Generation of Leukaemia-Derived Dendritic Cells (DC) to Improve Anti-Leukaemic Activity in AML: Selection of the Most Efficient Response Modifier Combinations.

Dendritic cells (DC) and leukaemia derived DC (DC) 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-generating protocols. With respect to future clinical applications though, DC/DC-generating protocols specifically designed for application in a whole-blood-(WB)-environment must be established. Therefore, we developed ten new DC/DC-generating protocols (kits; Kit-A/-C/-D/-E/-F/-G/-H/-I/-K/-M) for the generation of DC/DC from leukaemic WB, containing calcium-ionophore, granulocyte-macrophage-colony-stimulating-factor (GM-CSF), tumour-necrosis-factor-alpha, prostaglandin-E (PGE), prostaglandin-E (PGE) and/or picibanil (OK-432).

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).

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

Dendritic cells (DC) and T-cells are mediators of CTL-responses. Autologous (from patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS)) or allogeneic (donor)-T-cells stimulated by DC, gain an efficient lysis of naive blasts, although not in every case. CXCL8, -9, -10, CCL2, -5 and Interleukin (IL-12) were quantified by Cytometric Bead Array (CBA) in supernatants from 5 DC-generating methods and correlated with AML-/MDS-patients' serum-values, DC-/T-cell-interactions/antileukemic T-cell-reactions after mixed lymphocyte culture (MLC) and patients' clinical course.

Expression profiles of HMGB1 on B-CLL related leukocytes contribute to prediction of relapse.

Background: The High Mobility Group Box 1 (HMGB1) is a nuclear protein that is frequently overexpressed in hematologic diseases and might be of relevance in immunogenic cancer control thus correlating with patients' (pts.) prognosis in diseases such as acute myeloid, acute lymphatic and chronic lymphocytic leukemia.

Materials And Methods: Expression profiles of blasts from AML (n = 21), ALL (n = 16) and of B-lymphocytes of CLL (n = 9) pts.

WT1, PRAME, and PR3 mRNA Expression in Acute Myeloid Leukemia (AML).

Several tumor-associated antigens (TAAs) were recently identified, that could qualify as targets for immunotherapy, they could qualify (on RNA-level) for monitoring of tumor load. Here, we studied the expression levels of the immunogenic antigens PRAME (preferentially expressed antigen of melanoma), WT1 (Wilms' tumor gene), and PR3 (proteinase 3) on myeloid blasts by real-time quantitative polymerase chain reaction and correlated these data to the state and course of disease and to the defined subgroups of acute myeloid leukemia (AML). At first diagnoses, 41 of 47 patients tested showed overexpression of PRAME (87%), 38 of WT1 (81%), and 26 of PR3 (55%), with the highest expression levels for PRAME (2048-fold), followed by WT1 (486-fold) and PR3 (196-fold).

Serum Chemokine-release Profiles in AML-patients Might Contribute to Predict the Clinical Course of the Disease.

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.

Role of Interferon (IFN)α in "Cocktails" for the Generation of (Leukemia-derived) Dendritic Cells (DCleu) From Blasts in Blood From Patients (pts) With Acute Myeloid Leukemia (AML) and the Induction of Antileukemic Reactions.

Strategies to stabilize remissions by specific elimination of residual acute myeloid leukemia (AML) blasts are needed. Leukemia-derived dendritic cell (DCleu/DC) generated from myeloid blasts improve antileukemic T-cell reactivity and install T-cell memory. Interferon (IFN)α-DC methods produce DCleu from chronic myeloid leukemia-patients (pts') blood.

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 ('DC') 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.

Cytokine Release Patterns in Mixed Lymphocyte Culture (MLC) of T-Cells with Dendritic Cells (DC) Generated from AML Blasts Contribute to Predict anti-Leukaemic T-Cell Reactions and Patients' Response to Immunotherapy.

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.

Expression of 4-1BB and its ligand on blasts correlates with prognosis of patients with AML.

Costimulatory ligands (COLs) and their receptors (COR) regulate immune reactions and cellular survival and might be relevant in acute myeloid leukemia (AML). This study evaluated the clinical relevance of 4-1BBL, glucocorticoid-induced TNFR-related protein (GITR) and ligand (GITRL), CD80, and CD86 in case of expression on AML blasts. 98 patients were evaluated at initial diagnosis.

Expression of RANK-L and in part of PD-1 on blasts in patients with acute myeloid leukemia correlates with prognosis.

Background: Co-stimulatory receptor (COR) and ligand (COL) expression on immune effectors are known to be relevant for immunological interactions and might be of prognostic relevance if expressed on acute myeloid leukemia (AML) blasts as reported for receptors of the tumor necrosis factor receptor family.

Aim And Methods: Antigen expression profiling of COR (RANK, PD-1), COL (RANK-L, PD-1L), and HLA-ABC-antigens on blasts from 90 AML-patients at first diagnosis was performed by flow cytometry (SFI-Level characterization) and findings were correlated with clinical parameters.

Results: RANK expression was higher in immature compared to mature FAB groups (P = 0.

Expression of surface-associated 82kDa-proMMP-9 in primary acute leukemia blast cells inversely correlates with patients' risk.

With its ability to degrade extracellular matrix proteins and activate growth factors and cytokines, matrix metalloproteinase (MMP)-9 is an important regulator of cell function. Previously, we reported that myeloid leukemic cells express a unique 82kDa-proMMP-9 variant on their cell surface that is not affected by its natural inhibitor. In this study, we generated monoclonal antibodies that specifically recognize 82kDa-proMMP-9.

