[通过流式细胞术检测自然杀伤细胞和细胞毒性淋巴细胞上的CD107α来筛查细胞毒性缺陷]

[Screening for cytotoxic defects with flow cytometric detection of CD107α on natural killer cells and cytotoxic lymphocyte cells].

作者信息

Wang Jing, Liu Zheng, Jiang Li-ping, An Yun-fei, Zhao Xiao-dong

机构信息

P2 Laboratory, Clinical Immunology Laboratory, Children's Hospital of Chongqing Medical University, Chongqing 400014, China.

出版信息

Zhonghua Er Ke Za Zhi. 2012 May;50(5):386-91.

DOI:

PMID:22883044

Abstract

OBJECTIVE

To establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 (LAMP-1, CD107α) on the cell surface of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) and evaluate the screening value of this assay for cytotoxic defects-related diseases such as familial hemophagocytic lymphopro-liferative (FHL) syndrome.

METHOD

Three suspected Chediak-Higashi Syndrome (CHS) patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin (PHA) and CD8+T cells by anti-CD3, the CD107α expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the pathogenic genes via DNA-PCR/RT-PCR and direct sequencing.

RESULT

The CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [(0.18 ± 0.07)% vs. (4.47 ± 2.36)%, P < 0.05] and NK cells after activation by PHA [(0.27 ± 0.07)% vs. (5.80 ± 2.83)%, P < 0.05]. The frequency of CD107α-expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0.5%, 0.6% vs. (5.80 ± 2.83)%] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL cells also showed no significant difference [0.3%, 0.9%, 0.2% vs. (4.47 ± 2.36)%] in three patients. All of their mean fluorescence intensity (MFI) showed the same trend. Patient 1 and 3 were identified to have LYST mutations (Patient 1: c.5411-5414 del TTTC, L1741fsX1758 and c.7975 C > T, R2596X; Patient 3: c.4863G > A, R1563H and c.5392-5393delAA, E1739fsX1756). There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STX11 were identified.

CONCLUSION

We developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.

摘要

目的

建立一种基于流式细胞术的新方法，用于检测自然杀伤（NK）细胞和细胞毒性T淋巴细胞（CTL）细胞表面溶酶体相关膜蛋白1（LAMP-1，CD107α）的表达，并评估该方法对细胞毒性缺陷相关疾病（如家族性噬血细胞性淋巴组织细胞增生症（FHL）综合征）的筛查价值。

方法

2010年10月至2011年6月，招募了3例疑似Chediak-Higashi综合征（CHS）患者、3例疑似FHL患者和10名健康儿童。分离他们的外周血单个核细胞（PBMC），并用白细胞介素-2过夜激活。在植物血凝素（PHA）激活NK细胞和抗CD3激活CD8 + T细胞后，通过流式细胞术分析CD107α的表达。提取患者的外周血DNA和RNA，通过DNA-PCR/RT-PCR和直接测序分析致病基因。

结果

10名健康儿童中，抗CD3激活后CTL上的CD107α表达显著增加[（0.18±0.07）%对（4.47±2.36）%，P < 0.05]，PHA激活后NK细胞上的CD107α表达也显著增加[（0.27±0.07）%对（5.80±2.83）%，P < 0.05]。除患者2外，3例疑似CHS患者激活后CD107α表达的NK细胞频率与健康对照相比显著升高[0.5%，0.6%对（5.80±2.83）%]。抗CD3激活后，3例患者CTL细胞上CD107α表达频率也无显著差异[0.3%，0.9%，0.2%对（4.47±2.36）%]。它们的所有平均荧光强度（MFI）均呈现相同趋势。患者1和3被鉴定出有LYST突变（患者1：c.5411 - 5414 del TTTC，L1741fsX1758和c.7975 C > T，R2596X；患者3：c.4863G > A，R1563H和c.5392 - 5393delAA，E1739fsX1756）。患者2的LYST基因未发现突变。疑似FHL患者中NK细胞和CTL的CD107α表达以及相应地，未发现PRF、MUNC13 - 4和STX11的潜在基因变异。