CD8~+T细胞表位鉴定技术研究进展

CD8~+T细胞表位通过MHCⅠ分子呈递到细胞表面,激活CTL细胞从而介导并调节机体抗内源性抗原的免疫应答。MHC-peptide稳定性试验通过TAP缺陷的RMAS细胞转染克隆在细胞水平鉴定CD8~+T细胞表位与MHCⅠ分子的亲和力;酶联免疫斑点技术(ELISPOT)通过检测IFN-γ的分泌水平评估抗原免疫后机体的细胞免疫应答能力,具有较高的敏感性和高效性。四聚体技术通过流式细胞仪分析抗原特异性CD8~+T细胞的表型和功能特征。论文介绍了CD8~+T细胞表位的特征和上述3种表位鉴定方法的基本原理和研究进展,为CD8~+T细胞表位的鉴定和表位疫苗的研制提供参考。     

默沙东动物保健特约技术讲座——家禽免疫学知识连载 家禽免疫应答

家禽免疫应答即禽体如何识别外来的非自身物质,如何对“非己”物质做出反应以及如何消灭该物质的过程。家禽免疫应答分为三个阶段,前两个阶段为非特异性免疫应答,主要包括物理屏障、先天免疫细胞、抗微生物物质和补体系统。第三个阶段为特异性免疫应答,主要包括细胞免疫和体液免疫。细胞免疫的功能主要体现在致敏T细胞对抗原的直接杀伤作用及致敏T细胞所释放的细胞因子的协同杀伤作用。体液免疫主要通过浆细胞产生的抗体发挥作用。家禽的先天性免疫和特异性免疫相互作用,共同抵抗病原微生物的入侵,为禽体的健康保驾护航。     

口服重组沙门菌诱导的免疫应答动态分析

以融合表达绿色荧光蛋白(Green fluorescent protein,GFP)和OVA、LCMV NP CD8~ T细胞表位的重组减毒沙门菌SL7207(ptG2F)为免疫原,经口服途径接种BALB/c小鼠,每2周加强免疫1次,分别于第2次、第3次、第4次、第5次免疫后,以ELISPOT法分别测定免疫小鼠派伊尔氏结细胞中特异性针对OVA和LCMV NP CD8~ T细胞表位的IFN-γ分泌细胞和IL-4分泌细胞。同时,运用ELISA分别检测血清、肠黏膜中的GFP特异性抗体,分析了口服重组减毒沙门菌诱导的特异性免疫应答动态规律。实验表明SL7207(ptG2F)口服免疫BALB/c小鼠后,诱导产生了LCMV NP CD8~ T细胞表位(H-2~d)特异的细胞免疫应答。在第2次免疫后,即可检测到特异性IFN-γ分泌细胞和IL-4分泌细胞;在第3次免疫后,IFN-γ分泌细胞数量明显高于IL-4分泌细胞数。然而,在第4、5次免疫后,IFN-γ分泌细胞数和IL-4分泌细胞数相当,未见明显变化。试验中未能检测到针对OVA CD8~ T细胞表位(H-2~b)的细胞免疫应答。运用ELISA方法检测到SL7207(ptG2F)、SL7207(ptGFP)免疫组GFP特异性IgG和IgA抗体,与对照组相比较,差异显著(P<0.05)。结果表明重组减毒沙门菌口服免疫可以有效运送外源抗原至宿主免疫系统,并诱导产生特异性细胞免疫应答和黏膜免疫应答。     

肿瘤多肽疫苗研究进展

肿瘤的发生、发展和治疗与机体免疫系统功能密切相关,伴随着肿瘤抗原、抗原递呈、T细胞识别机制的突破性研究进展,研究者发现抗肿瘤多肽疫苗能够通过肿瘤抗原多肽识别抗原递呈细胞表面的主要组织相容性复合体(MHC)分子,形成肽-MHC-T细胞受体复合物,引起相应的细胞毒性T淋巴细胞免疫反应,从而杀伤肿瘤。因此,研制既能打破肿瘤患者存在的免疫耐受又能诱发针对肿瘤相关抗原特异性免疫应答的高效多肽疫苗已成为肿瘤免疫治疗研究的热点。论文综述了肿瘤多肽疫苗抗肿瘤相关机制及其在该领域所取得的最新临床研究进展。     

IL-18的免疫调节功能及其应用

IL-18是一种具有多向和多层次免疫调节功能的细胞因子,其细胞来源较广。它可通过NF-κB、p56LCK-MAPK、Perforin等多个信号传导途径参与细胞凋亡以及IFN-γ分泌诱导及功能性免疫细胞活性的激活,从而直接或间接激活T细胞、NK细胞、PBMC、DC等免疫细胞的增殖、分化、抗原呈递、CTL反应等,并诱导这些细胞分泌效应性细胞因子或延长免疫保护,进而促进免疫系统增强抗感染、抗肿瘤等免疫效应。IL-18除了主要介导细胞免疫外,在一定条件下,亦能促进抗原特异性中和抗体以及Th2型细胞因子的应答。在某些疾病的防治和诊断中,具有重要临床意义。     

MTT法评价猪口蹄疫合成肽疫苗的细胞免疫应答

为评价猪口蹄疫合成肽疫苗免疫猪后的细胞免疫应答,用含有E、F两种多肽抗原的猪口蹄疫合成肽疫苗免疫猪,二免后两周采血分离猪外周血淋巴细胞,再用E、F两种合成肽及二者混合物对淋巴细胞刺激培养48 h,用MTT法检测特异性T淋巴细胞增殖反应。结果显示,在抗原E、F混合物浓度为50μg/mL时,抗原对免疫组淋巴结细胞的刺激增殖作用显著高于未免疫对照组（P〈0.01）。表明,该猪口蹄疫合成肽疫苗免疫猪能有效引起特异性T淋巴细胞免疫应答。     

RHDV VP60“通用型”T细胞表位预测

兔病毒性出血症核衣壳蛋白VP60是兔出血症病毒的主要结构蛋白,具有很强的抗原性和免疫原性,可诱导细胞免疫,在抗病毒免疫中起着关键作用.确定T细胞所识别抗原分子上的短肽序列对T细胞表位进行定位,对于研究特异性免疫应答有着重要意义.本研究应用T细胞抗原表位分析的研究方法,预测VP60T细胞蛋白质抗原表位,为后期试验奠定了基础.     

中药抗应激冲剂对热应激小鼠小肠黏膜中IL-2水平的影响

随着我国畜禽集约化养殖业的不断发展,应激性疾病尤其是炎热季节高温应激已经成为我国养殖业中的重要疾病之一[1].研究表明,应激可抑制机体免疫,如淋巴细胞活性、吞噬功能、T淋巴细胞TCRξ链基因表达水平下降等[2].细胞因子IL-2在胃肠道黏膜免疫中具有重要调节作用[3].IL-2是机体内重要的淋巴因子,可维持体外培养的T细胞长期存活,传达免疫效应,增强细胞免疫,调节机体免疫状态.因此,检测IL-2水平是评价机体细胞免疫的主要指标之一.     

一些新型免疫佐剂的研究进展

免疫佐剂可以理解为能加强抗原的免疫原性和免疫保护效果的物质。这种加强的免疫功能,可表现为增强与其同时使用的特异性抗原的体液免疫反应,也可以表现为增强特异性抗原的细胞免疫反应。近20年来,由于免疫学的进展,亚单位疫苗的开发,特别是肿瘤免疫和疫苗的研究迅速发展,促进了新佐剂研究。传统佐剂虽然副反应低,但对某些抗原没有或仅有很低的免疫增强作用,因而对新型佐剂的研制和开发已成为当今新疫苗研究中的一个非常重要的领域。1细胞因子佐剂细胞因子(cytokine,ck)是由细胞分泌的、能够影响其他细胞功能的多肽,它产生于天然免疫和特异…     

肿瘤多肽疫苗的研究进展

肿瘤的发生、发展和治疗与机体免疫系统功能密切相关,伴随着肿瘤抗原、抗原呈递、T 细胞识别机制的突破性研究进展,研究者发现抗肿瘤多肽疫苗能够通过肿瘤抗原多肽识别抗原呈递细胞（antigen presenting cell ,APC）表面的主要组织相容性复合体（major histocompatibility complex ,M HC）分子,形成肽‐M HC‐T细胞受体（T cell receptor ,TCR）复合物,引起相应的细胞毒性 T 淋巴细胞（cytotoxic T lym‐phocyte ,CTL）免疫反应,从而杀伤肿瘤。因此,研制既能打破肿瘤患者存在的免疫耐受又能诱发针对肿瘤相关抗原（tumor‐associated antigen ,TAAs）特异性免疫应答的高效多肽疫苗已成为肿瘤免疫治疗研究的热点。文章综述了肿瘤多肽疫苗抗肿瘤相关机制及其在该领域所取得的最新临床研究进展。     

Quantitative analysis of the antigen-specific IFNgamma+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNgamma+ CD8+ T cell response

It is now well established that antigen-specific CD8⁺ T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNγ-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNγ⁺ T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNγ⁺ T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNγ⁺ responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8^bright pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.     

