Chronic myelogenous leukemia (CML) is characterized by the presence of a BCR-ABL fusion gene, which is the result of a reciprocal translocation between chromosomes 9 and 22. The BCR-ABL gene product exhibits constitutive tyrosine kinase activity, which plays a critical role in BCR-ABL-induced transformation. Imatinib mesylate represents a potent inhibitor of BCR-ABL tyrosine kinase activity and demonstrated high efficacy in inducing cytogenetic remissions in CML patients.1
Recent in vitro studies have investigated the impact of imatinib on phenotype and function of human dendritic cells (DCs), which play an important role for the induction of innate and adaptive antitumor immunity. Thus, it has been shown that CML-derived DCs display an improved ability of antigen presentation in the presence of imatinib.2 Further data revealed that imatinib does neither inhibit the immunophenotypic profile and migration of CML-derived DCs nor their ability to stimulate T-cell proliferation (below 2.5 μ M imatinib).3 More recently, it has been reported that exposure to imatinib only minimally affects the differentiation of bcr-abl+ and normal monocytes into DCs and does not impair the DC-mediated polarization of naive CD4+ T cells.4 In contrast, Appel et al.5, 6 demonstrated that imatinib efficiently inhibits the differentiation of CD34+ hematopoietic progenitors and monocytes into DCs and their ability to induce cytotoxic T-cell responses. Whereas all these reports are based on DCs that are generated after several days in the presence of various cytokines studies investigating the impact of imatinib on native human blood DCs are very limited.
SlanDCs (formerly termed M-DC8+ DCs) are a major subset of inflammatory myeloid human blood DCs, which mainly differ from other blood DC subpopulations by the selective expression of 6-sulfo LacNAc, representing a carbohydrate modification of P-selectin glycoprotein ligand-1.7 Recently, we documented that the majority of slanDCs display a spontaneous maturation after 6 h in the absence of exogeneous cytokines and that these cells represent principal producers of the immunomodulatory cytokine interleukin (IL)-12 and the cytotoxic effector molecule tumor necrosis factor (TNF)-α.7, 8 Additional data revealed that slanDCs efficiently induce neoantigen-specific CD4+ T cells, activate tumor-reactive CD8+ cytotoxic T cells and improve the tumoricidal potential of natural killer (NK) cells. In the present study, we investigated the effect of imatinib on maturation and cytokine production of slanDCs. In addition, we evaluated its impact on the capacity of slanDCs to promote proliferation and differentiation of CD4+ T-helper cells and to increase the tumoricidal potential of NK cells.
Blood samples were obtained from healthy donors and imatinib-treated CML patients in complete cytogenetic remission with informed consent. Immunomagnetic isolation of slanDCs at high purity (>90%) was performed as described.7 To determine the impact of imatinib on maturation and cytokine expression of slanDCs, freshly isolated DCs were cultivated in the presence of 5 μ M imatinib dissolved in dimethyl sulfoxide (DMSO) or equal amounts of DMSO as a control. SlanDCs from CML patients in complete cytogenetic remission under imatinib treatment were cultured without imatinib. After 6 h, expression levels of human leukocyte antigen (HLA)-DR, CD83 and CD86 were determined by fluorescence-activated cell sorting (FACS) analysis, which was performed as described.7 Imatinib did not influence the surface expression of HLA-DR, CD83 and CD86 on slanDCs from healthy donors (Figure 1a–i) or CML patients (data not shown). To investigate cytokine production, slanDCs were maintained for 6 h to allow spontaneous maturation, washed and subsequently stimulated with 1 μg/ml lipopolysaccharide (LPS) for additional 18 h. FACS analysis or enzyme-linked immunosorbent assay revealed that the capacity of LPS-activated slanDCs from healthy donors or CML patients to produce TNF-α and IL-12 was not impaired by imatinib (Figure 1j–n). The latter observation is in agreement with a previous report, indicating that plasmacytoid DCs from CML patients responding to imatinib can secrete significant amounts of interferon (IFN)-α, compared to healthy donors.9
To investigate the influence of imatinib on slanDC-mediated proliferation and programming of CD4+ T cells, DCs from healthy donors were maintained for 6 h in the presence of imatinib and washed. To determine T-cell proliferation, slanDCs were cocultured with immunomagnetically isolated allogeneic CD4+ T-helper cells in the presence of LPS for 4 days. 3H-thymidine (1 μCi) was added to each well for the last 18 h of culture. Cells were harvested and incorporation was determined in a beta counter. As shown in Figure 2a, slanDC-induced T-cell proliferation was not affected by imatinib. To analyze T-cell programming, healthy donor-derived slanDCs were incubated with immunomagnetically purified allogeneic naive CD45RA+ CD4+ T-helper cells in the presence of LPS for 10 days. Thereafter, T cells were stimulated with 10 ng/ml phorbol myristate acetate and 1 μg/ml ionomycin for 4 h and evaluated for IFN-γ and IL-4 production. FACS analysis revealed that the percentage of IFN-γ- or IL-4-producing CD4+ T-helper cells induced by LPS-activated slanDCs was not influenced by imatinib (Figure 2b–d).
