【作者】 李艳萍；

【导师】 徐惠绵；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2004， 博士

【摘要】 目的癌症免疫治疗的主要目的是诱导有效的抗肿瘤特异细胞毒T淋巴细胞(CTL)，而主要组织相容性复合物(MHC)Ⅰ类分子在一系列恶性肿瘤中往往低表达，不足以在体内引起有效的免疫应答。人癌症中的自身抗原是免疫系统识别的最普遍抗原，说明癌细胞源于宿主自身组织而非外源蛋白。识别自身抗原存在许多问题，如由于自身耐受难以引出抗肿瘤免疫。即使免疫系统能识别并与自身抗原反应，通常免疫力也不足以排斥肿瘤。FasL是肿瘤坏死因子(TNF)蛋白家族的一个成员，组织学和流式细胞仪分析提示FasL表达细胞能引起局部中性粒细胞浸润，产生强烈的炎症，使肿瘤细胞受损，之后依赖CD8~+T淋巴细胞的抗肿瘤免疫，导致肿瘤细胞的清除。B16F10恶性黑色素瘤免疫原性极弱，因此是研究提高肿瘤免疫策略的良好模式。恶性黑色素瘤最具特色的抗原是非突变分化抗原，不仅表达于恶性黑色素瘤细胞，也表达于正常黑色素细胞，如糖蛋白100(gp100)，酪氨酸酶、酪氨酸酶相关蛋白(TRP)1和2、黑素A(Melan-A)。从这些抗原中选择3个有肿瘤排斥功能的抗原决定簇：TRP2、鼠糖蛋白100(mgp100)和它更具免疫源性的对应物人gp100(hgp100)。此外，还选择了2个在B16F10中表达的内生性病毒抗原决定簇恶性黑色素瘤抗原肽(mdm)、逆病毒包膜蛋白(p15E)，合成抗原肽，制作Tetramer。MHC-Ⅰ类分子-肽四聚体复合物技术是近年牛津大学同美国STAN-FORD大学共同研制发明的。它可以通过荧光激活细胞分类仪(FACS)在单细胞水平直接检测抗原特异性CTL，并同时进行细胞定量。经放射并表达FasL的B16F10细胞皮下注射使鼠产生免疫，3周后野生型B16F10激发，保护鼠行tetramer分析。牛痘病毒不仅诱导体液免疫，且强烈地诱导细胞免疫。因此，为了提高免疫应答，部分保护鼠用含黑色素分化抗原的重组牛痘病毒(rVV)加强。体内抗原特异杀伤还用另一种新技术来检测。希望我们的观察能帮助确定新的肿瘤细胞表面标志物，提供一种刺激产生广谱抗肿瘤免疫的新途径。实验方法本实验用B16F10恶性黑色素瘤C57BL／6鼠模型。使用2种技术来检测免疫鼠中抗原特异CTL活性。1) MHCⅠ类分子-肽-tetramer。选择5种相关肿瘤抗原决定簇，合成抗原肽，其氨基酸序列如下：mdm(Kb)：YAMIYRNL，p15E(Kb)：KSPWFTTL，trp-2(v)(K~b)：VYDFFVWL，trp-2(s)(K~b)：SVYDFFVWL hgp100(D~b)：KVPKNQDWL，mgp100(D~b)：EGSRNQDWL，制作相应tetramer：BirA生物素酶识别位点的MHC-H-2K~b或H-2D~b的胞外区、β2m的cDNA被克隆进入表达质粒，蛋白以包涵体形式在大肠杆菌(E.coli)中表达。重链、β2m和合成抗原肽在4℃含蛋白酶抑制剂的缓冲液中折叠，形成稳定的三分子复合物。浓缩后，复合物酶标生物素酰基化，由快速蛋白液相色谱(FPLC)按照分子量大小纯化。生物素酰基化蛋白单体与结合荧光素的细菌蛋白以4∶1摩尔比率混合。2)体内杀伤分析。CFSE是一种分子探针染料，在488nm可激发绿色荧光，因此可通过流式细胞仪检测。树突状细胞(DC)如与荧光染料结合，可在引流淋巴结中被检出。DC与MHC-Ⅰ类分子抗原肽结合，可作为CTL的靶，如存在抗原特异CTL，结合抗原DC的特异杀伤就能在不同小鼠中被检测出来。结合CFSE的同源靶细胞(脾)加入相关抗原肽，尾静脉注入保护鼠，5～18小时采集鼠血或鼠脾，FACS分析。标记相关抗原肽的靶细胞由于被特异CTLs识别和杀伤，应该减少或消失。将实验与对照鼠分成4组，施以不同处理因素：FasL，rVV(mgp100，NP)，FasL+rVV组，对照组，用以初次免疫激活。用自然鼠脾细胞与抗原肽(hgp100，Np)加强一次，行tetramer检测及体内杀伤实验。体外培养各组脾细胞建立CTL细胞系，共分两组，1组用hgp100刺激，末次刺激后一周用hgp100，mgp100两种tetramer检测，另1组分别用6种抗原肽刺激，末次刺激后行tetramer交叉检测。实验结果我们建立起一种tetramer与碘化丙啶(PI)相结合的技术。排除PI阳性细胞，有助于减少死细胞非特异染色，提高染色质量。重复多次实验，除一只rVVNP处理鼠作为阳性对照，脾脏中检出抗原特异CTL，其他各组血液、淋巴结、脾tetramer检测均阴性。我们共进行了5次杀伤实验，均难以解释。由于没能从鼠中得到足够细胞，标记CFSE、结合抗原肽的对照峰和相关峰都较低。血液标本出现双峰，脾脏检测出现单峰。与其他组相比，FasL+rVVgp100组，峰高明显降低，但无抗原特异性，因为对照峰与相关峰均降低。体外细胞培养，第1组中rVV处理细胞、FasL+rVVgp100处理细胞，tetramer检测，出现抗原特异CTL，hgp100、mgp100 tetramer检测均阳性，其它组阴性。第2组中FasL+rVVgp100处理细胞，出现tetramer交叉检测阳性，其它处理因素脾细胞检测阴性，提示在一些标本中存在非特异背景染色。结论1．tetramer技术能精确检测肿瘤抗原特异CTL。2．FasL与rVVgp100两种处理因素联合能打破恶性黑色素瘤B16F10的自身耐受状态，产生抗原特异性CTL。3．FasL与rVVgp100两种处理因素联合，可能诱导固有免疫，产生非特异性杀伤。4．FasL与rVVgp100两种处理因素联合，能扩大免疫效应，产生抗原交叉反应。5．小鼠免疫成功与否，与不同小鼠不同免疫状态及反应性有关。将来有必要扩大每组小鼠数量。6．FACS检测体外培养细胞时，同时使用PI能去除死细胞干扰，提高染色质量。7．细胞体外培养，末次刺激后两周tetramer检测达高峰。8．本研究结果为研制肿瘤疫苗、制定肿瘤治疗策略提供了新思路。更多还原

【Abstract】 The purpose of immunotherapy in cancer is to induce an effective anti-tumorspecific cytotoxic T lymphocytes (CTL) response, however, major histocompati-bility complex (MHC) class I is usually expressed at low levels by tumors andcant elicit effective immune responses. Self-antigens are the most common anti-gens that are recognized in human cancer. Immune responses to self-antigens aredifficult to induce because of self-tolerance. Even if the immune system recogni-zes and makes a response to self antigens, it is often too weak to reject thetumor.FasL is a member of TNF protein family; expression of FasL