Hospital da Mulher Professor Doutor José Aristodemo Pinotti - Centro de Atenção Integral à Saúde da Mulher, Campinas University (UNICAMP), Campinas, BrazilDepartamento de Anatomia Patológica, Faculdade de Ciências Médicas, Campinas University (UNICAMP), Campinas, BrazilDepartamento de Tocoginecologia, Faculdade de Ciências Médicas, Campinas University (UNICAMP), Campinas, BrazilDepartamento de Clínica Médica, Faculdade de Ciências Médicas, Campinas University (UNICAMP), Campinas, Brazil.
Scand J Immunol. 2011 Sep;74(3):244-252. doi: 10.1111/j.1365-3083.2011.02576.x.
Methods for ex vivo expansion of natural killer (NK) cells have allowed obtaining enough numbers of human NK cells for clinical trials. However, the evaluation of these methods has been mostly limited to haematological malignancies. This study aimed at evaluating a method for selective expansion of NK cells when applied in peripheral blood mononuclear cells (PBMC) of patients with ovarian neoplasia. PBMC from 13 volunteer patients with ovarian neoplasia, seven benign and six malignant tumours, were cultured in CellGro medium supplemented with anti-CD3 (9-10 initial days), IL-2 and foetal bovine serum for 21 days. The resulting effector cells were evaluated for their phenotype, cytotoxicity and cytokine secretion. PBMC cultures resulted in multiple populations (NK, NKT and T) of effector cells, enriched with CD56(+) lymphocytes. NK cells from patients with benign and malignant ovarian neoplasia were expanded 139.6 ± 63.4 and 82.7 ± 25.3-fold, respectively, being the largest lymphocyte subtype among CD56(+) population. Effector cells expanded from patients with malignant ovarian neoplasia had higher proportion of T lymphocytes and altered cytokine production patterns, characterized by lower INF-γ, TNF-α and higher IL-4, compared with patients with benign ovarian neoplasia. Effector cells were cytotoxic against K562 and OVCAR3 cell lines. Cytotoxicity was significantly higher (P < 0.05) using magnetically separated CD56(+) effector cell fractions compared with CD56-deprived ones. The present study demonstrates the feasibility of the culture system employed to generate effector cells, enriched with CD56(+) lymphocytes, from PBMC of patients with ovarian neoplasia. NK cells were the largest lymphocyte subtype among the CD56(+) population and the main variable among the final effector cell preparation affecting target cell killing.
方法用于体外扩增自然杀伤 (NK) 细胞已允许获得足够数量的人类 NK 细胞进行临床试验。然而，这些方法的评估大多仅限于血液系统恶性肿瘤。本研究旨在评估一种方法，用于选择性扩增 NK 细胞，当应用于卵巢肿瘤患者的外周血单个核细胞 (PBMC) 时。来自 13 名卵巢肿瘤志愿者患者的 PBMC，7 例良性肿瘤和 6 例恶性肿瘤，在含有抗 CD3（最初 9-10 天）、IL-2 和胎牛血清的 CellGro 培养基中培养 21 天。评估所得效应细胞的表型、细胞毒性和细胞因子分泌。PBMC 培养物产生了多种效应细胞群体（NK、NKT 和 T），富含 CD56+淋巴细胞。良性和恶性卵巢肿瘤患者的 NK 细胞分别扩增了 139.6±63.4 和 82.7±25.3 倍，是 CD56+群体中最大的淋巴细胞亚型。与良性卵巢肿瘤患者相比，来自恶性卵巢肿瘤患者的效应细胞具有更高比例的 T 淋巴细胞和改变的细胞因子产生模式，其特征是 INF-γ、TNF-α 较低，IL-4 较高。效应细胞对 K562 和 OVCAR3 细胞系具有细胞毒性。与 CD56 耗尽的效应细胞相比，使用磁性分离的 CD56+效应细胞部分的细胞毒性显著更高（P<0.05）。本研究证明了从卵巢肿瘤患者的 PBMC 中生成富含 CD56+淋巴细胞的效应细胞的培养系统的可行性。NK 细胞是 CD56+群体中最大的淋巴细胞亚型，也是影响靶细胞杀伤的最终效应细胞制剂中的主要变量。
