【作者】 贾正才；

【导师】 吴玉章；

【作者基本信息】 第三军医大学 ， 免疫学， 2004， 博士

【摘要】 肿瘤免疫学研究领域最重要的进展之一是能被T细胞识别的肿瘤相关抗原的鉴定和定性。在已鉴定了的肿瘤相关抗原中,有些抗原(如MAGE家族编码的抗原)为非己抗原,由于不存在对这些抗原的免疫耐受,因而能诱导高效的抗肿瘤免疫应答;而大部分的肿瘤相关抗原都是天然表达的抗原,属于自身抗原,由于预先存在的免疫耐受,因而不能有效诱导产生抗肿瘤免疫。以前的研究证实,用抗体清除某一特定的细胞亚群(如,CD25+ T细胞)或阻断某一特定的信号传导通路(如,CTLA-4)能增强抗肿瘤免疫应答。这些发现提示:至少在某些情况下,对自身抗原的免疫耐受是肿瘤抑制的结果而不是缺乏先天免疫的协同刺激。由于肿瘤的生长,大量的对免疫系统有抑制作用的细胞因子被肿瘤细胞直接分泌或被宿主细胞分泌。在这些细胞因子中,最突出的是转化生长因子-β(TGF-β)。研究发现,在人类肿瘤中,TGF-β的mRNA和蛋白表达都显著增加;而且在许多肿瘤中,TGF-β的高表达与更多的恶性进展和存活率的降低有关。TGF-β是一种具有多重免疫抑制特性的多向性细胞因子,其免疫抑制特性包括:抑制树突状细胞(DC)的成熟和活化、降低DC的抗原提呈能力、抑制T细胞分化成细胞毒性T淋巴细胞(CTL)和Th细胞、抑制CD4+ T细胞分化为Th1细胞和Th2细胞、诱导活化T细胞的凋亡等。由于CTL和Th1型细胞因子的产生对免疫介导的肿瘤根除非常重要,因此TGF-β对这些功能的抑制可能是其抑制免疫应答的重要机制。研究报道,阻断T细胞内TGF-β的信号转导能导致免疫介导的肿瘤根除;用TGF-β反义寡核苷酸(ODN)转染表达TGF-β的肿瘤细胞能显著降低肿瘤细胞的TGF-β表达,而用这种转染了TGF-β反义寡核苷酸的肿瘤细胞免疫小鼠能诱导更强的抗肿瘤免疫应答;用TGF-β1中和性单克隆抗体或通过细胞融合来中和TGF-β可增强DC疫苗的免疫治疗效力。但TGF-β的中和是否能提高DNA疫苗的免疫效能还未见报道。为了在小鼠黑色素瘤模型上阐明这个问题,我们已经选择了主动免疫的方式来诱导TGF-β中和性抗体的产生。非洲爪蟾TGF-β5(aTGF-β5)为小鼠TGF-β1(mTGF-β1)的异种同源物,其成熟蛋白与mTGF-β1的成熟蛋白间在氨基酸水平上的同源性大约为76%。我们研究了是否用编码aTGF-β5的质粒DNA免疫小鼠能有效诱导mTGF-β1中<WP=14>和性抗体的产生。T细胞识别的黑色素瘤抗原-1(MART-1),gp100,酪氨酸酶,酪氨酸酶相关蛋白1(TRP-1)和酪氨酸酶相关蛋白2(TRP-2)等黑色素瘤相关抗原为非突变的黑色素细胞分化抗原,属自身抗原。而TRP-2属于酪氨酸酶家族,参与黑色素合成,表达于黑色素细胞和大部分黑色素瘤。位于小鼠TRP-2(mTRP-2)180~188位氨基酸的多肽mTRP-2aa180-188 (SVYDFFVWL)为mTRP-2的H-2Kb限制性CTL表位。过继转移mTRP-2特异性CTLs能抑制B16黑色素瘤细胞肺部转移灶的生长,表明mTRP-2为小鼠的一种肿瘤排斥抗原。重要的是,人TRP-2(hTRP-2)也能被人类黑色素瘤特异性CTLs所识别;多肽mTRP-2aa180-188 (SVYDFFVWL)序列也存在于hTRP-2蛋白中,并能被黑色素瘤病人的黑色素瘤反应性CTLs识别。可见,TRP-2为一种与人类肿瘤有关的模型抗原。但由于免疫耐受的存在,用编码mTRP-2的质粒DNA免疫小鼠很难诱导产生抗原特异性的细胞免疫应答和体液免疫应答;即使诱导产生了抗原特异性的CTLs,所诱导的免疫应答也不能抗内源性表达mTRP-2的B16黑色素瘤。因此,我们进一步选择了mTRP-2作为靶抗原,以研究是否用aTGF-β5和mTRP-2联合免疫能有效打破机体对mTRP-2的免疫耐受,是否能更有效地诱导产生mTRP-2特异性的抗肿瘤免疫。采用RT-PCR方法,我们从小鼠黑色素瘤细胞B16F10扩增出了mTGF-β1 cDNA克隆,从非洲爪蟾肺组织扩增出了aTGF-β5 cDNA克隆。扩增产物首先被克隆入pMD18-T载体,然后分别被亚克隆入pCI-neo真核表达载体。克隆了mTGF-β1的pCI-neo的被命名为pCI-mTGF-β1,克隆了aTGF-β5的pCI-neo的被命名为pCI-aTGF-β5。为了研究pCI-aTGF-β5免疫诱导mTGF-β1中和性抗体的能力及其对B16黑色素瘤体内生长的影响,分别用pCI-aTGF-β5、pCI-mTGF-β1、 pCI-neo和PBS对野生型C57BL/6小鼠进行了免疫,并于末次免疫后第7天,于小鼠背部左侧皮下接种了B16F10细胞。结果发现,于免疫后第7天从pCI-aTGF-β5免疫后小鼠获得的血清可与aTGF-β5反应,Western blot分析呈现2条带,其分子量分别为25 kDa和95 kDa,分别代表aTGF-β5的成熟形式和前体复合物;而且该血清可与mTGF-β1交叉反应。但从pCI-mTGF-β1、pCI-neo或PBS免疫后小鼠获得的血清却不能检测到aTGF-β5和mTGF-β1。免疫后小鼠的血清经纯化、获得免疫球蛋白(Ig)后,我们进一步研究了Ig对mTGF-β1生物学活性的影响。结果发现,只有来自pCI-aTGF-β5免疫后小鼠的Ig能抑制mTGF-β1的生物学活性,降低mTGF-β1对Mv 1 Lu细胞生长的抑制作用;而来自其他各组小鼠的Ig对mTGF-β1的生物学活性无影响。但无论是用pCI-aTGF-β5免疫还是用pCI-mTGF-β1或pCI-neo免疫都不影响肿瘤的生长。 <WP=15>采用同样的方法,我们从B16F10细胞扩增出了mTRP-2 cDNA克隆,构建了真核表达质粒pVAX1-mTRP-2。采用标准Fmoc方案合成了H-2Kb结合多肽mTRP-2aa180-188,多肽经HPLC纯化,纯度达90%以上。为了研究pCI-aTGF-β5免疫对编码mT更多还原

