MHC-肽四聚体技术在造血干细胞移植中的应用

MHC-肽四聚体技术是一种直接监测抗原特异性细胞毒性T细胞(CTL)的新方法,具有较强的特异性和敏感性。本文就MHC-肽四聚体技术在造血干细胞移植后巨细胞病毒特异性CTL、次要组织相容性抗原(mHags)特异性CTL以及肿瘤抗原特异性CTL的检测及其意义进行综述。     

热休克蛋白与肿瘤的研究进展

热休克蛋白(HSPs)是一组重要的应激蛋白(SP)，在正常状态下有少量表达，应激状态下则明显提高。研究表明，热休克蛋白在肿瘤组织中表达异常，具有抑制细胞凋亡作用。从肿瘤组织中提取的HSP-肽复合物携带有多个T细胞表位，通过与抗原呈递细胞(APC)上的受体结合而内化，经MHC-I类途径激活特异性CTL和记忆性T细胞，引发机体细胞免疫反应，产生针对某种肿瘤内所有肿瘤细胞杀伤的效力，从而解决肿瘤抗原调变、很难鉴定来自病人自身癌组织的特异性抗原等问题。HSP-肽复合物作为疫苗用于肿瘤特异性免疫治疗，临床应用前景广阔。     

MHC—肽四聚体技术在造血干细胞移植中的应用

MHC－肽四聚体技术是一种直接监测抗原特异性细胞毒性T细胞（CTL）的新方法，具有较强的特异性和敏感性。本文就MHC－肽四聚体技术在造血干细胞移植后巨细胞病毒特异性CTL、次要组织相容性抗原（mHags)特异性CTL以及肿瘤抗原特异性CTL的检测及其意义进行综述。     

CTL表位预测的研究进展

细胞毒性T淋巴细胞(CTL)表位是抗原蛋白中经抗原提呈细胞(APCs)处理,与MHC-Ⅰ类分子结合并共同被T细胞抗原受体(TCR)特异性识别从而诱导相应的CTL克隆产生免疫应答的短肽.     

肿瘤靶向治疗新抗原筛选与临床研究进展

肿瘤免疫治疗是近年来研究的热点,而基于靶向肿瘤新抗原的免疫治疗极具广阔前景,但是长期以来,缺乏肿瘤特异性抗原是阻碍肿瘤免疫治疗的瓶颈,因此,发现新的肿瘤特异性抗原一直是基础和临床肿瘤免疫学家梦寐以求的目标。二代测序技术的出现推动了肿瘤新抗原筛选与鉴定的发展。目前主要有全外显子结合转录组测序法、串联微基因序列法、靶基因测序法、共享新抗原肽库法和质谱法等筛选肿瘤新抗原的方法。肿瘤新抗原的发现为临床开展抗原疫苗、抗原特异性T细胞、T细胞受体工程T细胞(T cell receptor genetically engineered T cell,TCR-T)和嵌合抗原受体T细胞(chimeric antigen receptor T cell,CAR-T)等靶向肿瘤抗原的免疫治疗提供了优质的靶标。     

负载肝癌肿瘤抗原的B淋巴细胞诱导特异性抗肿瘤免疫的实验研究

目的:探讨体外经小鼠肝癌细胞不同抗原致敏的CD40配体活化的B淋巴细胞诱导的特异性细胞毒T淋巴细胞(CTL)抗肿瘤免疫的能力.方法:分离、纯化T、B混合淋巴细胞,并在CD40L、rmIL-4联合作用下培养.然后分离T、B淋巴细胞以备用.将凋亡的肝癌细胞及其冻融裂解物作为实验组,1640培养基作为空白对照组分别与B淋巴细胞共同培养,检测培养后各组B细胞表面抗原呈递细胞标记(CD40、CD80、CD86)及主要组织相容性抗原的表达情况.利用混合淋巴细胞增殖实验检测T细胞增殖情况.以负载肿瘤抗原的B淋巴细胞诱导的CTL作为效应细胞,肝癌细胞Hepal-6为靶细胞,LDH释放实验检测杀伤活性.结果:负载肿瘤抗原的B淋巴细胞具有刺激T细胞增殖的能力,实验组的B淋巴细胞,其组织相容性分子及其刺激分子表达明显高于空白对照组,其对靶细胞的杀伤率与空白对照组比较经统计学分析差异有显著性.结论:负载肝癌肿瘤抗原的B淋巴细胞作为抗原呈递细胞诱导的CTL可有效产生特异性抗肝癌免疫作用.     