Death Receptor Expression on Blasts in AML Is Associated with Unfavorable Prognosis.

Background: Tumor necrosis factor (TNF) receptor family members play a key role in the regulation of biological functions such as differentiation, proliferation and apoptosis of various cell types.

Materials And Methods: We studied co-expression profiles of death receptors from the TNF family [TNF-related apoptosis-inducing ligand receptor (TRAILR) 1 to 3, TNF receptor 1 (TNFR1) and FAS receptor (FAS)] on peripheral blood blasts from 46 patients with acute myeloid leukemia (AML) at first diagnosis by flow cytometry and correlated the obtained specific fluorescence indices (SFI) with morphological, cytogenetic and clinical parameters.

Results: We found that the expression of TRAILR2 and R3 was significantly increased in unfavorable risk groups, according to the National Comprehensive Cancer Network.

Profiles of activation, differentiation-markers, or β-integrins on T cells contribute to predict T cells' antileukemic responses after stimulation with leukemia-derived dendritic cells.

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.

CD4(+)and CD8(+)T-cell reactions against leukemia-associated- or minor-histocompatibility-antigens in AML-patients after allogeneic SCT.

T-cells play an important role in the remission-maintenance in AML-patients (pts) after SCT, however the role of LAA- (WT1, PR1, PRAME) or minor-histocompatibility (mHag, HA1) antigen-specific CD4(+) and CD8(+)T-cells is not defined. A LAA/HA1-peptide/protein stimulation, cloning and monitoring strategy for specific CD8(+)/CD4(+)T-cells in AML-pts after SCT is given. Our results show that (1) LAA-peptide-specific CD8+T-cells are detectable in every AML-pt after SCT.

Antileukemic T-cell responses can be predicted by the composition of specific regulatory T-cell subpopulations.

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.

Combined immunophenotyping and fluorescence in situ hybridization with chromosome-specific DNA probes allows quantification and differentiation of ex vivo generated dendritic cells, leukemia-derived dendritic cells and clonal leukemic cells in patients with acute myeloid leukemia.

Antileukemic T-cell responses induced by leukemia-derived dendritic cells (DC(leu)) are variable, due to varying DC/DC(leu) composition/quality. We studied DC/DC(leu) composition/quality after blast culture in four DC media by flow cytometry (FC) and combined fluorescence in situ hybridization/immunophenotyping analysis (FISH-IPA). Both methods showed that DC methods produce variable proportions of DC subtypes.

In vitro-induced response patterns of antileukemic T cells: characterization by spectratyping and immunophenotyping.

Myeloid leukemic cells can be induced to differentiate into leukemia-derived dendritic cells (DCleu) regaining the stimulatory capacity of professional DCs while presenting the leukemic antigen repertoire. But so far, the induced antileukemic T-cell responses are variable both in specificity and in efficacy. In an attempt to elucidate the underlying causes of different T-cell response patterns, T-cell receptor (TR) Vβ chain rearrangements were correlated with the T cells corresponding immunophenotypic profile, as well as their proliferative response and cytolytic capacities.

Expression and prognostic value of FAS receptor/FAS ligand and TrailR1/TrailR2 in acute myeloid leukemia.

We studied the expressions of FR, FL, TR1, and TR2 on blasts and T cells from 71 patients with acute myeloid leukemia (AML) and correlated expression rates with the clinical course. Compared to AML-blasts we found higher co-expressions on healthy myeloid and T cells. Expression of all markers on blasts and on T cells was similar in different subtypes and acute stages of AML.

Various 'dendritic cell antigens' are already expressed on uncultured blasts in acute myeloid leukemia and myelodysplastic syndromes.

Aim And Methods: Leukemia-derived dendritic cells (DC(leu)) potentially present the whole leukemic antigen repertoire. We studied antigen-expression profiles of blasts/dendritic cells (DCs) generated from 137 acute myeloid leukemia (AML)/49 myelodysplastic syndromes (MDS) patients with six different DC-generating media by flow-cytometry combining expression of blast/maturation and DC antigens (DCA:CD1a,b,c, CD25, CD40, CD80, CD83, CD86, CD137-L and CD206).

Results: First, DCA are regularly and variably expressed on uncultured blasts/mononuclear cells (MNCs).

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.

Myeloid leukemic cells can differentiate into leukemia-derived dendritic cells (DC(leu)), presenting known/unknown leukemic-antigens. Induced anti-leukemic T-cell-responses are variable. To further elicit DC/DC(leu)-induced T-cell-response-patterns we performed (functional)flow-cytometry/fluorolysis-assays before/after mixed lymphocyte cultures (MLC) 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)-containing DC ('DC').

The quality and quantity of leukemia-derived dendritic cells from patients with acute myeloid leukemia and myelodysplastic syndrome are a predictive factor for the lytic potential of dendritic cells-primed leukemia-specific T cells.

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.

Dendritic cells (DCs) can be successfully generated from leukemic blasts in individual patients with AML or MDS: an evaluation of different methods.

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").

Improved effector function of leukemia-specific T-lymphocyte clones trained with AML-derived dendritic cells.

Recently it was shown that myeloid leukemic cells can be induced to differentiate into leukemia-derived dendritic cells (DCleu), regaining the stimulatory capacity of professional DCs while presenting the leukemic antigen repertoire. But so far, the induced antileukemic T-cell responses have varied in specificity and efficacy, or have even mediated opposite effects. In an attempt to further characterize the DC/DCleu induced T-cell response pattern, immunoscope spectratyping, a novel and powerful tool to detect T-cell receptor (TCR) rearrangements was used in combination with functional flow cytometry and non-radioactive fluorolysis assays.