Cytokine production and cytolytic mechanism of CD4(+) cytotoxic T lymphocytes in ex vivo expanded therapeutic Epstein-Barr virus-specific T-cell cultures.
Cytokine production and cytolytic mechanism of CD4(+) cytotoxic T lymphocytes in ex vivo expanded therapeutic Epstein-Barr virus-specific T-cell cultures.
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DOI：
10.1182/blood.v99.9.3302
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发表时间：
2002-05
期刊：
影响因子：
20.3
通讯作者：
Qi Sun;R. Burton;K. Lucas
中科院分区：
文献类型：
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作者：
Qi Sun;R. Burton;K. Lucas
体外扩增eb病毒（EBV）特异性T细胞已成功应用于临床过继免疫治疗。然而，CD4(+) T细胞在治疗性T细胞培养中对ebv特异性免疫重建的作用尚未确定。我们从离体t细胞培养中分离并鉴定了CD4(+) t细胞系。单克隆系PD-F4和寡克隆系ND-R4和TD-B4为CD3(+)CD4(+)CD8（-）。以不匹配的主要组织相容性复合体（MHC）和抗MHC抗体为靶点的细胞溶解试验证实，这些CD4（+）细胞的细胞毒性受到MHC II类的限制。ND-R4单细胞表达干扰素- γ (ifn - γ)或白细胞介素4 (IL-4)，但很少共表达这两种细胞因子。相反，PD-F4共表达ifn - γ、IL-2和IL-4。PD-F4的动力学研究表明，这3种细胞因子的表达在刺激后5小时达到平稳状态，然后急剧下降，其模式与独立调制和微分离周期信号要求一致。这些CD4（+）细胞的细胞毒性在很大程度上抵抗brefeldin A，一种fas配体家族分子的细胞溶解途径抑制剂。尽管CD4（+）细胞毒性T淋巴细胞（ctl）对concanamycin A和乙二醇四乙酸敏感，但它们不表达穿孔素，表明其细胞毒性机制独立于穿孔素，尽管涉及胞吐。流式细胞术分析显示，CD4(+) ctl表达颗粒素，这是一种最近发现的与胞外细胞溶解颗粒相关的细胞溶解分子。这些数据表明，在治疗性b淋巴母细胞样细胞系启动的T细胞培养中，CD4(+) T细胞在产生T(H)1和T(H)2细胞因子方面存在差异，并可能通过颗粒素的胞吐作用发挥特异性细胞毒性。
Ex vivo expanded Epstein-Barr virus (EBV)-specific T cells have been successfully applied clinically for adoptive immunotherapy. However, the role of CD4(+) T cells in the therapeutic T-cell culture has not been established for the reconstitution of EBV-specific immunity. We isolated and characterized CD4(+) T-cell lines from the ex vivo T-cell cultures. Monoclonal line PD-F4 and oligoclonal lines ND-R4 and TD-B4 were CD3(+)CD4(+)CD8(-). Cytolytic tests with targets of mismatched major histocompatibility complex (MHC) and anti-MHC antibodies confirmed that the cytotoxicity of these CD4(+) cells was restricted by MHC class II. Single cells of ND-R4 expressed interferon-gamma (IFN-gamma, or interleukin 4 (IL-4), but rarely coexpressed these 2 cytokines. In contrast, PD-F4 coexpressed IFN-gamma, IL-2, and IL-4. Kinetic studies with PD-F4 showed that expression of the 3 cytokines plateaued 5 hours upon stimulation and was then drastically reduced, with a pattern consistent with independent modulation and differential off-cycle signal requirements. The cytotoxicity of these CD4(+) cells was largely resistant to brefeldin A, an inhibitor for cytolytic pathways by Fas-ligand family molecules. Although sensitive to concanamycin A and ethyleneglycotetraacetic acid, which inhibit cytotoxicity by granule exocytosis, the CD4(+) cytotoxic T lymphocytes (CTLs) did not express perforin, suggesting a cytotoxic mechanism independent of perforin although involving exocytosis. Flow cytometric analysis showed that the CD4(+) CTLs expressed granulysin, a recently identified cytolytic molecule associated with exocytotic cytolytic granules. These data suggested that CD4(+) T cells in the therapeutic B-lymphoblastoid cell lines-primed T-cell culture are diverse in producing T(H)1 and T(H)2 cytokines, and may exert specific cytotoxicity via exocytosis of granulysin.