膜型、可溶性Tim-3在抗肿瘤免疫应答中的作用及肿瘤基因治疗的研究
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摘要
Tim-3(T-cell immunoglobulin-and mucin-domain-containg molecule)分子的发现是源于寻找区分T_H1细胞和T_H2细胞的表面标志物。对其功能的研究揭示Tim-3在自身免疫性疾病和器官移植排斥反应性疾病中具有重要的免疫学作用。应用Tim-3的抗体和Tim-3-Ig融合蛋白能够影响免疫反应，加速疾病的发展。随后的研究发现编码Tim-3分子的基因通过选择性剪切产生一长一短两种形式的mRNA剪切体。长的mRNA指导生成膜型Tim-3(简称Tim-3)，膜型Tim-3由信号肽、IgV区和黏液区、跨膜区以及胞质区组成；短的mRNA指导生成包含信号肽、IgV区和胞质区的蛋白质，由于缺少黏液区，因此推测短的mRNA编码生成的蛋白质可能是可溶性的分子(sTim-3，soluble Tim-3)，但是目前缺乏直接的实验证据证实sTim-3蛋白分子的存在。
许多重要的膜型免疫调控分子如CD86、Fas、CTLA-4都有天然的可溶性的对照物。而且这些可溶性的对照物在免疫应答反应中也发挥重要的作用。Tim-3两种形式的mRNA剪切体的发现向我们提出以下几个方面的问题：(1)在免疫反应中Tim-3和sTim-3 mRNA的表达规律。(2)短的Tim-3 mRNA的是否指导生成sTim-3，sTim-3是否具有生物活性。(3) sTim-3和Tim-3在肿瘤免疫应答中的作用及可能的作用机制。
本研究的第一部分和第二部分试图回答以上几个方面的问题。
本论文的第三部分的内容是有关克服肿瘤免疫逃避基因治疗的研究。目前，以提高机体抗肿瘤免疫反应为主的治疗措施受到广泛的关注。但是，近年研究发现：受到免疫攻击的肿瘤细胞会迅速上调表达免疫负性共刺激分子—B7-H1(又被称为PD-L1)。B7-H1能够同表达在活化的T淋巴细胞表面的免疫抑制性受体PD-1(program death-1)结合进而抑制T淋巴细胞的杀伤作用。因此，肿瘤细胞表达B7-H1能够促进肿瘤的免疫逃逸。我们采用本研究小组构建的可溶性的PD-1(sPD-1，soluble PD-1，)封闭瘤细胞表面的B7-H1，联合HSP70-肿瘤抗原肽复合物能有效抑制肺部转移性肿瘤。我们还探讨了联合应用的可能作用机制，希望能为制定有效的肿瘤免疫治疗方案提供理论基础和实验依据。
第一部分异位表达Tim-3促进抗肿瘤免疫应答反应
目的：在肿瘤局部组织微环境中转染表达Tim-3，并研究Tim-3在调节淋巴细胞功能活性及抗肿瘤方面的作用和机制。方法：提取鼠脾脏总RNA，RT-PCR扩增出Tim-3编码序列，限制性内切酶消化，回收目的片断并插入到pcDNA3.1相应多克隆位点上。构建含有Tim-3全长cDNA序列的真核表达质粒，脂质体介导体外转染CHO细胞，G418筛选出阳性克隆，流式细胞仪分析Tim-3的表达。建立小鼠H22肝细胞癌移植瘤模型，肿瘤组织内注射pTim-3真核表达质粒，RT-PCR、免疫组织化学法检测在肿瘤细胞内Tim-3表达情况。并观察肿瘤的生长速度和荷瘤鼠的生存期。另外，利用H&E染色法分析瘤组织中淋巴细胞浸润情况。分离脾细胞体外测淋巴细胞的杀伤作用。流式细胞仪检测分析Tim-3在新鲜分离的CD3~+T细胞和CD8~+T细胞和接受HSP70-肿瘤抗原肽刺激的CD3~+T细胞和CD8~+T细胞表面的表达情况。结果：限制性酶切分析及序列测定证实成功构建了小鼠Tim-3真核表达质粒pTim-3。FACS检测分析显示转染pTim-3质粒的CHO细胞在表面表达Tim-3抗原。RT-PCR、免疫组织化学法检测肿瘤接种部位能够表达Tim-3 mRNA和Tim-3抗原。同生理盐水对照组和空载体对照组相比，肿瘤接种部位转染表达Tim-3，能显著抑制肿瘤生长速率并明显延长荷瘤鼠的生存期。H&E染色显示有大量的淋巴细胞浸润。接受pTim-3治疗的小鼠的脾细胞杀伤活性明显增强。流式细胞仪检测分析显示新鲜分离的CD3~+T和CD8~+T细胞几乎不表达Tim-3。然而，接受肿瘤抗原肽刺激的CD3~+T和CD8~+T细胞表面表达Tim-3。而且随着刺激次数的增加，CD3~+T和CD8~+T细胞上调表达Tim-3。结论：在肿瘤微环境中转染表达Tim-3，能够有效地促进淋巴细胞的抗肿瘤活性。Tim-3除去表达在活化的CD4~+T细胞表面，也可以表达在活化的CD8~+T表面细胞。
第二部分可溶性Tim-3抑制T淋巴细胞的免疫应答反应
首先，我们阐明了Tim-3 mRNA表达在骨髓细胞，胸腺细胞和脾细胞中，而sTim-3 mRNA仅表达在脾细胞中，而骨髓细胞，胸腺细胞和其它非免疫组织细胞不表达sTim-3 mRNA。我们采用Real-Time PCR的方法研究新鲜分离的脾细胞和接受刺激的脾细胞表达两型Tim-3 mRNA的表达情况。Real-Time PCR的结果显示新鲜分离的脾细胞主要表达Tim-3mRNA，而接受刺激的脾细胞主要表达sTim-3mRNA。提示Tim-3和sTim-3 mRNA的表达与脾细胞的免疫状态有关。
然后，我们用Western blot的方法证实sTim-3蛋白质分子存在于接受刺激的脾细胞的分泌液中。并且转染了sTim-3真核表达质粒的CHO细胞分泌液中也存在sTim-3蛋白质分子。荧光检测显示转染Tim-3-GFP真核表达质粒的CHO细胞显示绿色荧光。而转染sTim-3-GFP真核表达质粒的CHO细胞几乎不显示绿色荧光。间接提示sTim-3-GFP以可溶性蛋白的形式被分泌到细胞外。用转染sTim-3-GFP真核表达质粒的CHO细胞的上清液同T细胞共孵育。FACS检测显示T细胞表面的荧光强度明显高于对照组。证实sTim-3能够和T细胞表面的配体结合。
体外的实验结果显示sTim-3能够抑制anti-CD3和anti-CD28的刺激淋巴细胞增殖激作用。并且sTim-3同样能够抑制HSP70-肿瘤抗原肽复合物的特异性抗原刺激作用。应用Tim-3的抗体后能够阻断sTim-3抑制淋巴细胞增殖激作用。ELISA检测细胞因子的结果显示：sTim-3能明显抑制IL-2、IFN-γ的分泌，但不影响IL-4的分泌。我们进一步采用肿瘤排斥模型研究sTim-3在体内的免疫学活性。结果显示，肿瘤细胞在接受sTim-3治疗的小鼠体内生长更快一些，荷瘤鼠的存活期明显缩短。和对照组比，接受sTim-3治疗的小鼠的脾细胞增殖能力和杀伤活性明显降低。病理和免疫组化检测结果显示接受sTim-3治疗的瘤组织内浸润T淋巴细胞的数目明显低于对照组。证实sTim-3在体内抑制T细胞的抗肿瘤免疫应答反应。
我们的研究还试图理解sTim-3影响T细胞免疫应答反应的作用机制。因为CD4~+CD25~-T效应细胞和CD4~+CD25~+T调节细胞都表达Tim-3的配体。因此，sTim-3抑制T细胞的抗肿瘤免疫应答反应可能是通过抑制CD4~+CD25~-T效应细胞的活化，或者促进CD4~+CD25~+T调节细胞的活化。为了回答这个问题。我们采用Real-Time PCR的方法研究反应CD4~+CD25~-T效应细胞和CD4~+CD25~+T调节细胞活性状态的免疫分子表达情况。结果显示，sTim-3能够抑制与CD4~+CD25~-T效应细胞功能相关的细胞因子的表达，比如IL-2、IFN-γ、TNF-β。但不影响与CD4~+CD25~+T调节细胞功能相关的免疫分子(Foxp3、I1-10、TGF-β)的表达。这些结果提示sTim-3主要通过抑制T效应细胞的功能发挥作用。
第三部分HSP70-肿瘤抗原肽复合物联合sPD-1治疗肺部转移性肿瘤的研究