can confer im-mune privilege to some organs, allowing them to kill infiltrating lymphocytes andinflammatory cells. However, the expression of FasL in a number of instancesprovokes an intense neutrophil infiltrating. Neutrophils expressing Fas can be at-tracted and activated by FasL to become cytotoxic. It is believed that this neu-trophil activation impairs tumor growth, then induces an adaptive immune re-sponse possibly mediated by CTLs. In this study we tested whether FasL ex-pressed by tumors is able to induce a CTL response. TheB16F10 melanomamodel was used because it is a poorly immunogenic tumor and thus a good modelto test strategies to enhance tumor immunity. The best-characterized melanomaantigens are the non-mutated differentiation antigens expressed in both melanomaand melanocytes: gp100, Tyrosinase, Tyrosinase-Related-Protein (TRP) 1 and2 and Melan-A. Three epitopes were chosen from these antigens known to be in-volved in the rejection of the tumor: murine gp100 (mgp100) and its more im-munogenic counterpart in human (hgp100) and TRP2. Furthermore twoepitopes from endogenous virus described to be expressed by B16F10 were alsochosen, peptides were synthesized and tetramers generated. The MHC class I tetramer technology was recently developed by Oxford andStanford universities. It can quantify and analyze antigen specific CTL at a sin-gle cell level by FACS. Mice were immunized with irradiated B16F10 expressingFasL and then challenged with wild type B16F10. Protected mice were chosenfor tetramer analysis. To improve the response, protected mice were boostedwith recombinant vaccinia virus expressing the melanocyte differentiation anti-gens. It is known that vaccinia virus can strongly induce T cell-mediated immu-nity as well as humoral immune response. Furthermore, antigen specific killingwas measured in vivo using a novel technique. We hope our observations mayhelp for the definition of new cell surface tumor markers and may provide an ad-ditional route to stimulate a broader anti-tumor response.Material and MethodsWe used the B16F10 melanoma C57BL/6 model. Two techniques wereused to detect antigen-specific CTL activity in immunized mice: 1) MHC classI-peptide tetramers. The protein sequence of five relevant epitopes are as fol-lows: mdm(K~b):YAMIYRNL,p15E (K~b) : KSPWFTTL, trp-2 (v) (K~b): VYD-FFVWL,trp-2(s) (K~b) :SVYDFFVWL hgp100 (D~b) : KVPKNQDWL. Makingcorresponding tetramers :Briefly, the cDNA for the MHC class-I and beta 2 mi-croglobulin are cloned into expression plasmids, and protein expressed as inclu-sion bodies within E. coli. After solubilisation, the class I molecule,theβ2mand synthetic peptide are added in relatively large quantities(10-30mg) to abuffer solution containing protease inhibitors at 4℃, in which the formation of astable trimolecular complex can occur, after concentration, the complexes areenzymatically biotinylated and purified by size. Monomeric complexes may thenbe frozen for future use or immediately formed into tetramers around streptavidin-phycoerythrocin (SA-PE) conjugates. 