JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2017 Vol.18 No.7 P.605-614

A simple method for removing low-density granulocytes to purify T lymphocytes from peripheral blood mononuclear cells

Author(s): Si-gong Zhang, Yu-xin Song, Xiao-ming Shu, Hai-li Shen, Han-bo Yang, Rui-xue Duo, Guo-chun Wang
Affiliation(s): Department of Rheumatology, Lanzhou University Second Hospital, Lanzhou 730030, China; more
Corresponding email(s): sigongzhang@126.com, guochunwang@hotmail.com
Key Words: Low-density granulocyte, Neutrophil extracellular trap, Peripheral blood mononuclear cells, Autoimmune disease, T-SPOT.TB

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Abstract: Objective: low-density granulocytes (LDGs) can form neutrophil extracellular traps (NETs) spontaneously and excessively. When peripheral blood mononuclear cells (PBMCs) are used for studying T lymphocytes, LDGs contained in the PBMCs may decrease the threshold of activating T lymphocytes by forming NETs. This study focused on the profiles of LDGs in common autoimmune diseases and methods for removing LDGs from PBMCs. Methods: The percentages of LDGs in PBMCs from 55 patients with dermatomyositis (DM), 15 with polymyositis (PM), 42 with rheumatoid arthritis (RA), 25 with systemic lupus erythematosus (SLE), and 19 healthy controls were determined by flow cytometry. Three methods of removing LDGs were explored and compared. After removal, PBMCs from six patients with positive t-SPOT.TB were tested again to find out if LDGs contained in the PBMCs could influence T lymphocyte reactions. Results: Significantly higher LDG percentages were found in PBMCs from patients with DM ((8.41±10.87)%, P<0.0001), PM ((8.41±10.39)%, P<0.0001), RA ((4.05±6.97)%, P=0.0249), and SLE ((7.53±11.52)%, P=0.0006), compared with the controls ((1.28±0.73)%). The t-SPOT.TB values significantly decreased after LDGs were removed. Increasing relative centrifugal force (RCF) within a limited range can decrease the LDG percentage from an initial high level, but not markedly increase the LDG clearance rate. Compared with the whole blood sediment method, the PBMC adherence method can significantly remove LDGs yet scarcely influence the T lymphocyte percentage in PBMCs. Conclusions: The LDG percentage in PBMCs is significantly increased in patients with SLE, DM, PM, and RA. The influence of LDGs on T lymphocytes cannot be ignored in PBMC cultures. The adherence method is a simple and easy-to-use method for removing LDGs and purifying T lymphocytes from PBMCs.

一种从外周血单个核细胞中祛除低密度粒细胞并纯化T淋巴细胞的简易方法

目的：系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMCs）中的低密度粒细胞（LDGs）可以自发而过度地形成中性粒细胞胞外网状陷阱（NETs），而形成的NETs会降低T细胞激活的阈值。采用PBMCs做淋巴细胞研究时，所含的异常增多的LDGs可能会显著影响实验结果。因此需要了解其他自身免疫病中LDGs的比例，并需要一种简便地祛除PBMCs中LDGs的方法。
创新点：首次提出LDGs清除的方法，验证了离心后贴壁法清除LDGs的有效性和可靠性。
方法：采用流式细胞仪测定55例皮肌炎（DM）患者、15例多发性肌炎（PM）患者、42例类风湿关节炎（RA）患者、25例SLE患者（阳性对照）和19例健康对照（阴性对照）PBMCs中LDGs的比例。采用提高离心力、离心后贴壁和全血沉淀后再离心这三种方法祛除PBMCs中的LDGs，进一步比较各种方法的LDGs清除率和对PBMCs中T细胞的影响程度。清除T-SPOT.TB阳性患者PBMCs中LDGs后复测T-SPOT.TB，明确LDGs是否对T-SPOT.TB结果有影响。
结论：本研究中DM、PM、RA和SLE患者PBMCs中LDGs比例普遍升高（图1）。清除PBMCs中LDGs后，T-SPOT.TB显著降低（图2）。离心后贴壁法不但能最有效地清除LDGs，且对PBMCs中T细胞的比例影响最小（图3~5）。综上所述，PBMCs中LDGs的比例在DM、PM、RA和SLE患者中普遍增高；采用PBMCs做T细胞相关研究时LDGs通过形成NETs对T细胞激活的影响不能忽视；离心后贴壁法可以简单而高效地清除PBMCs中的LDGs。

关键词：低密度粒细胞；中性粒细胞胞外网状陷阱；外周血单个核细胞；自身免疫病；T-SPOT.TB

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一种从外周血单个核细胞中祛除低密度粒细胞并纯化T淋巴细胞的简易方法

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