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

Abstract

Objectives

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.

Conclusion

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.

中文概要

题目

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。