Extensive replicative capacity of human central memory T cells
Extensive replicative capacity of human central memory T cells
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DOI：
10.4049/jimmunol.172.11.6675
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发表时间：
2004-06-01
影响因子：
4.4
通讯作者：
June, CH
中科院分区：
文献类型：
--
作者：
Maus, MV;Kovacs, B;June, CH
为了表征人中枢记忆（T-CM）CD 4 T细胞的复制能力，我们已经开发了一种确定的培养系统，其优化用于Ag特异性CD 4（+）T细胞的离体扩增。制备由涂覆有针对HLA II类的Ab和针对CD 28的共刺激Ab的磁珠组成的人工APC（aAPC）;然后将带肽的HLA II类四聚体加载到珠上以提供Ag特异性。用流式细胞术从正常献血员外周血中分离流感特异性DR*0401 CD 4 T-CM。负载肽的aAPC不足以诱导静息CD 4 T-CM增殖。相反，我们发现珠粒有效地促进先前活化的CD 4 T-CM细胞的生长，在两次刺激后产生具有>80% Ag特异性CD 4细胞的培养物。用负载肽的aAPC进一步刺激增加纯度至>99% Ag特异性T细胞。体外培养3-12周后，流感特异性CD 4 T-CM的表面标志物与CD 8 T细胞所述的效应表型基本一致，但维持CD 28表达除外。T-CM能够在体外进行20-40次平均群体倍增，并且扩增的细胞响应于Ag产生IFN-γ、IL-2和TNF-α，并且一个亚群的细胞还在PMA/离子霉素处理下分泌IL-4。总之，aAPC扩增具有广泛复制能力的T-CM，并且在过继免疫治疗以及研究人MHC II类限制性T细胞的生物学中具有潜在的应用。
To characterize the replicative capacity of human central memory (T-CM) CD4 T cells, we have developed a defined culture system optimized for the ex vivo expansion of Ag-specific CD4(+) T cells. Artificial APCs (aAPCs) consisting of magnetic beads coated with Abs to HLA class II and a costimulatory Ab to CD28 were prepared; peptide-charged HLA class II tetramers were then loaded on the beads to provide Ag specificity. Influenza-specific DR*0401 CD4 T-CM were isolated from the peripheral blood of normal donors by flow cytometry. Peptide-loaded aAPC were not sufficient to induce resting CD4 T-CM to proliferate. In contrast, we found that the beads efficiently promoted the growth of previously activated CD4 T-CM cells, yielding cultures with >80% Ag-specific CD4 cells after two stimulations. Further stimulation with peptide-loaded aAPC increased purity to >99% Ag-specific T cells. After in vitro culture for 3-12 wk, the flu-specific CD4 T-CM had surface markers that were generally consistent with an effector phenotype described for CD8 T cells, except for the maintenance of CD28 expression. The T-CM were capable of 20-40 mean population doublings in vitro, and the expanded cells produced IFN-gamma, IL-2, and TNF-alpha in response to Ag, and a subset of cells also secreted IL-4 with PMA/ionomycin treatment. In conclusion, aAPCs expand T-CM that have extensive replicative capacity, and have potential applications in adoptive immunotherapy as well as for studying the biology of human MHC class II-restricted T cells.