【作者】 刘小倩；

【导师】 王欣；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 研究背景非霍奇金淋巴瘤(NHL)是一组起源于淋巴结或其他淋巴组织的常见恶性肿瘤,病因及发病机制至今仍不甚清,免疫耐受可能在肿瘤发生、发展和转移中起关键作用。以利妥昔单抗(抗CD20单抗)为代表的针对淋巴瘤的免疫靶向治疗已取得良好疗效。但仍存在一个重要问题就是无法克服肿瘤细胞形成的免疫耐受,机体免疫系统无法对肿瘤细胞进行有效的识别和杀伤。免疫耐受形成过程中涉及到的主要细胞包括：树突状细胞(dendritic cells, DCs)、调节性T细胞(regulatory T cells, Tregs)、肥大细胞、甚至是睹酸性粒细胞,肿瘤细胞亦可直接或间接通过树突状细胞等诱导肿瘤内部免疫耐受微环境。因此,研究NHL肿瘤细胞如何直接或间接通过抗原呈递细胞形成免疫耐受的肿瘤微环境,从而针对性地阻断肿瘤免疫耐受形成的关键通路,将对NHL治疗起到关键性的辅助作用。作为近年来的研究热点,色氨酸代谢在诱导免疫耐受形成中的作用不断见诸报道。吲哚胺2,3-双加氧酶(Indoleamine 2,3-dioxygenase, IDO)是色氨酸代谢中肝脏以外唯一可催化色氨酸分子中吲哚环氧化裂解,从而沿犬尿酸途径分解代谢的限速酶；主要表达于一些免疫耐受或免疫特赦组织中,如胸腺、胃肠道粘膜、附睾、胎盘及眼前房等,并且特异性地表达在巨噬细胞和树突状细胞上。通过对小鼠和人DCs的大量研究发现,高表达IDO的DCs亚群与机体的免疫耐受密切相关,如参与诱导T细胞凋亡、T细胞失能及活化调节性T细胞。研究表明,IDO在肿瘤和肿瘤转移淋巴结中高表达,从而被证实参与了肿瘤的免疫耐受机制,并在其中发挥重要作用。以1-甲基色氨酸(1-MT)为代表的IDO抑制剂已在国外用于Ⅰ期临床试验,并取得了良好的疗效。研究者发现,凡是IDO高表达的部位,如胎盘、肿瘤及转移淋巴结中,都存在大量调节性T细胞浸润的现象。调节性T细胞于1995年首次报道,其中转录因子FoxP3是Tregs的特异性标志,是发育的关键调节基因。Tregs同样在维持自身免疫耐受中起重要作用,其作用已在多种自身免疫、肿瘤模型中得到证实,而且最新研究显示Tregs的增殖及活化可能与IDO+DC亚群密切相关。已有研究证实：在多种肿瘤、肿瘤细胞系、急性白血病及成人T细胞白血病／淋巴瘤中存在着IDO的过表达,并通过对小鼠淋巴瘤模型的研究证实了IDO的过表达与Tregs增殖存在相关性。然而IDO上调Tregs的具体机制尚待进一步的研究。本课题旨在研究IDO在NHL患者组织水平上的表达情况,探讨其与Tregs的相关性及其在NHL发生、发展和预后中的作用。进而通过体外实验和建立NHL动物模型,验证IDO在NHL肿瘤诱导的免疫耐受中的作用,并对其作用机制进行细致的研究和探讨。为临床发掘新的判断NHL预后的指标,并为IDO抑制剂1-MT的临床应用提供有力的理论支持。第Ⅰ部分IDO和FoxP3在人非霍奇金淋巴瘤中的表达及意义研究目的以人NHL标本为研究对象,探讨IDO在NHL中的表达情况,分析其与临床特征以及与Tregs的相关性研究方法1.收集NHL空白石蜡切片(n=57),以反应性增生(n=18)为对照,通过免疫组织化学技术研究IDO和FoxP3在组织中的表达及部位。2.收集NHL新鲜标本(n=20),以实时定量RT-PCR技术比较IDO和FoxP3在mRNA水平的表达情况。3.收集NHL新鲜标本(n=20),利用western blot技术比较IDO和FoxP3在蛋白水平的表达情况。4.统计学分析IDO在NHL和反应性增生中的表达差异及其与FoxP3的相关性。探讨IDO与非霍奇金淋巴瘤临床病例资料如年龄、性别、临床分期、病理类型、LDH及预后等因素之间的关系。研究结果1.免疫组化结果显示,对照组IDO阳性细胞率均在30%以下,而57例NHL石蜡切片中,IDO+NHL(阳性细胞率在30%以上者)为18例(31.58%)。18例IDO+NHL石蜡切片的FoxP3阳性率显著高于对照组及39例IDO-NHL切片。IDO-NHL切片的FoxP3阳性率与对照组无显著差异。结合NHL患者临床病例资料,18例IDO+NHL患者的临床分期多为Ⅲ或Ⅳ期,而39例IDO-NHL患者临床分期相对较早,两者差异有统计学意义。IDO+NHL患者LDH值以及肿瘤直径同样显著大于IDO-NHL患者。以上数据均提示IDO与NHL预后密切相关。2.20例新鲜NHL标本经实时定量RT-PCR测定,IDOmRNA表达量显著高于对照组(2-dCt：0.00582～0.546 v.0～0.0103,P<0.05)。对于NHL组内进行IDO与FoxP3mRNA表达的相关性分析,两者呈正相关(r=0.447)。3.20例新鲜NHL标本经WB测定,IDO蛋白表达量显著高于对照组(0.77±0.25 vs.0.18±0.18,P<0.05)。NHL组内,IDO与FoxP3在蛋白水平上的表达呈正相关(r=0.613)。结论1.部分NHL组织内存在IDOmRNA和蛋白高表达。2.IDO的高表达与NHL临床分期、LDH值等密切相关,提示IDO可能指示了NHL患者预后不良。3.IDO的表达与FoxP3呈正相关,推测IDO+NHL肿瘤细胞可能与Tregs存在功能上的密切联系。第Ⅱ部分高表达IDO的淋巴瘤细胞株对Tregs调控作用的研究研究目的以高表达IDO的小鼠B系淋巴瘤细胞株A20及小鼠T细胞为研究对象,体外混合细胞培养,证实高表达IDO的淋巴瘤细胞株可上调Tregs的比例。研究方法1.免疫磁珠技术(MACS)分离BALB/c小鼠CD4+CD25- T细胞,并通过流式细胞术验证其纯度。2.选取高表达IDO的A20细胞系(源于BALB/c小鼠),将IDO+A20细胞系与BALB/c小鼠CD4+CD25-T细胞分为以下三组共同培养：①CD4+CD25- T细胞组；②CD4+CD25- T细胞+A20细胞组；③CD4+CD25- T细胞+A20细胞+1-甲基色氨酸组。3.培养24小时后,流式细胞术测定CD4+CD25+T细胞的比例。观察IDO+肿瘤细胞以及IDO抑制剂1-MT对Tregs增殖和功能的影响。4.MACS技术收集第②组中培养后的CD4+CD25+T细胞；MACS技术分离BALB/c小鼠脾脏中的DCs; Ficoll分离BALB/c小鼠脾脏中的单个核细胞(MNC)。分为以下4组进行混合淋巴细胞反应：①CD4+CD25+Tc+DCs+MNC；②MNC+DCs;③MNC组；④CD4+CD25+Tc组。培养72h后,MTT法测定其在570nm吸光度值(A值),计算CD4+CD25+T细胞的抑制率(supressive rate, SR)。SR=[1-(A1-A3-A4)/(A2-A3)]×100%研究结果1.流式细胞术验证MACS技术分离得到的CD4+CD25- T细胞纯度在98%以上。2.混合细胞培养24小时后,流式细胞术测定CD4+CD25+ T细胞比例：第①组为0；第②组为7.87±1.65%；第③组为3.32±1.19%,证实高表达IDO的A20细胞系可诱导CD4+CD25-T细胞向CD4+CD25+表型转化,而且1-MT可明显抑制该过程。3.MTT法分析测定转化后CD4+CD25+T细胞的抑制功能：阳性对照组A值明显高于阴性对照组(0.8657±0.0353 vs 0.3434±0.0319,P<0.05),说明T细胞在DCs的作用下增殖、活化；而实验组A值明显小于阳性对照组(0.4331±0.0280 vs0.8657±0.0353,P<0.05),证实转化后的Tregs具有抑制T细胞增殖的作用(抑制率为82.54±7.31%)。结论1.A20细胞系在体外可稳定高表达IDO。2.免疫磁珠技术是一种简单有效的细胞分离手段,可被广泛应用于临床和基础研究中。3.IDO+肿瘤细胞可在体外将CD4+CD25-T细胞向CD4+CD25+ T细胞转化,转化后的CD4+CD25-T细胞具有免疫抑制功能。4.IDO抑制剂1-MT可逆转体外IDO+肿瘤细胞将CD4+CD25-T细胞转化为CD4+CD25+T细胞的过程,说明这一转化具有IDO依赖性。第Ⅲ部分动物模型的建立及IDO对Tregs调控作用的探讨研究目的通过NHL动物模型,研究IDO+NHL小鼠Tregs在肿瘤、引流淋巴结及脾脏中的含量以及使用IDO抑制剂l-MT对Tregs的影响研究方法1.建立NHL动物模型：选6-8周龄左右BALB/c小鼠,皮下注射A20细胞悬液(5×106/只)。2.1周后选取肿瘤大小相近的小鼠入组实验,以同周龄的正常BALB/c小鼠为对照,分为以下三组：正常对照组(n=6),肿瘤组(n=6),1-MT干预组(n=6)。3.对于1-MT干预组,每日瘤内注射1-MT溶液,正常组和肿瘤组注射同体积PBS。3.2周后处死小鼠,收集肿瘤、脾脏、淋巴结,通过流式细胞术检测各组Tregs在肿瘤组织、淋巴结及脾脏中的含量；通过Western Blot检测IDO和FoxP3在组织中的表达。收集数据进行统计学分析,比较各组差异。研究结果1.A20 B细胞淋巴瘤模型成瘤率为95%,成瘤时间为7天左右。2.流式细胞术检测结果就CD+CD25+T细胞在肿瘤组织、脾脏、淋巴结中占淋巴细胞的比例而言,肿瘤组均明显高于1-MT干预组,也明显高于正常对照组。CD4+T细胞在肿瘤组织、脾脏内占淋巴细胞的比例,肿瘤组、1-MT组和对照组之间无差异。在肿瘤引流淋巴结和对照淋巴结内,CD4+ T细胞占淋巴细胞的比例肿瘤组明显低于正常对照组,但与1-MT干预组之间无明显差异,1-MT干预组同样明显低于正常对照组。肿瘤组织内,CD4+CD25+T细胞/CD4+ T细胞肿瘤组明显高于1-MT干预组。脾脏内,CD4+CD25+T细胞/CD4+T细胞在肿瘤组中的比例为明显高于正常组,也明显高于1-MT组。正常对照组和1-MT组之间差异无统计学意义。肿瘤引流淋巴结和对照淋巴结内,CD4+CD25+T细胞/CD4+T细胞在肿瘤组中的比例明显高于正常组(P<0.001),也明显高于1-MT组。1-MT组仍高于正常对照组。3. Western Blot结果显示,IDO在肿瘤组和1-MT组肿瘤、脾脏、淋巴结中表达均明显高于对照组。FoxP3在肿瘤组肿瘤、脾脏和淋巴结中的表达明显高于常对照组,也明显高于1-MT组。l-MT组与正常对照组之间无明显差异。结论1.A20 B细胞淋巴瘤是一种良好的NHL动物模型。2.IDO在动物模型的肿瘤、脾脏、肿瘤引流淋巴结内高表达,通过上调Tregs诱导免疫耐受。3.1-MT通过减弱动物模型中IDO对Tregs的上调作用,从而对淋巴瘤的化学和免疫治疗提供有效的协同作用,在抗肿瘤治疗中极具应用前景。更多还原

【Abstract】 BackgroundNon-Hodgkin’s lymphoma is a malignance of the immune system. Immune tolerance participates in tumor transformation and metastasis, and possesses close relationship with tumor therapy and prognosis. In recent years, increasing evidence has been accumulated that tryptophan catabolism takes part in tumor induced immune tolerance. Indoleamine 2,3-dioxygenase(IDO) is the key enzyme in the tryptophan metabolism, which catalyzes the initial rate-limiting step of tryptophan degradation along the kynurenine pathway and can be expressed in many tissues and cells, but mainly upon cytokine stimulation during infection and with less substrate specificity. IDO activation leads to a tryptophan starvation and an accumulation of downstream breakdown products. Tryptophan starvation by IDO consumption inhibits T-cell activation, while products of tryptophan catabolism, such as kynurenine derivatives and 02-free radicals, regulate T-cell proliferation and survival.In humans, IDO expression has been observed by immunohistochemistry in placenta, tumor-draining lymph nodes and primary tumors, which in turn, is infiltrated by CD4+CD25+ Treg cells. The close relationship between IDO activity and Treg cells have recently been the focus of intense studies due to a positive correlation between the increased numbers of Treg cells and IDO expression in experimental as well as clinical settings. It has been shown that both IDO-expressing dendritic cells(DCs) and IDO-expressing tumor cell can bias naive CD4+ T cells to differentiate into FoxP3+ Tregs in vitro. This important finding thus linked IDO to the potent Treg system, which is known to be a key mechanism of immunosuppression in tumorbearing hosts. A series of studies provided the first indirect evidence that IDO may play an important role in proliferation and conversion of Tregs in tumors. In the present study, we aim to measure the expressions of IDO and FoxP3 in NHL patients and perform in vitro and animal experiments to investigate whether T-cell tolerance in NHL may be induced by directly converting CD4+CD25- T cells into CD4+CD25+Treg cells through an IDO-dependent mechanism.Part I Expression and Significance of IDO and FoxP3 in NHLObjectiveIn this study, we collected samples of NHL and detected the expression of IDO and FoxP3 by IHC staining, western blot and q-PCR. Expression of IDO and FoxP3 were then made analyses with clinical features.MethodsUnstained paraffin sections of NHL(n=57) and normal control(n=18) were obtained from the Pathology Department of Shandong Provincial Hospital. Fresh tumor tissues(n=20) and normal lymph nodes(n=9) were collected during lymph node biopsy or laparotomy. The diagnosis of lymphoma was based on clinical features and pathology. Immunohistochemical (IHC) analyses were performed by the avidin-biotin peroxidase complex method using standard manual methods. Tissue sections were stained with antibodies directed against human IDO and FoxP3.Western blot analyses and Real Time RT-PCR were performed to investigate the protein and mRNA expression of IDO and FoxP3.ResultsIn the IDO-staining sections, positive-staining was observed in tumor cells. In addition, it was clearly demonstrated that IDO was highly expressed in cytoplasm. In contrast, cells of positive-staining in control group were markedly less than that in NHL. In all eighteen normal control sections, the positive cell rate was less than 30%, while in NHL group, eighteen in fifty-seven sections(31.6%) possessed a positive cell rate of no less than 30%. Patients with IDO strong-stained subtype were more likely to be diagnosed at a late phase(P<0.05), present with large tumor (P<0.05) and have a higher level of LDH in serum(P<0.05), all of which may indicate a worse prognosis. We also measured the IDO protein level by western blotting, which was coincidentally higher in NHL(n=20) than controls(n=9). The ratio of IDO toβ-actin were 0.77±0.25 in NHL group, while 0.18±0.18 in couterparts(p<0.05). Corresponding IDO mRNA was evaluated in NHL tumors(n=20) as well as normal control(n=9) with real time RT-PCR. Higher IDO mRNA expression was also observed in NHL group, whereas there was very little IDO mRNA detected in normal control(2-dct:0.00582～0.546 v.0-0.0103, p<0.05).The percentage of FoxP3+ cells was significantly increased in IDO strong-stained NHL samples compared with IDO weak-stained NHL or normal control (P<0.05). Statistics indicated that a positive corelationship existed between IDO and FoxP3 expression(WB:r=0.482; real time PCR:r=0.447).Conclusions1. Both expressions of IDO and FoxP3 are upregulated in NHL,2. Over-expression of IDO is corelated with a later clinical phases and higher LDH values, which indicate a worse prognosis.3. IDO level possess a close relationship with that of FoxP3.PartⅡPurification of Murine CD4+CD25-T-cell Subsets and T-cell Coculture with IDO-expressing A20 Cells in vitroObjectiveIn this part, we aimed to observe whether IDO+ tumor cells could convert CD4+CD25- T cells into Tregs.MethodsMurine CD4+CD25- T-cell was isolated from splenocyte of normal BALB/c mice by MACS. Purified T cells were used for phenotypic assays using Tregs flow cytometry kit. Purifed murine CD4+CD25- with IDO-expressing A20 cell line in the presence and absence of 1-MT, which specially inhibits IDO activity. After 24 hours, cells were collected and used for flow cytometry assays and MACS.The converted CD4+CD25+ T cells were isolated by MACS. To test its suppressive function, the MTT cell proliferation assays were performed. DCs isolated from splenocytes by Dynabeads Mouse DC Enrichment Kit. Splenocytes from naive BALB/c were cultured alone(negtive control group) or cocultured with DCs. The mixed cells were cultured in the presence(experimental group) or absence(positive control group) of converted CD4+CD25+ T cells. Converted CD4+CD25+ T cells was cultured alone as blank control group. The absorbence at 570nm(A value) was recorded by microtiter plate reader. Suppressive rate(SR) was calculated. SR=[1-(Aexperimental group-Ablank control group-Anegtive control group)/(Apositive control group-Anegtive control group)]×100%ResultsConversion of CD4+CD25" T cells to CD4+CD25+ T cells was observed after 24 hours. The mean percentage of CD4+ CD25+T cells was 7.87±1.65% in CD4+CD25-T cells cocultured with A20 cell line, whereas CD4+CD25+ T cells were nearly not detected in CD4+CD25- cells cultured without A20. The addition of 1-MT attenuated conversion of CD4+CD25- T cells into CD4+CD25+ T cells, as we detected the mean percentage of CD4+CD25+ T cells was 3.32±1.19%.In the 3-day MTT proliferation assays, compared with the negtive control group, Apositive control goup was significantly increased(0.8657±0.0353 vs 0.3434±0.0319, p<0.05), which indicated that lymphocyte were stimulated to proliferate. Meanwhile, Aexperimental goup was lower than Apositive control goup.The addition of converted CD4+CD25+ T cells lead to the decreasing of A value(0.4331±0.0280 vs 0.8657±0.0353, P<0.05). The suppressive rate(82.54±7.31%) indicated the suppressive function of converted CD4+CD25+ T cells.Conclusions1. A20 cell line spontaneously high-express IDO.2. MACS is a simple but effective technique to isolate cells into different groups.3. Cocultured with A20 cell line, CD4+CD25- T cells will be converted to CD4+CD25+ T cells which could suppress immune response. The addition of 1-MT could abrogate this conversion. PartⅢStudy on Tumor Model for NHL in MiceObjectiveIn this part, we set up a tumor model for NHL. The contribution of IDO in tumor induced tolerance and the anti-tumor effect of inhibitor 1-MT would be determined by detecting the Tregs in the tumors, lymph nodes and spleens.MethodsTo establish tumors,5×106 live A20 cells were s.c. injected into the right back flank of naive syngeneic BALB/c mice. Tumor growth was monitored three times per week using calipers. For our studies, tumorbearing animals were injected with 1-MT intratumorally every day as soon as the tumors were palpable. PBS was used as a control. Animals bearing tumors were euthanized when tumors reached a size of 20 mm in diameter or earlier if tumors ulcerated or animal showed sign of discomfort. Flow cytometry was performed to measure the percentage of Tregs in tumor, spleen and tumor-draining lymphoma.Results1. The percentage of CD4+CD25+ T cells in lymphocytes and the ratio of CD4+CD25+T cells and CD4+T cells were significantly higher in tumorbearing animals compared with normal controls. The addition of 1-MT could deduce the increasing ratio.2. Data of western blot showed that, IDO expression in tumors, lymph nodes and spleens was upregulated in tumorbearing animals. FoxP3 was over-expressed in tumorbearing animals, but the administration of 1-MT could abrogate this upregulation.Conclusions1. A20 B lymphoma is a useful tumor model in research on IDO and immune tolerance.2. Over-expression of IDO in tumor, lymph nodes and spleens of tumorbearing animals leads to immune tolerance by incresing the percentage of Tregs.3. Administration of 1-MT can decreace the percentage of Tregs and may serve as a potential antitumor agent in the future.更多还原