Background: Extracellular vesicles (EV) are cell-derived vesicles released by all cells in health and disease. Accordingly, EVs are also released by cells in acute myeloid leukemia (AML), a hematologic malignancy characterized by uncontrolled growth of immature myeloid cells, and these EVs likely carry markers and molecular cargo reflecting the malignant transformation occurring in diseased cells. Monitoring antileukemic or proleukemic processes during disease development and treatment is essential. Therefore, EVs and EV-derived microRNA (miRNA) from AML samples were explored as biomarkers to distinguish disease-related patterns ex vivo or in vivo. Methodology: EVs were purified from serum of healthy (H) volunteers and AML patients by immunoaffinity. EV surface protein profiles were analyzed by multiplex bead-based flow cytometry (MBFCM) and total RNA was isolated from EVs prior to miRNA profiling via small RNA sequencing. Results: MBFCM revealed different surface protein patterns in H versus AML EVs. miRNA analysis showed individual as well as highly dysregulated patterns in H and AML samples. Conclusions: In this study, we provide a proof-of-concept for the discriminative potential of EV derived miRNA profiles as biomarkers in H versus AML samples.

Although several (chemotherapeutic) protocols to treat acute myeloid leukemia (AML) are available, high rates of relapses in successfully treated patients occur. Strategies to stabilize remissions are greatly needed. The combination of the (clinically approved) immune-modulatory compounds Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) and Prostaglandine E1 (PGE-1) (Kit-M) converts myeloid blasts into dendritic cells of leukemic origin (DC_leu). After stimulation with DC_leu ex vivo, leukemia-specific antileukemic immune cells are activated. Therefore, Kit-M treatment may be an attractive immunotherapeutic tool to treat patients with myeloid leukemia. Kit-M-mediated antileukemic effects on whole bone marrow (WBM) were evaluated and compared to whole blood (WB) to evaluate the potential effects of Kit-M on both compartments. WB and WBM samples from 17 AML patients at first diagnosis, in persisting disease and at relapse after allogeneic stem cell transplantation (SCT) were treated in parallel with Kit-M to generate DC/DC_leu. Untreated samples served as controls. After a mixed lymphocyte culture enriched with patients’ T cells (MLC), the leukemia-specific antileukemic effects were assessed through the degranulation- (CD107a⁺ T cells), the intracellular IFNγ production- and the cytotoxicity fluorolysis assay. Quantification of cell subtypes was performed via flow cytometry. In both WB and WBM significantly higher frequencies of (mature) DC_leu were generated without induction of blast proliferation in Kit-M-treated samples compared to control. After MLC with Kit-M-treated vs. not pretreated WB or WBM, frequencies of (leukemia-specific) immunoreactive cells (e.g., non-naive, effector-, memory-, CD3⁺β7⁺ T cells, NK- cells) were (significantly) increased, whereas leukemia-specific regulatory T cells (T_reg, CD152⁺ T cells) were (significantly) decreased. The cytotoxicity fluorolysis assay showed a significantly improved blast lysis in Kit-M-treated WB and WBM compared to control. A parallel comparison of WB and WBM samples revealed no significant differences in frequencies of DC_leu, (leukemia-specific) immunoreactive cells and achieved antileukemic processes. Kit-M was shown to have comparable effects on WB and WBM samples regarding the generation of DC_leu and activation of (antileukemic) immune cells after MLC. This was true for samples before or after SCT. In summary, a potential Kit-M in vivo treatment could lead to antileukemic effects in WB as well as WBM in vivo and to stabilization of the disease or remission in patients before or after SCT. A clinical trial is currently being planned.

New therapies are highly needed to stabilize remission in patients with acute myeloid leukemia (AML). This study investigates the value of dendritic cells derived from leukemic blasts (DC_leu) to enhance anti-leukemic immunity after T-cell-enriched mixed lymphocyte cultures (MLCs). We correlated induced anti-leukemic activity with patient data, including biological, clinical and prognostic factors. Additionally, we correlated the frequencies of DC/DC_leu and leukemic-specific T cells with the achieved anti-leukemic activity after MLC. We show that mature DC/DC_leu can be generated using the immunomodulating Kit-M, which contains granulocyte–macrophage colony-stimulating-factor (GM-CSF) and prostaglandin E₁ (PGE₁), without inducing blast proliferation from leukemic whole blood (WB) samples. Activated leukemia-specific immune and memory cells increased after MLC with Kit-M-pretreated WB, leading to improved blast lysis. Enhanced anti-leukemic activity positively correlated with the frequencies of generated DC/DC_leu, proliferating leukemic-specific T cells and memory T cells, but not with leukemic blast counts, hemoglobin levels or platelet counts at diagnosis. No correlation was found between improved blast lysis and patients’ prognostic data, including age, gender, ELN risk groups, disease stage and response to induction chemotherapy. These findings underscore the potential of DC/DC_leu to evoke robust immune responses and potential immunological memory against AML. Overall, this innovative approach could pave the way for the development of improved immunotherapeutic strategies that function in vivo.

T-cell receptor gamma delta (TCR gamma delta) 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) TCR gamma delta 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 TCR gamma delta expressing leukemia-specific degranulation and intracellularly cytokine producing T-cells were found. The effect of Kit-M-treatment on frequencies of TCR gamma delta expressing cells and their degranulation could be correlated with the Kit-M-mediated blast lysis compared to control. We also found higher frequencies of TCR gamma delta 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 TCR gamma delta expressing T-cells: Moreover a quantification of TCR gamma delta expressing T-cells might contribute to predict prognosis of AML/MDS patients.

Background: Acute myeloid leukemia (AML) is characterized by reduced antileukemic effector cells and increased immunosuppressive cell populations. Leukemia-derived dendritic cells (DC_leu), generated from 18 leukemic whole blood (WB) ex vivo using ‘Kit-M’ (clinically approved: GM-CSF + PGE1), lead to improved cytotoxicity against autologous blasts after mixed lymphocyte culture (MLC) with patients’ T-cells. Methods: We studied Kit-M-mediated effects on frequencies of tolerogenic, immunosuppressive DC (DC_tol) and correlated findings with ex vivo-achieved antileukemic effects (increased intracellular IFNγ production/degranulation, blast lysis) and patients’ clinical characteristics. Results: We show significantly decreased frequencies of DC_tol (and increased frequencies of mature DC_leu) without induced blast proliferation in Kit-M treated vs. untreated WB samples. After T-cell-enriched MLC with Kit-M pretreated vs. not pretreated, WB frequencies of regulatory (CD152+ T-cells) were significantly decreased, while ‘activated’ (IFNγ+, degranulating) non-naive, proliferating, memory, CD154+) T-cells, as well as NK and CIK-cells were (significantly) increased. We found a (significant) positive correlation of achieved improved blast lysis, frequencies of DC_leu and ‘activated’ (IFNγ+/degranulating) T- or NK/CIK cells, and a (significant) negative correlation with frequencies of DC_tol and regulatory (CD152+ T-cells). Kit-M treatment of leukemic WB increases DC_leu and decreases DC_tol, correlating with improved immune reactions/improved cytotoxicity against autologous blasts, and downregulated suppressive T-cells in samples before or after MLC. Conclusions: These findings demonstrate the potential of Kit-M (using clinically approved drug compositions) to treat AML patients to potentially overcome the immunosuppressive tumor microenvironment, leading to improved antileukemic responses—thereby stabilizing remission of the disease in AML patients.