Salmon P, Arrighi J F, Piguet V, Chapuis B, Zubler R H, Trono D, Kindler V
Department of Genetics and Microbiology, Faculty of Medicine, Geneva University Hospital, Switzerland.
J Gene Med. 2001 Jul-Aug;3(4):311-20. doi: 10.1002/1521-2254(200107/08)3:4<311::AID-JGM198>3.0.CO;2-B.
Genetically engineered dendritic cells (DC) presenting specific antigens to T cells may be of great interest for immunotherapy. For this reason, the production of transgene-expressing DC derived from CD34 + cells transduced either shortly after ex vivo purification or during their differentiation into DC were evaluated.
CD34+ cells were transduced with lentivectors encoding for GFP before or after 21 days of culture with FLT3-ligand, thrombopoietin and stem cell factor and induction into DC with GM-CSF+IL-4 (G4) or G4+TNF (GT4). GFP and DC-specific marker expression was assessed by flow cytometry, and allostimulatory capacity was evaluated on GFP+ and GFP- sorted cells.
Immature (G4-induced) DC obtained from amplified CD34 + cells were transducible by lentiviral vectors while mature (GT4-induced) DC were rather refractory. Moreover, since differentiated DC did not proliferate, large quantities of vectors were required to generate transgene-expressing cells with this protocol. In contrast, greater numbers of both immature and mature GFP- expressing DC were obtained with CD34+ cells exposed to lentivector shortly after purification. By the time of DC induction, GFP+ cells had increased by approximately 170-fold. After DC induction with G4, 32% of CD1a+, HLA-DR+, or CD40+ cells expressed GFP. CD1a+E-cadherin+ GFP+ Langerhans-like DC were also obtained. Incubation with TNF induced mature CD83+GFP+ DC that displayed a higher allostimulatory capacity than cells induced with G4 alone.
The transduction of a small number of CD34+ cells with minimal doses of lentivector may allow for the production of a large number of DC expressing selected antigens useful for immunotherapy.
基因工程改造的树突状细胞（DC）向T细胞呈递特定抗原，这可能对免疫治疗具有重要意义。因此，我们评估了从CD34 +细胞衍生的表达转基因的DC的产生情况，这些CD34 +细胞在体外纯化后不久或在其分化为DC的过程中被转导。
在用FLT3配体、血小板生成素和干细胞因子培养21天并分别用GM-CSF+IL-4（G4）或G4+TNF（GT4）诱导分化为DC之前或之后，用编码绿色荧光蛋白（GFP）的慢病毒载体转导CD34+细胞。通过流式细胞术评估GFP和DC特异性标志物的表达，并对GFP+和GFP-分选细胞的同种异体刺激能力进行评估。
从扩增的CD34 +细胞获得的未成熟（G4诱导）DC可被慢病毒载体转导，而成熟（GT4诱导）DC则较难转导。此外，由于分化后的DC不增殖，因此需要大量载体才能通过该方案产生表达转基因的细胞。相比之下，在纯化后不久将CD34+细胞暴露于慢病毒载体，可获得更多数量的未成熟和成熟的GFP表达DC。到DC诱导时，GFP+细胞增加了约170倍。用G4诱导DC后，32%的CD1a+、HLA-DR+或CD40+细胞表达GFP。还获得了CD1a+E-钙黏蛋白+GFP+朗格汉斯样DC。用TNF孵育可诱导成熟的CD83+GFP+DC，其同种异体刺激能力高于单独用G4诱导的细胞。
用最小剂量的慢病毒载体转导少量CD34+细胞，可能有助于产生大量表达选定抗原的DC，用于免疫治疗。
