Ilse Houtenbos

Serum-free generation of antigen presenting cells from acute myeloid leukaemic blasts for active specific immunisation.

Purpose.Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore, we compared morphological, immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture.Methods.AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days, respectively, in FCS-containing medium (FCS), StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity.Results.Serum-free culture of AML-APCs resulted in comparable morphology, relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture.Conclusion.These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological, immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP).

Identification of CD14 as a predictor for leukemic dendritic cell differentiation in acute myeloid leukemia

Identification of CD14 as a predictor for leukemic dendritic cell differentiation in acute myeloid leukemia

TNF-alpha receptor 1 expression on acute myeloid leukemic blasts predicts differentiation into leukemic dendritic cells.

TNF- α receptor 1 expression on acute myeloid leukemic blasts predicts differentiation into leukemic dendritic cells

Leukemia-derived dendritic cells in acute myeloid leukemia exhibit potent migratory capacity

Leukemia-derived dendritic cells in acute myeloid leukemia exhibit potent migratory capacity

Flt-3 internal tandem duplication hampers differentiation of AML blasts towards leukemic dendritic cells

Flt-3 internal tandem duplication hampers differentiation of AML blasts towards leukemic dendritic cells

Quantification of T-cell–mediated apoptosis in heterogeneous leukemia populations using four-color multiparameter flow cytometry

The unique capacity of dendritic cells to present antigens to naive T cells is being increasingly utilized in cancer therapy. The efficacy of cell‐based immunotherapy can be analyzed by determination of cytotoxic activity of T cells toward tumor cells in vitro. This study supplies a flow cytometric method to analyze T‐cell–mediated cytotoxic activity toward heterogeneous leukemic cell populations at a single‐cell level.

The novel bispecific diabody alpha CD40/alpha CD28 strengthens leukaemic dendritic cell-induced T-cell reactivity

Dendritic cell (DC)‐based immunotherapy faces new challenges because the efficacy of DC vaccines in clinical trials has been inconsistent. Strategies to improve immune responses induced by DC are currently being explored. We have recently shown the feasibility of generating fully functional DC from acute myeloid leukaemic (AML) blasts, but with varying expression levels of the important costimulatory molecule CD86. To overcome this variability, we developed a novel bispecific diabody that simultaneously and agonistically targeted CD40 on AML‐DC and CD28 on naïve T cells. Beside optimization of CD28‐mediated signalling, the resulting cellular cross‐linking was also hypothesized to increase the strength and duration of T cell/AML‐DC interactions, thus increasing T‐cell responsiveness to AML antigens. The αCD40/αCD28‐bispecific diabody was found to bind to its target antigens and provoked increased T‐cell–DC cluster formation. The αCD40/αCD28 diabody is capable of increasing T‐cell proliferation induced by AML‐DC as well as the induction of DC maturation. Importantly, priming efficacy of tumour‐specific cytotoxic T cells can also be improved by cross‐linking AML‐DC and T cells with the αCD40/αCD28 diabody. We propose that the αCD40/αCD28‐bispecific diabody can serve as a potent therapeutic tool to effectively augment anti‐tumour T‐cell responses elicited by AML‐DC.

Maintained immunogenicity of chronic myeloid leukemia-derived dendritic cells in the presence of Imatinib mesylate: implication for vaccination regimens

Maintained immunogenicity of chronic myeloid leukemia-derived dendritic cells in the presence of Imatinib mesylate: implication for vaccination regimens

Divergent Autologous T Cell Responses to Leukaemic Dendritic Cells during Remission in Acute Promyelocytic Leukaemia

