CD4+CD25+T细胞在CD8+T细胞抗肿瘤免疫中的调节作用

实验旨在研究CD4^＋CD25^＋T细胞在CD8^＋T细胞抗肿瘤免疫中的调节作用。将小鼠脾脏中分离的单个核细胞分为两组．即去除CD4^＋CD25^＋T细胞组和未去除CD4^＋CD25^＋T细胞组,测定树突状细胞提呈的肿瘤抗原多肽刺激不同T细胞增殖活性、细胞因子IFN一1分泌,以及多肽特异性CD8^＋T细胞对同源性胃癌细胞株MFC的杀伤活性。结果显示预先去除未致敏T细胞中的CD4^＋CD25^＋T细胞,所诱导的特异性CD8^＋CTL对肿瘤细胞免疫应答增强,表现为反应性T细胞对树突状细胞提呈的肿瘤抗原多肽增殖反应增强,IFN-γ分泌量提高及CD8＋T细胞对MFC杀伤活性增强。这些结果表明。预先去除未致敏T细胞中的CD4^＋CD25^＋T细胞,肿瘤抗原多肽修饰的树突状细胞肿瘤疫苗效能可明显增加。CD4^＋CD25^＋T细胞在CD8^＋T细胞抗肿瘤免疫中起下调作用。     

CD4~+CD25~+T细胞在CD8~+T细胞抗肿瘤免疫中的调节作用

实验旨在研究CD4+CD25+T细胞在CD8+T细胞抗肿瘤免疫中的调节作用。将小鼠脾脏中分离的单个核细胞分为两组,即去除CD4+CD25+T细胞组和未去除CD4+CD25+T细胞组,测定树突状细胞提呈的肿瘤抗原多肽刺激不同T细胞增殖活性、细胞因子IFN-γ分泌,以及多肽特异性CD8+T细胞对同源性胃癌细胞株MFC的杀伤活性。结果显示预先去除未致敏T细胞中的CD4+CD25+T细胞,所诱导的特异性CD8+CTL对肿瘤细胞免疫应答增强,表现为反应性T细胞对树突状细胞提呈的肿瘤抗原多肽增殖反应增强,IFN-γ分泌量提高及CD8+T细胞对MFC杀伤活性增强。这些结果表明,预先去除未致敏T细胞中的CD4+CD25+T细胞,肿瘤抗原多肽修饰的树突状细胞肿瘤疫苗效能可明显增加。CD4+CD25+T细胞在CD8+T细胞抗肿瘤免疫中起下调作用。     

CTL识别的HLA-A2限制性人卵巢癌相关抗原OVA66表位的鉴定

目的：鉴定CTL识别的HLA—A2限制性人卵巢癌相关抗原OVA66表位。方法：以细胞因子从外周血单个核细胞(PBMC)中诱导树突状细胞(DC)，通过形态学观察和流式细胞术进行鉴定。用表位预测法选取并合成两种肽分子，分别脉冲成熟的DC，并刺激HLA—A2^ 健康人自体CD8^ T细胞，1wk后，用脉冲肽的自体PBMC以每7d的间隔刺激该CD8^ T细胞3次。以共接受4次抗原肽刺激的T细胞作为CTL，用乳酸脱氢酶(LDH)释放试验，检测CTL对靶细胞的杀伤效应。用酶联免疫斑点法(ELISPOT)．检测CTL中抗原特异性分泌IFN-γ的T细胞数。结果：形态学和流式细胞术的结果显示．PBMC可诱生成熟的DC。肽1235(FLPDHINIV)诱导的CTL．可特异性杀伤1235脉冲的T2细胞和OVA66^ 、HLA—A2^ 的SW480细胞，且L235诱导的特异性分泌IFN-γ的T细胞数增加。结论：卵巢癌相关抗原OVA66的HLA—A2限制性CTL表位1235．能激发对肿瘤抗原的特异性免疫应答，为制备肿瘤特异性肽疫苗奠定了实验基础。     