2) In vivo killing assay: CFSE is a mo-lecular probe that emits green fluorescence at 488nm, thus can be detected byFACS. Combine with DC, it can be found in draining lymphatic nodes. It canbe a target for CTL if DC integrates with MHC class I Label syngeneic targetcells (Spleen)with CFSE and pulse with relevant peptides, then inject into pro- tected mice, bleed mice 5～18 hours later and perform FACS analysis of bloodsamples. Target cells labeled with the relevant peptide should disappear, as theywill be specifically killed by CTLs recognizing the peptide. Mice were dividedinto 4 groups for priming and boosting 1) B16F10-FasL followed by rVV(mgp100, NP), B16F10-FasL, rVV ,naive mice. Sometimes mice were alsoboosted with spleen cells loaded with peptides one week later, Tetramer stainingand vivo killing were performed. Spleen cells of the different groups are culturedto set up CTL lines. All cells were divided into two groups, one group was stim-ulated with hgp100 and tested by two kinds of tetramers: hgp100, mgp100 aweek later after final in vitro stimulation. The other six CTL lines were stimula-ted by six peptides and crisscross staining was performed after finial stimulation.ResultsWe established a technique whereby tetramer staining was combined withPropidium iodide (PI). Excluding PI positive cells helped to reduce the unspe-cific staining of dead cells. No antigen specific CTLs were detected ex vivo, tes-ting blood, lymph nodes and spleens except a mouse that received vaccinia-NPand was stained with NP tetramer used as a positive control. We performed 5 vi-vo killings, which are difficult to interpret. Both the irrelevant control peak andthe relevant peak from the CFSE labeled and peptide-pulsed targets are verylow, as we did not retrieve enough cells from the mice. Blood testing yielded twopeaks, while spleen only one. Compare with other groups, the peaks of FasL+rVVgp100 disposing group are remarkably lower, but it is obvious that they arenot antigen specific killings as not only the peak with relevant peptide, but alsothe one with irrelevant peptide low. Tetramer positive CTLs were observed incell lines made from mice that received rVVgp100 and FasL+rVVgp100, testedby tetramer hgp100 and mgp100. All other groups are negative. The cell linemade from FasL+rVVgp100 mice also stained positive with irrelevant tetramer,while the other CTL lines negative, indicating that there is non-specific back-ground occurring in some of the samples. Conclusions1. Tetramer can detect antigen specific CTL accurately.2. Combination of FasL and rVVgp100 can break self-tolerance to B16F10melanoma.3. Combination of FasL and rVVgp100 induces possibly an innate immuneresponse, leading to unspecific killing.4. Combination of FasL and rVVgp100 augments the overall immune reac-tion, leading to non-specific binding of irrelevant tetramer.5. Success of immune response in mice varies among individual mice andtherefore bigger groups of mice need to be included in the future.6. In combination with PI, the results of tetramer staining of CTL lines byFACS axe remarkably improved.7. Best result was obtained after two weeks after final stimulation for theCTL lines.8. Our observation and results will provide a basis to develop new anti-tumor vaccine and immunotherapy.更多还原