外周血Treg细胞及NKG2D+NK细胞表达在结直肠癌患者中的临床意义
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摘要
研究背景：
研究统计,近年来结直肠癌(colorectal cancer, CRC)一直是世界范围内发病率和致死率位居前三的恶性肿瘤之一,每年全球都有数百万新发病例及约600,000人死于结直肠癌。随着饮食习惯和饮食结构的改变,我国结直肠癌发病率和死亡率也呈上升趋势。由于结直肠癌早期临床症状不明显,大多数患者确诊时已属中晚期。目前结直肠癌的发生发展及免疫逃逸机理仍不明确,对于丧失手术时机的结直肠癌患者仍缺乏更有效的治疗方法。因此,寻找理想的结直肠癌早期诊断和预后评价指标以及理想的治疗靶点十分必要。
自然杀伤(Natural killer, NK)细胞是机体重要的先天免疫效应细胞,能清除体内的肿瘤细胞、被病毒或细菌感染的细胞等,发挥天然免疫监视功能。NK细胞表面存在多种活化性和抑制性受体,其细胞毒作用的发挥就依赖于各种活化和抑制信号的综合。NKG2A(Natural-killer group2, member A)是NK细胞表面的一种重要的抑制性受体,特异性识别和结合其配体HLA-E,向NK细胞传递抑制信号。NKG2D(Natural-killer group2, member D)是NK细胞表面的一种主要的活化性受体,特异性识别和结合其配体MICA/B、ULBPs后,向NK细胞传递活化信号。多项研究表明,NKG2D介导的NK细胞毒作用下降可能是造成肿瘤细胞免疫逃逸的原因之一。
调节性T细胞(regulatory T cells, Tregs)是机体重要的免疫抑制性细胞,可抑制CD8+CTL细胞、NK细胞及树突状细胞(dendritic cells, DCs)等多种免疫效应细胞的功能,是肿瘤免疫逃逸的关键因素。目前天然CD4+CD25+Foxp3+Treg细胞是研究最为深入的一类Treg细胞。叉头转录因子(Forkhead box protein3, Foxp3)是Treg细胞最关键的细胞内标志物,对于Treg细胞的分化和功能维持具有重要作用。研究发现Treg细胞在结直肠癌、肝癌、卵巢癌及前列腺癌等患者外周血和肿瘤局部微环境中比例增高,且Treg细胞水平与患者肿瘤进展及预后呈负相关；在肿瘤切除后Treg细胞恢复到正常水平,而肿瘤复发时Treg细胞增多。
研究报道,Treg细胞可以通过细胞接触依赖机制(cell-contact-dependant mechanism)及分泌转化生长因子(transforming growth factor, TGF)β、IL-10等免疫抑制性细胞因子抑制NK细胞的活化,从而使肿瘤细胞逃避免疫监视。然而,Treg细胞与NK细胞受体NKG2A、NKG2D在结直肠癌中的表达、相互关系及临床意义尚不明了。
基于上述研究现状,为了深入认识Treg细胞、NK细胞及其受体NKG2A与NKG2D表达在结直肠癌患者中的临床意义,以便为结直肠癌的诊断和治疗提供新的思路和实验依据,本研究确定研究目的及研究方法如下。
研究目的：
1.观察CRC患者外周血中NK细胞抑制性受体NKG2A及活化性受体NKG2D在mRNA水平及蛋白水平的表达情况。
2.观察封闭纯化NK细胞受体NKG2D表达对NK细胞毒作用及脱颗粒作用的影响。
3.观察CRC患者外周血中Treg细胞水平及其与淋巴结转移、临床分期的关系。
4.平行测定CRC患者外周血中Treg细胞水平、NKG2D+NK细胞水平及血清癌胚抗原(carcino-embryonic antigen, CEA)水平,评价它们的相关性。
研究方法：
1.患者与对照CRC患者97例(男52例,女45例)收自山东省立医院胃肠外科。所有患者的确诊根据卫生部颁布的结直肠癌诊断标准(2010),并排除合并糖尿病、肾病、肝病或其它自身免疫性疾病,标本收集前均未接受放、化疗。健康对照48例(男26例,女22例)收自山东省立医院健康查体中心。本课题得到山东大学附属山东省立医院伦理委员会的批准。
2.细胞与细胞培养抽取研究个体15mL抗凝静脉血,用Ficoll-Hypaque密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)。采用免疫磁珠方法阴性分选NK细胞,流式细胞术测定CD3-CDC6+NK细胞纯度达95%。获得的NK细胞在含10%灭活胎牛血清(fetal calf serum, FCS)、1%青链霉素的RPMI1640培养基中培养,加入100U/mL重组人白介素(recombinant human interkin, rIL)2,在37℃及5%CO2孵箱中孵育。
人结肠癌细胞株HT29作为靶细胞,在含10%灭活FCS、1%青链霉素的RPMI1640培养基中培养,在37℃及5%CO2孵箱中孵育。2-3天更换1次培养基,细胞在使用前洗2次。
在抗体封闭试验中,NK细胞先与不同浓度抗NKG2D中和抗体孵育30min,然后与靶细胞HT29共同孵育,之后进行细胞毒试验及CD107a脱颗粒试验。
3.实时定量PCR总RNA根据厂家说明书用TRIzol试剂从PBMCs中提取。提取的总RNA的浓度和质量由测定的吸光度A260及A260/A280比率评价。反转录体系(20μL)包括：1μg总RNA,2μL的Maxima enzyme mix,4μL的5×PCRmix。反转录程序如下：25℃10min,55℃30min,85℃5min。获得的cDNA在用于PCR扩增前作10倍稀释。
引物包括：CD94, NKG2A, NKG2D及β-actin。PCR反应体系(20μL)包括：5gL的cDNA,5μL的0.8μM上下游引物,10μL的2×PCR mix。扩增程序如下：95℃5min,然后45个循环：95℃15sec,60℃30sec,72℃15sec。用96孔板在罗氏LightCycler480序列测定系统上合成PCR产物。最后,将PCR产物电泳以评价是否合成了要求的产物。
4.流式细胞术NKG2A和NKG2D检测属于表面抗原检测,用10μL抗人CD3抗体,5μL抗人CD56抗体,10μL抗人NKG2A抗体,10μL抗人NKG2D抗体对PBMCs染色,同时使用同型对照。