Most acute myeloid leukaemia (AML) blasts fail to present antigens to T cells due to, e.g., downregulation of MHC molecules and lack of expression of co-stimulatory molecules [7]. Lack of these molecules prevents cross priming, which may result in tolerance or energy. Acute promyelocytic leukaemia (APL), a subset of AML, is typified by a unique chromosomal translocation t(15;17)(q22;q21) generating the fusion of the promyelocytic leukaemia gene PML and the retinoic acid receptor RARα, a potential tumour target [10]. Although all trans-retinoic acid (ATRA) and anthracyclin containing cytoreduction regimens cure the majority of APL patients, relapses occur. Immunotherapy using dendritic cell (DC) based vaccines could provide an effective tool to control or eradicate minimal residual disease in AML. DCs have strong expression of MHC class I, MHC class II and costimulatory molecules. Moreover, leukaemia-derived DCs are thought to conserve the characteristic cytogenetic and phenotypic aberrations of the malignant cells relevant for antigen presentation. In a phase-I pilot study on DC vaccination in advanced chronic myeloid leukaemia (CML) strong DTH-responses could be detected representing autologous CML-specific T cell responses in vivo [9]. The aim of this study was to examine the immunogenicity of APL-derived antigen presenting cells (APCs) in an autologous setting. For this purpose, we obtained bone marrow of one particular patient, a 22-year-old female, diagnosed with AML classified as t(15;17)-positive APL. All samples were drawn after informed consent. Mononuclear cells, consisting of 90% blast cells, were isolated and cultured in serum-free StemSpanTM H2000 medium (Stemcell Technologies, Meylan, France) supplemented with calcium ionophore A23187 (375 ng/ml, Sigma, St. Louis, MO) and rhIL-4 (250 U/ml, Serva GmbH, Heidelberg, Germany) for two days as described previously [5]. Directly after achieving complete remission (CR) following two courses of chemotherapy and after prolonged remission duration (20 mo. CR), autologous peripheral blood mononuclear cells (PBMCs) were isolated. Plastic adherent monocytes were cultured into remission MoDCs (remMoDCs) by 7 days of culture in StemSpanTM H2000 medium supplemented with 800 U/ml rhGM-CSF (250 U/ml, Peprotech, Rocky Hill, NJ) and 500 U/ml rhIL-4. Non-adherent peripheral blood lymphocytes (PBLs) were used as source of remission T cells. Blast cells showed typical APL morphology: hypergranularity and bundles of Auer rods (Fig. 1A). Flowcytometric analysis revealed that none of the blasts expressed HLA-DR, co-stimulatory molecules CD40, CD80 and CD86, CD54, CD1a and CD83. Previous data showed that APL cells can be induced to significantly upregulate CD40, CD80, CD86 and CD54 (n = 7, p < 0.05). HLA-DR and CD83 were also upregulated although significance was not reached (n = 7, p = 0.09 and p = 0.07, respectively (data not shown) [5,14]. In the blasts of the particular patient in this study, culture with A23187 and IL-4 induced expression of CD40 (21%), CD80 (17%), CD86 (38%), CD54 (22%), HLA-DR (20%) and the maturation marker CD83 (16%); CD1a remained low. Persistent bundles of Auer rods in the cytoplasm of the APLAPCs indicated their leukaemic origin (Fig. 1B). This was confirmed by detection of the PML/RARα-fusion protein in sorted APCs (FACS-VANTAGE, Becton Dickinson) [14]. In 98 out of 100 sorted APCs the microgranular staining pattern of PML/RARα-fusion protein was detected by anti-PML monoclonal antibody PG-M3 (Santa Cruz Biotechnology, CA) thereby proving the presence of t(15;17) in these APCs (data not shown). The availability of APL cells as well as sampling of remission T cells from this patient immediately after achieving CR enabled the study of functional properties of APL-APCs. Non-leukemic remMoDCs were used as a control. Irradiated APL-APCs and remMoDCs were allowed to stimulate autologous PBLs

Dendritic Cell-Based Immunotherapy in Myeloid Leukaemia: Translating Fundamental Mechanisms into Clinical Applications

Immunotherapy for leukaemia patients, aiming at the generation of anti-leukaemic T cell responses, could provide a new therapeutic approach to eliminate minimal residual disease (MRD) cells in acute myeloid leukaemia (AML). Leukaemic blasts harbour several ways to escape the immune system including deficient MHC class II expression, low levels of co-stimulatory molecules and suppressive cytokines. Therapeutic vaccination with dendritic cells (DC) is now recognized as an important investigational therapy. Due to their unique antigen presenting capacity, immunosuppressive features of the leukaemic blasts can be circumvented. DC can be successfully cultured from leukaemic blasts in 60-70% of patients and show functional potential in vivo. Alternatively, monocyte derived DC obtained at time of complete remission loaded with leukaemia-specific antigens can be used as vaccine. Several sources of leukaemia-associated antigen and different methods of loading antigen onto DC have been used in an attempt to optimize antitumour responses including apoptotic cells, necrotic cell lysates and tumour-associated pep-tides. Currently, the AML-derived cell line MUTZ-3, an immortalized equivalent of CD34(+) DC precursor cells, is under investigation for vaccination purposes. For effective DC vaccination the intrinsic tolerant state of the patient must be overcome. Therefore, the development of efficient and safe adjuvants in antigen specific immunotherapeutic programs should be encouraged.