癌-睾丸抗原MAGEC2 HLA-A3限制性细胞毒性T淋巴细胞表位的鉴定

目的:预测并初步鉴定HLA-A3超型限制性MAGEC2抗原特异性细胞毒性T细胞(CTL)表位肽,为基于超型表位的MAGEC2治疗提供实验基础及新的候选靶标。方法:通过BIMAS、SYFPEITHI和IEDB软件预测打分来选取MAGEC2的HLA-A3限制性表位;结合力实验用于检测候选表位与T2A3细胞表面HLA-A3分子的结合能力,ELISPOT实验检测候选表位肽诱导的CTL分泌IFN-γ的能力,体外细胞毒实验检测侯选表位肽诱导的CTL杀伤靶细胞的能力。结果 :表位肽P147、P167、P196、P229和P251具有较好的HLA-A3结合力。ELISPOT实验结果显示表位肽P167、P196和P251诱导的CTL具有分泌IFN-γ的能力。细胞毒实验结果显示表位肽P196和P251诱导的CTL对靶细胞有一定的杀伤作用(P0.05或P0.01)。结论 :P196和P251有更高的HLA-A3分子亲和力,保留了原有的免疫原性,是优秀的MAGEC2抗原的HLA-A3限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

人食管癌TE-1细胞MHC-Ⅰ相关抗原肽的初步研究

目的寻找TE-1细胞MHC-Ⅰ类分子提呈的抗原肽。方法用弱酸洗涤法获得TE-1细胞表面与MHC-Ⅰ类分子结合的抗原肽，经SepPak-C18柱、Centrion-3超滤器和RP-HPLE去盐纯化后，用负载该抗原肽的树突状细胞刺激生成肿瘤特异性杀伤细胞，以Cr^51释放法测定CTL的杀伤活性。结果高效液相色谱图显示多个峰；负载抗原肽DC刺激的CTL比未负载抗原肽DC刺激的CTL对TE-1细胞杀伤率高，两者差异有统计学意义；负载抗原肽DC刺激的CTL对与抗原肽孵育的T2细胞比对未经与抗原肽孵育的T2细胞杀伤率高，两者差异有统计学意义。结论弱酸洗涤法可从TE-1细胞上获得有效的抗原肽；混合抗原肽中有可与HLA-A2分子结合并能激发CTL的抗原肽。     

IL-18基因修饰增强肿瘤抗原多肽致敏的树突状细胞体内诱导的抗肿瘤免疫反应

目的 :研究肿瘤抗原多肽致敏的白细胞介素 18(IL 18)基因修饰的树突状细胞体内诱导的抗肿瘤免疫反应。方法 :①以Lewis 3LL肺癌细胞特异性抗原肽mut1冲击致敏IL 18基因修饰的骨髓来源的树突状细胞 (DC IL 18 mut1) ,每次用其 1× 10 5 只皮下免疫小鼠 2次 ,然后测定脾细胞的NK活性及CTL杀伤活性 ;②以DC IL 18 mut1每次 2× 10 5 只皮下免疫 1次 ,然后再以 5× 10 53LL细胞攻击 ,在诱导及效应阶段分别以单抗阻断不同免疫成份 ,观察肿瘤的生长。结果 :以DC IL 18 mut1皮下免疫后可诱导出比DC mut1等免疫组更高水平的 3LL肺癌细胞特异性CTL ,并使NK活性明显增加 ;单抗体内阻断实验提示在DC IL 18 mut1免疫诱导阶段 ,CD4 + T细胞和抗原共刺激分子、IFN γ均起到重要作用 ,而效应阶段CD8+ T、IFN γ、NK起作用 ,而CD4 + T则是非必需的。结论 :DC IL 18 mut1皮下免疫后可诱导高水平的抗肿瘤免疫活性 ,其机理与抗原有效提呈、特异性CTL诱导、NK活性增加以及CD4 + 、CD8+ T、NK细胞、IFN γ参与密切相关。     

