ILT2在结核患者NK细胞的表达及作用研究
发布时间：2018-08-11 19:39
【摘要】：结核病是引起死亡人数高居第二位的感染性疾病。由于耐药结核的产生、艾滋病感染流行、人口流动和临床免疫抑制剂的应用增多等原因，全球结核病疫情非常严重，仅2012年，全球新发结核病例达860万，有130万人死于结核病，约45万人为耐多药结核病。我国现有活动性肺结核病人约500万，每年约有13万人死于结核病。尽管结核感染人数众多，但是仅有5-10%的感染者发展成为活动性结核。研究结果表明，结核病的发生、发展和转归不仅仅取决于结核分枝杆菌的数量和毒力,在很大程度上还取决于机体的免疫状态。 宿主固有免疫在结核病早期发挥抗结核分枝杆菌感染作用，而且对于激发宿主获得性免疫也发挥了重要作用。在结核分枝杆菌入侵早期，获得性免疫未被充分激活之前，固有免疫系统在结核分枝杆菌的控制和清除过程中发挥着重要作用。参与固有免疫细胞主要为NK细胞、巨噬细胞、单核细胞、嗜中性白细胞等，它们无须抗原预先致敏,主要通过释放细胞因子、穿孔素、颗粒酶、表达配体等发挥作用，诱导免疫细胞聚集，杀伤靶细胞。 NK细胞是参与抗结核固有免疫的重要细胞，具有细胞毒性效应，能分泌IFN-γ等细胞因子，同时具有免疫调节的功能。为了保持抗感染免疫的平衡，NK细胞的功能由位于细胞表面不同的激活和抑制型受体共同进行调节。免疫球蛋白样转录子2（ILT2，又称CD85j，LILRB1，LIR-1）是一种I型转膜蛋白，胞浆区含有四个免疫受体酪氨酸抑制性基序。该受体通过动员如SHP-1的Src同源结构域（SH2）蛋白参与ITIM酪氨酸磷酸化的负信号调节。ILT2在NK细胞、T细胞、B细胞、单核细胞和树突细胞上均有表达，与MHC-I类分子（HLA-A, HLA-B, HLA-C,HLA-E和HLA-G）结合，同时，ILT-2还能识别其他蛋白，，如人类巨细胞病毒（HCMV）编码的UL18蛋白。目前，ILT2在抗结核感染免疫中的作用尚不明确。为此，本论文开展了对ILT2在结核患者NK细胞的表达及作用的研究： 第一，研究NK细胞及亚群在肺结核患者外周血淋巴细胞的分布情况，研究ILT2在肺结核患者NK细胞上的表达及与结核病情严重程度的关系。研究对象包含肺结核组（n=70）和健康对照组（n=67），根据痰涂片或痰培养结果将肺结核组分为菌阳组（n=41）和菌阴组肺结核组（n=29）。 首先采集活动性肺结核组和健康对照组的新鲜外周EDTA抗凝血,密度梯度离心分离PBMCs，用荧光标记的抗人CD3、CD56、CD16进行染色，流式细胞术检测CD3-CD56+NK细胞及两个亚型CD3-CD56dimCD16+和CD3-CD56brightCD16+/-的分布比例。研究结果显示，活动性肺结核患者外周血淋巴细胞群中CD3-CD56+NK细胞的比例为2.3%(1.2%-4.0%)，明显低于健康对照组（7.8%；6.5%-11.9%)，p0.0001；活动性肺结核外周血淋巴细胞群中CD3-CD56dimNK细胞的比例(3.6%；2.2-6.7%)明显低于健康对照组（9.2%；6.2-14.1%；p0.0001），活动性肺结核组外周血淋巴细胞群中CD3-CD56brightNK细胞的比例(0.3%；0.2-0.6%)较健康对照组（0.5%；0.3-0.6%）显著下降（p=0.0002）。研究结果表明活动性肺结核患者外周血NK细胞及亚群的比例均明显下降。 其次，采集肺结核组和健康对照组的新鲜外周EDTA抗凝血,密度梯度离心分离PBMCs，用荧光标记的抗人CD3、CD56、CD16和ILT2染色，流式细胞术检测ILT2抗体在NK细胞不同亚群上的表达。实验结果显示，活动性肺结核患者ILT2+CD56dimCD16+NK细胞比例45.7%(33.9%-56.2%)明显高于健康对照者19.1%(9.9-30.0%)，(p0.0001)。ILT2在活动性肺结核组CD56brightCD16+/-NK细胞上的表达比例为2.6%（1.4-4.9%)，与健康对照组CD56brightCD16+/-NK细胞表达的比例2.4%（0.7-5.7%)相似，结果无统计学意义(p=0.5163)。研究结果表明活动性肺结核患者外周血ILT2+CD56dimCD16+NK细胞表达比例明显高于健康对照者。 此外，比较了ILT2在菌阳活动性肺结核组和菌阴活动性肺结核组D56dimCD16+-NK细胞上的表达，分析ILT2在肺结核患者NK细胞上的表达与结核病病情的关系。实验结果显示，ILT2在菌阳肺结核组CD56dimCD16+NK细胞上的表达比例(55.3%；45.4-59.9%)明显高于菌阴肺结核组(32.9%；23.3-42.0%)， p0.0001。实验结果表明ILT2在CD56dimCD16+NK细胞上的表达水平与肺结核病的病情严重程度相关。 第二，通过研究ILT2表达与NK细胞CD107a表达的关系及ILT2表达与NK细胞分泌IFN-γ的关系，探究ILT2对肺结核患者NK细胞的细胞毒作用和干扰素分泌的影响。同时通过检测ILT2+CD56dimNK细胞的自然凋亡与ILT2-CD56dimNK细胞的自然凋亡以研究ILT2对NK细胞的自然凋亡作用。研究对象为38例活动性结核患者，痰涂片或痰培养结果阳性。 首先，以K562细胞为靶细胞，以5:1比例将15例活动性结核患者外周血单个核细胞与靶细胞K562共孵育，加入抗人CD107a荧光抗体。采用流式细胞术分析CD107a在ILT2+CD56dimNK和ILT2-CD56dimNK细胞上的表达区别。实验结果显示，CD107a在ILT2+CD56dimCD16+NK细胞的表达比例显著低于ILT2-CD56dimNK细胞(p=0.0169)。