Conversion of AML-blasts to leukemia-derived dendritic cells (DCleu) in ‘DC-culture-media’ shifts correlations of released chemokines with antileukemic T-cell reactions

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.

Acute myeloid leukemia (AML) remains a devastating diagnosis in clear need of therapeutic advances. Both targeted dendritic cells (DC) and particularly leukemia-derived dendritic cells (DCleu) can exert potent anti-leukemic activity. By converting AML blasts into immune activating and leukemia-antigen presenting cells, DC/DCleu-generating protocols can induce immune responses against AML blasts. Such protocols combine approved response modifiers (i.e., GM-CSF and PGE1/OK-432/PGE2) that synergistically improve the conversion of AML blasts into (mature) DC/DCleu. To guide potential clinical application of these response modifiers, we analyzed three different DC-generating protocols that combine a constant GM-CSF dose with varying concentrations of PGE1 (Kit-M), OK-432 (Kit-I), and PGE2 (Kit-K). Here, we specifically aimed to assess how different response modifier concentrations impact DC/DCleu generation, immune cell activation and leukemic blast lysis. We found that all immunomodulatory kits were effective in generating mature and leukemia-derived DCs from healthy and leukemic whole blood. For Kit-M, we noted optimal generation of DC-subsets at intermediary concentration ranges of PGE1 (0.25-4.0 µg/mL), which facilitated upregulation of activated and memory T-cells upon mixed lymphocyte culture, and efficient anti-leukemic activity in cytotoxicity assays. For Kit-I, we observed DC/DCleu generation and enhanced T- and immune cell activation across a broader range of OK-432 concentrations (5-40 µg/mL), which also facilitated improved leukemic blast killing. In conclusion, our results highlight that Kit-mediated DC/DCleu generation, immune cell activation and blast lysis are dependent on the concentration of response modifiers, which will guide future clinical development. Overall, DCleu-based immunotherapy represents a promising treatment strategy for AML patients.

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 (DCleu) 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/DCleu and leukemic-specific T cells with the achieved anti-leukemic activity after MLC. We show that mature DC/DCleu 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/DCleu, 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/DCleu 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.

Quality of Leukemia-Derived Dendritic Cells (DC) and T-Cells Are Predictive for the Specific Anti-Leukemic Potential of DC-Trained T-Cells.

Quality of T-cells after stimulation with leukemia-derived dendritic cells (DC) from patients with acute myeloid leukemia (AML) or myeloid dysplastic syndrome (MDS) is predictive for their leukemia cytotoxic potential

Preliminary anti-leukemia activity from A phase 1 study of cln-049, a novel anti-FLT3 x anti-CD3 bispecific T-cell engager, in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) and myelodysplastic syndrome (MDS)

Hypoxia can modulate the immune system by affecting the function and activity of immune cells, potentially leading to altered immune responses. This study investigated the generation of leukemia-derived dendritic cells (DCleu) from leukemic blasts and their impact on immune cell activation under hypoxic (5-10% O2) compared to normoxic (21% O2) conditions using various immunomodulatory Kits. The results revealed that DC/DCleu-generation was similar under hypoxic and normoxic conditions, with no significant differences observed in frequencies of generated DC/DCleu. Furthermore, the study showed that the activation of immune cells and their anti-leukemic activity improved when T cell-enriched immunoreactive cells were co-cultured with DC/DCleu which were generated with Kit I and M compared to the control after mixed lymphocyte cultures. The anti-leukemic activity was improved under hypoxic compared to normoxic conditions after MLCWB-DC Kit M. These findings suggest that DC/DCleu-cultures of leukemic whole blood with Kits under hypoxic conditions yield comparable frequencies of DC/DCleu and can even increase the anti-leukemic activity compared to normoxic conditions. Overall, this research highlights the potential of utilizing DC/DCleu (potentially induced in vivo with Kits) as a promising approach to enhance immune response in patients with acute myeloid leukemia.

Impact of the hypomethylating agent 5-azacytidine on dendritic cells function

Invariant natural killer T (iNKT)/natural killer (NK)/cytokine-induced killer (CIK) cells are important for immune surveillance. (I) Novel combinations of antibody 6B11 (targeting the Vα24-Jα18-invariant T-cell receptor) with CD4/CD8/CD1d/Vα24 for iNKT subset detection and "T/NK cell-like"-iNKT subsets were defined. Compared with healthy peripheral blood mononuclear cells (MNC) (significantly) lower proportions of iNKT cells (6B11/6B11CD3/6B11CD161), NK cells (CD3CD56/CD3CD161), and CIK cells (CD3CD56/CD3CD161) were found in peripheral blood MNC from acute myeloid (AML)/acute myeloid, lymphoid (ALL)/chronic lymphoid leukemia (CLL) patients in acute disease stages. Subtyping of iNKT cells revealed (significantly) higher proportions of CD3 T cells and CD161 NK cells in AML/ALL/CLL expressing 6B11 compared with healthy MNC. Prognostic evaluations showed higher proportions of iNKT/NK/CIK cells in favorable AML subgroups (younger age, primary, no extramedullary disease, achievement/maintenance of complete remission) or adult ALL and CLL patients. (II) iNKT/NK/CIK cell frequencies increased after (vs. before) mixed lymphocyte cultures of T-cell-enriched immune reactive cells stimulated with MNC/whole blood with or without pretreatment with "cocktails" (dendritic cells generating methods/kits inducing blasts' conversion to leukemia-derived dendritic cells from AML patients). Individual "cocktails" leading to "highest" iNKT cell frequencies could be defined. Antileukemic blast lytic activity correlated significantly with frequencies of iNKT/NK/CIK cells. In summary healthy MNC show significantly more iNKT/NK/CIK cells compared with AML/ALL/CLL MNC, a shift in the iNKT cell composition is seen in healthy versus leukemic samples and iNKT/NK/CIK cell-proportions in AML/ALL/CLL MNC samples correlate with prognosis. "Cocktail"-treated AML blasts lead to higher iNKT/NK/CIK cell frequencies and samples with antileukemic activity show significantly higher frequencies of iNKT/NK/CIK cells. Proportions of iNKT/NK/CIK cells should regularly be evaluated in AML/ALL/CLL diagnosis panels for quantitative/prognostic estimation of individual patients' antileukemic potential and their role in dendritic cells/leukemia-derived dendritic cells triggered immune surveillance.

