Myeloid-leukemic cells (AML, MDS, CML) can be differentiated to leukemia-derived dendritic cell [DC (DCleu)] potentially presenting the whole leukemic antigen repertoire without knowledge of distinct leukemia antigens and are regarded as promising candidates for a vaccination strategy. We studied the capability of 6 serum-free DC culture methods, chosen according to different mechanisms, to induce DC differentiation in 137 cases of AML and 52 cases of MDS. DC-stimulating substances were cytokines ("standard-medium", "MCM-Mimic", "cytokine-method"), bacterial lysates ("Picibanil"), double-stranded RNA ["Poly (I:C)"] or a cytokine bypass method ("Ca-ionophore"). The quality/quantity of DC generated was estimated by flow cytometry studying (co) expressions of "DC"antigens, costimulatory, maturation, and blast-antigens. Comparing these methods on average 15% to 32% DC, depending on methods used, could be obtained from blast-containing mononuclear cells (MNC) in AML/MDS cases with a DC viability of more than 60%. In all, 39% to 64% of these DC were mature; 31% to 52% of leukemic blasts could be converted to DCleu and DCleu-proportions in the suspension were 2% to 70% (13%). Average results of all culture methods tested were comparable, however not every given case of AML could be differentiated to DC with 1 selected method. However performing a pre-analysis with 3 DC-generating methods (MCM-Mimic, Picibanil, Ca-ionophore) we could generate DC in any given case. Functional analyses provided proof, that DC primed T cells to antileukemia-directed cytotoxic cells, although an anti-leukemic reaction was not achieved in every case. In summary our data show that a successful, quantitative DC/DCleu generation is possible with the best of 3 previously tested methods in any given case. Reasons for different functional behaviors of DC-primed T cells must be evaluated to design a practicable DC-based vaccination strategy.

Adoptive immunotherapy is an important therapy option to reduce relapse rates after stem-cell transplantation in patients suffering from acute myeloid leukemia and myelodysplastic syndromes. Myeloid leukemic cells can regularly be induced to differentiate into leukemia-derived dendritic cells (DC(leu)), regaining the stimulatory capacity of professional dendritic cells (DCs) while presenting the known/unknown leukemic antigen repertoire. So far, induced antileukemic T-cell responses are variable or even mediate opposite effects. To further elicit DC/DC(leu)-induced T-cell-response patterns, we generated DC from 17 Acute myeloid leukemia (AML) and 2 myelodysplastic syndrome cases and carried out flowcytometry and (functional) nonradioactive fluorolysis assays before/after mixed lymphocyte cultures of matched (allogeneic) donor T cells (n=6), T cells prepared at relapse after stem-cell transplantation (n=4) or (autologous) patients' T cells (n=7) with blast containing mononuclear cells ("MNC") or DC(leu) ("DC"). Compared with "MNC", "DC" were better mediators of antileukemic-activity, although not in every case effective. We could define DC subtypes and cut-off proportions of DC subtypes/qualities (mature DC/DC(leu)) after "DC" priming, which were predictive for an antileukemic activity of primed T cells and the clinical course of the disease after immunotherapy (allogeneic stem-cell transplantation/donor lymphocytes infusion/therapy). In summary, our data show that the composition and quality of DC after a mixed lymphocyte culture-priming phase is predictive for a successful ex vivo antileukemic response, especially with respect to proportions of mature and leukemia-derived DC. These data contribute not only to predict DC-mediated functions or the clinical course of the diseases but also to develop and refine DC-vaccination strategies that may pave the way to develop and modify adoptive immunotherapy, especially for patients at relapse after allogeneic stem-cell transplantation.

Individual DCA-expression profiles should be evaluated before culture to evaluate DC counts/subtypes (mature/viableDC, DC(leu)) in individual patients.

No abstract

In cancer or hematologic disorders, chemokines act as growth-or survival factors, regulating hematopoiesis and angiogenesis, determining metastatic spread and controlling leukocyte infiltration into tumors to inhibit antitumor immune responses. The aim was to quantify the release of CXCL8, −9, −10, CCL2, −5, and IL-12 in AML/ MDS-pts' serum by cytometric bead array and to correlate data with clinical subtypes and courses. Minimal differences in serum-levels subdivided into various groups (e.g. age groups, FAB-types, blastproportions, cytogenetic-risk-groups) were seen, but higher release of CXCL8, −9, −10 and lower release of CCL2 and −5 tendentially correlated with more favorable subtypes (<50 years of age, <80% blasts in PB). Comparing different stages of the disease higher CCL5-release in persisting disease and a significantly higher CCL2release at relapse were found compared to first diagnosispointing to a change of 'disease activity' on a chemokine level. Correlations with later on achieved response to immunotherapy and occurrence of GVHD were seen: Higher values of CXCL8, −9, −10 and CCL2 and lower CCL5-values correlated with achieved response to immunotherapy. Predictive cut-off-values were evaluated separating the groups in 'responders' and 'non-responders'. Higher levels of CCL2 and −5 but lower levels of CXCL8, −9, −10 correlated with occurrence of GVHD. We conclude, that in AML-pts' serum higher values of CXCL8, −9, −10 and lower values of CCL5 and in part of CCL2 correlate with more favorable subtypes and improved antitumor'-reactive function. This knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune responses.

No abstract

The TNFR family member 4-1BB and its ligand 4-1BBL are involved in the costimulation of T-cells and tumor-derived soluble (s)4-1BBL may influence the interaction of malignant cells with the immune system. Here, we report that cell-surface-expressed (c)4-1BBL can be expressed on mononuclear blood cells from patients with acute myeloid leukemia (AML) (n = 35), myelodysplasia (n = 5) or non-Hodgkin lymphoma (n = 11) and can be coexpressed on varying proportions of lymphoid or myeloid malignant cells and on dendritic cells differentiated from AML-blasts. Direct correlations between c- and s4-1BBL were not found in the investigated cases. Up to now expression of 4-1BBL has not been described on primary myeloid malignant cells, but only on malignant cells of lymphoid or solid tumor origin or on tumor cell lines. With our work we further contribute to the understanding of the potential role of c/s4-1BBL in immune reactions and its influence on the interaction of tumor and immunoreactive cells.

To enlighten interactions between autologous, allogeneic or T-cells from patients after stem cell transplantation with leukaemia-derived-dendritic-cells containing dendritic cells or blast containing mononuclear cells (n ¼ 21, respectively), we determined cytokine-concentrations (interleukin 2, 4, 6, 10, tumor-necrosis-factor-a, interferon-c) in supernatants of mixed-lymphocyte-culture and in serum (n ¼ 16) of 20 patients with acute myeloid leukaemia and three patients with myelodysplastic syndromes by cytometric-bead-assay. We correlated our data with lytic capabilities of stimulated T-cells in a fluorolysis-assay and clinical data: Dendritic-cell-/mononuclear-cell-stimulation of T-cells resulted in increased cytokine-levels in culture-medium compared to serum. There were no significant differences between cytokine-patterns of cases with/without lytic T-cell-activity, response to immunotherapy (stem cell transplantation/donor-lymphocyte-infusion) or graft-versus-host-disease. However, some predictive cytokine-cut-off-values for antileukaemic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease could be defined. Cytokine-profiles alone, without functional assays, are no useful tool to predict antileukaemic T-cell-function, although they can indicate lytic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease. ARTICLE HISTORY