To evaluate whether imatinib affects the ability of slanDCs to promote the tumoricidal potential of NK cells, DCs from healthy donors were cultured for 6 h in the presence of imatinib. Subsequently, slanDCs were cocultured with immunomagnetically purified autologous CD56+ CD3− NK cells in the presence of LPS for 18 h. Then, NK cells were separated from adherent DCs by resuspension, and cytotoxic activity of NK cells was analyzed against the CML cell line K-562 in a 4 h 51Cr-release assay. As illustrated in Figure 2e, imatinib did not inhibit the capacity of slanDCs to improve the tumor-directed cytotoxicity of NK cells.
Taken together, we demonstrated that imatinib does not impair maturation and cytokine expression of slanDCs from healthy donors or CML patients in complete cytogenetic remission under treatment. Functional data revealed that the capacity of slanDCs to promote proliferation and differentiation of CD4+ T cells and to improve the tumoricidal potential of NK cells is not influenced by imatinib. These results provide evidence that imatinib as a potent therapeutic agent for CML patients does not impair the effectivity of native human myeloid blood DCs as important regulators of innate and adaptive antitumor immunity. Furthermore, our findings encourage the design of clinical trials combining immunotherapy and imatinib. This effort is also inspired by other reports,4, 9 particularly by a recent study demonstrating that peptide vaccination induced immunological and clinical responses in imatinib-treated CML patients.10
Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002; 346: 645–652.
Sato N, Narita M, Takahashi M, Yagisawa K, Liu A, Abe T et al. The effects of STI571 on antigen presentation of dendritic cells generated from patients with chronic myelogenous leukemia. Hematol Oncol 2003; 21: 67–75.
Westers TM, Janssen JJ, Houtenbos I, Snoijs NC, van de Loosdrecht AA, Ossenkoppele GJ . Maintained immunogenicity of chronic myeloid leukemia-derived dendritic cells in the presence of imatinib mesylate: implication for vaccination regimens. Leukemia 2006; 20: 154–157.
Boissel N, Rousselot P, Raffoux E, Cayuela JM, Soulier J, Mooney N et al. Imatinib mesylate minimally affects bcr-abl+ and normal monocyte-derived dendritic cells but strongly inhibits T cell expansion despite reciprocal dendritic cell-T cell activation. J Leukoc Biol 2006; 79: 747–756.
Appel S, Boehmler AM, Grünebach F, Müller MR, Rupf A, Weck MM et al. Imatinib mesylate affects the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells. Blood 2004; 103: 538–544.
Appel S, Rupf A, Weck MM, Schoor O, Brümmendorf TH, Weinschenk T et al. Effects of imatinib on monocyte-derived dendritic cells are mediated by inhibition of nuclear factor-κB and Akt signaling pathways. Clin Cancer Res 2005; 11: 1928–1940.
Schäkel K, Kannagi R, Kniep B, Goto Y, Mitsuoka C, Zwirner J et al. 6-Sulfo LacNAc, a novel carbohydrate modification of PSGL-1, defines an inflammatory type of human dendritic cells. Immunity 2002; 17: 289–301.
Schäkel K, von Kietzell M, Hänsel A, Ebling A, Schulze L, Haase M et al. Human 6-sulfo LacNAc-expressing dendritic cells are principal producers of early interleukin-12 and are controlled by erythrocytes. Immunity 2006; 24: 767–777.
Mohty M, Jourdan E, Mami NB, Vey N, Damaj G, Blaise D et al. Imatinib and plasmacytoid dendritic cell function in patients with chronic myeloid leukemia. Blood 2004; 103: 4666–4668.
Bocchia M, Gentili S, Abruzzese E, Fanelli A, Iuliano F, Tabilio A et al. Effect of a p210 multipeptide vaccine associated with imatinib or interferon in patients with chronic myeloid leukaemia and persistent residual disease: a multicentre observational trial. Lancet 2005; 365: 657–662.
The technical assistance of Karin Günther and Bärbel Löbel is greatly appreciated. This work was supported by grants from the German Ministry of Education and Science as well as the Medical Faculty, Technical University of Dresden.
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BCR–ABL-specific CD4+ T-helper cells promote the priming of antigen-specific cytotoxic T cells via dendritic cells
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