN- and GM-CSF differen- tiated into DCs. 19 Because IFN- is used as immunotherapy for various malignancies, including CML, it is possible that the therapeutic efficacy of this agent may be partially due to its affect on DC development. Therefore, the ability of IFN- to differentiate CML mononuclear cells into DCs was evaluated. Materials and Methods Culture methods Peripheral blood mononuclear cells were obtained from hep- arinized blood of CML patients and normal human donors by Ficoll–Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. The mononuclear cell fraction was washed three times with phosphate-buffered saline (PBS), resuspended in RPMI 1640 media (Gibco BRL, Gaithersburg, MD, USA) at 0.5–1.0 × 10 7 cells/ml, and seeded into 25 cm 2 flasks (Costar, Cambridge, MA, USA) at 8 ml/flask. The flasks were incubated at 37°C for 2 h, then the nonadherent cells were decanted. The adherent cell population was rinsed gently with RPMI 1640. ‘Complete medium’ containing peni- cillin–streptomycin (100 IU/ml and 100 g/ml, respectively; Mediatech, Herndon, VA, USA) and 10% AB serum (Gibco BRL) was added to the adherent cells. Optimal concentrations of cytokines determined by titration experiments were used except as indicated: IFN--2b 2000 U/ml (Schering, Kenil- worth, NJ, USA), GM-CSF 500 U/ml (10 4 U/mg; Pepro Tech, Rocky Hill, NJ, USA), and IL-4 1000 U/ml (2.9 × 10 4 U/mg; R&D Systems, Minneapolis, MN, USA). 18 The DCs were cul- tured for 7 days without the addition of supplemental media or cytokines. To induce maturation, DCs were incubated with mouse fibroblasts stably expressing human CD40 ligand for 12 h (gift of Gordon Freeman, Dana-Farber Cancer Institute, Boston, MA, USA).