不同浓度IL-2对体外诱导肽特异性细胞毒性T淋巴细胞培养体系的影响

摘要

Background and purpose:Cytotoxic T lymphocyte (CTL) plays a vital role in the process of anti-tumor immunology. The aim of this study was to investigate whether changes in concentration of IL-2 (50, 200 and 1 000 U/mL) would affect the sub-population and cytotoxic function of cells cultivated by peptide-specific CTL induction systemin vitro and also observe whether using the concentration of IL-2 at a range of 50-1 000 U/mL isbeneifcial to regulatory cells (Tregs) enrichment.Methods:Peripheral blood from 10 healthy donors and 10 cancer patients that were HLA-A2 positive, were collected in the study. HLA-A2 restricted CTL epitope P321 (ILIGETIKI) derived from COX-2 pulsed with different concentrations of IL-2 were used to induce peptides-speciifc CTLin vitro. Flow cytometry was performed to analyze the proliferative capability, the proportion of different T-cell subsets, and secretion of perforin, granzyme B and IFN-γ. IFN-γ secretion was assessed by ELISpot assay.Results:High concentration of IL-2 increased the proliferative activity. The percentage of CD4+ T cells of cancer patient group was signiifcantly higher than that of healthy donor group, while the percentage of CD8+ T cells of cancer patient group was signiifcantly lower than that of healthy donor group. And there was no signiifcant difference in the percentages of CD4+ T cells, CD8+ T cells and Tregs among groups with different IL-2 concentrations. No difference was seen in cytokine (perforin, granzyme B, IFN-γ) secretion capacity of CD8+ T cells. ELISpot study revealed that high-dose IL-2 resulted in the increasing of IFN-γ secretion.Conclusion:The sub-population and the function of cells cultured by peptide-speciifc CTL induction systemin vitro are not affected by different concentrations of IL-2. Furthermore, high concentrations of IL-2 (50-1 000 U/mL) do not provide the enrichment for Tregs. Higher concentration of IL-2 is likely to cause high secretion of IFN-γ in ELISpot assay. In order to exclude the distraction of NK cells or NKT cells, the concentration of 50 U/mL is better choice.%背景与目的：细胞毒性T淋巴细胞(cytotoxic T lymphocyte，CTL)介导的特异性细胞免疫在抗肿瘤免疫过程中发挥着主要作用。该研究探讨不同浓度IL-2(50、200和1000 U/mL)对体外诱导表位肽特异性CTL培养体系培养的细胞亚群比例和功能的影响，以及高剂量IL-2是否会诱导该体系中的调节性T细胞(regulatory cell，Treg)的富集。方法：选取HLA-A2超型的肿瘤患者和健康供者各10例，取外周血分离外周血单核细胞，使用环氧合酶-2(Cox-2)来源的CTL表位肽P321(ILIGETIKI)与不同浓度IL-2体外诱导肽特异性CTL。运用流式细胞仪检测细胞的增殖能力、CD4+T淋巴细胞和CD8+T淋巴细胞亚群的比例、Treg细胞亚群的比例，以及CD8+T淋巴细胞分泌穿孔素(perforin)、颗粒酶B(granzyme-B)、干扰素IFN-γ的能力。使用Elispot实验检测实验组细胞分泌IFN-γ的能力。结果：高浓度的IL-2有利于细胞的增殖。肿瘤患者组的CD4+T淋巴细胞比例高于健康供者组，而CD8+T淋巴细胞比例较健康供者组低；不同浓度IL-2对CD4+T淋巴细胞、CD8+T淋巴细胞和Treg细胞亚群比例以及CD8+T淋巴细胞分泌穿孔素、颗粒酶B、IFN-γ的能力没有影响。随着IL-2浓度的增高，Elispot实验出现的阳性斑点数越多。结论：不同浓度IL-2条件对体外诱导表位肽特异性CTL培养体系培养出的细胞亚群比例和功能没有影响，在50~1000 U/mL IL-2浓度范围内，高剂量的IL-2不会诱导该体系中的Treg的富集。但高浓度的IL-2可以提高细胞的增殖能力，并且促进细胞分泌IFN-γ，而高浓度的IL-2可以使培养体系中存在的NK细胞或NKT细胞能够非特异性产生IFN-γ，从而对Elispot实验产生干扰。因此，在体外诱导肽特异性CTL时选用50 U/mL的IL-2浓度可以很好地维持T细胞的增殖和存活，并且最大程度地降低对Elispot实验的干扰。