Transduction of dendritic cell progenitors with a retroviral vector encoding viral interleukin-10 and enhanced green fluorescent protein allows purification of potentially tolerogenic antigen-presenting cells.
Transduction of dendritic cell progenitors with a retroviral vector encoding viral interleukin-10 and enhanced green fluorescent protein allows purification of potentially tolerogenic antigen-presenting cells.
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DOI：
10.1097/00007890-199912270-00015
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发表时间：
1999-12
期刊：
影响因子：
6.2
通讯作者：
T. Takayama;H. Tahara;A. Thomson
中科院分区：
文献类型：
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作者：
T. Takayama;H. Tahara;A. Thomson
树突状细胞（DC）是重要的抗原呈递细胞，在免疫应答的启动和调节中起着关键作用。DC基因工程表达免疫抑制分子是抑制同种异体移植排斥反应的新途径。逆转录病毒将病毒白细胞介素(vIL)-10传递给复制的髓系DC祖细胞（DCp），会损害它们的t细胞刺激能力，并促进抗原特异性t细胞的低反应性诱导。然而，逆转录病毒载体的转导效率相对较低。增强型绿色荧光蛋白（EGFP）作为基因转导的标志和转导细胞的选择是重要的。我们的目的是构建一个同时编码vIL-10和EGFP的逆转录病毒载体，对转导的DC进行阳性选择，并评估这些高度纯化的、分泌vIL-10的抗原呈递细胞对异体t细胞反应的影响。方法以粒细胞-巨噬细胞集落刺激因子(GM-CSF)+IL-4转染C57BL10 （H2b）小鼠骨髓，用编码il -10和EGFP的逆转录病毒载体离心增强转导DCp。流式细胞术检测基因转移效率。转导后的细胞进行流式分选，ELISA法测定细胞中il -10的分泌量。DC功能通过细胞诱导幼稚异体（C3H; H2k） T细胞增殖和细胞毒性T淋巴细胞生成的能力来评估。结果逆转录病毒转导的DC均表达了vIL-10和EGFP基因产物。通过流式细胞术测定，约20%的未分选细胞表达EGFP。il -10的平均产率为31 ng/40 hr/10(6)个细胞。分选后，EGFP+ DC的发生率急剧增加至至少95%，vIL-10的产量增加了约3至4倍，平均为107 ng/40 hr/10(6)个细胞。这些高度纯化的分泌vil -10的DC诱导同种异体t细胞增殖和细胞毒性反应的能力明显减弱。结论逆转录转导表达il -10和EGFP的DCp可快速鉴定和分选，纯度高。高富集的vil -10转导DC制剂的可用性促进了其免疫调节功能的研究，并可能增强其在移植或自身免疫性疾病中的治疗潜力。
BACKGROUND Dendritic cells (DC) are important antigen-presenting cells that play critical roles in the initiation and modulation of immune responses. Genetic engineering of DC to express immunosuppressive molecules is a novel approach to the inhibition of allograft rejection. Retroviral delivery of viral interleukin (vIL)-10 to replicating myeloid DC progenitors (DCp) impairs their T-cell stimulatory capacity and promotes the induction of antigen-specific T-cell hyporesponsiveness. However, transduction efficiency with retroviral vectors is comparatively low. Enhanced green fluorescent protein (EGFP) is important both as a marker of gene transduction and for the selection of transduced cells. Our aims were to construct a retroviral vector encoding both vIL-10 and EGFP, to positively select transduced DC, and to assess the impact of these highly purified, vIL-10-secreting antigen-presenting cells on allogeneic T-cell responses. METHODS DCp propagated from bone marrow of C57BL10 (H2b) mice in granulocyte-macrophage colony-stimulating factor (GM-CSF)+IL-4 were transduced with a retroviral vector encoding both vIL-10 and EGFP by centrifugal enhancement. Gene transfer efficiency was determined by flow cytometry. Transduced cells were flow sorted, and vIL-10 secretion was quantified by ELISA. DC function was assessed by the ability of the cells to induce naive allogeneic (C3H; H2k) T-cell proliferation and cytotoxic T lymphocyte generation. RESULTS Retrovirally transduced DC expressed both vIL-10 and EGFP gene products. Approximately 20% of unsorted cells expressed EGFP, as determined by flow cytometry. vIL-10 was produced at a mean rate of 31 ng/40 hr/10(6) cells. After sorting, the incidence of EGFP+ DC was increased dramatically to at least 95%, and the production of vIL-10 was increased approximately three- to fourfold, to a mean of 107 ng/40 hr/10(6) cells. These highly purified, vIL-10-secreting DC exhibited markedly diminished capacity to induce allogeneic T-cell proliferative and cytotoxic responses. CONCLUSIONS DCp retrovirally transduced to express both vIL-10 and EGFP can be rapidly identified and sorted to high levels of purity. The availability of highly enriched preparations of vIL-10-transduced DC facilitates studies of their immunoregulatory function and may enhance their therapeutic potential in transplantation or autoimmune disease.