Leukemia‐Derived Dendritic Cells can be Generated from Blood or Bone Marrow Cells from Patients with Acute Myeloid Leukaemia: A Methodological Approach under Serum‐Free Culture Conditions

Abstract

Functional dendritic cells (DC) are professional antigen-presenting cells (APC) and can be generated in vitro from healthy as well as from leukaemic cells from acute myeloid leukemia (AML) patients giving rise to APC of leukaemic originpresenting leukaemic antigens. We describe the generation and characterization of DC from different mononuclear cell (MNC) fractions from 50 AML patients under different serum-free culture conditions, determine the optimal culture conditions and compare the results with that from 23 healthy donors. In parallel cultures, we compared DC harvests after 7-or 14-day culture, with total or adherent MNC or T-cell depleted MNC or peripheral blood (PB) or bone marrow-MNC (BM-MNC), thawn or fresh MNC, in Xvivo or CellGro serumfree media, AE10% autologous plasma or AEFL. In detail, we could show that AML-DC harvests were higher after 10-14 days culture (healthy DC: 7 days); total or adherent PB or BM-MNC fractions yield comparable DC counts, however, from magnetic cell sorting (MACS)-depleted MNC fractions or thawn MNC lower DC counts can be generated. Whereas the addition of FL increases the DC harvest, the addition of autologous plasma in many cases has inhibitory influence on DC maturation. CellGro and Xvivo media yield comparable DC counts. Optimal harvest of vital and mature DC from AML samples was obtained with a granulocyte/macrophage-colony stimulating factor, interleukin-4, FL and tumour necrosis factor-a-containing serum-free Xvivo medium after 10-14 days of culture (36/26% DC; 38/64% vital DC; 46/51% mature DC were generated from AML/healthy MNC samples). Surface marker profiles (e.g. costimulatory antigen expressing) of DC obtained from AML samples were comparable with that of healthy DC. The leukaemic derivation of AML-DC was demonstrated by the persistence of the clonal cytogenetic aberration in the DC or by coexpression of leukaemic antigens on DC. Autologous T-cell activation of leukaemia-derived DC was demonstrated in cases with AML. Autologous T cells proliferate and upregulate DC-contact-relevant antigens. We demonstrate that the generation of leukaemia-derived DC is feasable in AML under serum-free culture conditions giving rise to DC with comparable characteristics as healthy DC and offering an anti-leukaemia-directed immunotherapeutical vaccination strategy in AML.

Aberrant Phenotype and Function of Myeloid Dendritic Cells in Systemic Lupus Erythematosus

“…In addition, their cells were cultured for longer periods of time and under conditions that were not serum-free. It has been demonstrated that serum contains growth factors that could affect DC development, differentiation and maturation in vitro (87,88) and that serum-free conditions like the ones used in our study lead to more optimal DC harvest that non-serum free conditions (89). Furthermore, Koller et al (54) used irradiated DCs for their mixed-lymphocyte reaction studies, while we used nonirradiated DCs.…”

Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response

“…26,27 Immunological and also clinical responses observed in these patients prompted researchers in the field to extend the concept of DC vaccination also to leukemia patients. [28][29][30][31] In our previous study with B-CLL patients, 12 we showed that immune and to some extent clinical responses could be achieved in B-CLL patients using allogeneic DCs as anticancer vaccine. Since DC precursor cells were obtained from HLAunmatched donors, one might speculate whether immune and clinical responses in the vaccinated patients might have been related to a specific antigen recognition or rather to an unspecific allo-reaction.…”

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

“…However, in a few cases, T-cell clones mediating anergy or even blast proliferation could be observed and it already became obvious that both quantity and quality of the generated DC leus as well as the induced microenvironment might be crucial for the induction of a protective T-cell response [20,31,37].…”

“…But in some cases, T cells are generated mediating anergy or even tumor cell growth [20,31,37]. To further elucidate the underlying cause for this opposite T-cell response pattern, we studied the impact of blast-or DC/DC leu -mediated stimulation on T-cell differentiation in context of an autologous (#546), human leukocyte antigen (HLA)-identical (#502) and haplo-identical (#538) experimental setting.…”

“…To evaluate amounts of viable (7AAD -) target T cells and to quantify the cell loss after the corresponding incubation time, cells were harvested, washed in PBS and resuspended in a FACS flow solution containing 7AAD (BD, Biosciences Pharmingen, Heidelberg, Germany) and a defined number of fluorosphere beads (Becton-Dickinson, Heidelberg, Germany). Viable, 7AAD negative cells coexpressing specific blast marker (combinations) were quantified taking into account defined counts of calibration beads as described [29][30][31]. Cells were analyzed in a BD FACSCalibur flow cytometer using CellQuest software (Becton Dickinson, Heidelberg, Germany).…”