肝素酶HLA-A2限制性CTL表位肽的筛选及其活性鉴定

目的 预测并鉴定新的人乙酰肝素酶(Hpa)抗原的HLA-A2限制性CTL表位,为恶性肿瘤多肽疫苗的免疫治疗提供依据. 方法 采用SYFPEITHI和BIMAS软件预测方法,对肝素酶HLA-A2限制性CTL表位进行预测,合成候选表位肽;利用T2细胞特点,对合成的候选肽与HLA-A2分子进行亲和力分析;利用乳酸脱氢酶释放试验检测待检肽特异性CTL诱导活性;利用ELISPOT检测T细胞活性. 结果 在所筛选的6条候选CTL表位肽中,Hpa(310～318)FLNPDVLDI与HLA-A2分子具有高亲和力,在体外可有效诱导肝素酶特异性CTL的产生,对肝素酶阳性且HLA-A2限制性的HCC-LM6肝癌细胞及SW-480结肠癌细胞具有明显的杀伤效应,且能有效诱导IFN-γ分泌,增强免疫活性. 结论 首次发现Hpa(310～318)FLNPDVLDI可能是肿瘤肝素酶抗原的HLA-A2限制性CTL表位.     

人乳头状瘤病毒11型E7抗原HLA-A*0201限制性细胞毒性T淋巴细胞表位的功能鉴定

目的 筛选和鉴定人乳头状瘤病毒11型E7抗原(HPVllE7)HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)表位.方法 预测HPVllE7抗原HLA-A*0201限制性CTL表位并合成相对应的表位多肽和四聚体(tetramer),即HPVllE7 7-15(TLKDIVLDL)、15-23(LQPPDPVGL)、47-55(PLTQHYQIL)、81-89(DLLLGTLNI)和82-90(LLLGTLNIV).从健康HLA-A*0201成人外周血单一核细胞诱导树突状细胞(DC)并负载上述表位多肽,流式细胞技术检测DC成熟分化标记及ELISA法检测DC分泌的IL-12;成熟DC负载各组多肽后观察DC激活T淋巴细胞的效应,ELISA法检测T细胞分泌的IFN-γ;四聚体检测抗原特异性CD8+ T细胞及乳酸脱氢酶(LDH)释放法评价DC诱导的CTL对靶细胞的特异性体外杀伤效应.结果 预测的5条HPVllE7表位多肽均能诱导DC的成熟分化;E7 7-15、82-90和15-23多肽负载的DC能激活T淋巴细胞分泌高水平IFN-γ;E7 7-15多肽负载的DC能刺激特异性tetramer+CD8+细胞增殖且其诱导的CTL对HPVllE7/293细胞产生高效率的特异性杀伤作用(P<0.05).结论 筛选并鉴定出1条HPVllE7HLA-A*0201限制性CTL表位E7 7-15(TLKDIVLDL),负载该表位肽的DC体外可诱导高效、特异性的CTL效应,抗原性较强,有可能作为HPV感染治疗用肽疫苗的候选表位.     

负载不同形式肝癌抗原的树突状细胞抑瘤功能的比较

目的：探讨不同形式的肝癌抗原修饰的树突状细胞(DC)的抑瘤功能。方法：分别用肝癌H22冻融抗原、H22小分子抗原肽和Hsp70-H22抗原肽复合物修饰DC；用MTT比色法分析DC激活的CTL对H22细胞的杀伤能力，并用RT-PCR法测定脾脏T细胞中IFN-γ mRNA的表达水平；用不同修饰的DC免疫小鼠，观察其对H22肝癌的生长抑制作用。结果：单独的H22肝癌抗原肽修饰的DC不能激活CTL。Hsp70-H22肽复合物修饰的DC激活CTL的能力强于H22肝癌冻融抗原修饰的DC，对H22细胞的杀伤率分别为47．3％和18．3％。各组T细胞中IFN-γ表达水平的变化与杀伤率的变化相一致。用H22肝癌冻融抗原和Hsp70-H22肽复合物修饰的DC免疫小鼠后，均可抑制H22细胞生长，但后者的抑制作用更强，成瘤率仅40％。其他各组的成瘤率均为100％。结论：Hsp70-H22肽复合物是一种DC的强致敏物．可通过激活CTL、诱导CD4^ T细胞分化成Th1型细胞而参与肝癌的免疫排斥。     

EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。     

Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells

Allospecific immune responses against the MHC of another individual are remarkably strong, due t a high number of responding T cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class I molecules, it has remained unclear whether MHC molecules can be recognized directly. We used the H-2b-derived murine lymphoma mutant RMA-S, which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Type A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse RMA-S. Up-regulation of the levels of H-2Kb density improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CTL clones apparently can recognize class I molecules devoid of peptides, while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.     

Recruitment of alloreactive cytotoxic T lymphocytes by an antigenic peptide

To investigate the requirements for induction of cytotoxic T lymphocytes (CTL) by peptides we chose the 16-residue nucleoprotein peptide (NPP; 365-380) from the influenza virus A/NT/60/68 as model substrate that is recognized in conjunction with major histocompatibility complex H-2d. Here we present that CTL can be raised from naive animals by repeated in vitro stimulation with high concentrations of peptide. The frequency of this response can be boosted by immunization of the animals with NPP-conjugated to ovalbumin as a carrier. However, in contrast to NPP-specific CTL lines raised from virus-primed animals none of the peptide-induced CTL lines were able to lyse virus-infected targets. Although they did not show an apparent difference in fine specificity of the peptide recognized, their affinity to the target cells was 100-fold lower than that of CTL from virus-primed animals as estimated from the peptide concentration needed to achieve significant lysis. In addition, the activity of peptide-induced CTL was very sensitive to blocking by anti-CD8 antibodies as compared to virus-specific CTL. Furthermore, all peptide-induced CTL showed a high second reactivity for allogeneic H-2k targets. Therefore, it is argued that high epitope density achieved by high peptide concentrations can in vitro recruit lymphocytes of another specificity. For the tested peptide the reactive T lymphocytes showed high alloreactivity.     

肿瘤抗原MAGE-3 HLA-A2限制性CTL表位的预测

目的 预测肿瘤抗原ＭＡＧＥ－３的ＨＬＡ－Ａ２限制性ＣＴＬ表位。方法 以肿瘤特异性抗原ＭＡＧＥ－３为研究目标,采用基序方案和二级锚点相结合的ＣＴＬ表位预测方法。结果 预测出了ＭＡＧＥ－３的６个ＨＬＡ－Ａ２限制性ＣＴＬ表位。结论：所预测出的６个ＨＬＡ－Ａ２限制性ＣＴＬ表位经后续实验筛选、鉴定后,可用于基于ＭＡＧＥ－３的肿瘤治疗性多肽疫苗的设计研究。     

Specific suppression of antibody responses by soluble protein-specific, class II-restricted cytolytic T lymphocyte clones

Antigenic stimulation with exogenous soluble proteins induces cytotoxic T lymphocytes (CTL) recognizing antigenic peptides presented on class II major histocompatibility complex (MHC) molecules. Such CTL have been shown to lyse preferentially B cells expressing immunoglobulin receptors reactive with the relevant antigens, presumably because such B cells can efficiently trap and present the antigen. Therefore, possible involvement of soluble protein antigen-specific CTL in specific suppression of antibody responses has been hypothesised. In this report, keyhole limpet hemocyanin and ovalbumin-specific, class II-restricted CD4+ CTL clones established from lymph nodes of immunized mice were examined for their suppressive activities on antibody production. When these CTL clones were added to in vitro secondary cultures, genetically restricted, carrier-specific suppression of anti-2,4,6-trinitrophenyl antibody production was observed. These data therefore demonstrate that CTL directed toward soluble antigens are capable of mediating specific suppression of antibody responses. Furthermore, the antibody response of MHC-heterozygous F1 lymphocytes was almost completely suppressed by a CTL clone restricted to one parental class II MHC antigen, indicating that the mechanism of suppression by these CTL is distinct from that by classical suppressor T cells.     

Comparison of the frequency of peptide-specific cytotoxic T lymphocytes restricted by self- and allo-MHC following in vitro T cell priming

T cell recognition of antigenic peptides is thought to occur preferentially in the context of self-MHC. Here, we have tested the ability of four different K(b)-peptide combinations to stimulate self- and allo-restricted CTL responses in three different mouse stains. Responder T cells were primed in vitro with peptide-loaded stimulator cells, followed by limiting dilution assays to measure the number of peptide-specific cytotoxic T lymphocytes (CTL). For three peptides the number of CTL restricted by self-MHC was higher than for allo-MHC-restricted responses, although the difference was surprisingly small (3- to 5-fold). For the fourth peptide there was no detectable difference in the number of self- and allo-restricted CTL. Peptide titration experiments revealed that high avidity CTL were present in both the self- and allo-restricted setting. These data showed that the bias for preferred peptide recognition in the context of self-MHC imposed by positive thymic selection seems marginal. This raised the possibility that the TCR repertoire is inherently biased towards MHC restriction, independent of MHC-guided thymic selection. This was supported by the analysis of mature T cells generated from the thymus of MHC-deficient mice by lectin stimulation. K(b)-restricted CTL were found amongst these T cells at numbers similar to those of allo-restricted CTL. In summary, the data suggest that MHC-restricted peptide recognition is an inherent feature of the TCR repertoire and does not require thymic selection by MHC molecules.     

