【作者】 李卫泊；

【导师】 蔡建辉；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 博士

【摘要】 目前肿瘤免疫治疗的研究重点集中在打破肿瘤免疫耐受和提高效应性T细胞反应。各种类型的肿瘤疫苗和过继性细胞免疫治疗逐渐进入临床应用阶段,并取得了阶段性的成果。其具有代表性的治疗如树突状细胞(dendritic cells, DCs)疫苗和体外培养的各类效应性T细胞的过继回输。然而在临床实践中,特别是对于那些已长成的血管化实体瘤,其单独应用的实际总体有效率均未超过30%。故目前国内外的肿瘤免疫治疗多停滞于Ⅰ、Ⅱ期临床试验。这是因为肿瘤微环境中复杂多变的免疫抑制网络限制了免疫治疗的实际效果,其中的抑制因素是困扰肿瘤免疫治疗的关键。近年来随着肿瘤免疫基础研究的发展,虽然仍未完整阐明肿瘤的免疫抑制网络,但其中的重要节点——CD4+CD25+FOXP3+调节性T细胞(regulatory T cells, Treg)的作用日益受到重视。在荷瘤机体内,CD4+CD25+FOXP3+Treg数量明显升高。越来越多的证据表明了无论在肿瘤环境下还是在抗肿瘤免疫中,Treg都扮演了负面角色。荷瘤机体内Treg的增加成为肿瘤免疫耐受,导致肿瘤免疫逃逸和限制肿瘤免疫治疗疗效的重要原因之一。所以对Treg进行针对性地调控成为肿瘤免疫治疗的重要环节。目前在欧美日等发达国家,调控Treg的措施已进入临床试验阶段,而国内的相关研究基本处于极少量的临床前实验阶段。环磷酰胺作(cyclophosphamide, CTX)为常规化疗药,被广泛应用于临床。CTX除了直接的细胞毒作用以外,还同时具有免疫调节方面的功能。近年来国外相关动物实验研究证实低剂量的CTX能够选择性地删除CD4+CD25+FOXP3+Treg,其删除作用具有时间和剂量依赖性。目前较一致的研究结果认为单次应用CTX后3-4天,Treg数量下降至最低水平,7天后逐渐恢复至正常水平。但CTX的应用都局限为单次给药,且有关CTX的最佳剂量、与免疫治疗的具体联合方式不尽相同。单次应用CTX(single administration)清除Treg的时间是短暂的,而在临床的免疫治疗中多为连续或循环应用疫苗或过继回输效应性T细胞,如果能够延长抑制Treg的时间(即在连续的免疫治疗时间段内保持Treg的相对低水平)可能将取得更好的效果。为此本研究以黑色素瘤的C57BL/6荷瘤小鼠为模型,筛选出CTX能够清除Treg的最佳剂量；采用循环应用CTX (cyclical administration),即间隔规律的多次给药方式,观察其对Treg的调控作用,能否延长对Treg的抑制时间；观察循环应用CTX联合DC疫苗和/或CTLs过继免疫治疗的抗瘤效果；同时观察自身免疫病的发生情况。第一部分循环应用低剂量环磷酰胺对荷瘤小鼠Treg的影响目的：观察循环应用低剂量CTX对荷瘤鼠Treg的影响，从免疫调节的角度探讨CTX的抗瘤作用。方法：通过皮下接种瘤细胞制备黑色素瘤荷瘤鼠模型；以25mg/kg到200mg/kg的剂量对小鼠腹腔注射CTX,流式细胞术检测小鼠脾脏中CD4+CD25+FOXP3+Treg的比例,筛选出CTX的最佳剂量；以最佳剂量腹腔注射CTX,每隔7天给药一次,共3次；应用流式细胞术检测Treg的变化,同时观测小鼠肿瘤的大小,绘制肿瘤生长曲线；培养小鼠骨髓来源的树突状细胞(DCs),将DCs与脾T淋巴细胞混合培养,应用ELISA法检测T淋巴细胞干扰素-γ(IFN-γ)的分泌量。结果：1、随着荷瘤时间的延长,荷瘤鼠脾脏CD4+CD25+FOXP3+/CD4+比例逐渐升高,至荷瘤后14d明显高于对照组(P<0.05)。2、当CTX的剂量为100mg/kg时,荷瘤鼠CD4+CD25+FOXP3+/CD4+比例明显降低(P<0.05)。3、单次应用CTX抑制Treg的时间较短,而循环应用CTX能够延长对Treg的抑制,维持其在相对较低水平(P<0.05)。4、循环应用CTX显著提高了荷瘤鼠脾T淋巴细胞IFN-γ的分泌水平(P<0.05),其中负载肿瘤细胞裂解物的DCs组(TL-DC)明显高于未负载肿瘤细胞裂解物的DCs组(Unpulsed DC)(P<0.05)。5、单次应用CTX和循环应用CTX均未能延缓肿瘤生长,均未导致免疫性白斑及明显化疗毒副反应。结论：1、随着肿瘤的生长进程,荷瘤鼠脾脏中Treg比例逐渐升高。2、对于C57BL/6J小鼠,CTX能够删除Treg的最佳剂量可能是100mg/kg。3、循环应用CTX能够使Treg在较长的时间里维持在相对较低水平,循环应用CTX更具有实用价值。4、循环应用CTX能够更加有效调控Treg,从而促进DCs对T淋巴细胞的抗原特异性激活,这将会提高DCs疫苗或过继性细胞免疫治疗的抗瘤效果。第二部分循环应用低剂量环磷酰胺联合树突状细胞疫苗的抗瘤作用目的：观察低剂量循环应用CTX联合树突状细胞疫苗的实际抗瘤效果,并探讨其中可能的机制。方法：通过皮下接种瘤细胞制备黑色素瘤荷瘤鼠模型,并于体外培养制备负载肿瘤抗原的DCs (TL-DCs)疫苗。根据不同的CTX给药方式,将小鼠随机分4组：①循环CTX+DCs疫苗治疗组；②单次CTX+DCs疫苗组治疗；③单纯DCs疫苗治疗组；④单纯循环CTX治疗组。联合治疗的方式为在腹腔注射CTX (100mg/kg)后4天,瘤周皮下注射TL-DCs疫苗。绘制肿瘤生长曲线和小鼠生存曲线。采用ELISA法检测各组小鼠血清IFN-γ水平,并应用免疫组化染色观察小鼠淋巴结内的CD8+T细胞。结果：1、循环CTX+DCs疫苗治疗组的抑瘤作用最为明显,与单次CTX+DCs疫苗组、单纯DCs疫苗治疗组和单纯循环CTX组比较均有统计学意义(P<0.05)。2、循环CTX+DCs疫苗治疗组的累积生存率和平均生存期明显高于其它各组,其差异均有统计学意义(P<0.05)。3、循环CTX+DCs疫苗组小鼠的血清IFN-γ水平均明显高于其它各组,其差异均有统计学意义(P<0.05)。4、循环CTX+DCs疫苗治疗组淋巴结内CD8+细胞数量和平均光密度值均高于其它各组,其差异均有统计学意义(P<0.05)。5、应用CTX的各治疗组中均未见免疫性白斑等自身免疫病及明显化疗毒副反应结论：1、循环应用低剂量CTX通过规律调控Treg,能显著提高DCs疫苗的疗效,为CTX调控Treg的基础上联合DCs疫苗免疫治疗的临床应用提供必要的理论依据。2、循环应用低剂量CTX联合DCs疫苗治疗未导致自身免疫病的发生,是安全可靠的。第三部分循环应用低剂量环磷酰胺联合CTLs过继免疫治疗的抗瘤作用目的：观察低剂量循环应用CTX联合CTLs过继免疫治疗的实际抗瘤效果并探讨其中可能的机制。方法：通过皮下接种瘤细胞制备黑色素瘤荷瘤鼠模型。体外培养制备肿瘤抗原特异性的CTLs,用CCK-8法检测CTLs的体外杀伤效力。根据不同的CTX给药方式,将小鼠随机分4组：①循环CTX+CTLs治疗组；②单次CTX+CTLs治疗组；③单纯CTLs治疗组；④单纯循环CTX治疗组。联合治疗的方式为在腹腔注射CTX 100mg/kg)后4天,鼠尾静脉注射CTLs。绘制肿瘤生长曲线和小鼠生存曲线。采用ELISA法检测各组小鼠血清IFN-y水平。结果：1、TL-DCs诱导的CTLs对B16细胞具有强烈的杀伤作用,其杀伤率(76.4±11.4)%远高于对CT26细胞(15.3±6.5)%和BGC-823(9.8±4.3)%细胞的杀伤率,也高于对照CTLs对B16细胞(35.2±7.8)%的杀伤率(P<0.05)。2、循环CTX+CTLs组的抑瘤作用最为明显,与单纯循环CTX组、单纯CTLs治疗组单次CTX+CTLs组比较均有统计学意义(P<0.05)。3、循环CTX+CTLs治疗组的累积生存率和生存期明显高于其它各组,其差异均有统计学意义(P<0.05)。4、循环CTX+CTLs组小鼠的血清IFN-y水平均明显高于其它各组,其差异均有统计学意义(P<0.05)。5、应用CTX的各治疗组中均未见免疫性白斑等自身免疫病及明显化疗毒副反应。结论：1、循环应用低剂量CTX通过规律调控Treg,能显著提高CTLs过继免疫治疗的疗效,为CTX调控Treg的基础上过继性细胞免疫治疗的临床应用提供必要的临床前理论依据。2、低剂量循环应用CTX联合CTLs过继免疫治疗未导致自身免疫病的发生,是安全可靠的。第四部分循环应用低剂量环磷酰胺联合DCs疫苗、CTLs过继免疫治疗的抗瘤作用目的：观察低剂量循环应用CTX调控Treg的基础上联合DCs疫苗和CTLs过继免疫治疗的实际抗瘤效果,并探讨其中可能的机制。方法：建立黑色素瘤荷瘤鼠模型,制备负载肿瘤抗原的DCs (TL-DCs)疫苗及肿瘤抗原特异性的CTLs。根据不同的CTX给药方式和不同的联合方式,将小鼠随机分4组：①循环CTX+DCs疫苗+CTLs治疗组；②单纯DCs疫苗+CTLs组；③循环CTX+DCs疫苗组；④循环CTX+CTLs组。联合治疗的方式为在腹腔注射CTX 100mg/kg)后4天,瘤周皮下注射TL-DCs疫苗,同时鼠尾静脉注射CTLs。绘制肿瘤生长曲线和小鼠生存曲线。采用ELISA法检测各组小鼠血清IFN-y水平。观察各组荷瘤鼠免疫性白斑的发生情况及化疗的毒副作用。结果：1、循环CTX+DCs+CTLs组取得最佳的抑瘤效果,与其它各组比较均有统计学意义(P<0.05),而单纯循环CTX+CTLs组、循环CTX+DCs疫苗组和单纯DCs+CTLs组的抑瘤效果则基本相同,其差异均无统计学意义(P＞0.05)。2、循环CTX+DCs+CTLs治疗组的累积生存率和生存期高于其它各组,其差异均有统计学意义(P<0.05)。单纯循环CTX+CTLs组、循环CTX+DCs疫苗组和单纯DCs+CTLs组之间比较差异均无统计学意义(P>0.05)3、循环CTX+DCs+CTLs治疗组小鼠的血清IFN-γ水平均明显高于明显高于其它各组,其差异均有统计学意义(P<0.05)。4、应用CTX的各治疗组中均未见免疫性白斑等自身免疫病及明显化疗毒副反应。结论：1、循环应用低剂量CTX通过规律调控Treg,能显著提高DCs疫苗和CTLs过继免疫治疗的疗效,为调控Tregs的基础上免疫治疗的临床应用提供了必要的理论依据。2、针对肿瘤免疫耐受的多个环节采取综合的手段才可能取得更佳的抗瘤疗效。更多还原

【Abstract】 The focus of tumor immunotherapy is currently how to break through tumor immune tolerance and enhence the effective T cell response. Various types of tumor vaccine and adoptive cellular immunotherapy have gradually entered into the period of clinical application. And the phased Achievement have been got that was represented by DCs vaccine and the adoptive reinfusion of effective T cell cultured in vitro. However the total effective rate of individual use of these therapies was less than 30% in clinical practice, especially for these established solid tumors which are rich in vascular. Thus the application of immunotherapy were blocked in the stage of Phase I or II clinical trial. The reason is because the complicated immunosuppression network in tumor microenvironment limits the practical effect of immunotherapy, in which these inhibitory factors are the key to bother immunotherapy. With the development of the fundamental research on tumor immune, more attention has been increasingly paid to the Key-point, CD4+CD25+FOXP3+regulatory T cells (Treg) in the recent years.In tumor bearing host, the percentage of Treg is increasing significantly. More and more evidences have shown that Treg plays a negative role in tumor environment or in antitumor immune. Increased numbers of Treg have been one of important reasons of tumor immune tolerance, which leads to tumor immune escape and limits the immunotherapy effect. Therefore regulating Treg has been the crucial part of tumor immunotherapy. Now in Japan or European and American Countries, regulation of Treg have gradually entered into the period of clinical trial. But the related research in our country still rests in few preclinical experiments.Cyclophosphamide (CTX) has been widely used in clinic for cancer therapy, as a Conventional chemotherapeutic alkylating drugs. Besides its derect cytotoxic effect, CTX also plays a role of immunomodulation. Recently foreign researches in murine models have proved that low-dose CTX could