【作者】 张镭；

【导师】 储以微；

【作者基本信息】 复旦大学 ， 免疫学， 2008， 硕士

【摘要】 肿瘤是威胁人类健康和生命的最重要疾病之一。抗肿瘤化疗药因其对肿瘤细胞的杀伤而成为常用的治疗手段。然而,由于抗肿瘤化疗药在杀伤肿瘤细胞的同时对机体的免疫细胞也产生损伤,使淋巴细胞数量减少,因此,以往认为,抗肿瘤化疗不利于进行后续的主动免疫生物治疗。然而,近年来研究发现,抗肿瘤化疗药作用后过继转输自体肿瘤特异性CTL细胞,获得优于单一使用过继转输淋巴细胞的抗肿瘤效果。我们课题组的前期动物研究也发现:以抗肿瘤化疗药泰素注射荷瘤小鼠后再给予荷有肺癌抗原多肽的DC瘤苗,对荷瘤小鼠具有显著的治疗效应,效果明显优于单一肿瘤疫苗的使用。这些实验结果充分提示抗肿瘤化疗药的使用不仅没有抑制机体的免疫应答,反而能诱导出有利于抗肿瘤免疫应答的微环境。然而,国内外对于抗肿瘤化疗药发挥免疫学效应的机制尚无深入的研究。鉴于此,我们推测以泰素为代表的抗肿瘤化疗药除具有直接杀伤肿瘤细胞的作用外,还影响免疫细胞及免疫应答,通过作用于机体的免疫细胞,改变机体的免疫格局与功能,为荷瘤机体进一步开展免疫生物治疗提供益处。在本研究中,我们从已有基础研究出发,着手临床病例,通过对16例肺癌手术患者以及50例肺癌化疗患者的手术标本或外周血淋巴细胞的检测,研究以泰素为代表的抗肿瘤化疗对肿瘤患者外周免疫细胞格局的改变,联合体外实验研究泰素作用机体免疫细胞的作用机制,进一步探讨以泰素为代表的抗肿瘤化疗药除细胞毒作用外的潜在作用,为抗肿瘤化疗联合主动生物治疗的临床应用提供理论和实验基础,同时也为肿瘤的治疗提供新思路和新策略。第一部分肺癌患者体内的免疫格局肿瘤的发生发展和机体对其的免疫监视始终处于动态改变中,当免疫正效应发挥作用,可以抑制肿瘤生长;相反,当免疫负效应占据主导地位,则促使机体发生免疫抑制并使肿瘤发生免疫逃逸而持续生长。因此,以非小细胞肺癌患者为研究对象,分析患者的免疫细胞亚群有利于了解荷瘤机体的免疫状态,为改善和优化抗肿瘤免疫提供对策。已有研究证明调节性T细胞在抗肿瘤免疫应答中起抑制作用,其在体内的比例改变与抗肿瘤免疫效应的发挥以及肿瘤生长密切相关。因此,我们检测了66例非小细胞肺癌患者及20例正常对照外周血中免疫细胞亚群,特别是调节性T细胞亚群的比例变化。结果显示:与正常对照比较,肿瘤患者的外周血白细胞,淋巴细胞绝对数,CD3⁺T淋巴细胞亚群、CD19⁺B淋巴细胞亚群,CD8⁺T淋巴细胞亚群百分比都没有明显改变,但肺癌病人CD4⁺T淋巴细胞的比例明显低于正常对照。在此基础上,对肿瘤患者CD4⁺CD25⁺Foxp3⁺淋巴细胞亚群检测结果显示,其百分比明显高于正常对照;而CD4⁺CD25⁺Foxp3^-T淋巴细胞与正常对照比较无明显改变。提示肿瘤患者外周血淋巴细胞中效应T细胞并没有增加,增多的CD4⁺CD25⁺T细胞主要为CD4⁺CD25⁺Foxp3⁺调节性T细胞。为探讨肺癌患者调节性T细胞亚群比例升高在肿瘤发生发展中的作用,我们将肿瘤患者外周血中调节性T淋巴细胞的改变与肿瘤患者的年龄、性别,以及肿瘤病理等进行相关性比较,结果发现外周血中调节性T淋巴细胞的升高与肿瘤患者的年龄、性别,以及肿瘤病理等不存在相关性,但是与肿瘤的临床分期密切相关。根据临床TNM分期发现,肿瘤患者CD4⁺CD25⁺Foxp3⁺调节性T细胞的比例在癌症各个时期均显著高于正常对照,其中癌症晚期明显高于癌症早期,存在显著性差异。上述结果表明:1)肿瘤患者机体外周血中Treg细胞亚群占外周淋巴细胞的比例明显升高;2)随着肿瘤病程的不断进展,Treg细胞亚群也不断增多,Treg细胞的数量与肿瘤病程进展密切相关。肿瘤的免疫逃逸除了肿瘤细胞本身免疫原性低下、缺乏表达协同刺激分子使得机体免疫系统难以诱生有效的免疫应答以外,机体的免疫负调控效应也在其中扮演重要角色。故推测Treg细胞亚群的增多可能是诱导肿瘤发生免疫逃逸的重要因素之一。第二部分抗肿瘤化疗改变肺癌患者的免疫格局抗肿瘤化疗因其对肿瘤细胞的直接杀伤作用成为当前最为常用治疗肿瘤的方法之一。传统理念认为抗肿瘤化疗在杀伤肿瘤细胞的同时,也杀伤机体正常细胞,使机体的免疫功能严重受损。但近来,有研究发现在淋巴细胞减少的状态下,机体发生免疫格局的改变,有利于免疫应答的产生。基于上述研究结果,我们设想:抗肿瘤化疗能使肿瘤患者淋巴细胞的比例发生重新分配,使机体重建免疫格局。因此,我们共收集了66例非小细胞肺癌病人,其中50位患者接受了1～5次不等的以泰素为主的抗肿瘤化疗方案,获取总共70次化疗的外周血标本。且这些患者在化疗过程中未接受任何与免疫相关的治疗。通过分析其抗肿瘤前后不同淋巴细胞亚群的比例、细胞膜表面相关CD分子的变化,研究抗肿瘤化疗对免疫格局的影响。结果显示:抗肿瘤后白细胞和淋巴细胞显著降低,在淋巴细胞亚群中CD3⁺T细胞亚群比例明显下降,但同时CD19⁺B细胞在外周淋巴细胞中所占的百分比例未发生明显改变。聚焦T细胞亚群的改变,我们发现,CD4⁺T细胞亚群在抗肿瘤化疗后占CD3⁺T细胞比例明显减少,而CD8⁺T细胞亚群在CD3⁺T细胞中所占比例没有发生明显改变。在CD4⁺T细胞中,我们着重关注了CD25⁺细胞亚群比例的变化,发现其中CD25⁺在CD4⁺细胞中所占的百分比例明显下降,而CD25^-亚群所占比例有所上升。更进一步,我们关注抗肿瘤化疗后CD25⁺T细胞亚群,尽管Teff和Treg细胞化疗后绝对数明显减少,但是Teff在CD4⁺T细胞中所占的百分比未发生明显改变,而Treg细胞亚群所占的百分比也显著降低。这表明Treg细胞的减少幅度明显大于Teff细胞亚群,以泰素为代表的抗肿瘤化疗能选择性减少Treg细胞亚群。上述结果表明:1)以泰素为代表的抗肿瘤化疗可作用于机体的免疫细胞,使机体的免疫细胞亚群比例发生重新分配;2)在各个T细胞亚群中,抗肿瘤化疗药泰素能选择性下调Treg细胞亚群的比例,一定程度削弱由于Treg细胞增加而诱导的肿瘤发生免疫逃逸。第三部分抗肿瘤化疗选择性下调Treg亚群比例及功能泰素是紫杉醇化疗药家族中的一员,它通过抑制微管形成,直接杀伤肿瘤细胞发挥抗肿瘤效应。我们的研究已证明泰素不仅能作用于机体的免疫细胞,并且其对不同细胞亚群的影响存在差异。由此我们假设,泰素除直接发挥细胞毒作用外,是否还存在免疫作用机制。我们通过体外分选正常人外周血获得Treg亚群细胞及作为对照的T效应细胞亚群（Teff）,体外实验研究泰素的可能的作用机制,并在临床病例中进行实验验证,探讨泰素诱导机体免疫细胞数量及功能改变的可能性机制。结果显示:相较于未使用泰素化疗的Treg细胞,化疗后的Treg细胞的凋亡显著增加,细胞表面的CD95表达明显上调,但Teff细胞亚群在泰素的作用下,凋亡比例未发生明显上调,且细胞表面的CD95表达未发生改变。我们在临床病例的实验中也获得了类似的实验结果。由此假设,泰素是通过增强Treg细胞亚群表面的CD95的表达,Fas/FasL途径诱导Treg细胞凋亡,选择性杀伤Treg细胞亚群。为进一步了解泰素化疗影响Treg亚群的数量同时,是否可以影响Treg和Teff细胞亚群的功能,我们通过体外增殖实验来检测Treg和Teff亚群的功能,发现泰素化疗后的Treg细胞的免疫抑制功能明显下调。