A MAGE-6-encoded peptide is recognized by expanded lymphocytes infiltrating a spontaneously regressing human primary melanoma lesion

In recent years, experiments based on the in vitro stimulation of either autologous peripheral blood lymphocytes or tumor-infiltrating lymphocytes with melanoma cells have shown that distinct members of the large MAGE gene family encode tumor-associated antigenic peptides. However, little is still known about natural anti-MAGE responses in vivo. We have studied a case of spontaneously regressing human melanoma, hypothesizing that in this unique situation, the host immune system had developed an efficient cytotoxic T lymphocyte (CTL) response against the cancer cells. Amongst the dense tumor infiltrate, certain clonal populations of T cells were shown to be amplified, thereby suggesting that an antigen-driven selection had occurred at the tumor site. One of the expanded tumor-infiltrating lymphocytes was shown to be a Vbeta13+ CD8+ CTL displaying a strong and selective cytotoxic activity against the autologous melanoma cells. Here we show that this cytotoxic T cell clone recognizes a MAGE-6-encoded peptide. MAGE-6 is therefore the fourth gene of the MAGE family shown to encode antigenic peptide recognized by T cells. Together, these data provide further evidence that T cell responses against MAGE antigens may naturally develop in vivo.     

H-2-linked murine cytotoxic T cell responses specific for sendai virus-infected cells.

CBA (H-2k) mouse-derived lymphochoriomeningitis virus and herpes simplex virus-specific cytotoxic T lymphocytes lyse virus-infected target cells compatible on either the H-2k or H-2D region. In contrast, CBA, C3H and AKR (H-2k) mouse-derived sendai virus-specific cytotoxic T lymphocytes (CTL) fail to lyse H-2D-compatible virus-infected cells. A similar lack of H-2D region-associated lytic activity was found with C57BL/6 and C57BL/10 (H-2b) mice as well as with the recombinants B10.A (2R) [Kb-Db] and B10.A (4R) [Kk-Db]. On the other hand, BALB/c (H-2d) mice and A/J (H-2a) mice do generate H-2Dd-associated sendai virus-specific CTL. These results are in contrast to those obtained with (CBA X BALB/c)F1 and B10.HTT [Ks-Dd] mice, which failed to mount Dd region-associated CTL responses. It is concluded that D region-associated sendai virus-specific CTL responsiveness varies with the H-2 genotype of the responder cells.     

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein–Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivities directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416–424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC : peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95·8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.     

A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3

The human MAGE-3 gene is expressed in many tumors of several histological types but it is silent in normal tissues, with the exception of testis. Antigens encoded by MAGE-3 may, therefore, be useful targets for specific anti-tumor immunization of cancer patients. We reported previously that MAGE-3 codes for an antigenic peptide recognized on a melanoma cell line by autologous cytolytic T lymphocytes (CTL) restricted by HLA-A1. Here we report that the MAGE-3 gene also codes for another antigenic peptide that is recognized by CTL restricted by HLA-A2. MAGE-3 peptides bearing consensus anchor residues for HLA-A2 were synthesized and tested for binding. T lymphocytes from normal individuals were stimulated with autologous irradiated lymphoblasts pulsed with each of three peptides that showed strong binding to HLA-A2. Peptide FLWGPRALV was able to induce CTL. We obtained CTL clones that recognized not only HLA-A2 cells pulsed with this peptide but also HLA-A2 tumor cell lines expressing the MAGE-3 gene. The proportion of melanoma tumors expressing this antigen should be approximately 32 % in Caucasian populations, since 49 % of individuals carry the HLA-A2 allele and 65 % of melanomas express MAGE-3.