delete Treg cells selectively in both dose-dependent and time-dependent manner. And current research Results showed consistently that the number of Treg declined to the lowest at 3rd to 4th day after CTX administration, while recoverd to the normal level after about 7th day. The application of CTX in those researches was single administration, meawhile there was much defferent about the optimal dosage of CTX deleting Treg cells and combination with immunotherapy. On the other hand, the duration of single CTX administration deleting Treg remains shorter, which is not enough to the consecutive or cyclical use of DCs vaccination and T cell adoptive reinfusion in clinical practice. If the duration of CTX deleting Treg remain more longer, more better result would be achieved. So in this study, C57BL/6 mice inoculated B16 cells was used as tomor model to select the optimal dosage of CTX. The effect of cyclical CTX administration referred to repeated administration at rhythmic interval on Treg was observed. Subsequently the antitomor effect of cyclical CTX administration combined with DCs vaccine or adoptive CTLs therapy was analyzed. And autoimmune Diseases of mice were also investigated after those therapies.Part 1 Influence of cyclical administration of low-dose cyclophosphamide on regulatory T cell in melanoma-bearing miceObjective:To observe the influence of cyclical low-dose cyclophosphamide administration on regulatory T cell of melanoma-bearing mice and explore its antitumor effect from the view of immunoregulation.Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). mice were injected intraperitoneally with CTX at the dose of 25mg/kg to 200mg/kg, then the CD4+CD25+Foxp3+Treg in spleens were detected by flow cytometry to select the optimal dosage of CTX. The mice were treated with CTX at the optimal dose every 7 days, which were repeated 3 cycles. The changes of the CD4+CD25+Foxp3+Treg were detected by flow cytometry. The dynamic changes of tumor size were observed and the growth curves were drawn. The dendritic cells derived from the bone marrow of mice were cultured with spleen T lymphocytes, and the interferon-γ(IFN-γ) levels in culture supernatant were detected by ELISA.Results:1. the proportion of CD4+CD25+Foxp3+/CD4+was increased gradually in spleens of mice after tumor inoculation, which were significantly higher than those in mormal group at the 14th day.2. When the CTX was used at the dose of 100mg/kg, the proportion of CD4+CD25+Foxp3+/CD4+in tumor-bearing mice was decreased significantly (P<0.05).3. Single administration of CTX only inhibited Treg in short time, while cyclical administration of CTX could prolong the period in which Treg was kept at low level.4. Cyclical administration of CTX enhanced the IFN-γproduction of T lymphocytes in tumor-bearing mice higher than that in control group (P<0.05). And the IFN-γproduction in TL-DC group was higher than that in Unpulsed DC group.5. Neither single nor cyclical administration of CTX could delay the growth of tumor. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:1. With the process of tomor growth, the proportion of Treg gradually increased in spleens of tumor-bearing mice.2. The optimal dosage of CTX regulating Treg cells may be 100mg/kg for the C57BL/6J mice.3. Cyclical administration of low-dose CTX may be of more clinical value, which could prolong the period in which Treg was kept at low level.4. Cyclical administration of low-dose CTX could regulate the level of Treg more effectively, and enhance the antigen-specific activation of T cells by DCs, which may enhence antitumor effect of DCs vaccine or ACI.Part 2 Anti-tumor effect of cyclical administration of low-dose cyclophosphamide combined with DCs vaccineObjective:To observe the actual Anti-tumor effects of cyclical administration of low-dose cyclophosphamide with DCs vaccine and explore its possible mechanism.Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). The tumor lysate-pulsed dendritic cells (TL-DCs) were prepared for DCs vaccine by cultivation in vitro. Mice were were randomly divided into 4 groups:①cyclical CTX+DCs group,②single CTX+DCs group,③DCs group,④cyclical CTX group. mice were injected intraperitoneally with CTX at the dose of 100mg/kg, then received vaccination by subcutaneous inoculation with TL-DCs 4 days later. The growth curves of tumor and the survival curve of mice were drawn. The interferon-y (IFN-y) levels in serum were detected by ELISA. And CD8+T cells in lymph nodes were detected by immunohistochemical method.Results:1. The tumor growth was delayed more significantly in cyclical CTX+DCs group, compared with single CTX+DCs group, DCs group and cyclical CTX group (P<0.05).2. The cumulative survival rate and mean survival time in cyclical CTX+DCs group were significantly higher than other groups(P<0.05).3. The IFN-y levels in serum from cyclical CTX+DCs group increased significantly, compared with other groups(P<0.05).4. The number of CD8+T Cells and its average optical density were higher than other groups(P<0.05).5. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:1. Cyclical administration of low-dose CTX could enhence the anti-tumor effect of DCs vaccine by regulating Treg, which provided a theoretical basis for the clinical application of DCs vaccine based on CTX regulating Treg.2. The combined treatment of CTX and DCs vaccine did not lead to the autoimmune disease such as vitiligo, that is safe.Part 3 Anti-tumor effect of cyclical administration of low-dose cyclophosphamide combined with CTLs adoptive transfusion.Objective:To observe the actual Anti-tumor effects of cyclical administration of low-dose cyclophosphamide with CTLs adoptive transfusion and explore its possible mechanism.Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). The tumor antigen-specific CTLs were prepared by cultivation in vitro. And the killing effect of CTLs was detected by CCK-8. Mice were were randomly divided into 4 groups:①cyclical CTX+CTLs group,②single CTX+CTLs group,③CTLs group,④cyclical CTX group. The mice in combined treatment group were injected intraperitoneally with CTX at the dose of 100mg/kg, then received CTLs by veinous transfusion 4 days later. The growth curves of tumor and the survival curve of mice were drawn. The interferon-y (IFN-y) levels in serum were detected by ELISA.Results:1. The tumor antigen-specific CTLs induced by TL-DCs could kill the B16 cells specifically and strongly. Its killing rate for B16 cells (76.4±11.4)% was significantly higher than that for CT26 cells (15.3±6.5)% and BGC-823 cells (9.8±4.3)%, also higher than the killing rate of control CTLs (35.2±7.8)% (P<0.05).2. The tumor growth was delayed more significantly in cyclical CTX+CTLs group, compared with single CTX+ CTLs group, CTLs group and cyclical CTX group (P<0.05).3. The cumulative survival rate and mean survival time in cyclical CTX+CTLs group were significantly higher than other groups(P<0.05).4. The IFN-y levels in serum from cyclical CTX+ CTLs group increased significantly, compared with other groups(P<0.05).5. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:Cyclical administration of low-dose CTX could enhence the anti-tumor effect of CTLs adoptive immunotherapy by regulating Treg, which provided a theoretical basis for the clinical application of CTLs adoptive immunotherapy based on CTX regulating Treg.2. The combined treatment of CTX and CTLs adoptive immunotherapy did not lead to the autoimmune disease such as vitiligo, that is safe.Part 4 Anti-tumor effect of cyclical administration of low-dose cyclophosphamide combined with DCs vaccine and CTLs adoptive transfusionObjective:To observe the actual Anti-tumor effects of cyclical administration of low-dose cyclophosphamide combined with DCs vaccine and CTLs adoptive transfusion, and explore its possible mechanism. Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). The tumor lysate-pulsed dendritic cells (TL-DCs) and tumor antigen specific CTLs were prepared by cultivation in vitro. Mice were were randomly divided into 4 groups:①cyclical CTX+DCs+CTLs group,②cyclical CTX+CTLs group,③cyclical CTX+DCs group,④DCs+CTLs group. The mice in combined treatment group were injected intraperitoneally with CTX at the dose of 100mg/kg, then received CTLs by veinous transfusion and TL-DCs by subcutaneous inoculation 4 days later. The growth curves of tumor and the survival curve of mice were drawn. The IFN-y levels in serum were detected by ELISA.Results:1. The tumor growth was delayed most significantly in cyclical CTX+DCs+CTLs group, compared with other 3 groups (P<0.05). There were no significant difference among cyclical CTX+CTLs group, cyclical CTX+DCs and DCs+CTLs group (P>0.05).2. The cumulative survival rate and mean survival time in cyclical CTX+DCs+CTLs group were significantly higher than other groups(P<0.05). There were no significant difference among cyclical CTX+CTLs group, cyclical CTX+DCs and DCs+CTLs group (P>0.05).3. The interferon-γ(IFN-y) levels in serum from CTX+DCs+CTLs group increased significantly, compared with other groups(P<0.05).4. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:1. Cyclical administration of low-dose CTX could enhence the anti-tumor effect of DCs vaccine and CTLs adoptive transfusion by regulating Treg, which provided a theoretical basis for the clinical application of immunotherapy based on CTX regulating Treg.2. The optimal anti-tumor effect may be achieved only when synthetic methods were adopted according to many links of tumor immune tolerance.更多还原