应用免疫球蛋白重链可变区上框架区来源的抗原九肽获得的细胞毒T细胞功能分析

目的明确存B淋巴细胞肿瘤表面的免疫球蛋白重链可变区(IgHV)的框架区(FR)上是否存在可以激发细胞毒T淋巴细胞(CTL)的抗原表位，这些来源于FR的抗原肽是否能够激发家族特异性的免疫反应，探讨按照B淋巴细胞肿瘤的IgHV基因分型进行家族性的免疫治疗的可能性，方法利用生物信息学系统预测了40例B淋巴细胞肿瘤表面的免疫球蛋白序列的抗原表位，人工合成不同基因家族的抗原几肽，利用1、2细胞结合实验检测预测的抗原肽和HLA分子的亲合力利用体外CTL刺激扩增体系。诱导特异性的CTL细胞增殖，用肽／HLA四聚体检测特异性CTL的数目，应用LDH释放实验检测CTL的细胞毒活性并验证其家族特异性。结果在B淋巴细胞肿瘤的7个IgHV基因家族中找到了12个高亲和力的抗原九肽，其中10个(83％)位于FR，以HLA-A*0201正常供的外周血单个核细胞(PBMNC)负荷该九肽作为抗原呈递细胞去刺激自身PBMNC，经过4轮刺激，CD8和肽／HLA四聚体双阳性细胞从0．38％上升到49．38％LDH释放实验证明对HLA—A*0201( )、IgHV1( )靶细胞有明显的杀伤作用，并且被IgHV1肽刺激出的CTL不能识别负荷IgHV3肽的靶细胞：结论IgHV基因FR抗原儿肽可以刺激产生具有HLA限制性的并且肽特异性的CTL，同时这样的杀伤作用具有家族特异性，以此为靶位有望对B淋巴细胞肿瘤进行家族特异性的免疫治疗。     

肿瘤病人自体CIK和ECTL细胞治疗的护理

CIK为细胞因子诱导的杀伤细胞,是一种新型、高效,具有广谱杀瘤活性的免疫效应细胞,ECTL为肿瘤抗原特异性杀伤T淋巴细胞,是通过树突状细胞(DC) 摄取、加工肿瘤抗原为抗原多肽提呈给 T细胞,使 T细胞被充分激活、增殖为 CTL,启动细胞免疫机制,特异性杀伤肿瘤细胞.CIK和ECTL治疗均为免疫细胞治疗,是肿瘤治疗的重要综合方法之一,并在临床上显示了一定的应用前景[1].     

外周血单核细胞来源树突状细胞体外扩增及诱导抗乳腺癌免疫的研究

目的研究外周血来源树突状细胞(DC)的体外扩增及诱导特异性抗乳腺癌细胞的免疫效应。方法(1)从外周血中分离单个核细胞后,获得单核细胞(monocyte,Mo)。粒-单集落刺激因子(GM-CSF)和白介素4(IL-4)诱导分化扩增培养7d后,应用流式细胞术进行表型分析。(2)诱导单核细胞分化的第3天加入人乳腺癌细胞株3A0的冻融抗原培养4d后,获得负载肿瘤抗原的成熟DC;将致敏DC与从外周血中分离的同种异体T淋巴细胞共培养3d,获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝法检测CTL及上清液对人乳腺癌细胞株3A0、人胚肾细胞株293T(对照细胞)、人肝癌细胞株HCCC-9810的细胞毒作用。结果(1)外周血来源Mo在GM-GSF和IL-4作用下,第7天后可分化生成成熟的DC,高表达DC特异性抗原CDla、CD80(B7-1)、CD86(B7-2)HLA-DR、CD83。(2)DC可负载并递呈肿瘤抗原,激活同种异体T淋巴细胞,诱导肿瘤特异性CTL产生。不同浓度CTL及上清液对乳腺癌细胞3A0有特异性杀伤、抑制作用(P<0.05)。结论外周血中Mo可体外分化扩增为成熟的功能性DC,并诱导出特异性杀伤乳腺癌细胞的免疫效应。     

MAGE-12的HLA-A2限制性CTL表位肽预测及其三维结构构建

目的从理论上分析、预测黑色素瘤抗原(MAGE)-12的人类白细胞抗原(HLA)-A2限制性细胞毒性T淋巴细胞(CTL)表位肽,并构建其三维结构。方法以肿瘤特异性抗原MAGE-12为研究目标,采用超基序法、量化基序法、多项式法、延展基序法与三维结构构建相结合的CTL表位预测方法。结果预测出的MAGE-12的表位中有4个符合HLA-A2限制性CTL表位要求。结论预测出的4个HLA-A2限制性CTL表位为MAGE-12的表位的可能性较大,经后续实验筛选、鉴定后,可用于基于MAGE-12的肿瘤治疗性多肽疫苗的设计研究。     

A fail-safe mechanism for maintaining self-tolerance.

Using cytotoxic T lymphocyte (CTL) responses to the class I histocompatibility antigen Qa1 and to the minor histocompatibility antigen H-Y, we show that the immune system maintains a peripheral screening process that is able to tolerize a wide variety of potentially autoimmune CTL. The critical factor is the presence or absence of specific T helper cells. If T help is available, CTL precursors that recognize antigen are activated. In the absence of help, they are tolerized. Thus, T helper cells are guardians of peripheral tolerance in CTL.     

自身肺癌抗原致敏DCs的抗肺癌体外免疫效应研究

目的探讨自身肺癌抗原致敏树突状细胞（DendriticCeil，DC）的体外免疫效应。方法体外原代培养肺癌细胞，并制备冻融抗原，冲击DCs，致敏肿瘤浸润淋巴细胞（TIL）和外周血T淋巴细胞，MTT法检测DCs瘤苗体外联合抗肺腺癌细胞效应。结果采用效靶比为30：1和20：1的比例，30：1和20：1的CTL杀伤率为（18．21±2．89）和（24．69±3．51）；30：1和20：1的CTIL杀伤率为（25．46±3．02）和（27．52±2．68），CTIL比CTL杀伤率高（P〈0．05）。结论负载自身肺癌抗原的DCs致敏的肿瘤浸润淋巴细胞TIL比外周血T淋巴细胞杀伤率高，为肺癌的生物治疗提供了新的思路。     

Antitumor immunization with a minimal peptide epitope (G9-209-2M) leads to a functionally heterogeneous CTL response.

The goal of experimental clinical protocols using peptide antigen for active vaccination and treatment of patients with metastatic cancer is to induce a vigorous cytotoxic T lymphocyte (CTL) response against the immunizing antigen, and thereby against tumor cells expressing the antigen. However, the magnitude and breadth of human CTL responses induced by peptide immunization, and in particular against antigens expressed by normal tissues as well as tumors, is not well characterized. This issue was examined by characterizing CTL cloids derived from peripheral blood mononuclear cells of three patients who received peptide immunization as treatment for metastatic melanoma. All patients received G9-209-2M peptide, a modified epitope of the gp100 melanoma-associated antigen. The results indicated that the CTL response induced by this peptide antigen was highly heterogeneous both in terms of avidity toward the peptide antigen and recognition of tumor cell lines. Furthermore, avidity of each CTL cloid for the native peptide was highly predictive of tumor reactivity. These results are discussed in terms of their implications for peptide vaccination and adoptive tumor immunotherapy.     

Immunogenicity of premalignant lesions is the primary cause of general cytotoxic T lymphocyte unresponsiveness

Cancer is sporadic in nature, characterized by an initial clonal oncogenic event and usually a long latency. When and how it subverts the immune system is unknown. We show, in a model of sporadic immunogenic cancer, that tumor-specific tolerance closely coincides with the first tumor antigen recognition by B cells. During the subsequent latency period until tumors progress, the mice acquire general cytotoxic T lymphocyte (CTL) unresponsiveness, which is associated with high transforming growth factor (TGF) beta1 levels and expansion of immature myeloid cells (iMCs). In mice with large nonimmunogenic tumors, iMCs expand but TGF-beta1 serum levels are normal, and unrelated CTL responses are undiminished. We conclude that (a) tolerance to the tumor antigen occurs at the premalignant stage, (b) tumor latency is unlikely caused by CTL control, and (c) a persistent immunogenic tumor antigen causes general CTL unresponsiveness but tumor burden and iMCs per se do not.     