Beta-glucan enhancement of T cell IFNgamma response in swine

Beta-glucan has been shown to enhance anti-tumor and anti-infection functions in animals. Pigs at 4 months of age were infected with porcine reproductive and respiratory syndrome virus (PRRSV), and peripheral blood monocytes (PBMC) were isolated for the detection of interferon gamma (IFNgamma)-producing cells. We found that soluble high molecular weight beta-glucan could increase IFNgamma-producing cell frequency in a dose-dependent manner in the enzyme-linked immunospot assay (ELISPOT) in the absence of antigenic restimulation. A concentration as low as 1.6 microg/ml gave a significant increase and a similarly high enhancement was achieved at concentrations from 3.2 to 100 microg/ml. In PRRSV-specific IFNgamma ELISPOT, soluble beta-glucan elicited increased PRRSV-specific responses at concentrations from 3.2 to 50 microg/ml, but not at 100 microg/ml, whereas insoluble beta-glucan had no effect. Soluble beta-glucan augmented the porcine cellular immune response in an antigen-independent fashion, whereas insoluble beta-glucan had no activity. This finding suggests that soluble beta-glucan may enhance innate antiviral immunity against PRRSV.     

Auricular chondritis caused by metal ear tagging in C57BL/6 mice

Although it has been shown that auricular chondritis in rats is caused by the use of metal identification ear tags, the pathogenesis remains unclear. Based on the hypothesis that the auricular chondritis is caused by metal ions released from metal identification ear tags, we investigated the pathogenesis in male C57BL/6 mice tagged with metal identification ear tags. Twenty-six weeks after the attachment of the ear tags, visible increases in the thickness of the auricle were observed, and the concentrations of copper and iron in the tagged ears were significantly increased (P < .05) in the tagged ears compared with the untagged ears. There was up-regulation of metallothionein (MT)-I and MT-II mRNA in the tagged ears, and this was confirmed by immunohistologic staining of the destroyed cartilage. Histopathologically, there were observed severe chondritis with extensive granulomatous inflammation, newly formed cartilage nodules, and osseous metaplasia accompanied by cellular infiltrates, such as CD4 T lymphocyte, macrophages, neutrophils, and mast cells, and expression of Th1 cytokines, such as interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 in the tagged ear. Based on these results, we concluded that the release of copper and iron ions from the metal ear tags played a major role in the onset of auricular chondritis. Subsequent cellular interactions, such as CD4 T cells, macrophages, fibroblasts, and mast cells, mediated by cytokines, such as tumor necrosis factor-alpha and interferon-gamma, caused an autoimmune response that may have led to the progression of auricular chondritis as an autoimmune disease.     

Generation of anti‐porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis

T cell‐mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti‐porcine CD69 mAb‐producing mouse hybridomas, 01‐14‐22‐51 (IgG2b–κ) and 01‐22‐44‐102 (IgG2a–κ), both showing fine reactivity with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin‐stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti‐interferon‐γ mAb and anti‐tumor necrosis factor‐α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.     

应用ＥＬＩＳＰＯＴ试验测定重组卡介苗诱导的Ｔ细胞应答

应用免疫磁性分离技术去除免疫小鼠脾脏细胞中ＣＤ４^ 或ＣＤ8^ Ｔ细胞后，在酶免疫斑点试验中证实表达大肠杆菌ＭalE蛋白的重组卡介苗（rBCG.MalE)诱导的Ｔ细胞应答是ＣＤ４^ t细胞依赖的对MalE、PPD持异Ｔ细胞应答的动态分析结果表明，，rBCG.MalE、ＢＣＧ诱导的特异ＣＤ４^ T细胞应答存在Ｔh1/Th2平衡转换现象，即起始阶段为Ｔh1应答，一段时间后出现Ｔh2应答，并逐步形成Ｔh1/Th２混合应答。这些结果，分为枝杆菌Ｔ细胞应答规律提供了新的认识，同时，亦表明ＢＣＧ是优良的外源抗原表达与运送载体。     

