Generation of Leukaemia-Derived Dendritic Cells (DCleu) to improve anti-leukaemic activity in AML: selection of the most efficient response modifier combinations

Dendritic cells (DC) and leukaemia derived DC (DC(leu)) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated from mononuclear cells in vitro following standard DC/DC(leu)-generating protocols. With respect to future clinical applications though, DC/DC(leu)-generating protocols specifically designed for application in a whole-blood-(WB)-environment must be established. Therefore, we developed ten new DC/DC(leu)-generating protocols (kits; Kit-A/-C/-D/-E/-F/-G/-H/-I/-K/-M) for the generation of DC/DC(leu) from leukaemic WB, containing calcium-ionophore, granulocyte-macrophage-colony-stimulating-factor (GM-CSF), tumour-necrosis-factor-alpha, prostaglandin-E(1) (PGE(1)), prostaglandin-E(2) (PGE(2)) and/or picibanil (OK-432). All protocols were evaluated regarding their performance in generating DC/DC(leu) using refined classification and/or ranking systems; DC/DC(leu) were evaluated regarding their performance in stimulating anti-leukaemic activity using a cytotoxicity fluorolysis assay. Overall, we found the new kits capable to generate (mature) DC/DC(leu) from leukaemic WB. Through refined classification and ranking systems, we were able to select Kit-I (GM-CSF + OK-432), -K (GM-CSF + PGE(2)) and -M (GM-CSF + PGE(1)) as the most efficient kits in generating (mature) DC/DC(leu), which are further competent to stimulate immunoreactive cells to show an improved anti-leukaemic cytotoxicity as well. This great performance of Kit-I, -K and -M in mediating DC/DC(leu)-based anti-leukaemic immunity in a WB-environment in vitro constitutes an important and directive step for translating DC/DC(leu)-based immunotherapy of AML into clinical application

In vitro generated dendritic cells of leukemic origin predict response to allogeneic stem cell transplantation in patients with AML and MDS

Allogeneic stem cell transplantation (alloSCT) is the treatment of choice for many patients with acute myeloid leukemia (AML) and myelodysplastic syndrome. The presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. Conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (alloSCT). Our purpose was to test, whether the ability of malignant blasts to differentiate in vitro toward dendritic cells of leukemic origin (DCleu) is associated with clinical outcome. We isolated leukemic blasts from peripheral blood or bone marrow of AML and myelodysplastic syndrome patients before alloSCT (n=47) or at relapse after alloSCT (n=22). A panel of 6 different assays was used to generate DCleu in vitro. Results were correlated with clinical outcome. DCleu could be generated from all 69 samples. Significantly higher mean frequencies of DCleu were found in clinical long-term responders versus nonresponders to SCT (76.8% vs. 58.8%, P=0.006). Vice versa, the chance for response to SCT was significantly higher, if a DCleu+/dendritic cells (DC) ratio of >50% could be reached in vitro (P=0.004). Those patients were characterized by a longer time to relapse (P=0.04) and by a higher probability for leukemia-free survival (P=0.005). In vitro generation of DC and DCleu from leukemic blasts correlated with the clinical outcome. This observation may support a role of leukemic antigen presentation by "leukemia-derived DC" for the stimulation of an allogeneic immune response in AML

Paramunity-inducing Factors (PINDs) in dendritic cell (DC) cultures lead to impaired antileukemic functionality of DC-stimulated T-cells

Introduction: Paramunity-inducing-Factors (PINDs) consist of attenuated/inactivated viruses of various poxvirus-genera, used in veterinary medicine as non-antigen-specific, non-immunising stimulators of the innate immune system against infectious and malignant diseases. Their danger-signaling-interactions were tested for their capacity to improve leukemic antigen-presentation on DC generated from AML-patients' blasts ('DCleu') and DC-stimulation/activation of antileukemic T-cells. Methods: We analyzed, whether the addition of PINDs during DC cultures (15 healthy, 22 leukemic donors) and mixed lymphocyte culture (MLC, n = 15) with autologous (n = 6), allogeneic (n = 2) or T-cells after stem cell transplantation (SCT; n = 7) would alter the quality and quantity of DC, the composition of T-cell-subsets, and/or their antileukemic functionality (AF) as studied by FACS and functional Fluorolysis-cytotoxicity-assays. Results: Effects on 1. DC-cultures: PINDs in DC-cultures lead to increased proportions of mature DC and DCleu but reduced proportions of viable and overall, as well as TLR4- and TLR9-expressing DC. 2. MLC: PINDs increased early (CD8+) T-cell activation (CD69+), but reduced proportions of effector-T-cells after MLC 3. AF: Presence of PINDs in DC- and MLC-cultures reduced T-cells' as well as innate cells' antileukemic functionality. 4. Cytokine-release profile: Supernatants from PIND-treated DC- and MLC-cultures resembled an inhibitory microenvironment, correlating with impaired blast lysis. Conclusions: Our data shows that addition of PINDs to DC-cultures and MLC result in a blast-protective-capacity leading to impaired AF, likely due to changes in the composition of T-/innate effector cells and the induction of an inhibitory microenvironment. PINDs might be promising in treating infectious diseases, but cannot be recommended for the treatment of AML-patients due to their inhibitory influence on antileukemic functionality