Regulatory T cells (Treg) are important regulators of immune responses. In acute myeloid leukemia (AML) patients before/after immunotherapy (stem cell transplantation or donor lymphocyte infusion), their suppressive role can contribute to suppress severe graft-versus-host reactions, but also to impair antileukemic reactions. As leukemia-derived dendritic cells (DCleu) are known to improve the antileukemic functionality of T cells, we evaluated the composition and development of distinct Treg subtypes in AML patients (n=12) compared with healthy probands (n=5) under unstimulated conditions and during stimulation with DCleu-containing DC (DC) or blast-containing mononuclear cells (MNC) in 0- to 7-day mixed lymphocyte cultures by flow cytometry. T-cell subgroups in AML patients were correlated with antileukemic functionality before and after DC or MNC stimulation by functional fluorolysis assays. (1) AML patients' T cells presented with significantly higher frequencies of Treg subgroups in unstimulated T cells compared with healthy probands. (2) After 7 days of DC or MNC stimulation, all Treg subtypes generally increased; significantly higher frequencies of Treg subtypes were still found in AML patients. (3) Antileukemic cytotoxicity was achieved in 36% of T cells after MNC compared with 64% after DC stimulation. Antileukemic activity after DC but not after MNC stimulation correlated with significantly lower frequencies of Treg subtypes (CD8Treg/Teff/em reg). Furthermore, cut-off values for Treg subpopulations could be defined, allowing a prediction of antileukemic response. We demonstrate a crucial role of special Treg subtypes in the mediation of antileukemic functionality. High CD8 Treg, Teff/em reg, and CD39 T cells correlated clearly with a reduced antileukemic activity of T cells. DC stimulation of T cells contributes to overcome impaired antileukemic T-cell reactivity. Refined analyses in the context of clinical responses to immunotherapies and graft versus host reactions are required.

Stem cell transplantations and donor lymphocyte infusions are promising immunotherapies to cure acute myeloid leukemia (AML). Leukemia-derived dendritic cells are known to improve antileukemic functionality of T cells. We evaluated the composition and development of distinct T-cell subtypes in AML patients (n=12) compared with healthy probands (n=5) before and during stimulation with leukemia-derived dendritic cells-containing DC (DC) or blast-containing mononuclear cells (MNC) in 0-7 days mixed lymphocyte cultures (MLC) by flow cytometry. AML patients' T-cell subgroups were correlated with antileukemic functionality before and after DC/MNC stimulation by functional fluorolysis assays. (1) Unstimulated T cells from AML patients presented with significantly lower proportions of activated, Tcm, CD137, and β-integrin T cells, and significantly higher proportions of Tnaive and Teff compared with healthy probands. (2) After 7 days of DC or MNC stimulation, T-cell profiles were characterized by (significantly) increased proportions of activated T cells with effector function and significantly decreased proportions of β-integrin T cells. (3) Antileukemic cytotoxicity was achieved in 40% of T cells after MNC stimulation compared with 64% after DC stimulation. Antileukemic activity after DC stimulation but not after MNC stimulation correlated with higher proportions of Tcm and Tnaive before stimulation, as well as with significantly higher proportions of activated and β-integrin T cells. Furthermore, cutoff values for defined T-cell activation/differentiation markers and β-integrin T cells could be defined, allowing a prediction of antileukemic reactivity. We could demonstrate the potential of the composition of unstimulated/DC-stimulated T cells for the lysis of AML blasts. Especially, AML patients with high numbers of Tnaive and Tcm could benefit from DC stimulation; proportions of activated and β-integrin T cells correlated with increased antileukemic functionality and could serve to predict T cells' reactivity during stimulation. Refined analyses in the context of responses to immunotherapies are required.

Deficit of microRNA (miR)-142 Promotes Transformation of Clonal Myeloid Disorder into Acute Myeloid Leukemia (AML)

Dendritic Cell Subsets in Bone Marrow and Peripheral Blood of Patients with Myelodysplastic Syndromes Display Numeric and Functional Defects

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 = 21, respectively), we determined cytokine-concentrations (interleukin 2, 4, 6, 10, tumor-necrosis-factor-α, interferon-γ) in supernatants of mixed-lymphocyte-culture and in serum (n = 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.

Immunogenicity and Antileukemic Activity of Dendritic Cells Electroporated with Wilms' Tumor WT1 mRNA: A Phase I/II Trial in Acute Myeloid Leukemia