title: Serum-free generation and quantification of functionally active Leukemia-derived DC is possible from malignant blasts in acute myeloid leukemia and myelodysplastic syndromes.
abstract: Functional dendritic cells (DC) are professional antigen presenting cells (APC) and can be generated in vitro from leukemic cells from acute myeloid leukemia AML patients, giving rise to APC of leukemic origin presenting leukemic antigens (DC(leu)). We have already shown that DC can be successfully generated from AML and myeloplastic syndromes (MDS) cells in serum-free 'standard' medium (X-vivo + GM-CSF + IL-4 +TNFalpha + FL) in 10-14 days. In this study, we present that DC counts generated from mononuclear cells (MNC) varied between 20% (from 55 MDS samples), 34% (from 100 AML samples) and 25% (from 38 healthy MNC samples) medium. Between 53% and 58% of DC are mature CD83+ DC. DC harvests were highest in monocytoid FAB types (AML-M4/M5, MDS-CMML) and independent from cytogenetic risk groups, demonstrating that DC-based strategies can be applied for patients with all cytogenetic risk groups. Proof of the clonal derivation of DC generated was obtained in five AML and four MDS cases with a combined FISH/immunophenotype analysis (FISH-IPA): The clonal numerical chromosome aberrations of the diseases were regularly codetectable with DC markers; however, not with all clonal cells being convertible to leukemia-derived DC(leu) (on average, 53% of blasts in AML or MDS). To the contrary, not all DC generated carried the clonal aberration (on average, 51% of DC). In 41 AML and 13 MDS cases with a suitable antigen expression, we could confirm FISH-IPA data by Flow cytometry: although DC(leu) are regularly detectable, on average only 57% of blasts in AML and 64% of blasts in MDS were converted to DC(leu). After coculture with DC in mixed lymphocyte reactions (MLR), autologous T cells from AML and MDS patients proliferate and upregulate costimulatory receptors. The specific lysis of leukemic cells by autologous T cells could be demonstrated in three cases with AML in a Fluorolysis assay. In six cases with only few DC(leu) or few vital T cells available after the DC/MLR procedure, no lysis of allogeneic or autologous leukemic cells was seen, pointing to the crucial role of both partners in the lysis process. We conclude: (1) the generation of DC is regularly possible in AML and also in MDS under serum-free conditions. (2) Clonal/leukemia-derived DC(leu) can be regularly generated from MDS and AML-MNC; however, not with all blasts being converted to DC(leu) and not all DC generated carrying leukemic markers. We recommend to select DC(leu) for vaccinations or ex vivo T-cell activations to avoid contaminations with non-converted blasts and non-leukemia-derived DC and to improve the harvest of specific, anti-leukemic T cells. DC and DC-primed T cells could provide a practical strategy for the immunotherapy of AML and MDS.
authors.affiliation: Medical Department III, Klinikum Grosshadern, University of Munich, Marchioninistr 15, 81377 Munich, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Kufner
authors.forename: R Pelka
authors.initials: RP
authors.lastname: Fleischer
authors.forename: T
authors.initials: T
authors.lastname: Kroell
authors.forename: C
authors.initials: C
authors.lastname: Schmid
authors.forename: H
authors.initials: H
authors.lastname: Zitzelsberger
authors.forename: H
authors.initials: H
authors.lastname: Salih
authors.forename: F
authors.initials: F
authors.lastname: de Valle
authors.forename: W
authors.initials: W
authors.lastname: Treder
authors.forename: H M
authors.initials: HM
authors.lastname: Schmetzer
mesh_terms: D000208:Acute Disease; D000328:Adult; D000368:Aged; D000938:Antigen-Presenting Cells; D015703:Antigens, CD; D018122:B7-1 Antigen / Q000276:immunology / Q000378:metabolism; D001752:Blast Crisis; D018929:Cell Culture Techniques; D016895:Culture Media, Serum-Free; D003713:Dendritic Cells / Q000276:immunology*; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology / Q000378:metabolism; D015847:Interleukin-4 / Q000494:pharmacology; D007951:Leukemia, Myeloid / Q000276:immunology* / Q000628:therapy; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology*; D008213:Lymphocyte Activation; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D008875:Middle Aged; D009190:Myelodysplastic Syndromes / Q000276:immunology* / Q000628:therapy; D013601:T-Lymphocytes / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000097802:CD83 Antigen
chemical_list: D015703:Antigens, CD; D018122:B7-1 Antigen; D016895:Culture Media, Serum-Free; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007136:Immunoglobulins; D015847:Interleukin-4; D008562:Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D000097802:CD83 Antigen