B7-H1(又被称为PD-L1)是表达在膜表面的糖蛋白分子，属于共刺激分子B7家族。其受体是PD-1(programmed death-1)，PD-1是在淋巴细胞活化过程中诱导表达的一个免疫抑制性受体。B7-H1和其受体PD-1结合后传导的抑制性信号参与了肿瘤的免疫逃避机制。在此研究中，我们发现这一抑制性信号也参与了HSP70-肿瘤抗原肽复合物治疗过程中肿瘤的免疫逃避。我们在用HSP70-肿瘤抗原肽抑制黑色素瘤肺转移的研究中发现，HSP70-肿瘤抗原肽复合物能够有效地始动免疫应答反应，表现为黑色素瘤结节处有大量的淋巴细胞浸润，血清中IL-2、IFN-γ水平明显升高。和对照组相比，HSP70-肿瘤抗原肽复合物治疗组在早期肺部黑色素瘤结节数目明显减少。但是，出乎意料的是，随着观察时间的延长HSP70-肿瘤抗原肽复合物治疗组有大量的黑色素瘤结节生成。在体外，用IFN-γ同黑色素瘤细胞共孵育，流式细胞仪检测发现随着IFN-γ的浓度增加黑色素瘤细胞表面的B7-H1表达显著升高。而未经IFN-γ处理的黑色素瘤细胞不表达B7-H1。Real-Time PCR检测发现B7-H1基因高表达在体内黑色素瘤细胞上。淋巴细胞杀伤实验证实受到IFN-γ处理的黑色素瘤细胞能有效的抵抗淋巴细胞的杀伤。当我们用本研究小组构建的可溶性的PD-1(sPD-1，soluble PD-1)封闭瘤细胞表面的B7-H1，淋巴细胞的杀伤活性得到了提高。提示表达在黑色素瘤细胞表面的B7-H1能够有效抑制淋巴细胞的杀伤活性。在体内，HSP70-肿瘤抗原肽复合物和sPD-1联合应用时观察到明显的协同治疗作用，联合治疗能显著抑制黑色素瘤结节的生成、明显延长小鼠的生存时间。并增强肿瘤特异的CTL对肿瘤细胞的杀伤活性。我们还采用Real-Time PCR检测肿瘤浸润淋巴细胞中相关细胞因子的表达情况。在HSP70-肿瘤抗原肽复合物与sPD-1联合应用组，IFN-γ和IL-2等T_H1型的细胞因子表达明显升高。IL-10、TGF-β和foxp3等免疫负调控分子表达下降。提示联合治疗能够有效的克服黑色素的免疫抵抗作用。在这部分的研究中，我们还探索了应用sPD-1的剂量-效应关系。一次尾静脉注射10μg的sPD-1的表达质粒，在血清中可连续7天检测到sPD-1的存在。每周两次或每周一次应用10μg的sPD-1的表达质粒联合HSP70-肿瘤抗原肽复合物能有效抑制黑色素结节的生成。每周两次5μg的sPD-1的表达质粒联合HSP70-肿瘤抗原肽复合物也能有效抑制黑色素结节的生成。但是每周一次应用5μg的sPD-1的表达质粒联合HSP70-肿瘤抗原肽复合物不能有效抑制黑色素结节的生成。这一研究结果不仅揭示了肿瘤免疫逃逸的一个新的机制，而且也开辟了一条肿瘤免疫治疗的新路径：封闭肿瘤细胞表面的免疫负性共刺激分子—B7-H1，保护CTL。
The search for cell-surface markers that can distinguish T_H1 from T_H2 cells has lead to the identification of a new molecular---Tim-3. It has been reported that administration of anti-Tim-3 antibody and Tim-3 Ig fusion protein can abrogate the induction of peripheral tolerance and promote T_H1-mediated autoimmune and alloimmune disease, which indicated that Tim-3 has important function in the regulation of immune-mediated disease. It was found that Tim-3 mRNA could be alternatively spliced to produce two mRNA molecules. The longer one directs the synthesis of full-length Tim-3 (i.e., Tim-3), the shorter one, without the region encoding the mucin domain and transmembrane domain, was supposed to direct the synthesis of a splice variant of sTim-3.
Many co-stimulatory molecules and immunoregulatory receptors, such as CD86, Fas and CTLA-4, have native soluble molecules, and these soluble variants were important in controlling immune response as well as in susceptibility and resistance to autoimmune disease. The discovery of sTim-3 raised several question to be answer. The first one was the expression pattern of Tim-3 and sTim-3 mRNA in immune response. The second one was the existence of the translated product of sTim-3 and whether sTim-3 could bind to Tim-3 ligands. The third one was the regulatory role of Tim-3 and sTim-3 in antitumor immune response.
At present, tumor immunotherapy efforts are focused on the generation of strong T cell response against tumor antigens. However, strong T cell response does not always coincide with tumor rejection, for which upregulated expression of irnmunoinhibitory molecules--- may be responsible. Emerging evidences suggest that B7 homolog 1 (B7-H1) is important in the mechanisms of resistance against tumor associated antigen-specific immunity. Blockade of B7-H1 by sPD-1 can enhance tumor specific CTL response and cause tumor rejection.
Part I
Ectopic expression of Tim-3 induces tumor-specific antitumor immunity
Objective: To express Tim-3 in tumor microenvironment and investigate the role of Tim-3 in regulation of lymphocyte activity and antitumor effect. Methods: Total RNA of concanavalin A stimulated splenocytes was extracted and Tim-3 cDNA was amplified by RT-PCR. The amplified fragment was digested by restriction enzymes and inserted into compatible enzyme restriction sites of pcDNA3.1 to generate murine Tim-3 eukaryotic expression plasmid (pTim-3). CHO cells were transfected with pTim-3 with Dosper liposomal transfection reagent, the transfected cells were screened by G418. And the expression of Tim-3 in the transfected cells were detected by FACS. Tumors were established by injection of H22 into right hind thigh muscle and pTim-3 plasmid was injected intratumorally. The expression of Tim-3 on H22 cells was examined by immunohistochemistry, and tumor volume and the survive was recorded. Hematoxylin and eosin (H&E) staining was performed to detect tumor-infilating T lymphocytes. Cytolytic activity against H22 was determined in 4h - ~(51)Cr release assay. Tim-3 expression on freshly isolated CD3~+ T and CD8~+ T cells and HSP70-tumor peptide complex stimulated CD3~+ T and CD8~+ T cells were examined by FACS analysis. Results: The constructed plasmid, designated as pTim-3, was confirmed by restriction enzymes digestion and DNA sequencing. Flow cytometric analysis revealed that Tim-3 antibody stained the surface of cells transfected with pTim-3 but not the surface of cells transfected with mock plasmid (pcDNA3.1). Intratumor injection of pTim-3 plasmid potently inhibited tumor growth and results in prolonged survival. There was microscopic evidence of dense lymphocyte infiltration in pTim-3 treated mice. In addition, Splenocytes from the pTim-3 treated group showed a strong CTL response against H22 tumor cells, which suggested that ectopic Tim-3-mediated antitumor responses were dependent on a functional adaptive lymphocyte response. We also demonstrate endogenous Tim-3 expression on lymphocyte is relevant in antitumor immune response. Since considerable number of CD3~+ T cell including CD8~+ T cell can express Tim-3 after stimulate with tumor-peptide complex. Conclusion: Expression of Tim-3 on H22 tumor cells can enhance the proliferation and other functions of T lymphocytes. Tim-3 expression is not limit on CD4~+ T cell but also on CD8~+ T cells after stimulation with tumor- peptides complex. Our results thus gain further insight into the function of Tim-3 expressed on nonlymphoid tissues in regulating antitumor immune response. Part II
Soluble Form of Tim-3 Is An Inhibitory Molecule in T Cell-Mediated Immune Response
In initial experiments, we demonstrated that Tim-3 mRNA was existent in the cells from thymus, bone marrow and spleen, whereas sTim-3 mRNA was expressed only in spleen cells but not in bone marrow, thymus and non-lymphoid organs. To investigate the expression pattern of sTim-3 and Tim-3 mRNA in immune response, we examined the expression of these mRNAs in freshly isolated and activated splenocytes. Real-Time PCR revealed that both Tim-3 mRNA and sTim-3 mRNA were significantly increased after each rounds of stimulation, whereas there appeared to be a higher increase of sTim-3 product than that of Tim-3 product, suggesting that the expression pattern of Tim-3 mRNA and sTim-3 relied on the activation of splenic lymphocyte.
We next proceed to examine the existence of sTim-3's protein product by Western blot. The results showed that sTim-3's protein was detected in the cell-free supernatants of the stimulated splenocyts and CHO cells transfected with sTim-3 eukaryotic expression plasmid. Fluorescence microscope analysis revealed that Green fluorescence was found in CHO cells transfected with pTim-3-GFP but scarcely in CHO cells transfected with psTim-3-GFP, indicating that sTim-3 is mostly secreted out, which further conformed that sTim-3 is a soluble molecule. The binding of sTim-3-GFP to its putative ligand(s) on splenic T cells was determined by FACS analysis. The fluorescence intensity on T cells incubated with sTim-3-GFP was significantly increased as compared with control. These results clearly indicated that sTim-3 could bind to putative Tim-3 ligand(s).
In vitro, the response of T cells to antigen-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation was significantly suppressed in the presence of sTim-3, and the blockade of sTim-3 with anti-Tim-3 antibody recovered the response of T cells to these stimuli, supporting the notion that sTim-3 mediates the inhibitory effect in immune response. We then attempt to further understand the function of sTim-3 in vivo, in tumor rejection model, sTim-3 facilitated the growth of tumor in vivo and shorten survival of tumor-bearing mice. The proliferation of T cells from sTim-3 treated mice was significantly reduced when they were re-stimulated by antigen-loaded DC in vitro, Moreover, the antitumor CTL activity was significantly reduced by sTim-3 and the amount of T cells in tumor tissue was also significantly decreased. Pathologic and immunohistological findings revealed the reduced tumor-infilating T lymphocytes. To sum up, these results confirmed the inhibitory effect of sTim-3 on the generation of antigen-specific T cell immune response.
Since both effector CD4~+CD25~- T cells and regulatory CD4~+CD25~+ T cells express a putative Tim-3 ligand, the impairment of antitumor immunity by sTim-3 might be the result of either impairing the function of effector CD4~+ T cells or activating the function of CD4~+ regulatory T cells. To understand the underlying cellular mechanism contributing to the impaired antitumor immunity by sTim-3, we analyzed the expression of genes related to the function of tumor-infiltrating lymphocytes by real-time quantitative PCR. The results indicated that, in sTim-3 treated mice, the expression of IL-2, IFN-γ and TNF-β mRNA was down-regulated, whereas, the expression of forkhead transcription factor 3 (Foxp3), IL-10 and TGF-p mRNA was not influenced as compared with control, theses results suggested that sTim-3 didn't influence Treg cells but otherwise resulted in the profound immune unresponsiveness of CD4~+ T effector cells. Part III
HSP70 Vaccine in Combination with Gene Therapy with Plasmid
DNA Encoding sPD-1 Overcomes Immune Resistance and Suppresses
the Progression of Pulmonary Metastatic Melanoma
B7-H1 (also known as PD-L1) is a cell surface glycoprotein belonging to the B7 family of costimulatory molecules. It was founded that PD-1 (programmed death-1), which is expressed on the activated lymphocytes, is the receptor of B7-H1. B7-H1 is thought to contribute to immune escape of cancer by interacting with PD-1 receptor. In this study, the treatment with HSP70 vaccine induced an infiltration of T cells into the tumor site as well as the expression of IFN-γ and IL-2, and delayed lung metastases of tumor, but the tumor progression nonetheless occur finally. We incubated B16F1 cells with different doses of IFN-γ in vitro. FACS analysis revealed that the percentage of B7-H1-positive B16F1 cells was significantly increased by IFN-γ stimulation. By contrast, B7-H1 was hardly detectable on the untreated B16F1 cells. Consistent with this result, the upregulatd expression of B7-H1 in vivo in tumor microenvironment was also confirmed by Real-time PCR. We demonstrated that B7-H1 expressed by residual tumor cells was responsible for the resistance of tumor to the therapy with HSP70 vaccine. Blockade of B7-H1 by i.v. injection pPD-1A, a plasmid encoding the extracellular domain of PD-1 (sPD-1) could reverse this resistance and enhance the therapeutic efficacy. To complement these findings, we investigated the gene expression of TILs by Real-time PCR analysis, which revealed that the expression of TH1 cytokines IFN-γ and IL-2 by TIL in the mice treated with HSP70 vaccine in combination with sPD-1 was increased and the expression of negative regulatory molecules IL-10, TGF-β and foxp3 was decreased, demonstrating that multifunctional properties afforded by the combination therapy can effectively overcome tumor resistance and promote effective antitumor immunity. In vivo transfection with pPD-1A could be performed as infrequently as once a week and still produce a significant antitumor effect. These findings suggest that the treatment with HSP70 vaccine followed by blockade of tumor-B7-H1 with sPD-1 may provide a promising approach for tumor immunotherapy.
许多重要的膜型免疫调控分子如CD86、Fas、CTLA-4都有天然的可溶性的对照物。而且这些可溶性的对照物在免疫应答反应中也发挥重要的作用。Tim-3两种形式的mRNA剪切体的发现向我们提出以下几个方面的问题：(1)在免疫反应中Tim-3和sTim-3 mRNA的表达规律。(2)短的Tim-3 mRNA的是否指导生成sTim-3，sTim-3是否具有生物活性。(3) sTim-3和Tim-3在肿瘤免疫应答中的作用及可能的作用机制。
本研究的第一部分和第二部分试图回答以上几个方面的问题。
本论文的第三部分的内容是有关克服肿瘤免疫逃避基因治疗的研究。目前，以提高机体抗肿瘤免疫反应为主的治疗措施受到广泛的关注。但是，近年研究发现：受到免疫攻击的肿瘤细胞会迅速上调表达免疫负性共刺激分子—B7-H1(又被称为PD-L1)。B7-H1能够同表达在活化的T淋巴细胞表面的免疫抑制性受体PD-1(program death-1)结合进而抑制T淋巴细胞的杀伤作用。因此，肿瘤细胞表达B7-H1能够促进肿瘤的免疫逃逸。我们采用本研究小组构建的可溶性的PD-1(sPD-1，soluble PD-1，)封闭瘤细胞表面的B7-H1，联合HSP70-肿瘤抗原肽复合物能有效抑制肺部转移性肿瘤。我们还探讨了联合应用的可能作用机制，希望能为制定有效的肿瘤免疫治疗方案提供理论基础和实验依据。
第一部分异位表达Tim-3促进抗肿瘤免疫应答反应
目的：在肿瘤局部组织微环境中转染表达Tim-3，并研究Tim-3在调节淋巴细胞功能活性及抗肿瘤方面的作用和机制。方法：提取鼠脾脏总RNA，RT-PCR扩增出Tim-3编码序列，限制性内切酶消化，回收目的片断并插入到pcDNA3.1相应多克隆位点上。构建含有Tim-3全长cDNA序列的真核表达质粒，脂质体介导体外转染CHO细胞，G418筛选出阳性克隆，流式细胞仪分析Tim-3的表达。建立小鼠H22肝细胞癌移植瘤模型，肿瘤组织内注射pTim-3真核表达质粒，RT-PCR、免疫组织化学法检测在肿瘤细胞内Tim-3表达情况。并观察肿瘤的生长速度和荷瘤鼠的生存期。另外，利用H&E染色法分析瘤组织中淋巴细胞浸润情况。分离脾细胞体外测淋巴细胞的杀伤作用。流式细胞仪检测分析Tim-3在新鲜分离的CD3~+T细胞和CD8~+T细胞和接受HSP70-肿瘤抗原肽刺激的CD3~+T细胞和CD8~+T细胞表面的表达情况。结果：限制性酶切分析及序列测定证实成功构建了小鼠Tim-3真核表达质粒pTim-3。FACS检测分析显示转染pTim-3质粒的CHO细胞在表面表达Tim-3抗原。RT-PCR、免疫组织化学法检测肿瘤接种部位能够表达Tim-3 mRNA和Tim-3抗原。同生理盐水对照组和空载体对照组相比，肿瘤接种部位转染表达Tim-3，能显著抑制肿瘤生长速率并明显延长荷瘤鼠的生存期。H&E染色显示有大量的淋巴细胞浸润。接受pTim-3治疗的小鼠的脾细胞杀伤活性明显增强。流式细胞仪检测分析显示新鲜分离的CD3~+T和CD8~+T细胞几乎不表达Tim-3。然而，接受肿瘤抗原肽刺激的CD3~+T和CD8~+T细胞表面表达Tim-3。而且随着刺激次数的增加，CD3~+T和CD8~+T细胞上调表达Tim-3。结论：在肿瘤微环境中转染表达Tim-3，能够有效地促进淋巴细胞的抗肿瘤活性。Tim-3除去表达在活化的CD4~+T细胞表面，也可以表达在活化的CD8~+T表面细胞。
第二部分可溶性Tim-3抑制T淋巴细胞的免疫应答反应
首先，我们阐明了Tim-3 mRNA表达在骨髓细胞，胸腺细胞和脾细胞中，而sTim-3 mRNA仅表达在脾细胞中，而骨髓细胞，胸腺细胞和其它非免疫组织细胞不表达sTim-3 mRNA。我们采用Real-Time PCR的方法研究新鲜分离的脾细胞和接受刺激的脾细胞表达两型Tim-3 mRNA的表达情况。Real-Time PCR的结果显示新鲜分离的脾细胞主要表达Tim-3mRNA，而接受刺激的脾细胞主要表达sTim-3mRNA。提示Tim-3和sTim-3 mRNA的表达与脾细胞的免疫状态有关。
然后，我们用Western blot的方法证实sTim-3蛋白质分子存在于接受刺激的脾细胞的分泌液中。并且转染了sTim-3真核表达质粒的CHO细胞分泌液中也存在sTim-3蛋白质分子。荧光检测显示转染Tim-3-GFP真核表达质粒的CHO细胞显示绿色荧光。而转染sTim-3-GFP真核表达质粒的CHO细胞几乎不显示绿色荧光。间接提示sTim-3-GFP以可溶性蛋白的形式被分泌到细胞外。用转染sTim-3-GFP真核表达质粒的CHO细胞的上清液同T细胞共孵育。FACS检测显示T细胞表面的荧光强度明显高于对照组。证实sTim-3能够和T细胞表面的配体结合。
体外的实验结果显示sTim-3能够抑制anti-CD3和anti-CD28的刺激淋巴细胞增殖激作用。并且sTim-3同样能够抑制HSP70-肿瘤抗原肽复合物的特异性抗原刺激作用。应用Tim-3的抗体后能够阻断sTim-3抑制淋巴细胞增殖激作用。ELISA检测细胞因子的结果显示：sTim-3能明显抑制IL-2、IFN-γ的分泌，但不影响IL-4的分泌。我们进一步采用肿瘤排斥模型研究sTim-3在体内的免疫学活性。结果显示，肿瘤细胞在接受sTim-3治疗的小鼠体内生长更快一些，荷瘤鼠的存活期明显缩短。和对照组比，接受sTim-3治疗的小鼠的脾细胞增殖能力和杀伤活性明显降低。病理和免疫组化检测结果显示接受sTim-3治疗的瘤组织内浸润T淋巴细胞的数目明显低于对照组。证实sTim-3在体内抑制T细胞的抗肿瘤免疫应答反应。
我们的研究还试图理解sTim-3影响T细胞免疫应答反应的作用机制。因为CD4~+CD25~-T效应细胞和CD4~+CD25~+T调节细胞都表达Tim-3的配体。因此，sTim-3抑制T细胞的抗肿瘤免疫应答反应可能是通过抑制CD4~+CD25~-T效应细胞的活化，或者促进CD4~+CD25~+T调节细胞的活化。为了回答这个问题。我们采用Real-Time PCR的方法研究反应CD4~+CD25~-T效应细胞和CD4~+CD25~+T调节细胞活性状态的免疫分子表达情况。结果显示，sTim-3能够抑制与CD4~+CD25~-T效应细胞功能相关的细胞因子的表达，比如IL-2、IFN-γ、TNF-β。但不影响与CD4~+CD25~+T调节细胞功能相关的免疫分子(Foxp3、I1-10、TGF-β)的表达。这些结果提示sTim-3主要通过抑制T效应细胞的功能发挥作用。
第三部分HSP70-肿瘤抗原肽复合物联合sPD-1治疗肺部转移性肿瘤的研究