靶向VEGF基因的siRNA联合树突状细胞介导的CTL对MCF-7细胞作用的研究

目的探讨靶向血管内皮细胞生长因子(VEGF)基因的小干扰RNA(siRNA)联合负载肿瘤抗原的树突状细胞(DC)致敏的细胞毒性T淋巴细胞(CTL)的联合抗瘤作用。方法体外应用靶向VEGF基因最佳转染浓度(100nmol/L,增加浓度不再提高沉默作用)的siRNA联合负载人乳腺癌细胞(MCF-7细胞)抗原的DC介导的CTL(siRNAVEGF+CTL组)共同作用于MCF-7细胞,同时设立空白对照组和空白+CTL组(只加无血清无抗生素培养基);脂质体组和脂质体+CTL组(只加LipofectamineTM2000);siRNAVEGF-组(100nmol/LsiRNA);siRNASCR组和siR-NASCR+CTL组(100nmol/LsiRNASCR),每组做6个平行孔,四甲基偶氮唑蓝(MTT)法检测siRNAVEGF+CTL组和各对照组肿瘤杀伤活性(n=6),Hoechst33258核染色观察细胞的凋亡(n=3)。结果siRNAVEGF+CTL组、siR-NAVEGF-组siRNASCRCTL组肿瘤杀伤活性分别为99.37%,51.17%和43.94%,siRNAVEGF+CTL组与对照组比较可明显杀伤肿瘤细胞,瘤细胞几乎完全溶解,Hoechst33258显示细胞核呈明显的细胞凋亡改变。结论体外实验显示靶向VEGF的siRNA联合负载肿瘤抗原DC致敏的CTL能有效抑制乳腺癌MCF-7细胞的生长,二者的联合应用抗瘤效果显著。     

LPS刺激来自人单核细胞的树突状细胞在调节自体CD4+CD25+T细胞活性作用研究

树突状细胞（DC）是现今被认为最具潜能的专职抗原呈递细胞.应用不同方法在体内诱导细胞毒T细胞（CTL）来识别肿瘤相关抗原的肿瘤免疫治疗研究已有报告.然而,在体内免疫治疗的有效性可能仅限制在局部或系统抑制CTL产生和功能.为了检测LPS刺激人单核细胞的DC来抑制自体CD4^＋CD25^＋T细胞能力,使用HLA-A2限制性p53264-272肽作为肿瘤抗原,用LPS（DC-LPS^＋）或不用LPS（DC-LPS^-）产生的DC分别与自体T细胞共同培养.结果显示：在DC-LPS^＋活化的T细胞的CD4^＋CD25^＋T细胞群比DC-LPS^-活化的T细胞要低.这个结果提示,DC-LPS^＋与CD4^＋CD25^＋T细胞群有关联,而且这种特性可能是由于T细胞对肿瘤相关抗原的调节作用.     

Inhibition of MHC class II-restricted T cell response by Lyt-2 alloantigen

T cell hybridomas were established by fusing a CD8+ V beta 8.1+ CTL clone and a CD4+ V beta 8.1+ helper T lymphocyte (HTL) clone to the thymoma cell line BW5147. In contrast to the HTL x BW hybridomas, which retain the same antigen specificity as the original T cell clone, the CTL x BW hybridomas lost the class I MHC-restricted antigen response but acquired a new specificity to Mlsa antigen. Mlsa reactivity of CTL x BW hybridomas was shown to be mediated by the CTL TCR as assayed by inhibition using an anticlonotypic antibody to the CTL clone. Since hybridomas established with BW5147 lose CD8 expression, we have introduced the CD8 molecule into CTL x BW5147 hybridomas by gene transfection. The CD8+ V beta 8.1+ hybridoma was no longer capable of reacting to Mlsa antigen but exhibited the same antigen specificity as the parental CTL clone. Furthermore, the presence of the transfected CD8 molecule in the HTL x BW hybridomas was found to be inhibitory to class II MHC-restricted antigen reactivity. These results demonstrate that, besides its role in increasing the overall avidity of T cell-class I MHC/antigen interaction, the CD8 molecule inhibits T cell-class II MHC gene product/antigen interaction. This negative effect of the CD8 molecule on a class II MHC-restricted response may account for the failure of CD8+ T cells using either V beta 8.1 or V beta 6, which impart reactivity to the Mlsa antigen on CD4+ T cells, to respond to the Mlsa antigen.     

Avoiding tolerance against prostatic antigens with subdominant peptide epitopes

A potential novel therapy for prostate cancer is the induction of immune responses to normal prostate-associated antigens (PAA). One approach is to use synthetic peptides from PAA to educate T cells as a means of developing a defined and specific immunotherapy for prostate cancer. A likely major hurdle when using normal PAA for this type of therapy is the tolerance that the immune system may already have for PAA. To evaluate mechanisms for overcoming tolerance, the authors assessed the level of tolerance to SV40T antigen in a transgenic mouse. The SV40T antigen is selectively expressed in the prostates of mice from the transgenic adenocarcinoma mouse prostate (TRAMP) model. The authors have shown that TRAMP mice are tolerant to a dominant cytotoxic T-lymphocyte (CTL) epitope from the SV40T antigen compared with nontransgenic littermates. The tolerance was exhibited as early as 4 weeks and as late as 24 weeks. The use of multiple injections of an oligonucleotide that contains an unmethylated CpG induced high levels of hematopoiesis but did not overcome the tolerance. Injection of an antibody to activate CD40 increased the CTL response in normal mice but also did not overcome tolerance. However, tolerance in the TRAMP mice was avoided when an epitope that had previously been characterized as a subdominant epitope was administered. The authors are investigating the potential of subdominant epitopes to induce prostatitis and antitumor responses. The results of this work should facilitate the development of immune-based therapies for prostate cancer.     

Helper T cells for cytotoxic T lymphocytes need not be I region restricted

We investigated the antigenic requirements for restimulation of H-2- restricted cytolytic T lymphocytes (CTL) in vitro to determine whether H-2 I region-restricted helper T cells are required in these responses. In one set of experiments, we studied the in vitro response of (responder x nonresponder)F(1) female T cells to the male antigen H-Y. We chose to examine this response because it has been suggested that the defect in nonresponder strains is a failure of helper T cells to recognize H-Y in association with nonresponder I region determinants. However, we find that nonresponder male stimulator cells are as effective as F(1) male stimulator cells at inducing H-Y-specific CTL responses. This finding calls into question reports that secondary CTL responses to H-Y are dependent upon the activation of H-Y- specific helper T cells restricted to responder type I region determinants. In a second set of experiments, we examined the requirements for restimulation of H-2-restricted T cells specific for minor-histocompatibility antigens from long-term mixed lymphocyte cultures. These cultures were established by repeatedly restimulating cultures of specific T cells with H- 2-matched stimulator cells expressing foreign minor histocompatibility antigens. We found that H-2D-restricted T ceils, including CTL, could be restimulated with cells that were matched with the responding cells at only the D region genes. This response did not appear to result from positive allogeneic effects or from antigen processing and “representation” by responder type APC that might contaminate the cultures. Thus, we find no evidence for a requirement for I region-restricted helper T cells in these CTL responses. However, helper T cells are required because we find that CTL lines derived by limit-dilution cloning from these long-term MLC are absolutely dependent upon exogenous helper factors for growth. The most simple interpretation of these results is that the helper cells are restricted to H-2 antigens other than I region antigens or to antigens that code outside of the H-2 complex. Finally, we show that factor-dependent CTL lines must recognize their specific antigen to proliferate, even in the presence of exogenous factors. The requirement of activated CTL for antigen to proliferate provides an explanation for how specific CTL can be selectively enriched in MLC by specific antigen stimulation. Furthermore, it is at variance with reports that memory CTL or activated CTL require only interleukin 2 for restimulation.     

Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes

The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.