【Abstract】 One of the most significant advances in the field of tumor immunology has been the identification and characterization of the tumor-associated antigens that are recognized by T cells. Some of them, such as the antigens encoded by MAGE family, are nonself-antigens and induce vigorous antitumor immune responses due to the lack of tolerance. By contrast, most of the naturally expressed tumor-associated antigens belong to self-antigens, and therefore may elicit an inefficient immunity owing to preexisting tolerance. Previous studies demonstrate that depletion of a particular cellular subset (for example, CD25+ T cells) or blockade of a particular signaling pathway (for example, CTLA-4) by antibody administration can augment generation of an antitumor response. These observations suggest that, at least in some cases, the tolerance to self-antigens could be a result of the suppression mediated by the tumor and not by the lack of costimulation from innate immunity. A number of factors either secreted directly by tumor cells or secreted by host cells as a result of tumor growth have been shown to exert suppressive effects on the immune system. Among such factors the most prominent is transforming growth factor-beta (TGF-β).It has been found that there is a marked increase in the expression of TGF-β mRNA and protein in human cancers (in vivo). Moreover, in many cancers high expression of TGF-β correlates with more advanced stages of malignancy and decreased survival. In untreated breast cancer patients, plasma TGF-β1 levels were elevated and normalized after surgery. TGF-β is a pleiotropic cytokine with multiple immunosuppressive properties. These properties include inhibition of maturation and activation of dendritic cell (DC), reducing of antigen-presenting capacity of DC, inhibition of T cell differentiation into cytotixic T lymphocytes (CTLs) and Th cells, inhibition of CD4+ T cell differentiation into <WP=9>Th1 cells and Th2 cells, and induction of apoptosis of activated T cells. Because CTLs and T helper type 1 cytokine production are important for effective immune-mediated tumor eradication, suppression of these functions by TGF-β could be an important mechanism whereby it inhibits immune response. It has been reported that blockade of TGF-β signaling in T cells results in the immune-mediated eradication of tumors. Modification with TGF-β antisense oligonucleotide significantly decreases the amounts of TGF-β produced by modified tumor cells and immunization with TGF-beta antisense oligonucleotide-modified autologous tumor vaccine enhances the antitumor immunity of tumor-bearing mice. Neutralization of TGF-β1 using TGF-β1-neutralizing monoclonal antibody or produced by the fusion cells enhances the effectiveness of DC-based immunotherapy. It is presently unclear whether neutralization of TGF-β can enhance the efficacy of DNA vaccines.To address the question in an experimental murine model of melanoma, we have chosen active immunization to induce the generation of TGF-β-neutralizing antibodies and investigated whether genetic immunization of mice with plasmid DNA encoding Xenopus laevis transforming growth factor-beta 5 (aTGF-β5), of which mature chain is approximately 76% identical with that of murine TGF-β1 (mTGF-β1) at the amino acid level, would be effective in inducing the production of mTGF-β1-neutralizing antibodies. The melanoma-associated antigens, including melanoma antigen recognized by T cell 1 (MART-1), gp100, tyrosinase, tyrosinase-related protein 1 (TRP-1) and TRP-2, are derived from normal nonmutated melanocyte lineage differentiation antigens and belong to self-antigens. TRP-2 belongs to the tyrosinase family of melanosomal enzymes involved in melanin synthesis and is expressed in melanocytes and most melanomas. The peptide SVYDFFVWL (mTRP-2aa180-188) corresponding to amino acids 180-188 of murine TRP-2 (mTRP-2) was identified by H-2Kb-restricted CTL specific for B16 melanoma cells. Adoptive transfer of mTRP-2-specific CTLs inhibits the growth of experimental melanoma lung metastas更多还原