Influence of physiological hypoxia on whole blood (WB) from healthy donors and AML-patients: the (leukemic) antigen-presentation on blasts, on leukemia-derived dendritic cells (DCleu) after stimulation with "DC-generating cocktails" as well as the antileukemic functions of DC/ DCleu-stimulated immune reactive cells

DCleu generation in vivo and transferring to the patients could qualify as a novel treatment option. Furthermore, blasts modulated to leukemia-derived DC (DCleu) in vivo could result in T-cell activation. We have already established a method using a combination of GM-CSF+ PICIBANIL (I), or +PGE2 (K) or + PGE1 (M) kits to convert leukemic blasts into DCleu without induction of blasts’ proliferation in a WB-culture model (in the presence of individual patients’ soluble and cellular components). Such DCleu cells can strongly induce antileukemic and memory T-cells. To simulate physiological conditions, we compared Normoxic (No) standard- (21% O2) vs. physiological Hypoxic (Hy) (10% O2) culture conditions. In the first part of my work, DC-generation was done in AML cell lines to set up the physiological conditions. AML cell lines were cultured with immunomodulatory Kits (-I, -K, -M) under Normoxic condition (21% Oxygen) as well as under Hy (approx. 6% Oxygen). Our cell line results showed no significant changes in DCleu (frequencies of DCleu/Bla+) or the proliferation of blasts in Hy condition under the stimulation of Kits compared to control and also as compared to No condition. In the second part of my work, samples from AML-patients and healthy blood donors (peripheral blood mononuclear cells (PBMNC) and heparinized whole blood (WB)) were cultured in parallel under No (21% Oxygen) and Hy (10% Oxygen) using Pici-PGE1, Pici-PGE2 and Kit-I, Kit-K and Kit-M DC-generating media which followed by MLC (mixed lymphocyte culture) using patients’ or healthy donors’ T-cells and functional (blast-cytotoxicity- and cytokine secretion) assays in AML-samples. Compositions of DC-, blast-subtypes, the immune status, and results of functional assays were compared for all tested conditions. We found that DCs and their subtypes can be generated in comparable frequencies from AML and healthy PBMNCs samples using pici-PGE1 and pici-PGE2 under No and Hy conditions. Comparable frequencies of DC were generated with Kits (-I, -K, -M) from 35 AML and 16 healthy WB -samples. DC generation was comparable under No vs. Hy in AML and healthy samples. Further, as control when there was no added response modifier (w/o), no or low production of DC/DCleu was detected. Interestingly, no induction of blasts’ proliferation was shown under the influence of kits under No or Hy conditions. After MLC of T-cells with kit-treated AML-WB under No vs. Hy (with Kit-I, -K, -M), we found a significant increase of activated T-cells, CD4 +CD3+/CD3+, Tprol, Tnon-naive, Teff-em as well as of NK and iNKT cells and low frequencies of Treg, Tcm DC, T-cells, NK, iNKT, and CIK cells after MLC under Hy as well as after No conditions. Finally, we demonstrated an increase of antileukemic activity after the stimulation of patients’ T-cells in MLC with (DC/DCleu containing) Kit-I or -M treated (vs. untreated) WB in No as well as under Hy conditions. Average blast lysis was increased in MLCWB-DC Kit-I and MLCWB-DC Kit-M compared to MLCWB, with equal average lysis in MLCWB-DC Kit-I and MLCWB-DC Kit-M. We further found significantly higher amounts of INFγ secreting cells of the innate and adaptive immune system after MLCWB-DC Kit-I and MLCWB-DC Kit-M compared to MLCWB. Results were comparable under No and Hy conditions. We found a significant positive correlation between the DC/WB produced with Kit-M and the antileukemic reactivity of T ‘effector-cells’ after MLCWB-DC kit-M. Furthermore, we found a significant correlation between DC+/WB and DCleu /WB generated with kit-M and the best anti-leukemic reactivity of T-cells’ MLCWB-DC kit-M in Hy conditions

Interferon gamma secretion of adaptive and innate immune cells as a parameter to describe leukaemia-derived dendritic-cell-mediated immune responses in acute myeloid leukaemia in vitro

INTRODUCTION: Myeloid leukaemic blasts can be converted into leukaemia-derived dendritic cells (DCleu), characterised by the simultaneous expression of dendritic- and leukaemia-associated antigens, which have the competence to prime and enhance (leukaemia-specific) immune responses with the whole leukaemic antigen repertoire. To display and further specify dendritic cell (DC)- and DCleu-mediated immune responses, we analysed the interferon gamma (IFNy) secretion of innate and adaptive immune cells. METHODS: DC/DCleu were generated from leukaemic whole blood (WB) with (blast)modulatory Kit-I (granulocyte-macrophage colony-stimulating factor [GM-CSF] + Picibanil [OK-432]) and Kit-M (GM-CSF + prostaglandin E1) and were used to stimulate T cell-enriched immunoreactive cells. Initiated anti-leukaemic cytotoxicity was investigated with a cytotoxicity fluorolysis assay. Initiated IFNy secretion of T, NK, CIK, and iNKT cells was investigated with a cytokine secretion assay (CSA). IFNy positivity was additionally evaluated with an intracellular cytokine assay (ICA). Recent activation of leukaemia-specific cells was verified through addition of leukaemia-associated antigens (LAA; WT-1 and Prame) RESULTS: We found Kit-I and Kit-M competent to generate mature DC and DCleu from leukaemic WB without induction of blast proliferation. Stimulation of immunoreactive cells with DC/DCleu regularly resulted in an increased anti-leukaemic cytotoxicity and increased IFNy secretion of T, NK, and CIK cells, pointing to the significant role of DC/DCleu in leukaemia-specific alongside anti-leukaemic reactions. Interestingly, an addition of LAA did not further increase IFNy secretion, suggesting an efficient activation of leukaemia-specific cells. Here, both the CSA and ICA yielded comparable frequencies of IFNy-positive cells. Remarkably, the anti-leukaemic cytotoxicity positively correlated with the IFNy secretion in TCD3+, TCD4+, TCD8+, and NKCD56+ cells. CONCLUSION: Ultimately, the IFNy secretion of innate and adaptive immune cells appeared to be a suitable parameter to assess and monitor the efficacy of in vitro and potentially in vivo acute myeloid leukaemia immunotherapy. The CSA in this regard proved to be a convenient and reproducible technique to detect and phenotypically characterise IFNy-secreting cells. In respect to our studies on DC-based immunomodulation, we were able to display the potential of DC/DCleu to induce or improve leukaemia-specific and anti-leukaemic activity

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

Dendritic/antigen presenting cell mediated provision of T-cell receptor gamma delta (TCRγδ) expressing cells contributes to improving antileukemic reactions ex vivo

T-cell receptor gamma delta (TCRγδ) expressing T-cells are known to mediate an MHC-independent immune response and could therefore qualify for immune therapies. We examined the influence of dendritic cells(DC)/antigen presenting cell (APC) generated from blast-containing whole blood (WB) samples from AML and MDS patients on the provision of (leukemia-specific) TCRγδ expressing T-cells after mixed lymphocyte culture (MLC). Kit-M (granulocyte-macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) were used to generate leukemia derived APC/DC (DCleu)from WB, which were subsequently used to stimulate T-cell enriched MLC. Immune cell composition and functionality were analysed using degranulation- (DEG), intracellular cytokine- (INTCYT) and cytotoxicity fluorolysis- (CTX) assays. Flow cytometry was used for cell quantification. We found increased frequencies of APCs/DCs and their subtypes after Kit-treatment of healthy and patients´ WB compared to control, as well as an increased stimulation and activation of several types of immune reactive cells after MLC. Higher frequencies of TCRγδ expressing leukemia-specific degranulation and intracellularly cytokine producing T-cells were found. The effect of Kit-M-treatment on frequencies of TCRγδ expressing cells and their degranulation could be correlated with the Kit-M-mediated blast lysis compared to control. We also found higher frequencies of TCRγδ expressing T-cells in AML patients´ samples with an achieved remission (compared to blast persistence) after induction chemotherapy. This might point to APC/DC-mediated effects resulting in the provision of leukemia-specific TCRγδ expressing T-cells: Moreover a quantification of TCRγδ expressing T-cells might contribute to predict prognosis of AML/MDS patients

Dendritic Cell-Triggered Immune Activation Goes along with Provision of (Leukemia-Specific) Integrin Beta 7-Expressing Immune Cells and Improved Antileukemic Processes

Integrin beta 7 (β7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell–cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific β7 expression in immune cells after T-cell-enriched mixed lymphocyte culture—finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients’ whole blood (WB) was treated with Kit-M (granulocyte–macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DCleu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DCleu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of β7-expressing T-cells, degranulating and intracellular cytokine-producing β7-expressing immune cells and, in patients’ samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of β7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) β7-expressing immune cells. Furthermore, β7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies