Kufner S, Zitzelsberger H, Kroell T, Pelka-Fleischer R, Salem A, de Valle F, Schmid C, Schweiger C, Kolb H J, Schmetzer H M
Medical Department III, Klinikum Grosshadern, University of Munich, Munich, Germany.
Scand J Immunol. 2005 Jul;62(1):75-85. doi: 10.1111/j.1365-3083.2005.01631.x.
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 AML patients giving rise to APC of leukaemic origin presenting leukaemic antigens. In a comparative methodological analysis of 50 AML samples, we could already show that leukaemia-derived DC can regularly be generated under serum-free culture conditions. In this study, we describe the generation and characterization of DC from different mononuclear cell (MNC) fractions from 24 myelodysplastic syndrome (MDS) patients under those 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 PB or BM-MNC, thawn or fresh MNC, in Xvivo or CellGro serum-free media, +/-10% autologous plasma or +/-FL. In detail, we could show that MDS-DC harvests compared to healthy DC were higher after 10- to 14-day culture; total or adherent PB or BM-MNC fractions yield comparable DC counts; however, from 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 MDS samples was obtained with a GM-CSF, IL-4, FL and TNF-alpha containing serum-free Xvivo medium after 10-14 days of culture (18/26% DC; 54/64% vital DC; 59/51% mature DC were generated from MDS/healthy MNC samples). Surface marker profiles (e.g. costimulatory antigen expression) of DC obtained from MDS samples were comparable with that of healthy DC. The leukaemic derivation of MDS-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 MDS. Autologous T cells proliferate and upregulate DC-contact-relevant antigens. We are the first who demonstrate that the generation of leukaemia-derived DC is feasible not only in AML but also in MDS under serum-free culture conditions giving rise to DC with comparable characteristics as healthy DC and offering an antileukaemia-directed immunotherapeutical vaccination strategy in AML and MDS.
功能性树突状细胞（DC）是专业的抗原呈递细胞（APC），可在体外从健康细胞以及急性髓系白血病（AML）患者的白血病细胞中生成，从而产生呈递白血病抗原的白血病来源的APC。在对50份AML样本进行的比较方法分析中，我们已经能够证明在无血清培养条件下可常规生成白血病来源的DC。在本研究中，我们描述了在这些不同的无血清培养条件下，从24例骨髓增生异常综合征（MDS）患者的不同单核细胞（MNC）组分中生成DC并进行表征，确定最佳培养条件，并将结果与23名健康供体的结果进行比较。在平行培养中，我们比较了在7天或14天培养后，使用总MNC或贴壁MNC或T细胞去除的MNC或外周血（PB）或骨髓（BM）-MNC、解冻或新鲜的MNC，在Xvivo或CellGro无血清培养基中，±10%自体血浆或±FL时的DC收获量。详细而言，我们能够证明，与健康DC相比，MDS-DC在培养10至14天后收获量更高；总PB或BM-MNC组分或贴壁PB或BM-MNC组分产生的DC数量相当；然而，从MACS去除的MNC组分或解冻的MNC中产生的DC数量较低。虽然添加FL可增加DC收获量，但在许多情况下添加自体血浆对DC成熟有抑制作用，CellGro和Xvivo培养基产生的DC数量相当。培养10 - 14天后，使用含有GM-CSF、IL-4、FL和TNF-α的无血清Xvivo培养基可从MDS样本中获得活力和成熟DC的最佳收获量（从MDS/健康MNC样本中产生18/26%的DC；54/64%的活力DC；59/51%的成熟DC）。从MDS样本中获得的DC的表面标志物谱（如共刺激抗原表达）与健康DC相当。通过DC中克隆细胞遗传学异常的持续存在或DC上白血病抗原的共表达证明了MDS-DC的白血病来源。在MDS病例中证明了白血病来源的DC的自体T细胞激活。自体T细胞增殖并上调与DC接触相关的抗原。我们是首个证明在无血清培养条件下，白血病来源的DC不仅在AML中而且在MDS中均可生成，所产生的DC具有与健康DC相当的特征，并为AML和MDS提供了一种抗白血病导向的免疫治疗疫苗策略的研究团队。