结果表明ILT2在CD56dimNK细胞的表达与NK细胞细胞毒作用缺陷相关。 其次，采用胞内染色方法，将10例活动性肺结核患者单个核细胞与靶细胞K562混合孵育，加入monensin，细胞表面荧光染色后，行细胞破膜，加入FITC-标记抗人IFN-γ单抗，流式细胞术检测IFN-γ在ILT2+CD56dimNK细胞和ILT2-CD56dimNK细胞内的表达。实验结果显示，IFN-在ILT2+CD56dimNK细胞的表达明显少于ILT2-CD56dimNK细胞（p=0.0038）。结果表明ILT2在CD56dimNK细胞的表达与NK细胞IFN-γ分泌水平降低相关。 此外，加入抗ILT2的阻断抗体及同型对照抗体，将CD56dimCD16+NK细胞与靶细胞K562进行孵育，流式细胞术检测阻断ILT2信号通路后CD107a在CD56dimCD16+NK细胞上表达情况。实验结果显示，阻断ILT2信号通路后，CD107a在CD56dimCD16+NK细胞上表达的比例较加入同型对照后明显升高（p=0.0223）。结果表明阻断ILT2信号通路可提高CD56dimCD16+NK细胞的细胞毒作用。 最后，采用Annexin V与PI双染方法判定NK细胞自然凋亡，采用流式细胞术检测，比较分析ILT2+CD56dimNK细胞和ILT2-CD56dimNK细胞的凋亡比率。实验结果显示，ILT2+CD56dimNK细胞的自然凋亡率显著高于ILT2-CD56dimNK细胞(p=0.0335)。研究结果表明ILT2可促进活动性肺结核患者CD56dimCD16+NK细胞的自然凋亡。 本研究采用流式细胞术,开展了对ILT2在结核患者NK细胞的表达及作用的研究。研究发现，活动性肺结核患者外周血中NK细胞及亚群的分布比例均显著下降；ILT2在活动性肺结核患者CD56dimCD16+NK细胞上的表达频率升高，并与活动性结核病的病情严重程度相关；ILT2对活动性结核患者CD56dimNK细胞的细胞毒作用和细胞因子分泌的功能具有抑制作用；ILT2可促进活动性肺结核患者CD3-CD56dimNK细胞的自然凋亡。本研究为更深入了解NK细胞抗结核感染免疫理论，建立基于增强NK细胞功能的抗结核免疫治疗提供了新的思路。
[Abstract]:Tuberculosis is the second leading cause of death. Due to the emergence of drug-resistant tuberculosis, the epidemic of AIDS infection, population mobility and the increasing use of clinical immunosuppressants, the global epidemic of tuberculosis is very serious. In 2012 alone, 8.6 million new cases of tuberculosis worldwide, 1.3 million people died of tuberculosis, about 450,000 people are resistant to tuberculosis. There are about 5 million active tuberculosis patients in China, and about 130,000 people die of tuberculosis every year. Despite the large number of tuberculosis infections, only 5-10% of the infected people develop into active tuberculosis. The results show that the occurrence, development and outcomes of tuberculosis not only depend on the number and virulence of Mycobacterium tuberculosis. To a large extent, it also depends on the immune state of the body.
The innate immune system plays an important role in the control and clearance of Mycobacterium tuberculosis before the acquired immunity is fully activated in the early stage of the invasion of Mycobacterium tuberculosis. Inherent immune cells are mainly NK cells, macrophages, monocytes, neutrophils, etc. They do not need antigen pre-sensitization, mainly through the release of cytokines, perforin, granzymes, expression ligands and other functions, induce immune cells to aggregate, kill target cells.