B7-H1(又被称为PD-L1)是表达在膜表面的糖蛋白分子，属于共刺激分子B7家族。其受体是PD-1(programmed death-1)，PD-1是在淋巴细胞活化过程中诱导表达的一个免疫抑制性受体。B7-H1和其受体PD-1结合后传导的抑制性信号参与了肿瘤的免疫逃避机制。在此研究中，我们发现这一抑制性信号也参与了HSP70-肿瘤抗原肽复合物治疗过程中肿瘤的免疫逃避。我们在用HSP70-肿瘤抗原肽抑制黑色素瘤肺转移的研究中发现，HSP70-肿瘤抗原肽复合物能够有效地始动免疫应答反应，表现为黑色素瘤结节处有大量的淋巴细胞浸润，血清中IL-2、IFN-γ水平明显升高。和对照组相比，HSP70-肿瘤抗原肽复合物治疗组在早期肺部黑色素瘤结节数目明显减少。但是，出乎意料的是，随着观察时间的延长HSP70-肿瘤抗原肽复合物治疗组有大量的黑色素瘤结节生成。在体外，用IFN-γ同黑色素瘤细胞共孵育，流式细胞仪检测发现随着IFN-γ的浓度增加黑色素瘤细胞表面的B7-H1表达显著升高。而未经IFN-γ处理的黑色素瘤细胞不表达B7-H1。Real-Time PCR检测发现B7-H1基因高表达在体内黑色素瘤细胞上。淋巴细胞杀伤实验证实受到IFN-γ处理的黑色素瘤细胞能有效的抵抗淋巴细胞的杀伤。当我们用本研究小组构建的可溶性的PD-1(sPD-1，soluble PD-1)封闭瘤细胞表面的B7-H1，淋巴细胞的杀伤活性得到了提高。提示表达在黑色素瘤细胞表面的B7-H1能够有效抑制淋巴细胞的杀伤活性。在体内，HSP70-肿瘤抗原肽复合物和sPD-1联合应用时观察到明显的协同治疗作用，联合治疗能显著抑制黑色素瘤结节的生成、明显延长小鼠的生存时间。并增强肿瘤特异的CTL对肿瘤细胞的杀伤活性。我们还采用Real-Time PCR检测肿瘤浸润淋巴细胞中相关细胞因子的表达情况。在HSP70-肿瘤抗原肽复合物与sPD-1联合应用组，IFN-γ和IL-2等T_H1型的细胞因子表达明显升高。IL-10、TGF-β和foxp3等免疫负调控分子表达下降。提示联合治疗能够有效的克服黑色素的免疫抵抗作用。在这部分的研究中，我们还探索了应用sPD-1的剂量-效应关系。一次尾静脉注射10μg的sPD-1的表达质粒，在血清中可连续7天检测到sPD-1的存在。每周两次或每周一次应用10μg的sPD-1的表达质粒联合HSP70-肿瘤抗原肽复合物能有效抑制黑色素结节的生成。每周两次5μg的sPD-1的表达质粒联合HSP70-肿瘤抗原肽复合物也能有效抑制黑色素结节的生成。但是每周一次应用5μg的sPD-1的表达质粒联合HSP70-肿瘤抗原肽复合物不能有效抑制黑色素结节的生成。这一研究结果不仅揭示了肿瘤免疫逃逸的一个新的机制，而且也开辟了一条肿瘤免疫治疗的新路径：封闭肿瘤细胞表面的免疫负性共刺激分子—B7-H1，保护CTL。
The search for cell-surface markers that can distinguish T_H1 from T_H2 cells has lead to the identification of a new molecular---Tim-3. It has been reported that administration of anti-Tim-3 antibody and Tim-3 Ig fusion protein can abrogate the induction of peripheral tolerance and promote T_H1-mediated autoimmune and alloimmune disease, which indicated that Tim-3 has important function in the regulation of immune-mediated disease. It was found that Tim-3 mRNA could be alternatively spliced to produce two mRNA molecules. The longer one directs the synthesis of full-length Tim-3 (i.e., Tim-3), the shorter one, without the region encoding the mucin domain and transmembrane domain, was supposed to direct the synthesis of a splice variant of sTim-3.
Many co-stimulatory molecules and immunoregulatory receptors, such as CD86, Fas and CTLA-4, have native soluble molecules, and these soluble variants were important in controlling immune response as well as in susceptibility and resistance to autoimmune disease. The discovery of sTim-3 raised several question to be answer. The first one was the expression pattern of Tim-3 and sTim-3 mRNA in immune response. The second one was the existence of the translated product of sTim-3 and whether sTim-3 could bind to Tim-3 ligands. The third one was the regulatory role of Tim-3 and sTim-3 in antitumor immune response.
At present, tumor immunotherapy efforts are focused on the generation of strong T cell response against tumor antigens. However, strong T cell response does not always coincide with tumor rejection, for which upregulated expression of irnmunoinhibitory molecules--- may be responsible. Emerging evidences suggest that B7 homolog 1 (B7-H1) is important in the mechanisms of resistance against tumor associated antigen-specific immunity. Blockade of B7-H1 by sPD-1 can enhance tumor specific CTL response and cause tumor rejection.
Part I
Ectopic expression of Tim-3 induces tumor-specific antitumor immunity
Objective: To express Tim-3 in tumor microenvironment and investigate the role of Tim-3 in regulation of lymphocyte activity and antitumor effect. Methods: Total RNA of concanavalin A stimulated splenocytes was extracted and Tim-3 cDNA was amplified by RT-PCR. The amplified fragment was digested by restriction enzymes and inserted into compatible enzyme restriction sites of pcDNA3.1 to generate murine Tim-3 eukaryotic expression plasmid (pTim-3). CHO cells were transfected with pTim-3 with Dosper liposomal transfection reagent, the transfected cells were screened by G418. And the expression of Tim-3 in the transfected cells were detected by FACS. Tumors were established by injection of H22 into right hind thigh muscle and pTim-3 plasmid was injected intratumorally. The expression of Tim-3 on H22 cells was examined by immunohistochemistry, and tumor volume and the survive was recorded. Hematoxylin and eosin (H&E) staining was performed to detect tumor-infilating T lymphocytes. Cytolytic activity against H22 was determined in 4h - ~(51)Cr release assay. Tim-3 expression on freshly isolated CD3~+ T and CD8~+ T cells and HSP70-tumor peptide complex stimulated CD3~+ T and CD8~+ T cells were examined by FACS analysis. Results: The constructed plasmid, designated as pTim-3, was confirmed by restriction enzymes digestion and DNA sequencing. Flow cytometric analysis revealed that Tim-3 antibody stained the surface of cells transfected with pTim-3 but not the surface of cells transfected with mock plasmid (pcDNA3.1). Intratumor injection of pTim-3 plasmid potently inhibited tumor growth and results in prolonged survival. There was microscopic evidence of dense lymphocyte infiltration in pTim-3 treated mice. In addition, Splenocytes from the pTim-3 treated group showed a strong CTL response against H22 tumor cells, which suggested that ectopic Tim-3-mediated antitumor responses were dependent on a functional adaptive lymphocyte response. We also demonstrate endogenous Tim-3 expression on lymphocyte is relevant in antitumor immune response. Since considerable number of CD3~+ T cell including CD8~+ T cell can express Tim-3 after stimulate with tumor-peptide complex. Conclusion: Expression of Tim-3 on H22 tumor cells can enhance the proliferation and other functions of T lymphocytes. Tim-3 expression is not limit on CD4~+ T cell but also on CD8~+ T cells after stimulation with tumor- peptides complex. Our results thus gain further insight into the function of Tim-3 expressed on nonlymphoid tissues in regulating antitumor immune response. Part II
Soluble Form of Tim-3 Is An Inhibitory Molecule in T Cell-Mediated Immune Response
In initial experiments, we demonstrated that Tim-3 mRNA was existent in the cells from thymus, bone marrow and spleen, whereas sTim-3 mRNA was expressed only in spleen cells but not in bone marrow, thymus and non-lymphoid organs. To investigate the expression pattern of sTim-3 and Tim-3 mRNA in immune response, we examined the expression of these mRNAs in freshly isolated and activated splenocytes. Real-Time PCR revealed that both Tim-3 mRNA and sTim-3 mRNA were significantly increased after each rounds of stimulation, whereas there appeared to be a higher increase of sTim-3 product than that of Tim-3 product, suggesting that the expression pattern of Tim-3 mRNA and sTim-3 relied on the activation of splenic lymphocyte.
We next proceed to examine the existence of sTim-3's protein product by Western blot. The results showed that sTim-3's protein was detected in the cell-free supernatants of the stimulated splenocyts and CHO cells transfected with sTim-3 eukaryotic expression plasmid. Fluorescence microscope analysis revealed that Green fluorescence was found in CHO cells transfected with pTim-3-GFP but scarcely in CHO cells transfected with psTim-3-GFP, indicating that sTim-3 is mostly secreted out, which further conformed that sTim-3 is a soluble molecule. The binding of sTim-3-GFP to its putative ligand(s) on splenic T cells was determined by FACS analysis. The fluorescence intensity on T cells incubated with sTim-3-GFP was significantly increased as compared with control. These results clearly indicated that sTim-3 could bind to putative Tim-3 ligand(s).
In vitro, the response of T cells to antigen-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation was significantly suppressed in the presence of sTim-3, and the blockade of sTim-3 with anti-Tim-3 antibody recovered the response of T cells to these stimuli, supporting the notion that sTim-3 mediates the inhibitory effect in immune response. We then attempt to further understand the function of sTim-3 in vivo, in tumor rejection model, sTim-3 facilitated the growth of tumor in vivo and shorten survival of tumor-bearing mice. The proliferation of T cells from sTim-3 treated mice was significantly reduced when they were re-stimulated by antigen-loaded DC in vitro, Moreover, the antitumor CTL activity was significantly reduced by sTim-3 and the amount of T cells in tumor tissue was also significantly decreased. Pathologic and immunohistological findings revealed the reduced tumor-infilating T lymphocytes. To sum up, these results confirmed the inhibitory effect of sTim-3 on the generation of antigen-specific T cell immune response.
Since both effector CD4~+CD25~- T cells and regulatory CD4~+CD25~+ T cells express a putative Tim-3 ligand, the impairment of antitumor immunity by sTim-3 might be the result of either impairing the function of effector CD4~+ T cells or activating the function of CD4~+ regulatory T cells. To understand the underlying cellular mechanism contributing to the impaired antitumor immunity by sTim-3, we analyzed the expression of genes related to the function of tumor-infiltrating lymphocytes by real-time quantitative PCR. The results indicated that, in sTim-3 treated mice, the expression of IL-2, IFN-γ and TNF-β mRNA was down-regulated, whereas, the expression of forkhead transcription factor 3 (Foxp3), IL-10 and TGF-p mRNA was not influenced as compared with control, theses results suggested that sTim-3 didn't influence Treg cells but otherwise resulted in the profound immune unresponsiveness of CD4~+ T effector cells. Part III
HSP70 Vaccine in Combination with Gene Therapy with Plasmid
DNA Encoding sPD-1 Overcomes Immune Resistance and Suppresses
the Progression of Pulmonary Metastatic Melanoma
B7-H1 (also known as PD-L1) is a cell surface glycoprotein belonging to the B7 family of costimulatory molecules. It was founded that PD-1 (programmed death-1), which is expressed on the activated lymphocytes, is the receptor of B7-H1. B7-H1 is thought to contribute to immune escape of cancer by interacting with PD-1 receptor. In this study, the treatment with HSP70 vaccine induced an infiltration of T cells into the tumor site as well as the expression of IFN-γ and IL-2, and delayed lung metastases of tumor, but the tumor progression nonetheless occur finally. We incubated B16F1 cells with different doses of IFN-γ in vitro. FACS analysis revealed that the percentage of B7-H1-positive B16F1 cells was significantly increased by IFN-γ stimulation. By contrast, B7-H1 was hardly detectable on the untreated B16F1 cells. Consistent with this result, the upregulatd expression of B7-H1 in vivo in tumor microenvironment was also confirmed by Real-time PCR. We demonstrated that B7-H1 expressed by residual tumor cells was responsible for the resistance of tumor to the therapy with HSP70 vaccine. Blockade of B7-H1 by i.v. injection pPD-1A, a plasmid encoding the extracellular domain of PD-1 (sPD-1) could reverse this resistance and enhance the therapeutic efficacy. To complement these findings, we investigated the gene expression of TILs by Real-time PCR analysis, which revealed that the expression of TH1 cytokines IFN-γ and IL-2 by TIL in the mice treated with HSP70 vaccine in combination with sPD-1 was increased and the expression of negative regulatory molecules IL-10, TGF-β and foxp3 was decreased, demonstrating that multifunctional properties afforded by the combination therapy can effectively overcome tumor resistance and promote effective antitumor immunity. In vivo transfection with pPD-1A could be performed as infrequently as once a week and still produce a significant antitumor effect. These findings suggest that the treatment with HSP70 vaccine followed by blockade of tumor-B7-H1 with sPD-1 may provide a promising approach for tumor immunotherapy.
引文
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