新型共刺激分子CD137抗白血病作用的体外研究

Antileukemic Effects In Vitro of New Co-stimulatory Molecule CD137

本研究探讨新型共刺激分子CD137(4-1BB)在人T淋巴细胞上的表达特点及其单克隆抗体hCD137mAb在刺激T淋巴细胞增殖、促进细胞因子分泌、增强细胞杀伤作用的抗白血病功能。应用FACS及间接免疫荧光法分别检测正常人T淋巴细胞上加入植物血凝素(PHA)前后不同时相CD137的表达。在HL-60细胞和T淋巴细胞体外培养体系中,用MTT比色法测定hCD137mAb、PHA对T淋巴细胞增殖作用的影响,用FACS及间接免疫荧光法检测其对T细胞表面上IFN-γ和IL-4分泌水平的影响。在体外混合淋巴细胞肿瘤细胞培养(MLTC)体系中,研究不同效靶比例时hCD137mAb增强T细胞杀伤白血病细胞的抗白血病作用。结果表明:①T细胞未经PHA刺激几乎不表达hCD137,经PHA活化后开始表达,且随着时间延长表达率逐渐增高,第7天为高峰(FACS法为56.4%±1.98%,间接免疫荧光法为52.8%±2.01%)。②MTT法检测提示,单独使用hCD137mAb不能刺激T细胞增殖(增殖指数1.002±0.011),但其与PHA伍用可增强后者刺激活性(增殖指数2.161±0.102)约2倍(增殖指数4.705±0.133)。③在PHA存在时,hCD137mAb可使IFN-γ在T细胞上表达增加到大约2倍,而对IL-4几无影响。④hCD137mAb明显增强T细胞对白血病细胞株HL-60的杀伤活性,且共刺激活性在40:1效靶比时最强,杀伤百分比增加到大约2倍。结论:新型共刺激分子CD137具有显著的抗白血病作用,应用hCD137mAb是一种有效排斥白血病的免疫策略,且安全、简便,本研究为其用于临床免疫治疗提供了实验依据。

This study was aimed to investigate the expression characteristics of new co-stimulatory molecule CD137 （4-1BB）on human T lymphocytes and antileukemic effects of monoclonal antibody hCD137mAb in stimulating the T lymphocyte proliferation, promoting the cytokine secretion, enhancing the cell killing effect and so on. The expression of CD137 on normal T lymphocytes treated with phytohemagglutinin （PHA） was detected by FACS and indirect immunofluorescence. In HL-60 and T lymphocyte system in vitro, the effect of hCD137mAb and PHA on T lymphocyte proliferation was tested by MTr colorimetric assay. The IFN--γ and IL-4 expression levels on the surface of T cells were detected by FACS and indirect immunofluorescence. In vitro mixed lymphocyte tumor cell culture （MLTC） system, the function of hCD137mAb enhancing toxicity killing leukemic cells at different effect-target ratio were studied. The results showed that almost no expression of hCD137 was found in T cells without PHA stimulation, but after activation of T cells by PHA, the expression gradually increased with a peak at 7th day （FACS 56. 4% ± 1. 98%, indirect immunofluorescence 52.8% ±2.01% ）. CD137mAb alone could not stimulate T cell proliferation （proliferation index 1. 002±0. 011 ）, but could enhance PHA stimulating activity （proliferative index of 2. 161± 0. 102 ） about 2-folds （proliferation index 4. 705 ± 0. 133）. Moreover, hCD137mAb increased expression of IFN-γ high by about 3-fold in presence of PHA, but did not effect on IL-4. The hCD137mAb markedly enhanced T cell killing activity on HL-60 cell line and its co-stimulatory effect was best at the effect-target ratio of40：1 with increasing of killing percentage by about 2-fold. It is concluded that the new co-stimulatory molecule CD137 has significant antileukemic effect, use of hCD137mAb is an effective, safe and simple immunization strategy for leukemia therapy, this study provides some experimental basis for clinical immunotherapy with CD137 mAb.