CD4+CD25+ regulatory T cells are infected and activated during acute FIV infection

HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Treg cells). Treg cells have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Treg cells become infected and activated during the acute infection with FIV leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU1 and blood and lymph node cells were collected at weekly intervals following inoculation. Real-time RT-PCR was used to determine plasma viremia and the relative expression of FIV, FoxP3, TGF-beta, and GAPDH mRNA copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of surface TGF-beta and intracellular FoxP3 in CD4+CD25+ and CD4+CD25- T cells at each time-point. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays. Our results showed that peak viremia occurred at 2 weeks post infection and correlated with maximal infectivity in CD4+CD25+ T cell populations. FIV-gag-mRNA levels were higher in CD4+CD25+ T cells than CD4+CD25- T cells throughout the acute phase of infection. Induction of FoxP3 and TGF-beta indicated activation of Treg cells during the acute stage infection, which was confirmed by Treg cell suppression of IL-2 production by CD4+ Th cells in an ELISPOT assay. Our findings support the hypothesis that early activation of Treg immunosuppressor function may limit an effective anti-FIV response, contributing to the establishment of chronic infection and the immunodeficiency caused by this virus.     

Induction of remission results in spontaneous enhancement of anti-tumor cytotoxic T-lymphocyte activity in dogs with B cell lymphoma

Characterization of the tumor microenvironment, particularly the immune cells that infiltrate tumors, provides important predictive and prognostic information in humans with lymphoma and other types of cancer. Tumor associated T lymphocytes have not been previously described in dogs with lymphoma. Therefore, we investigated the phenotype and function of T cells in the lymph nodes of dogs with B cell Non-Hodgkin's lymphoma (NHL), as well as the function of T cells in circulation of these dogs. We found that CD4+ and CD8+ T lymphocytes were few in number and minimally responsive to mitogenic stimuli compared to T cells in lymph nodes of normal dogs. Additionally, regulatory T cells (Treg) were significantly increased in tumor tissues compared to lymph nodes of healthy dogs. To better understand cell mediated antitumor immune responses we developed a non-radioactive assay to measure cytotoxic T lymphocyte (CTL) mediated killing of autologous tumor cells. Using this assay, we found that spontaneous CTL activity in the blood of dogs with lymphoma improved significantly following induction of tumor remission using doxorubicin. Coincident with the improvement in CTL activity, circulating Treg numbers were significantly decreased compared to pretreatment levels. We conclude from these studies that CTL activity in dogs with lymphoma can be significantly improved following induction of tumor remission using chemotherapy, as assessed using a new non-radioactive CTL assay.     

Interleukin 4-producing CD4+ T cells in the skin of cats with allergic dermatitis

Lesional skin of cats with allergic dermatitis has a cellular infiltrate and a CD4/CD8 ratio comparable to that in humans with atopic dermatitis. CD4+ helper T cells and in particular cells belonging to the Th2 subset play an important role in disease pathogenesis in humans. We investigated the cytokine pattern of CD4+ T cells in situ, with special emphasis on the putative presence of cells producing interleukin 4 (IL4), in cats with allergic dermatitis. Immunohistochemical procedures were used to determine that CD4+ T cells in lesional and nonlesional skin of cats with allergic dermatitis can produce IL4, as occurs in humans. Lesional and nonlesional skin of cats with allergic dermatitis had significantly more IL4+ T cells (P = 0.001) than did skin of healthy control cats. Double staining indicated that all IL4+ cells were positive for pan-T or CD4 markers. Double labeling for mast cell chymase and IL4 stained primarily different cells. Western blotting demonstrated cross-reactivity between the antibody against human IL4 and a feline recombinant IL4. These results indicate that IL4 is primarily produced by CD4+ T cells and is also present in clinically uninvolved skin, indicating a role in the pathogenesis of allergic dermatitis in cats.     

Ovine and murine T cell epitopes from the non-structural protein 1 (NS1) of bluetongue virus serotype 8 (BTV-8) are shared among viral serotypes

Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2^b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4⁺ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4⁺ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals.