吲哚胺2，3-双加氧酶在非霍奇金淋巴瘤免疫耐受中的作用及机制探讨

发布时间：2018-01-11 10:39

本文关键词：吲哚胺2，3-双加氧酶在非霍奇金淋巴瘤免疫耐受中的作用及机制探讨　出处：《山东大学》2010年博士论文　论文类型：学位论文

【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,P0.05)。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,P0.05)。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,P0.05),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,P0.05),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(P0.001),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。
[Abstract]:Research background
Non Hodgkin's lymphoma (NHL) is a common malignant tumor originated from lymph node or other lymphoid tissues, the etiology and pathogenesis of the disease is still not very clear, immune tolerance may play a key role in tumorigenesis, development and metastasis. With rituximab (anti CD20 antibody) as the representative of the target for immune lymphoma the treatment has achieved good curative effect. But there is still an important problem is to overcome the formation of tumor cell immune tolerance, immune system is unable to effectively recognize tumor cells and kill. The formation of immune tolerance cells mainly involved in the process include: dendritic cells (dendritic cells, DCs), regulatory T cells (regulatory T cells, Tregs), mast cells, and even see the eosinophil, tumor cells can directly or indirectly through dendritic cells induced tumor immune tolerance by micro environment. Therefore, research To investigate how NHL tumor cells directly or indirectly form immune tolerance tumor microenvironment through antigen presenting cells, so as to block the key pathway of tumor immune tolerance, it will play a key role in NHL treatment.
As a research hotspot in recent years, the role of tryptophan metabolism in inducing immune tolerance in constantly reported. Indoleamine 2,3- dioxygenase (Indoleamine 2,3-dioxygenase IDO) is a liver tryptophan metabolism only can catalyze tryptophan indole ring oxidation cracking, thus limiting enzyme along the kynurenine catabolism pathway; mainly expressed in some immune tolerance or immune amnesty, such as thymus, gastrointestinal mucosa, epididymis, placenta and anterior chamber, and is specifically expressed in macrophages and dendritic cells. Through a lot of research on mouse and human DCs found that the high expression of IDO and DCs subsets the immune tolerance is closely related, such as participation in inducing T cell apoptosis, T cell anergy and activation of regulatory T cells. The results show that IDO high expression in tumor and tumor metastasis in lymph nodes, which proved the argument and tumor free Immune tolerance mechanism, and play an important role in it. The 1- methyltryptophan (1-MT) as the representative of the IDO inhibitor has been used in phase I clinical trials in foreign countries, and achieved good results.
The researchers found that high expression of all IDO parts, such as the placenta, tumors and lymph node metastases, there are a lot of T cell infiltration phenomenon. Regulation of regulatory T cells was first reported in 1995, the transcription factor FoxP3 is a specific marker of Tregs, is the development of key regulatory gene.Tregs plays an important role in the same maintaining self tolerance, its role has been confirmed in a variety of immune, tumor models, and the latest research shows that Tregs may be closely related to the proliferation and activation of IDO+DC subsets. Studies have confirmed: in a variety of tumors, tumor cell lines, acute leukemia and adult T cell leukemia / lymphoma there too the expression of IDO, and through the research of the murine lymphoma model confirmed the correlation between IDO expression and Tregs proliferation. However the specific mechanism of IDO regulation of Tregs remains to be further studied.
The purpose of this study is to investigate the expression of IDO in patients with NHL in the tissues, and explore the correlation between Tregs and NHL in the occurrence, development and prognosis effect. Then through experiments in vitro and animal model of NHL, IDO in NHL verification of tumor induced immune tolerance, and the mechanism of detailed study for the clinical prognosis of NHL. To explore new targets, and provide strong theoretical support for the clinical application of IDO inhibitor 1-MT.
Expression and significance of part I IDO and FoxP3 in human non Hodgkin's lymphoma
research objective
Taking human NHL as a study object, the expression of IDO in NHL was discussed, and the correlation with the clinical features and the correlation with Tregs was analyzed.
research method
1. the NHL blank paraffin section (n=57) was collected and the reactive hyperplasia (n=18) was used as the control. The expression and location of IDO and FoxP3 in the tissues were studied by immunohistochemistry.
2. NHL fresh specimens (n=20) were collected, and the expression of IDO and FoxP3 at mRNA level was compared by real-time quantitative RT-PCR technique.
3. the fresh specimens of NHL (n=20) were collected, and the expression of IDO and FoxP3 at protein level was compared by Western blot technique.
4. statistical analysis of IDO expression difference in NHL and reactive hyperplasia and its correlation with FoxP3. To explore the relationship between IDO and clinical data of non Hodgkin lymphoma, such as age, gender, clinical stage, pathological type, LDH and prognosis.
Research results
1. immunohistochemistry results showed that IDO positive cells in control group were below 30%, and 57 cases of NHL paraffin sections, IDO+NHL (positive rate in more than 30%) 18 cases (31.58%) the positive rate of FoxP3.18 cases of IDO+NHL paraffin section was significantly higher than the control group and 39 cases of.IDO-NHL positive IDO-NHL slice slice the rate of FoxP3 had no significant difference with control group. The clinical data of patients with NHL, 18 patients with clinical IDO+NHL staging for stage III or IV, and 39 cases of IDO-NHL patients with clinical stage relatively early, the difference was statistically significant in patients with.IDO+NHL LDH and the same diameter of the tumor was significantly higher than IDO-NHL patients. The above data indicate that IDO and NHL is closely related to the prognosis.
The expression of IDOmRNA in 2.20 fresh NHL specimens was significantly higher than that in the control group (2-dCt:0.00582 ~ 0.546 v.0 ~ 0.0103, P0.05). The expression of IDO and FoxP3mRNA in NHL group was positively correlated (r=0.447). The expression level of IDOmRNA was significantly higher in the NHL group than that in the control group.
The expression of IDO protein in 3.20 fresh NHL specimens was significantly higher than that in the control group (WB, 0.77 + 0.25 vs.0.18 + 0.18, P0.05). The expression of IDO and FoxP3 on protein level was positively correlated (r=0.613) in the.NHL group.
conclusion
There is a high expression of IDOmRNA and protein in the 1. part of NHL.
The high expression of 2.IDO is closely related to the clinical staging of NHL and the value of LDH, suggesting that IDO may indicate poor prognosis in patients with NHL.
The expression of 3.IDO is positively related to FoxP3, and it is suggested that the IDO+NHL tumor cells may be closely related to the function of Tregs.
Study on the regulation of Tregs in part II of the lymphoma cell line with high expression of IDO
research objective
The B lymphoma cell line A20 and mouse T cells with high expression of IDO were selected as the research objects. In vitro mixed cell culture, it was confirmed that the high expression of IDO lymphoma cell line could increase the proportion of Tregs.
research method
1. immunomagnetic bead technique (MACS) was used to isolate CD4+CD25- T cells from BALB/c mice and the purity of the cells was verified by flow cytometry.
The selection of 2. A20 high expression cell line IDO (from BALB/c mice), IDO+A20 cells and BALB/c mice CD4+CD25-T cells co culture into the following three groups: CD4+CD25- group, CD4+CD25- T T cells; +A20 cells; the CD4+CD25- T +A20 cell +1- methyltryptophan group.
After 24 hours of culture, the proportion of CD4+CD25+T cells was measured by flow cytometry. The effects of IDO+ tumor cells and IDO inhibitor 1-MT on the proliferation and function of Tregs were observed.
4.MACS collection of cultured CD4+CD25+T cells of the second group; separation of BALB/c mice spleen DCs MACS; mononuclear cells were isolated from mouse spleen BALB/c in Ficoll (MNC). Divided into the following 4 groups of mixed lymphocyte reaction: CD4+CD25+Tc+DCs+MNC; MNC+DCs; MNC group; CD4+CD25+Tc group. After 72h of culture determination of the absorbance at 570nm, MTT (A), calculate the inhibition rate of CD4+CD25+T cells (supressive, rate, SR).SR=[1- (A1-A3-A4) / (A2-A3)] x 100%
Research results
1. flow cytometry proved that the purity of CD4+CD25- T cells isolated by MACS technology was more than 98%.
2. mixed cell culture after 24 hours, determination of the proportion of T cells CD4+CD25+ by flow cytometry: the first group was 0; the second group is 7.87 + 1.65%; the third group is 3.32 + 1.19%, confirmed that the high expression of A20 in IDO cell line can be transformed to the CD4+CD25+ phenotype of CD4+CD25-T cells induced by 1-MT, and can inhibit the process of.
Determination of inhibitory function of CD4+CD25+T cells transformed by 3.MTT: positive control group A was significantly higher than the negative control group (0.8657 + 0.0353 vs 0.3434 + 0.0319, P0.05), the proliferation of T cells that, under the effect of DCs activation; and A value of experimental group was significantly less than the positive control group (0.4331 + 0.0280 vs0.8657 + 0.0353. P0.05), confirmed after the conversion of Tregs can inhibit the proliferation of T cells (the inhibition rate was 82.54 + 7.31%).
conclusion
1.A20 cell line is stable and high expression of IDO. in vitro
2. immunomagnetic bead technology is a simple and effective method of cell separation, which can be widely used in clinical and basic research.
The 3.IDO+ tumor cells can transform the CD4+CD25-T cells into CD4+CD25+ T cells in vitro, and the transformed CD4+CD25-T cells have the immunosuppressive function.
4.IDO inhibitor 1-MT can reverse the transformation of CD4+CD25-T cells into CD4+CD25+T cells by IDO+ tumor cells in vitro, indicating that this transformation is IDO dependent.
The establishment of Part III animal model and the study of the effect of IDO on the regulation of Tregs
research objective
The content of Tregs in the tumor, drainage lymph node and spleen of IDO+NHL mice and the effect of IDO inhibitor l-MT on Tregs were studied by NHL animal model.
research method
1. the animal model of NHL was established: 6-8 weeks old BALB/c mice were selected and A20 cell suspension was injected subcutaneously (5 x 106/).
After 2.1 weeks, the mice with similar tumor size were enrolled in the experiment. The normal BALB/c mice of the same age were divided into the following three groups: the normal control group (n=6), the tumor group (n=6) and the 1-MT intervention group (n=6).
3. for the 1-MT intervention group, 1-MT solution was injected daily in the tumor, and the normal group and the tumor group were injected with the same volume of PBS..
After 3.2 weeks, the mice were killed, and the tumor, spleen and lymph nodes were collected. The content of Tregs in tumor tissues, lymph nodes and spleen was detected by flow cytometry. The expression of IDO and FoxP3 in tissues was detected by Western Blot. Data were collected and analyzed statistically.
Research results
The tumor formation rate of 1.A20 B cell lymphoma model is 95%, and the time of tumor formation is about 7 days.
Results of 2. flow cytometry
In terms of the proportion of CD+CD25+T cells in tumor tissue, spleen and lymph nodes in the proportion of lymphocytes, the tumor group was significantly higher than that in the 1-MT intervention group, which was also significantly higher than that in the normal control group.
CD4+T cells in the tumor, accounting for the proportion of lymphocytes in spleen, tumor group, no difference between 1-MT group and control group. The tumor draining lymph node and control lymph nodes, CD4+ T cell percentage of lymphocytes in tumor group was significantly lower than that in normal control group, but no significant difference between the intervention group and 1-MT intervention group, 1-MT the same was significantly lower than the normal control group.
In tumor tissue, CD4+CD25+T cells of /CD4+ T cell tumor group was significantly higher than that of 1-MT group. In the spleen, the proportion of CD4+CD25+T cells and /CD4+T cells in tumor group was significantly higher than the normal group, were significantly higher than those in 1-MT group. The difference between normal control group and 1-MT group had no statistical significance. The tumor draining lymph node and control lymph nodes. The proportion of CD4+CD25+T cells, /CD4+T cells in the tumor group was significantly higher than that in normal group (P0.001), was significantly higher than 1-MT group.1-MT group was still higher than the normal control group.
3. Western Blot
The results showed that the expression of IDO in tumor, spleen and lymph nodes in tumor group and 1-MT group was significantly higher than that in control group.
The expression of FoxP3 in tumor group, spleen and lymph node was significantly higher than that of normal control group, but it was obviously higher than that of group 1-MT and normal control group, no significant difference was found between.L-MT group and normal control group.
conclusion
1.A20 B cell lymphoma is a good animal model of NHL.
2.IDO is highly expressed in the tumor, spleen, and tumor drainage lymph nodes of animal models, and induces immune tolerance by up regulation of Tregs.
3.1-MT, by reducing the up regulation effect of IDO on Tregs in animal models, provides an effective synergistic effect on the chemical and immunotherapy of lymphoma, and has great potential in anti-tumor therapy.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【参考文献】