Treg细胞用调节性T细胞试剂盒参照厂家说明书测定。对于表面抗原染色,在100μL全血中加入10μL抗人CD4抗体和10μL抗人CD25抗体,4℃避光孵育30min。然后室温加入1×RBC Lysis Buffer溶解红细胞。对于细胞内抗原染色,需要先破坏细胞膜,在细胞中加入fixation/permeabilization workingsolution,避光孵育60min。然后加入10μL抗人Foxp3抗体染色。同时使用同型对照抗体。
对于CD107a脱颗粒测定,将NK细胞与HT29在37℃混合培养,培养体积为200μL,然后每孔加入10μL抗人CD107a抗体。37℃孵育1h后,加入1.7μL莫能霉素(0.1mg/mL),再孵育3h。同时设对照孔。收集细胞后,再加入10gL抗人CD3抗体及5gL抗人CD56抗体进行表面抗原染色。
用EPICS XL流式细胞仪(Beckman Coulter)或BD FACS II流式细胞仪(BDBiosciences)至少计数10,000个细胞。
5.细胞毒测定采用51Cr释放试验,纯化NK细胞作为效应细胞,HT29作为靶细胞。HT29与S1Cr同位素在37℃孵育1h。被标记的靶细胞与NK细胞以不同效靶比共孵育4h。收集上清,用y-counter(Packard Cobra Ⅱ5002)分析。计算51Cr释放百分率。自发性释放应小于最大释放的15%。
6.血清CEA测定非特异性肿瘤标志物CEA作为CRC患者的诊断、疗效监测和预后判断指标,在我院生化室采用罗氏Cobas e601系统检测。在健康人群中,CEA的参考范围是0-10ng/L。
7.统计学分析采用GraphPad Prism软件对数据进行统计学分析。t test用于比较2个组之间的可数变量结果。one-way Anova用于比较3或4组之间的可数变量。Spearman correlation analysis用于判断2个变量之间的相关性。p<0.05被认为具有统计学意义。
结果：
1.CRC患者外周血NKG2A在mRNA水平、蛋白水平及NK细胞表面的表达水平均与健康对照相似；而NKG2D在mRNA水平、蛋白水平及NK细胞表面的表达水平均显著低于健康对照。
2.用抗NKG2D中和抗体封闭NKG2D路径后,NK细胞毒作用及CD107a脱颗粒均随着抗NKG2D中和抗体浓度的增加而降低。
3. NKG2A和NKG2D也可以在NKT细胞及T细胞上表达。但NKG2A在外周血NK细胞、NKT细胞及T细胞上的表达依次降低；NKG2D在外周血T细胞上的表达显著低于NK细胞和NKT细胞。
4.CRC患者外周血CD4+CD25+Foxp3+及CD4+CD25highFoxp3+Treg细胞均比健康对照显著升高,但与淋巴结转移、临床分期不相关。
5.在CRC患者和健康对照外周血中,CD4+CD25highFoxp3+细胞水平均显著高于CD4+CD25lowFoxp3+细胞水平。
6.CRC患者上调的CD4+CD25+Foxp3+及CD4+CD25highFoxp3+Treg细胞水平与下调的NKG2D+NK细胞水平无统计学相关性。
7.CRC患者CD4+CD25+Foxp3+及CD4+CD25highFoxp3+Treg细胞水平与血清CEA水平之间未发现统计学相关性,尽管一些严重的进展期CRC患者Treg细胞和血清CEA同时增高。
结论：
结直肠癌患者抑制性受体NKG2A和活化性受体NKG2D在转录水平及翻译水平的表达失衡,可能与NK细胞活性抑制相关。我们推论CRC肿瘤细胞可以经由NKG2路径逃避NK细胞免疫监视；然而,其潜在的机制需要进一步探究。CRC患者外周血免疫抑制性Treg细胞表达显著增高,而外周血NKG2D+NK细胞表达显著降低,但均与CRC患者是否合并淋巴结转移及临床分期不相关。受到研究样本量的限制,在上调的Treg细胞与下调的NKG2D+NK细胞之间我们未发现显著的统计学相关性；在Treg细胞与血清CEA水平之间也未发现统计学相关性。需要更多研究以探明Treg细胞与NKG2D+NK细胞参与结直肠癌免疫逃逸的机制。
Background:
Recent statistics show that colorectal cancer (CRC) is both the third most commonly diagnosed cancer and the third most fatal cancer worldwide with millions of new cancer cases being reported annually, causing approximately600,000deaths annually. With the changes of diet customs and diet structures, the morbidity and mortality of CRC are increasing in China. Because the early clinical symptoms of CRC are not obvious, most patients were diagnosed as the middle and advanced stages of CRC. At present the precise mechanisms of pathogenesis and immune escape of CRC are not yet completely understood. For the CRC patients having lost the chance of surgery, there are short of more efficient treatments. Therefore, it is so necessary to look for ideal indicators for early diagnosis, prognostic evaluation and therapeutic targets of CRC.
Natural killer (NK) cells are a type of important innate immune effector cells in organisms, which can eliminate tumor cells and infectious cells invaded by virus or bacteria in organisms and exert capability of immunological surveillance. There are many kinds of activating and inhibitory receptors on the surface of NK cells. It is depending on the synthesis of all kinds of activating and inhibitory signals that NK cells exert their cytotoxicity. Natural-killer group2, member A (NKG2A) is an important inhibitory receptor in NK cells, which specially recognizes and binds its ligand HLA-E and transfers inhibitory signals to NK cells. Natural-killer group2, member D is a major activating receptor in NK cells, which specially recognizes and binds its ligands MICA/B, ULBPs and transfers activating signals to NK cells. Several studies have shown that the decline of NK cytotoxicity mediated by NKG2D might be one of reasons that resulted in tumor immune escape.
Regulatory T cells (Tregs) are a type of important immune suppressive cells in organisms and may suppress the functions of some immunologic effector cells such as CD8+CTL cells, natural killer (NK) cells and dendritic cells. Tregs are one of the key elements of tumor immune escape. At present natural CD4+CD25+Foxp3+Tregs are studied most deeply among Tregs. Forkhead box protein3(Foxp3) is the key intracellular marker of Tregs and is important for Tregs'developments and functions. Studies have shown an increase in populations of Tregs in peripheral blood as well as tumor microenvironment in patients with different cancers such as colorectal cancer, hepatocellular cancer, ovarian cancer and prostate cancer. Moreover, the populations of Tregs were negatively correlated with the tumor developments and prognosis of patients. The population of Tregs recovered to normal levels after tumor resection, while it increased when tumor recurrence.
Studies have shown that Tregs may suppress the activation of NK cells by cell-contact-dependent mechanism and secreting immunosuppressive cytokines such as TGF-β, IL-10, which consequently make tumor cells evade immunological surveillance. However, the relationships and clinical significances of Tregs and NK cells' receptors NKG2A and NKG2D expressions in CRC remain obscure.
On the basis of above situations as well as in order to further understand the relationships and clinical significances of Tregs, NK cells and NK receptors NKG2A and NKG2D expressions in CRC, we formulate the following objectives and methods so as to supply new thoughts and experimental basis for CRC dignosis and therapy.
Objectives:
1. To observe both the mRNA transcription and protein expression levels of inhibitory receptor NKG2A and activating receptor NKG2D in peripheral NK cells in CRC patients.
2. To observe how blocking NKG2D receptor in purified NK cells affect NK cytotoxicity and CD107a degranulation.
3. To observe the level of peripheral Tregs in CRC patients and the relationships between Tregs and lymph node transfer or clinical stages of CRC.
4. To assay peripheral Tregs, NKG2D+NK cells and serum carcino-embryonic antigen (CEA) levels in CRC patients parallelly and evaluate their relations.
Methods:
1. Patients and controls Ninety-seven patients (52men and45women) with primary CRC were recruited from the gastrointestinal surgery ward of Shandong Provincial Hospital affiliated to Shandong University. These patients were diagnosed with CRC on the basis of colorectal cancer diagnosis standard (2010) issued by Ministry of Health. The patients had no history of other diseases such as diabetes, kidney disease, hepatic disease, or autoimmune disease. All patients were not received with radiotherapy or chemotherapy before sample collection. Forty-eight healthy subjects (26men and22women) from the physical examination centre of Shandong Provincial Hospital. The study was approved by Shandong Provincial Hospital affiliated to Shandong University Ethics Committee, and informed consent was acquired from each individual.
2. Cells and cell culture Peripheral blood mononuclear cells (PBMCs) were isolated from15mL venous blood obtained from study subjects by using Ficoll-Hypaque density gradient centrifugation. NK cells were isolated from PBMCs by negative selection using magnetic cell separation. Flow cytometry revealed the purity of CD3-CD56+NK cells to be greater than95%. The obtained NK cells were cultured in RPMI1640medium with10%heat-inactivated fetal calf serum (FCS),1%penicillin-streptomycin and100U/mL recombinant human interleukin2(rIL-2) in37℃and5%CO2incubator.
Human colon carcinoma cell line HT29cells were cultured in RPMI1640supplemented with10%FCS and were used as target cells. The medium was regularly changed, and the cells were always washed twice before use.
In antibody-blocking experiments, purified NK cells were pre-incubated with different concentrations of anti-NKG2D neutralizing antibodies for30min before they were cultured with the target cells. Then, the cytotoxicity and CD107a degranulation assays were performed.
3. Real-time PCR Total cellular RNA was extracted from PBMCs by using TRIzol reagent. Concentration and quality of the extracted total RNA were determined by measuring its light absorbance at260nm (A260) and the ratio of (A260/A280). A1μg of total RNA was reverse transcribed in a20-μl reaction mixture containing2μl of Maxima enzyme mix and4μl of5×PCR mix. The procedure for reverse transcription was performed as follows:10min at25℃,30min at55℃, and then5min at85℃.
The obtained cDNA was diluted1:10before PCR analysis.
The primers included CD94, NKG2A, NKG2D, and β-actin. Reactions were performed in a total volume of20μl, which included5μl of cDNA sample,5μl Of0.8μM primer pair, and10μl of2×PCR mix. PCR was performed as follows:5min at95℃and45cycles of15s at95℃,30s at60℃, and15s at72℃. Incubation and on-line detection of the PCR products were carried out using optical96-well plates and the LightCycler480sequence detection system (Roche, Germany). Finally, the PCR products were subjected to electrophoresis to determine whether the required products were formed.
4. Flow cytometry PBMCs were stained with10μl anti-human CD3,5μl anti-human CD56,10μl anti-human NKG2A, and10μl anti-human NKG2D antibodies to assay NKG2A and NKG2D expression on the surface of NK cells. Isotype controls were used meanwhile.
Tregs were assayed with regulatory T cells kit according to the manufacturer's instructions. For surface antigens staining, added10μl anti-human CD4,10μl anti-human CD25antibodies to100μl of the whole blood and incubated for30min in the dark at4℃. Then1×RBC Lysis Buffer was added to the whole blood in the room temperature in order to lyse RBCs. For intracellular antigen staining, fixation/permeabilization working solution was added to the cells and incubated for60min in the dark before the cells were stained with anti-human Foxp3antibody. Isotype controls were used at the same time.
For CD107a degranulation assays, NK cells and HT29cells were co-cultured at37℃. The volume of per well was200μl, then10μl anti-human CD107a antibodies were added into the well. After incubating for1h at37℃,1.7μl monensin (0.1mg/ml) was added into the well and the plate was incubated for another3h. Control wells were used meanwhile. After collecting the cells, they were stained with10ul anti-human CD3,5μl anti-human CD56antibodies to assay the expression of CD107a on NK cells.
At least10,000cells were analyzed using a3-color EPICS XL flow cytometer (Beckman coulter, USA) or BD FACSⅡ flow cytometer (BD Bioscences, USA).5. Cytotoxicity assays HT29cells were used as target cells, and the purified NK cells were used as effector cells in the51Cr release assay. The HT29cells were labeled by incubating them with the51Cr isotope for1h at37℃. The labeled targets were then co-incubated with NK cells at different ratios for4h. The supernatant was harvested and analyzed using a y-counter (Packard Cobra Ⅱ5002, USA). The percentage of51Cr released was calculated. Spontaneous release was less than15%of the maximum release.
6. Serum CEA assay Serum CEA protein in CRC patients was assayed using electrochemiluminescence method with Roche cobas e601(Roche, Germany) at the Department of Medical Biochemistry, Shandong Provincial Hospital. In healthy subjects, CEA<10ng/ml was considered as normal.
7. Statistical analysis Data were analyzed using the GraphPad Prism (GraphPad Software, USA). A t test was used for comparing quantitative variables of two different groups. A one-way Anova was used for comparing the data of three or four groups. Spearman correlation analysis was performed to determine the association between two variables. Probability values were considered statistically significant if p <0.05.
Results:
1. The levels of peripheral NKG2A mRNA trscription, NKG2A protein in PBMCs and NKG2A expression in NK cells were similar between from patients with CRC and from healthy controls. However, the levels of peripheral NKG2D mRNA trscription, NKG2D protein in PBMCs and NKG2D expression in NK cells from CRC patients were significantly downregulated compared with healthy controls.
2. After blocking NKG2D signaling with anti-NKG2D antibodies ex vivo, cytotoxicity and CD107a degranulation were gradually decreased along with the increasing concentration of anti-NKG2D neutralizing antibodies.
3. NKG2A and NKG2D were also expressed on the membranes of CD3+CD56+NKT cells and CD3+CD56-T cells. However, NKG2A expression levels decreased successively in NK cells, NKT cells, and T cells. The NKG2D expression levels in T cells were lower than the corresponding levels in NK cells and NKT cells.
4. CD4+CD25+Foxp3+and CD4+CD25highFoxp3+Tregs were significantly up-regulated in peripheral blood in CRC patients compared to healthy controls, while the numbers of Tregs were not related with lymph node transfer or clinical stages.
5. The levels of CD4+CD25highFoxp3+cells were much higher than those of CD4+CD25lowFoxp3+cells in both CRC patients and healthy controls.
6. Increased CD4+CD25+Foxp3+as well as CD4+CD25highFoxp3+Tregs were not statistically correlated with decreased NKG2D expression in NK cells in peripheral blood from CRC patients.
7. Peripheral CD4+CD25+Foxp3+as well as CD4+CD25high Foxp3+Tregs were not correlated with serum CEA protein in CRC patients although there were elevated levels of both Treg and CEA in severe advanced CRC patients.
Conclusions:
The imbalance of inhibitory receptor NKG2A and activating receptor NKG2D expresions in transcription and translation level in CRC patients may be associated with suppression of NK cell activity. We inferred that tumor cells may escape NK cell surveillance via the NKG2pathway in CRC patients; however, the underlying mechanism needs to be investigated further in detail. Immunosuppressive Tregs were significantly up-regulated in peripheral blood in CRC patients; however, peripheral NKG2D+NK cells were significantly down-regulated. Yet both Tregs and NKG2D+NK cells were not correlated with lymph node metastasis or clinical stages of CRC. Limited by the small sample size, we did not find significant correlations between increased Tregs and decreased NKG2D+NK cells. Similarly, no correlation was observed between Tregs and serum CEA protein. More studies are needed to explore the mechnism that Tregs and NKG2D+NK cells participate in immune escape of CRC.
研究统计,近年来结直肠癌(colorectal cancer, CRC)一直是世界范围内发病率和致死率位居前三的恶性肿瘤之一,每年全球都有数百万新发病例及约600,000人死于结直肠癌。随着饮食习惯和饮食结构的改变,我国结直肠癌发病率和死亡率也呈上升趋势。由于结直肠癌早期临床症状不明显,大多数患者确诊时已属中晚期。目前结直肠癌的发生发展及免疫逃逸机理仍不明确,对于丧失手术时机的结直肠癌患者仍缺乏更有效的治疗方法。因此,寻找理想的结直肠癌早期诊断和预后评价指标以及理想的治疗靶点十分必要。
自然杀伤(Natural killer, NK)细胞是机体重要的先天免疫效应细胞,能清除体内的肿瘤细胞、被病毒或细菌感染的细胞等,发挥天然免疫监视功能。NK细胞表面存在多种活化性和抑制性受体,其细胞毒作用的发挥就依赖于各种活化和抑制信号的综合。NKG2A(Natural-killer group2, member A)是NK细胞表面的一种重要的抑制性受体,特异性识别和结合其配体HLA-E,向NK细胞传递抑制信号。NKG2D(Natural-killer group2, member D)是NK细胞表面的一种主要的活化性受体,特异性识别和结合其配体MICA/B、ULBPs后,向NK细胞传递活化信号。多项研究表明,NKG2D介导的NK细胞毒作用下降可能是造成肿瘤细胞免疫逃逸的原因之一。
调节性T细胞(regulatory T cells, Tregs)是机体重要的免疫抑制性细胞,可抑制CD8+CTL细胞、NK细胞及树突状细胞(dendritic cells, DCs)等多种免疫效应细胞的功能,是肿瘤免疫逃逸的关键因素。目前天然CD4+CD25+Foxp3+Treg细胞是研究最为深入的一类Treg细胞。叉头转录因子(Forkhead box protein3, Foxp3)是Treg细胞最关键的细胞内标志物,对于Treg细胞的分化和功能维持具有重要作用。研究发现Treg细胞在结直肠癌、肝癌、卵巢癌及前列腺癌等患者外周血和肿瘤局部微环境中比例增高,且Treg细胞水平与患者肿瘤进展及预后呈负相关；在肿瘤切除后Treg细胞恢复到正常水平,而肿瘤复发时Treg细胞增多。
研究报道,Treg细胞可以通过细胞接触依赖机制(cell-contact-dependant mechanism)及分泌转化生长因子(transforming growth factor, TGF)β、IL-10等免疫抑制性细胞因子抑制NK细胞的活化,从而使肿瘤细胞逃避免疫监视。然而,Treg细胞与NK细胞受体NKG2A、NKG2D在结直肠癌中的表达、相互关系及临床意义尚不明了。
基于上述研究现状,为了深入认识Treg细胞、NK细胞及其受体NKG2A与NKG2D表达在结直肠癌患者中的临床意义,以便为结直肠癌的诊断和治疗提供新的思路和实验依据,本研究确定研究目的及研究方法如下。
研究目的：
1.观察CRC患者外周血中NK细胞抑制性受体NKG2A及活化性受体NKG2D在mRNA水平及蛋白水平的表达情况。
2.观察封闭纯化NK细胞受体NKG2D表达对NK细胞毒作用及脱颗粒作用的影响。
3.观察CRC患者外周血中Treg细胞水平及其与淋巴结转移、临床分期的关系。
4.平行测定CRC患者外周血中Treg细胞水平、NKG2D+NK细胞水平及血清癌胚抗原(carcino-embryonic antigen, CEA)水平,评价它们的相关性。
研究方法：
1.患者与对照CRC患者97例(男52例,女45例)收自山东省立医院胃肠外科。所有患者的确诊根据卫生部颁布的结直肠癌诊断标准(2010),并排除合并糖尿病、肾病、肝病或其它自身免疫性疾病,标本收集前均未接受放、化疗。健康对照48例(男26例,女22例)收自山东省立医院健康查体中心。本课题得到山东大学附属山东省立医院伦理委员会的批准。
2.细胞与细胞培养抽取研究个体15mL抗凝静脉血,用Ficoll-Hypaque密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)。采用免疫磁珠方法阴性分选NK细胞,流式细胞术测定CD3-CDC6+NK细胞纯度达95%。获得的NK细胞在含10%灭活胎牛血清(fetal calf serum, FCS)、1%青链霉素的RPMI1640培养基中培养,加入100U/mL重组人白介素(recombinant human interkin, rIL)2,在37℃及5%CO2孵箱中孵育。
人结肠癌细胞株HT29作为靶细胞,在含10%灭活FCS、1%青链霉素的RPMI1640培养基中培养,在37℃及5%CO2孵箱中孵育。2-3天更换1次培养基,细胞在使用前洗2次。
在抗体封闭试验中,NK细胞先与不同浓度抗NKG2D中和抗体孵育30min,然后与靶细胞HT29共同孵育,之后进行细胞毒试验及CD107a脱颗粒试验。
3.实时定量PCR总RNA根据厂家说明书用TRIzol试剂从PBMCs中提取。提取的总RNA的浓度和质量由测定的吸光度A260及A260/A280比率评价。反转录体系(20μL)包括：1μg总RNA,2μL的Maxima enzyme mix,4μL的5×PCRmix。反转录程序如下：25℃10min,55℃30min,85℃5min。获得的cDNA在用于PCR扩增前作10倍稀释。
引物包括：CD94, NKG2A, NKG2D及β-actin。PCR反应体系(20μL)包括：5gL的cDNA,5μL的0.8μM上下游引物,10μL的2×PCR mix。扩增程序如下：95℃5min,然后45个循环：95℃15sec,60℃30sec,72℃15sec。用96孔板在罗氏LightCycler480序列测定系统上合成PCR产物。最后,将PCR产物电泳以评价是否合成了要求的产物。
4.流式细胞术NKG2A和NKG2D检测属于表面抗原检测,用10μL抗人CD3抗体,5μL抗人CD56抗体,10μL抗人NKG2A抗体,10μL抗人NKG2D抗体对PBMCs染色,同时使用同型对照。
Treg细胞用调节性T细胞试剂盒参照厂家说明书测定。对于表面抗原染色,在100μL全血中加入10μL抗人CD4抗体和10μL抗人CD25抗体,4℃避光孵育30min。然后室温加入1×RBC Lysis Buffer溶解红细胞。对于细胞内抗原染色,需要先破坏细胞膜,在细胞中加入fixation/permeabilization workingsolution,避光孵育60min。然后加入10μL抗人Foxp3抗体染色。同时使用同型对照抗体。
对于CD107a脱颗粒测定,将NK细胞与HT29在37℃混合培养,培养体积为200μL,然后每孔加入10μL抗人CD107a抗体。37℃孵育1h后,加入1.7μL莫能霉素(0.1mg/mL),再孵育3h。同时设对照孔。收集细胞后,再加入10gL抗人CD3抗体及5gL抗人CD56抗体进行表面抗原染色。
用EPICS XL流式细胞仪(Beckman Coulter)或BD FACS II流式细胞仪(BDBiosciences)至少计数10,000个细胞。
5.细胞毒测定采用51Cr释放试验,纯化NK细胞作为效应细胞,HT29作为靶细胞。HT29与S1Cr同位素在37℃孵育1h。被标记的靶细胞与NK细胞以不同效靶比共孵育4h。收集上清,用y-counter(Packard Cobra Ⅱ5002)分析。计算51Cr释放百分率。自发性释放应小于最大释放的15%。
6.血清CEA测定非特异性肿瘤标志物CEA作为CRC患者的诊断、疗效监测和预后判断指标,在我院生化室采用罗氏Cobas e601系统检测。在健康人群中,CEA的参考范围是0-10ng/L。
7.统计学分析采用GraphPad Prism软件对数据进行统计学分析。t test用于比较2个组之间的可数变量结果。one-way Anova用于比较3或4组之间的可数变量。Spearman correlation analysis用于判断2个变量之间的相关性。p<0.05被认为具有统计学意义。
结果：
1.CRC患者外周血NKG2A在mRNA水平、蛋白水平及NK细胞表面的表达水平均与健康对照相似；而NKG2D在mRNA水平、蛋白水平及NK细胞表面的表达水平均显著低于健康对照。
2.用抗NKG2D中和抗体封闭NKG2D路径后,NK细胞毒作用及CD107a脱颗粒均随着抗NKG2D中和抗体浓度的增加而降低。
3. NKG2A和NKG2D也可以在NKT细胞及T细胞上表达。但NKG2A在外周血NK细胞、NKT细胞及T细胞上的表达依次降低；NKG2D在外周血T细胞上的表达显著低于NK细胞和NKT细胞。
4.CRC患者外周血CD4+CD25+Foxp3+及CD4+CD25highFoxp3+Treg细胞均比健康对照显著升高,但与淋巴结转移、临床分期不相关。
5.在CRC患者和健康对照外周血中,CD4+CD25highFoxp3+细胞水平均显著高于CD4+CD25lowFoxp3+细胞水平。
6.CRC患者上调的CD4+CD25+Foxp3+及CD4+CD25highFoxp3+Treg细胞水平与下调的NKG2D+NK细胞水平无统计学相关性。
7.CRC患者CD4+CD25+Foxp3+及CD4+CD25highFoxp3+Treg细胞水平与血清CEA水平之间未发现统计学相关性,尽管一些严重的进展期CRC患者Treg细胞和血清CEA同时增高。
结论：
结直肠癌患者抑制性受体NKG2A和活化性受体NKG2D在转录水平及翻译水平的表达失衡,可能与NK细胞活性抑制相关。我们推论CRC肿瘤细胞可以经由NKG2路径逃避NK细胞免疫监视；然而,其潜在的机制需要进一步探究。CRC患者外周血免疫抑制性Treg细胞表达显著增高,而外周血NKG2D+NK细胞表达显著降低,但均与CRC患者是否合并淋巴结转移及临床分期不相关。受到研究样本量的限制,在上调的Treg细胞与下调的NKG2D+NK细胞之间我们未发现显著的统计学相关性；在Treg细胞与血清CEA水平之间也未发现统计学相关性。需要更多研究以探明Treg细胞与NKG2D+NK细胞参与结直肠癌免疫逃逸的机制。
Background:
Recent statistics show that colorectal cancer (CRC) is both the third most commonly diagnosed cancer and the third most fatal cancer worldwide with millions of new cancer cases being reported annually, causing approximately600,000deaths annually. With the changes of diet customs and diet structures, the morbidity and mortality of CRC are increasing in China. Because the early clinical symptoms of CRC are not obvious, most patients were diagnosed as the middle and advanced stages of CRC. At present the precise mechanisms of pathogenesis and immune escape of CRC are not yet completely understood. For the CRC patients having lost the chance of surgery, there are short of more efficient treatments. Therefore, it is so necessary to look for ideal indicators for early diagnosis, prognostic evaluation and therapeutic targets of CRC.
Natural killer (NK) cells are a type of important innate immune effector cells in organisms, which can eliminate tumor cells and infectious cells invaded by virus or bacteria in organisms and exert capability of immunological surveillance. There are many kinds of activating and inhibitory receptors on the surface of NK cells. It is depending on the synthesis of all kinds of activating and inhibitory signals that NK cells exert their cytotoxicity. Natural-killer group2, member A (NKG2A) is an important inhibitory receptor in NK cells, which specially recognizes and binds its ligand HLA-E and transfers inhibitory signals to NK cells. Natural-killer group2, member D is a major activating receptor in NK cells, which specially recognizes and binds its ligands MICA/B, ULBPs and transfers activating signals to NK cells. Several studies have shown that the decline of NK cytotoxicity mediated by NKG2D might be one of reasons that resulted in tumor immune escape.
Regulatory T cells (Tregs) are a type of important immune suppressive cells in organisms and may suppress the functions of some immunologic effector cells such as CD8+CTL cells, natural killer (NK) cells and dendritic cells. Tregs are one of the key elements of tumor immune escape. At present natural CD4+CD25+Foxp3+Tregs are studied most deeply among Tregs. Forkhead box protein3(Foxp3) is the key intracellular marker of Tregs and is important for Tregs'developments and functions. Studies have shown an increase in populations of Tregs in peripheral blood as well as tumor microenvironment in patients with different cancers such as colorectal cancer, hepatocellular cancer, ovarian cancer and prostate cancer. Moreover, the populations of Tregs were negatively correlated with the tumor developments and prognosis of patients. The population of Tregs recovered to normal levels after tumor resection, while it increased when tumor recurrence.
Studies have shown that Tregs may suppress the activation of NK cells by cell-contact-dependent mechanism and secreting immunosuppressive cytokines such as TGF-β, IL-10, which consequently make tumor cells evade immunological surveillance. However, the relationships and clinical significances of Tregs and NK cells' receptors NKG2A and NKG2D expressions in CRC remain obscure.
On the basis of above situations as well as in order to further understand the relationships and clinical significances of Tregs, NK cells and NK receptors NKG2A and NKG2D expressions in CRC, we formulate the following objectives and methods so as to supply new thoughts and experimental basis for CRC dignosis and therapy.
Objectives:
1. To observe both the mRNA transcription and protein expression levels of inhibitory receptor NKG2A and activating receptor NKG2D in peripheral NK cells in CRC patients.
2. To observe how blocking NKG2D receptor in purified NK cells affect NK cytotoxicity and CD107a degranulation.
3. To observe the level of peripheral Tregs in CRC patients and the relationships between Tregs and lymph node transfer or clinical stages of CRC.
4. To assay peripheral Tregs, NKG2D+NK cells and serum carcino-embryonic antigen (CEA) levels in CRC patients parallelly and evaluate their relations.
Methods:
1. Patients and controls Ninety-seven patients (52men and45women) with primary CRC were recruited from the gastrointestinal surgery ward of Shandong Provincial Hospital affiliated to Shandong University. These patients were diagnosed with CRC on the basis of colorectal cancer diagnosis standard (2010) issued by Ministry of Health. The patients had no history of other diseases such as diabetes, kidney disease, hepatic disease, or autoimmune disease. All patients were not received with radiotherapy or chemotherapy before sample collection. Forty-eight healthy subjects (26men and22women) from the physical examination centre of Shandong Provincial Hospital. The study was approved by Shandong Provincial Hospital affiliated to Shandong University Ethics Committee, and informed consent was acquired from each individual.
2. Cells and cell culture Peripheral blood mononuclear cells (PBMCs) were isolated from15mL venous blood obtained from study subjects by using Ficoll-Hypaque density gradient centrifugation. NK cells were isolated from PBMCs by negative selection using magnetic cell separation. Flow cytometry revealed the purity of CD3-CD56+NK cells to be greater than95%. The obtained NK cells were cultured in RPMI1640medium with10%heat-inactivated fetal calf serum (FCS),1%penicillin-streptomycin and100U/mL recombinant human interleukin2(rIL-2) in37℃and5%CO2incubator.
Human colon carcinoma cell line HT29cells were cultured in RPMI1640supplemented with10%FCS and were used as target cells. The medium was regularly changed, and the cells were always washed twice before use.
In antibody-blocking experiments, purified NK cells were pre-incubated with different concentrations of anti-NKG2D neutralizing antibodies for30min before they were cultured with the target cells. Then, the cytotoxicity and CD107a degranulation assays were performed.
3. Real-time PCR Total cellular RNA was extracted from PBMCs by using TRIzol reagent. Concentration and quality of the extracted total RNA were determined by measuring its light absorbance at260nm (A260) and the ratio of (A260/A280). A1μg of total RNA was reverse transcribed in a20-μl reaction mixture containing2μl of Maxima enzyme mix and4μl of5×PCR mix. The procedure for reverse transcription was performed as follows:10min at25℃,30min at55℃, and then5min at85℃.
The obtained cDNA was diluted1:10before PCR analysis.
The primers included CD94, NKG2A, NKG2D, and β-actin. Reactions were performed in a total volume of20μl, which included5μl of cDNA sample,5μl Of0.8μM primer pair, and10μl of2×PCR mix. PCR was performed as follows:5min at95℃and45cycles of15s at95℃,30s at60℃, and15s at72℃. Incubation and on-line detection of the PCR products were carried out using optical96-well plates and the LightCycler480sequence detection system (Roche, Germany). Finally, the PCR products were subjected to electrophoresis to determine whether the required products were formed.
4. Flow cytometry PBMCs were stained with10μl anti-human CD3,5μl anti-human CD56,10μl anti-human NKG2A, and10μl anti-human NKG2D antibodies to assay NKG2A and NKG2D expression on the surface of NK cells. Isotype controls were used meanwhile.
Tregs were assayed with regulatory T cells kit according to the manufacturer's instructions. For surface antigens staining, added10μl anti-human CD4,10μl anti-human CD25antibodies to100μl of the whole blood and incubated for30min in the dark at4℃. Then1×RBC Lysis Buffer was added to the whole blood in the room temperature in order to lyse RBCs. For intracellular antigen staining, fixation/permeabilization working solution was added to the cells and incubated for60min in the dark before the cells were stained with anti-human Foxp3antibody. Isotype controls were used at the same time.
For CD107a degranulation assays, NK cells and HT29cells were co-cultured at37℃. The volume of per well was200μl, then10μl anti-human CD107a antibodies were added into the well. After incubating for1h at37℃,1.7μl monensin (0.1mg/ml) was added into the well and the plate was incubated for another3h. Control wells were used meanwhile. After collecting the cells, they were stained with10ul anti-human CD3,5μl anti-human CD56antibodies to assay the expression of CD107a on NK cells.
At least10,000cells were analyzed using a3-color EPICS XL flow cytometer (Beckman coulter, USA) or BD FACSⅡ flow cytometer (BD Bioscences, USA).5. Cytotoxicity assays HT29cells were used as target cells, and the purified NK cells were used as effector cells in the51Cr release assay. The HT29cells were labeled by incubating them with the51Cr isotope for1h at37℃. The labeled targets were then co-incubated with NK cells at different ratios for4h. The supernatant was harvested and analyzed using a y-counter (Packard Cobra Ⅱ5002, USA). The percentage of51Cr released was calculated. Spontaneous release was less than15%of the maximum release.
6. Serum CEA assay Serum CEA protein in CRC patients was assayed using electrochemiluminescence method with Roche cobas e601(Roche, Germany) at the Department of Medical Biochemistry, Shandong Provincial Hospital. In healthy subjects, CEA<10ng/ml was considered as normal.
7. Statistical analysis Data were analyzed using the GraphPad Prism (GraphPad Software, USA). A t test was used for comparing quantitative variables of two different groups. A one-way Anova was used for comparing the data of three or four groups. Spearman correlation analysis was performed to determine the association between two variables. Probability values were considered statistically significant if p <0.05.
Results:
1. The levels of peripheral NKG2A mRNA trscription, NKG2A protein in PBMCs and NKG2A expression in NK cells were similar between from patients with CRC and from healthy controls. However, the levels of peripheral NKG2D mRNA trscription, NKG2D protein in PBMCs and NKG2D expression in NK cells from CRC patients were significantly downregulated compared with healthy controls.
2. After blocking NKG2D signaling with anti-NKG2D antibodies ex vivo, cytotoxicity and CD107a degranulation were gradually decreased along with the increasing concentration of anti-NKG2D neutralizing antibodies.
3. NKG2A and NKG2D were also expressed on the membranes of CD3+CD56+NKT cells and CD3+CD56-T cells. However, NKG2A expression levels decreased successively in NK cells, NKT cells, and T cells. The NKG2D expression levels in T cells were lower than the corresponding levels in NK cells and NKT cells.
4. CD4+CD25+Foxp3+and CD4+CD25highFoxp3+Tregs were significantly up-regulated in peripheral blood in CRC patients compared to healthy controls, while the numbers of Tregs were not related with lymph node transfer or clinical stages.
5. The levels of CD4+CD25highFoxp3+cells were much higher than those of CD4+CD25lowFoxp3+cells in both CRC patients and healthy controls.
6. Increased CD4+CD25+Foxp3+as well as CD4+CD25highFoxp3+Tregs were not statistically correlated with decreased NKG2D expression in NK cells in peripheral blood from CRC patients.
7. Peripheral CD4+CD25+Foxp3+as well as CD4+CD25high Foxp3+Tregs were not correlated with serum CEA protein in CRC patients although there were elevated levels of both Treg and CEA in severe advanced CRC patients.
Conclusions:
The imbalance of inhibitory receptor NKG2A and activating receptor NKG2D expresions in transcription and translation level in CRC patients may be associated with suppression of NK cell activity. We inferred that tumor cells may escape NK cell surveillance via the NKG2pathway in CRC patients; however, the underlying mechanism needs to be investigated further in detail. Immunosuppressive Tregs were significantly up-regulated in peripheral blood in CRC patients; however, peripheral NKG2D+NK cells were significantly down-regulated. Yet both Tregs and NKG2D+NK cells were not correlated with lymph node metastasis or clinical stages of CRC. Limited by the small sample size, we did not find significant correlations between increased Tregs and decreased NKG2D+NK cells. Similarly, no correlation was observed between Tregs and serum CEA protein. More studies are needed to explore the mechnism that Tregs and NKG2D+NK cells participate in immune escape of CRC.
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