NK cells are important cells involved in the innate immunity against tuberculosis. They have cytotoxic effects, secrete cytokines such as IFN-gamma, and have immunoregulatory functions. In order to maintain the balance of anti-infective immunity, NK cells are regulated by different activation and inhibition receptors located on the cell surface. (ILT2, also known as CD85j, LILRB1, LIR-1) is a type I transmembrane protein with four tyrosine inhibitory motifs in the cytoplasm. The receptor is involved in negative signal regulation of ITIM tyrosine phosphorylation by mobilizing Src homologous domain (SH2) proteins such as SHP-1. ILT2 is expressed in NK cells, T cells, B cells, monocytes and dendritic cells. It binds to MHC-I molecules (HLA-A, HLA-B, HLA-C, HLA-E and HLA-G) and recognizes other proteins, such as UL18 protein encoded by human cytomegalovirus (HCMV). At present, the role of ILT2 in anti-tuberculosis immunity is not clear.
Firstly, the distribution of NK cells and their subsets in peripheral blood lymphocytes of pulmonary tuberculosis patients was studied, and the expression of ILT2 in NK cells of pulmonary tuberculosis patients and its relationship with the severity of tuberculosis were studied. =41) and pulmonary tuberculosis group (n=29).
Fresh peripheral EDTA anticoagulants were collected from active pulmonary tuberculosis group and healthy control group. PBMCs were separated by density gradient centrifugation. Anti-human CD3, CD56 and CD16 were stained with fluorescent labels. The distribution of CD3-CD56 + NK cells and two subtypes of CD3-CD56 dimCD16 + and CD3-CD56 bright CD16 + /- were detected by flow cytometry. The percentage of CD3-CD56+NK cells in peripheral blood lymphocyte population of patients with pulmonary tuberculosis was 2.3% (1.2% - 4.0%) significantly lower than that of healthy control group (7.8%; 6.5% - 11.9%) and p0.0001; the percentage of CD3-CD56 dim NK cells in peripheral blood lymphocyte population of active pulmonary tuberculosis (3.6%; 2.2-6.7%) was significantly lower than that of healthy control group (9.2%; 6.2-14.1%; p0.0001), and the activity of active pulmonary tuberculosis The percentage of CD3-CD56 bright NK cells (0.3%; 0.2-0.6%) in peripheral blood lymphocyte population of tuberculosis group was significantly lower than that of healthy control group (0.5%; 0.3-0.6%) (p = 0.0002). The results showed that the percentage of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients was significantly decreased.