我们同时发现相较于未化疗的Treg细胞,化疗后Treg细胞表面Foxp3平均荧光强度明显下降,由此我们推测,泰素能通过降低Treg细胞Foxp3的表达,来抑制Treg细胞的负调功能。上述结果表明:1)泰素能通过选择性上调Treg细胞表面的CD95的表达,诱导Treg细胞亚群凋亡;2)泰素能通过下调Treg的Foxp3表达强度,削弱Treg细胞发挥免疫抑制功能。因此泰素不论从数量上还是功能上,都对Treg产生抑制作用,通过下调Treg细胞亚群在抗肿瘤免疫中发挥免疫抑制效应。第四部分抗肿瘤化疗诱导肿瘤免疫微环境朝向Th1免疫格局在之前的研究中已证实Treg在肿瘤免疫逃逸中发挥重要的作用,且泰素化疗能选择性作用于Treg细胞亚群,解除Treg细胞亚群在抗肿瘤免疫中发挥的免疫抑制效应,那么此时机体的免疫情况又是如何呢?我们选择在肿瘤免疫中发挥重要作用的Th1细胞亚群进行研究,我们通过流式细胞术胞内染色的方法检测了肿瘤患者抗肿瘤前后CD4⁺T细胞分泌IL-2的表达量以及CD8⁺T细胞分泌IFN-γ的表达量。我们发现抗肿瘤化疗后,肿瘤患者外周淋巴细胞中具有分泌IL-2能力的CD4⁺T细胞和具有分泌IFN-γ能力的CD8⁺T细胞不论是绝对数量还是所占的百分比都明显增加。另一方面,我们同时检测了细胞亚群的活化和记忆标志。我们以CD44为活化标记,CD62L为记忆标记检测了肿瘤患者外周淋巴细胞的情况。发现CD4⁺和CD8⁺T细胞高表达CD44^high活化标志,CD62L^low记忆CD4⁺和CD8⁺T细胞在抗肿瘤化疗后,其所占的外周淋巴细胞百分比都有明显上升。上述结果表明:Treg细胞的作用被削弱后机体的Th1细胞免疫应答作用增强;2)发挥抗肿瘤作用的功能细胞群比例也有所升高。因此在抗肿瘤化疗的作用下,由于Treg的肿瘤免疫逃逸的作用被解除,机体的免疫格局发生重建,为进一步进行主动免疫提供了基础和保障。综上所述,本研究我们以抗肿瘤化疗作用肺癌患者的免疫细胞亚群为切入点,以肺癌患者手术切除肿瘤样本及肺癌化疗患者外周血为研究对象,通过对以泰素为代表的紫杉醇类抗肿瘤化疗药的研究,分析抗肿瘤化疗后淋巴细胞数量及各亚群比例,发现抗肿瘤化疗药作用后能使机体的免疫细胞各亚群的比例发生重新分配,并且其中诱导肿瘤发生免疫逃逸的重要因素之一的Treg细胞亚群的减少最为显著。更为重要的是,我们通过实验发现泰素能通过选择性上调Treg表面的CD95诱导Treg凋亡,同时还能通过下调Treg的Foxp3表达强度削弱Treg的抑制功能,进而抑制了因Treg增多而诱导肿瘤发生的免疫逃逸,调整机体的免疫格局,为进一步主动免疫治疗的使用提供了机体的保证。本研究部分修正了以往认为抗肿瘤化疗在杀伤肿瘤细胞时会严重损伤机体的免疫力的观点,为以泰素为代表的抗肿瘤化疗药的潜在抗肿瘤免疫作用提供理论和实验依据,为抗肿瘤化疗药的临床应用提供新思路,具有潜在的应用前景和广泛的社会价值。更多还原

【Abstract】 Cancer has become one of the most debilitating and life-threatening diseases. Owing to their tumoricidal effects,antineoplastic chemotherapeutic drugs have become a mainstay of anticancer therapy.However,while anti-tumor chemotherapy destructs tumor cells,the immune cells are simultaneously destroyed,resulting in a state of immunodeficiency.Hence,the postchemotherapeutic state had generally been regarded as an obstacle for active immune therapy to be carried out effectively. However,recent research showed that anti-tumor efficacy is more superior when adoptive cell transfer of autologous tumor-specific CTL was performed after anti-tumor chemotherapy,compared to that without anti-tumor chemotherapy. Similar findings were also discovered by our research group in preliminary animal studies:anti-tumor effect for lung cancer is more marked when tumor-peptide-pulsed DC vaccine was given to tumor-bearing mice after paclitaxel was injected,in comparison to the less marked anti-tumor efficacy when only tumor vaccine was used.These research findings suggest that the use of chemodrugs did not inhibit immune response;rather,it created a microenvironment which facilitated anti-tumor immune response.However,in-depth investigations into how such biological effect is brought about by chemodrugs are still unavailable.For these reasons,we postulate that chemodrugs,represented by paclitaxel,exert their anti-tumor effects via mechanisms other than direct tumoricidal actions,which include actions on immune cells and immune responses.By acting on immune cells, chemodrugs modulate the immune profile,creating a milieu in tumor-bearing individuals for further active immunotherapy to take effect.In this study,based on findings from our previous animal research,we carried out studies on clinical cases.We examined