Secondly, fresh peripheral EDTA anticoagulants were collected from pulmonary tuberculosis group and healthy control group, and PBMCs were separated by density gradient centrifugation. The expression of ILT2 antibody in different subsets of NK cells was detected by fluorescent staining of anti-human CD3, CD56, CD16 and ILT2. The results showed that the proportion of ILT2 + CD56dimCD16 + NK cells in active pulmonary tuberculosis patients was 45.7%. (33.9% - 56.2%) was significantly higher than that of healthy controls (19.1% (9.9-30.0%) (p0.0001). The expression of ILT2 on CD56 bright CD16 + / - NK cells was 2.6% (1.4-4.9%) in active pulmonary tuberculosis group and 2.4% (0.7-5.7%) in healthy control group. The results showed no statistical significance (p = 0.5163). The expression ratio of ILT2+CD56dimCD16+NK cells in peripheral blood of patients with pulmonary tuberculosis was significantly higher than that of healthy controls.
In addition, the expression of ILT2 on D56dimCD16 + - NK cells in bacterial positive active pulmonary tuberculosis group and bacterial negative active pulmonary tuberculosis group was compared, and the relationship between the expression of ILT2 on NK cells and tuberculosis was analyzed. The expression level of ILT2 on CD56 dim CD16 + NK cells was correlated with the severity of tuberculosis.
Secondly, by studying the relationship between the expression of ILT2 and the expression of CD107a in NK cells, and the relationship between the expression of ILT2 and the secretion of IFN-gamma in NK cells, the cytotoxic effect of ILT2 on NK cells and the secretion of interferon were investigated. The natural apoptosis of cells was observed in 38 patients with active tuberculosis. The results of sputum smear or sputum culture were positive.
Firstly, the peripheral blood mononuclear cells of 15 active tuberculosis patients were co-incubated with the target cell K562 at a ratio of 5:1 and added with anti-human CD107a fluorescent antibody. The expression of CD107a in ILT2+CD56dimNK and ILT2-CD56dim NK cells was analyzed by flow cytometry. The expression of ILT2 in CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0169).
Secondly, the mononuclear cells of 10 patients with active pulmonary tuberculosis were incubated with target cell K562 by intracellular staining. Monensin was added to the cells. After fluorescence staining, the membrane was broken and FITC-labeled anti-human IFN-gamma monoclonal antibody was added. The expression of IFN-gamma in ILT2+CD56dim NK cells and ILT2-CD56dim NK cells was detected by flow cytometry. The results showed that the expression of IFN-in ILT2+CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0038). The results showed that the expression of ILT2 in CD56dim NK cells was correlated with the decrease of IFN-gamma secretion of NK cells.
In addition, CD56 dimCD16 + NK cells were incubated with K562 cells by adding anti-ILT2 blocking antibodies and homo-control antibodies. The expression of CD107a on CD56 dimCD16 + NK cells was detected by flow cytometry after blocking the ILT2 signaling pathway. The results showed that the expression of CD107a on CD56 dimCD16 + NK cells was higher after blocking the ILT2 signaling pathway. The results showed that blocking ILT2 signaling pathway could enhance the cytotoxicity of CD56 dim CD16 + NK cells.
Finally, the natural apoptosis of NK cells was determined by Annexin V staining and PI staining. The apoptosis rates of ILT2+CD56dim NK cells and ILT2-CD56dim NK cells were compared and analyzed by flow cytometry. The results showed that the natural apoptosis rate of ILT2+CD56dim NK cells was significantly higher than that of ILT2-CD56dim NK cells (p=0.0335). Spontaneous apoptosis of CD56dimCD16+NK cells in patients with active pulmonary tuberculosis.