surgical specimens from 16 cases of lung cancer surgical patients and peripheral blood lymphocytes from 50 cases of lung cancer patients receiving anti-tumor chemotherapy.We analyzed how chemodrugs, with paclitaxel as the prototype,brought about alterations in the peripheral immune cell profile of non-small cell lung cancer patients.Further,in combination with in vitro studies we probed into the mechanisms behind the actions of paclitaxel on immune cells.Our aim is to elucidate the potential effects of anti-neoplastic chemodrugs aside from their cytotoxic actions,with paclitaxel as our prototype,to provide better theoretical and experimental basis for their clinical application,and novel strategies for the treatment of cancer.1.The Immune Profile of Cancer PatientsTumor genesis and tumor development,versus tumor immunosurveillance by the immune system,are constantly undergoing dynamic changes.When positive immunity is taking effect,tumor growth is inhibited.In contrast,when negative immunity is dominant,immune escape is resulted,prompting persistent tumor growth.Therefore,we selected non-small cell lung cancer as our research subjects, analyzed the immune cell profiles in these patients to understand the immune status in tumor-bearing individuals,in order to suggest strategies to improve and optimize anti-tumor immunity.Current researches showed that regulatory T cells exert negative regulatory effect in anti-tumor immune response.Changes in its frequency closely correlated with efficacy of anti-tumor immune response and tumor growth. Hence,we detected the changes in the frequencies of immune cell subsets and regulatory T cells in the peripheral blood of 66 cases of non-small cell lung cancer patients and 20 normal controls.Results showed no significant changes in the size of the peripheral leukocyte,lymphocyte,CD3⁺T lymphocyte,CD19⁺B lymphocyte,and CD8⁺T lymphocyte populations.The number and frequencies of CD4⁺T cells were significantly decreased among the peripheral lymphocytes of lung cancer patients compared to healthy controls.Further,analysis of the CD4⁺ CD25⁺ Foxp3⁺ T lymphocyte subset showed that its percentage was significantly higher than that in normal controls whereas no significant change was found in CD4⁺ CD25⁺ Foxp3^- T lymphocytes.These results suggest that effector T cells were not increased in lung cancer patients,increased CD4+CD25+ T cells were mainly attributed to increased CD4⁺ CD25⁺ Foxp3⁺ T cells.In our attempt to examine the regulatory actions of the regulatory T cell subset in tumor growth and anti-tumor immune response,we discovered that peripheral blood Treg in cancer patients did not vary with patient age,gender or tumor pathology,but correlated closely with clinical tumor staging.According to clinical TNM staging, the frequency of CD4⁺ CD25⁺ Foxp3⁺ Treg was significantly lower in early tumor stages compared to advanced tumor stages.Particularly in advanced stages,the frequency of Treg was markedly higher compared with normal controls.These findings suggested that:1) Frequency of peripheral blood Treg among peripheral lymphocytes were markedly elevated in tumor patients.2) The Treg subset increased with advancement of tumor stage.Among the mechanisms of tumor immune escape,other than reduced tumor cell immunogenenicity and lack of co-stimulating molecules,which render the immune system difficulty in launching effective immune responses,negative immune regulation also played a significant role.Therefore,we postulate that the increase in the Treg subset may be an important factor in the immune escape of tumor.2.Anti-tumor Chemotherapy Induced a Redistribution of Lymphocyte Subsets Proportions in Cancer PatientsAnti-tumor chemotherapy is currently the most widely adopted treatment modality.It is generally believed that anti-tumor chemotherapy does not only destroy tumor cells,but also destructs normal somatic cells,severely compromising immune functions.However,recent researches show that during a lymphopenic state,changes in immune profiles occur which facilitates effective immune responses.Based on these findings,we hypothesize that anti-tumor chemotherapy may bring about a redistribution of lymphocyte subset proportions in cancer patients,leading to reconstitution of the body’s immune profile.Therefore,we recruited 66 non-small cell lung cancer patients.Among them 50 patients underwent 1 to 5 courses of paclitaxel-based anti-tumor chemotherapy, hence we collected 70 peripheral blood specimens in total.These patients did not receive other immune therapies or therapies which may interfere with our experiment during the course of anti-tumor chemotherapy.By analyzing the frequencies of different lymphocyte subsets and changes in cell surface CD molecules before and after anti-tumor chemotherapy,we examined the effects of chemot anti-tumor chemotherapy herapy on their immune profiles.Our findings showed that:after anti-tumor chemotherapy,leukocytes and lymphocytes were significantly decreased.Within the lymphocyte subset,CD3⁺ T cell subset percentage decreased significantly,but at the same time CD19⁺ B cell percentages among peripheral lymphocytes did not show significant changes.Focusing on changes within the CD3⁺ T cell subset,we discovered that the percentage of CD4+ T cell subset decreased significantly,while the proportion of CD8⁺T cell subset did not undergo significant changes.Within the CD4⁺T cell subset,we focused on changes in the frequencies of the CD25⁺ subset.We found that the percentage of CD25⁺ cells decreased markedly among CD4⁺ cells,while the percentage of CD25^- cells increased significantly.Further,we focused on CD25⁺ subset,although the absolute numbers of Teff and Treg decreased significantly after anti-tumor chemotherapy,the percentage of Teff did not change significantly,while that of Treg decreased markedly.This indicated that the decline in Treg subset was significantly greater than that of Teff.Chemodrugs,represented by paclitaxel,could selectively reduce the frequency of Treg.These findings implied that:1) Anti-tumor chemotherapy,represented by paclitaxel,does not merely act on normal immune cells,but also induces a redistribution of immune cell subsets.2) Among the various immune cell subsets, chemodrugs（represented by paclitaxel） selectively downregnlates the frequencies of Treg,to some degree abrogating an important factor promoting tumor immune escape.3.Anti-tumor Chemotherapy Selectively Downregulate the Frequency And Function of the Treg SubsetPaclitaxel is a member of the taxanes chemodrugs.By inhibiting microtubule formation,paclitaxel directly destroys tumor cells and thereby exerts its antineoplastic actions.Our research had shown that not only does paclitaxel affects the body’s immune cells,but it also acts differentially on different cell subsets. Based on this finding we postulate that paclitaxel acts through some other mechanisms aside from direct cytotoxicity.We collected Treg and Teff as our control by in vitro cell sorting.We investigated the possible mechanisms by which paclitaxel affect immune cells by in vitro experiments.We also performed analysis in clinical cases to verify our findings.Our aim is to examine the mechanisms behind the redistribution of immune cell subsets induced by paclitaxel.Our findings showed:Compared to untreated Treg,taxol-treated Treg underwent significant apoptosis,and showed increased cell surface expression of CD95.On the other hand,apoptotic frequency of taxol-treated Teff did not increase significantly; neither did CD95 expression increased in this subset.We obtained similar experiment findings in our clinical cases.Based on these findings,we deduce that by enhancing cell surface expression of CD95 in the Treg subset,paclitaxel induces Treg cell apoptosis through Fas/FasL mediated pathway,thereby selectively destroys the Treg subset.To understand further whether paclitaxel could also affect the functions of Treg and Teff,we examined their functions by in vitro proliferation assays.We found that the negative regulatory function of Treg was significantly inhibited after paclitaxel anti-tumor chemotherapy.At the same time we discovered that,although after anti-tumor chemotherapy Foxp3 was still highly expressed among Treg, compared to untreated Treg,the mean fluorescence intensity of Foxp3 decreased significantly.Therefore,we hypothesize that by decreasing Foxp3 expression, paclitaxel inhibits the negative regulatory function of Treg.The above findings indicated that:1) Paclitaxel could selectively upregulate CD95 expression on the cell surface of Treg,thereby inducing Treg apoptosis.2) By downregulating the expression intensity of Foxp3 in Treg,paclitaxel inhibits the negative regulatory function of Treg.Hence,paclitaxei downregulates Treg both in terms of its number and function,which abrogates the role Treg plays in the immune escape of tumors from anti-tumor immunity.4.Anti-tumor Chemotherapy Drives Immune Microenvironment Towards a Th1-dominant Immune ProfileThe aforementioned findings showed that Treg played an important role in tumor immune escape,and paclitaxel-based chemotherapy selectively acts on Treg,thereby partially abrogating one significant means of tumor immune escape.What then is the immune status of the body under such conditions?We focused on the Th1 subset,which played a significant role in anti-tumor immunity.Through flow cytometric staining we detected the expression,of IL-2 secreted by CD4⁺ T cells and that of IFN-γsecreted by CD8⁺ T cells in the peripheral blood of non-small cell lung cancer patients before and after anti-tumor chemotherapy. We discovered that after anti-tumor chemotherapy,both absolute numbers and percentage of IL-2-secreting CD4+ T ceils and IFN-γ-secreting CD8+ T cells increased significantly among the peripheral lymphocytes of lung cancer patients.On the other hand,we also detected activation and memory markers of these cell subsets. With CD44 as the activation marker and CD62L as the memory marker,we discovered that the percentage of activated CD44^high CD4⁺ and memory CD8⁺ T cells and CD62L^low CD4⁺ and CD8⁺ T cells increased markedly after anti-tumor chemotherapy.The above findings showed that:After the actions of Treg have been abrogated,1) Th1-mediated immune response was enhanced;2) Functional cell subsets mediating anti-tumor immunity was also increased.Therefore,under the influence of antineoplastic anti-tumor chemotherapy,due to the abrogation of the actions of Treg in tumor immune escape,the body’s immune profile underwent reconstitution,which provides a foundation for further active immunotherapy.In conclusion,in this study we looked into the actions of antineoplastic anti-tumor chemotherapy on the immune cells of cancer patients,and examined tumor specimens surgically resected from lung cancer surgical patients and peripheral blood samples from lung cancer patients undergoing anti-tumor chemotherapy.We focused on taxanes chemodrugs with paclitaxel as our prototype,and analyzed the absolute numbers and percentages of various lymphocyte subsets after anti-tumor chemotherapy.We discovered that after anti-tumor chemotherapy the frequencies of immune cells were redistributed,among them the most marked was Treg,a subset which played a significant role in the immune escape of tumors.More importantly,we found that other than direct cytotoxicity,paclitaxel could selectively upregulate cell surface CD95 in Treg and induce Treg apoptosis,thereby abrogating the part Treg played in tumor immune escape,modulating the immune profile of the body,as a result providing a sound basis for further active immunotherapy to be performed.Our findings modified the past concept that anti-tumor chemotherapy severely compromised the body’s immune function by destroying immune cells.This study provided theoretical and experimental basis for potential alternative antineoplastic actions of chemodrugs represented by paclitaxel,and proposed novel potentials for their clinical applications.We hope that new strategies for the treatment of lung cancer could be derived from these findings,which carry important treatment prospects and profound social implications.更多还原