In this study, flow cytometry was used to study the expression and role of ILT2 in NK cells of tuberculosis patients. The results showed that the distribution of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients were significantly decreased; the expression of ILT2 in CD56 dimCD16 + NK cells of active pulmonary tuberculosis patients was increased, and it was associated with active tuberculosis. The severity of the disease is related; ILT2 inhibits the cytotoxicity and cytokine secretion of CD56dim NK cells in active tuberculosis patients; ILT2 promotes the natural apoptosis of CD3-CD56dim NK cells in active tuberculosis patients. Cell function provides a new idea for anti TB immunotherapy.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R52
本文编号：2178044
[Abstract]:Tuberculosis is the second leading cause of death. Due to the emergence of drug-resistant tuberculosis, the epidemic of AIDS infection, population mobility and the increasing use of clinical immunosuppressants, the global epidemic of tuberculosis is very serious. In 2012 alone, 8.6 million new cases of tuberculosis worldwide, 1.3 million people died of tuberculosis, about 450,000 people are resistant to tuberculosis. There are about 5 million active tuberculosis patients in China, and about 130,000 people die of tuberculosis every year. Despite the large number of tuberculosis infections, only 5-10% of the infected people develop into active tuberculosis. The results show that the occurrence, development and outcomes of tuberculosis not only depend on the number and virulence of Mycobacterium tuberculosis. To a large extent, it also depends on the immune state of the body.
The innate immune system plays an important role in the control and clearance of Mycobacterium tuberculosis before the acquired immunity is fully activated in the early stage of the invasion of Mycobacterium tuberculosis. Inherent immune cells are mainly NK cells, macrophages, monocytes, neutrophils, etc. They do not need antigen pre-sensitization, mainly through the release of cytokines, perforin, granzymes, expression ligands and other functions, induce immune cells to aggregate, kill target cells.
NK cells are important cells involved in the innate immunity against tuberculosis. They have cytotoxic effects, secrete cytokines such as IFN-gamma, and have immunoregulatory functions. In order to maintain the balance of anti-infective immunity, NK cells are regulated by different activation and inhibition receptors located on the cell surface. (ILT2, also known as CD85j, LILRB1, LIR-1) is a type I transmembrane protein with four tyrosine inhibitory motifs in the cytoplasm. The receptor is involved in negative signal regulation of ITIM tyrosine phosphorylation by mobilizing Src homologous domain (SH2) proteins such as SHP-1. ILT2 is expressed in NK cells, T cells, B cells, monocytes and dendritic cells. It binds to MHC-I molecules (HLA-A, HLA-B, HLA-C, HLA-E and HLA-G) and recognizes other proteins, such as UL18 protein encoded by human cytomegalovirus (HCMV). At present, the role of ILT2 in anti-tuberculosis immunity is not clear.
Firstly, the distribution of NK cells and their subsets in peripheral blood lymphocytes of pulmonary tuberculosis patients was studied, and the expression of ILT2 in NK cells of pulmonary tuberculosis patients and its relationship with the severity of tuberculosis were studied. =41) and pulmonary tuberculosis group (n=29).
Fresh peripheral EDTA anticoagulants were collected from active pulmonary tuberculosis group and healthy control group. PBMCs were separated by density gradient centrifugation. Anti-human CD3, CD56 and CD16 were stained with fluorescent labels. The distribution of CD3-CD56 + NK cells and two subtypes of CD3-CD56 dimCD16 + and CD3-CD56 bright CD16 + /- were detected by flow cytometry. The percentage of CD3-CD56+NK cells in peripheral blood lymphocyte population of patients with pulmonary tuberculosis was 2.3% (1.2% - 4.0%) significantly lower than that of healthy control group (7.8%; 6.5% - 11.9%) and p0.0001; the percentage of CD3-CD56 dim NK cells in peripheral blood lymphocyte population of active pulmonary tuberculosis (3.6%; 2.2-6.7%) was significantly lower than that of healthy control group (9.2%; 6.2-14.1%; p0.0001), and the activity of active pulmonary tuberculosis The percentage of CD3-CD56 bright NK cells (0.3%; 0.2-0.6%) in peripheral blood lymphocyte population of tuberculosis group was significantly lower than that of healthy control group (0.5%; 0.3-0.6%) (p = 0.0002). The results showed that the percentage of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients was significantly decreased.
Secondly, fresh peripheral EDTA anticoagulants were collected from pulmonary tuberculosis group and healthy control group, and PBMCs were separated by density gradient centrifugation. The expression of ILT2 antibody in different subsets of NK cells was detected by fluorescent staining of anti-human CD3, CD56, CD16 and ILT2. The results showed that the proportion of ILT2 + CD56dimCD16 + NK cells in active pulmonary tuberculosis patients was 45.7%. (33.9% - 56.2%) was significantly higher than that of healthy controls (19.1% (9.9-30.0%) (p0.0001). The expression of ILT2 on CD56 bright CD16 + / - NK cells was 2.6% (1.4-4.9%) in active pulmonary tuberculosis group and 2.4% (0.7-5.7%) in healthy control group. The results showed no statistical significance (p = 0.5163). The expression ratio of ILT2+CD56dimCD16+NK cells in peripheral blood of patients with pulmonary tuberculosis was significantly higher than that of healthy controls.
In addition, the expression of ILT2 on D56dimCD16 + - NK cells in bacterial positive active pulmonary tuberculosis group and bacterial negative active pulmonary tuberculosis group was compared, and the relationship between the expression of ILT2 on NK cells and tuberculosis was analyzed. The expression level of ILT2 on CD56 dim CD16 + NK cells was correlated with the severity of tuberculosis.
Secondly, by studying the relationship between the expression of ILT2 and the expression of CD107a in NK cells, and the relationship between the expression of ILT2 and the secretion of IFN-gamma in NK cells, the cytotoxic effect of ILT2 on NK cells and the secretion of interferon were investigated. The natural apoptosis of cells was observed in 38 patients with active tuberculosis. The results of sputum smear or sputum culture were positive.
Firstly, the peripheral blood mononuclear cells of 15 active tuberculosis patients were co-incubated with the target cell K562 at a ratio of 5:1 and added with anti-human CD107a fluorescent antibody. The expression of CD107a in ILT2+CD56dimNK and ILT2-CD56dim NK cells was analyzed by flow cytometry. The expression of ILT2 in CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0169).
Secondly, the mononuclear cells of 10 patients with active pulmonary tuberculosis were incubated with target cell K562 by intracellular staining. Monensin was added to the cells. After fluorescence staining, the membrane was broken and FITC-labeled anti-human IFN-gamma monoclonal antibody was added. The expression of IFN-gamma in ILT2+CD56dim NK cells and ILT2-CD56dim NK cells was detected by flow cytometry. The results showed that the expression of IFN-in ILT2+CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0038). The results showed that the expression of ILT2 in CD56dim NK cells was correlated with the decrease of IFN-gamma secretion of NK cells.
In addition, CD56 dimCD16 + NK cells were incubated with K562 cells by adding anti-ILT2 blocking antibodies and homo-control antibodies. The expression of CD107a on CD56 dimCD16 + NK cells was detected by flow cytometry after blocking the ILT2 signaling pathway. The results showed that the expression of CD107a on CD56 dimCD16 + NK cells was higher after blocking the ILT2 signaling pathway. The results showed that blocking ILT2 signaling pathway could enhance the cytotoxicity of CD56 dim CD16 + NK cells.
Finally, the natural apoptosis of NK cells was determined by Annexin V staining and PI staining. The apoptosis rates of ILT2+CD56dim NK cells and ILT2-CD56dim NK cells were compared and analyzed by flow cytometry. The results showed that the natural apoptosis rate of ILT2+CD56dim NK cells was significantly higher than that of ILT2-CD56dim NK cells (p=0.0335). Spontaneous apoptosis of CD56dimCD16+NK cells in patients with active pulmonary tuberculosis.
In this study, flow cytometry was used to study the expression and role of ILT2 in NK cells of tuberculosis patients. The results showed that the distribution of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients were significantly decreased; the expression of ILT2 in CD56 dimCD16 + NK cells of active pulmonary tuberculosis patients was increased, and it was associated with active tuberculosis. The severity of the disease is related; ILT2 inhibits the cytotoxicity and cytokine secretion of CD56dim NK cells in active tuberculosis patients; ILT2 promotes the natural apoptosis of CD3-CD56dim NK cells in active tuberculosis patients. Cell function provides a new idea for anti TB immunotherapy.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R52
本文编号：2178044
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