人外周血T细胞系间接识别SLAⅠ类分子引起的异种细胞增殖反应

目的 确认猪→人异种移植中存在针对猪白细胞抗原(swine leukocyte antigen，SLA)Ⅰ类分子的间接识别而引起的急性细胞性排斥。方法 用纯化的中国版纳猪SLA Ⅰ类P1基因工程蛋白为抗原，体外反复刺激健康人外周血T细胞，建立P1特异性T细胞系。观察抗原特异性CD4 T细胞系在自身APC存在下对版纳猪外周血单个核细胞与软骨细胞的反应性，以及HLA-DR单抗与氯喹的阻断作用。结果 建立10株SLA Ⅰ类P1抗原特异性T细胞系，其中8株以TCRαβ CD4 为主要表型，将其合并使用。该CD4 P1特异性T细胞系在自身APC存在下对相同品系版纳猪PBMC与软骨细胞均产生显著增殖反应。经抗人HLA-DR单抗与氯喹处理均能明显阻断其增殖反应。结论 健康人外周血T细胞可通过间接识别SLAⅠ类抗原对版纳猪细胞产生明显应答。     

建立猪MHCⅠ类抗原特异性人T细胞系的探讨

目的　建立间接识别中国版纳猪SLAⅠ类P1分子的特异性人T细胞系的方法 ,以进一步研究SLAⅠ类分子的间接识别在猪→人异种细胞性排斥中的作用。方法　以E·coli中表达并纯化的SLAⅠ类分子P1为抗原 ,体外反复刺激健康人PBMC ,3H TdR掺入法筛选特异性增殖的T细胞 ,FACS作表型初步分析。结果　初步测定健康人外周血版纳猪SLAⅠ类P1分子特异性T细胞反应频率约 6 .67× 1 0 - 7,所建 4个T细胞系表型均为TCRαβ+ ,其中 3株以CD4+ 为主 ,1株以CD8+ 为主。结论　利用E .coli表达的纯蛋白抗原可建立间接识别版纳猪SLAⅠ类P1分子的特异性人T细胞系。     

人T细胞对猪白细胞抗原的直接识别

猪已被认为是异种移植的最理想供体。有关人 猪器官间超急性排斥研究已有所突破 ,而细胞性排斥报道尚少。本文用混合淋巴细胞反应体系研究异种移植细胞性排斥机制 ,发现异种反应明显弱于同种反应 ,而且以直接识别为主 ,这和同种移植细胞性排斥机制一致。抗人CD4单抗和抗猪白细胞抗原SLA DR、DQ单抗都能明显地抑制 (XMLR) ,而抗人CD8单抗无抑制功能 ,表明CD4+T细胞对SLA的识别在异种排斥应答中起重要作用     

内源性IL-12决定人PBMC产生干扰素γ的水平

目的：IFN－γ是由被有丝分裂原或抗原所激活的T细胞和NK细胞所产生,它具有广泛的免疫调节活性,现认为IL－12（外源性）是诱导IFN－γ产生的强诱导剂,并可促进静息CD4^ T细胞朝向Th1表型分化,即诱导细胞免疫。目的是为了解PBMC产生的内源性IL－12是否在体外可诱导IFN－γ的产生及通过何机制诱导细胞免疫。方法：用抗CD3抗体、PHA、抗CD3抗体加抗CD28抗体和抗原（MLC）来检测被刺激的PBMC细胞的IFN－γ的产生。同时也用IL－12和IL－12Rβ1的中和抗体来抑制IFN－γ的产生。结果：活的人PBMC中IFN－γ分泌依赖于内源性IL－12的产生,而且激活的T细胞可诱导APC细胞产生IL－2,此过程是通过T细胞表面的CD40L和APC的CD40相互作用而实现。结论：这些结果显示,内源性IL－12在正常罕主抗细胞内抗原的感染反应中起重要作用,在某些形式的自身免疫性疾病和移植排斥反应的免疫病理发生中也起中心作用。     

T细胞直接活化与交叉活化研究进展

T细胞介导的免疫应答在机体排斥肿瘤的过程中起核心主导作用 ,原始的CD8+ T细胞分化成效应细胞 ,在自身MHCⅠ类分子存在的条件下能识别特异性抗原肽 ,CD8+ CTL可直接识别、杀伤表达特异性抗原的肿瘤细胞。多数抗原加工通道能产生有效的CD8+ T细胞介导的免疫反应     

EB病毒转化B淋巴母细胞在汉滩病毒CTL表位研究中的应用

目的:建立EB病毒转化B淋巴母细胞系(B-LCL),作为抗原提呈细胞(APC)提呈抗原肽,刺激短期培养的特异性T细胞活化并分泌IFN-γ,从而应用于T细胞表位鉴定中.方法:用B95-8细胞培养上清中的EB病毒转化肾综合征出血热(HFRS)患者PBMC,建立HFRS患者的B-LCL,以自身BLCL为APC,加载抗原肽后,刺激短期培养的G9L特异的HFRS患者CD8+ T细胞系,应用ELISPOT测定CD8+ T细胞受到抗原肽刺激后产生IFN-γ的能力.结果:加载过抗原肽G9L或V15R的B-LCL可刺激G9L特异的CD8+ T细胞活化并产生IFN-γ,而与G9L无同源序列的115P则不能刺激G9L特异的CD8+ T细胞活化.结论:B-LCL可作为非专职APC有效地将抗原肽提呈给特异性T细胞.     

抗CD4人—鼠嵌合抗体的鉴定及对PBMC增殖反应的影响

目的对已构建表达的抗 CD4人 -鼠嵌合抗体的生物学特性进行鉴定 ,比较嵌合抗体与鼠源性单抗对抗 CD3Mc Ab和 EBV转化细胞诱导的外周血单个核细胞 (PBMC)增殖反应的影响 ,以探讨抗 CD4抗体的增殖抑制作用机制。方法采用间接免疫荧光竞争抑制实验比较两种抗体对 CD4抗原的相对亲和力 ,MTT法检测抗 CD4抗体的增殖抑制作用。结果转染瘤细胞具有稳定表达及分泌特异性抗 CD4人 -鼠嵌合抗体的能力 ;两种抗体对 CD4抗原的相对亲和力相同 ;对经 TCR途径刺激诱导的 PBMC增殖反应均有抑制作用 ,嵌合抗体的增殖抑制作用较鼠源性单抗强。结论抗 CD4抗体的抑制作用很大程度上直接作用于 TCR诱导的活化信号。     

CD4~+T细胞识别的肿瘤抗原的研究进展

在以往的肿瘤免疫研究中 ,人们往往关注肿瘤特异性CD8+ T细胞的抗肿瘤效应 ,并由此鉴定出许多CD8+ T细胞识别的肿瘤抗原。随着研究的深入 ,CD4 + T细胞在抗肿瘤免疫中的作用逐渐为人们所关注。联合应用MHCⅠ类和MHCⅡ类限制性抗原的肿瘤疫苗将有可能取得更好的抗肿瘤效果。本文就近年来鉴定的CD4 + T细胞识别的肿瘤特异性抗原进行了综述。     

人外周血中SLA-P1抗原特异性T细胞前体频率及其细胞因子分泌格局分析

用基因工程表达并纯化的版纳猪SLAI类P1为抗原 ,经两轮刺激PBMC以3H TdR法检测 1 0名健康人外周血中的特异性T细胞前体频率在 6 67× 1 0 7～ 1 0 8× 1 0 6 之间。用ELISA与RT PCR法进一步分析了抗原特异性细胞的细胞因子分泌特点 ,发现所有抗原阳性孔都呈现IFN γ (+) /IL 4( )的典型Th1型格局。以上结果部分明确了外周血SLA P1特异性T细胞的特征 ,为揭示SLA P1特异性T细胞在猪 人急性细胞性排斥中的作用提供了实验依据     

032 CD4+T细胞识别的肿瘤抗原的研究进展

在以往的肿瘤免疫研究中，人们往往关注肿瘤特异性CD8+T细胞的抗肿瘤效应，并由此鉴定出许多CD8+T细胞识别的肿瘤抗原。随着研究的深入，CD4+T细胞在抗肿瘤免疫中的作用逐渐为人们所关注。联合应用MHCⅠ类和MHCⅡ类限制性抗原的肿瘤疫苗将有可能取得更好的抗肿瘤效果。本文就近年来鉴定的CD4+T细胞识别的肿瘤特异性抗原进行了综述。     

识别猪SLA的特异性人T细胞系TCR BV基因取用格局和CDR3结构多样性分析

不同T细胞克隆TCR分子的序列不同 ,所识别的抗原特异性也不同。其中第三互补决定区 (CDR3)变异最大 ,是TCR主要的抗原结合部位。本文采用荧光标记半定量PCR技术 ,用DNA测序仪作程序分析 ,了解猪细胞抗原致敏前后的人T细胞群和 5个T细胞系 2 4个TCRBV基因家族取用格局 ,并以TCRα链C区的基因片断作为内参对取用情况作定量估计。发现首次抗原致敏后培养 2周的T细胞除了BV2 4、BV8和BV10未能检测出 ,其它BV基因都有不同程度的取用。然而 ,5个细胞系的TCRBV基因呈现十分有限的取用格局 ,其中两个CD4+ T细胞系都取用BV12和BV14;3个CD8+ T细胞系中都优势取用BV1,有两个还取用BV19。CD4+ T细胞系和CD8+ T细胞系之间TCRBV无交叉取用 ,提示两类细胞识别的抗原表位存在差异。进一步用变性凝胶扫描分析上述T细胞系取用TCRBV中的CDR3的多样性 ,发现未经抗原致敏的T细胞BV的CDR3结构为多峰型且呈正态分布 ,表明涉及多种结构不同的细胞克隆 ;而抗原特异性T细胞系CDR3除了一个CD8+ T细胞系BV1有两个主峰外其它无例外地都显示单峰或者仅一个主峰 ,这从另一个角度证明建系T细胞的单克隆性。     

Oligopeptide Induction of a Secondary Cytotoxic T-cell Response to Epstein-Barr Virus In Vitro

Three Epstein-Barr virus (EBV) nuclear antigen (EBNA)-encoded oligopeptide epitopes have been mapped, each capable of acting as a recognition determinant for class I-restricted lysis by CD8+ cytotoxic T lymphocytes (CTL). This report shows that each peptide, when presented on an appropriate autologous antigen-presenting cell (APC), also stimulates EBV-specific memory T cells present in peripheral blood mononuclear cell (PBMC) populations to develop in vitro into peptide-specific CTL. These CTL specifically lysed autologous EBV-infected lymphoblastoid cell lines (LCL) and peptide-sensitized uninfected targets. Identical viral oligopeptides could therefore function as recognition determinants for both the induction and commission of class I-restricted specific cytotoxicity. A model system is described in which autologous phytohaemagglutinin (PHA) blasts present exogenous peptide during the stimulation phase. The magnitude of the peptide-specific CTL response was dependent on the concentration of peptide added to the APC and specific lysis was inhibited by anti-class I monoclonal antibody (MoAb) but not anti-class II MoAb. Cultures depleted of CD8+ T cells by cell separation with immunomagnetic beads prior to stimulation invariably failed to generate a peptide-specific CTL response. However, the effect of CD4 depletion on CTL activity was equivocal and indicated that a need for CD4+ T cells as accessory helper cells may depend on the efficiency of the APC to elaborate their own help. This model has advantages in the analysis of events involved in the development of CTL activity in vitro.     

Xenogeneic recognition of soluble and cell surface HLA class II antigens by proliferative murine T cells

In order to characterize the murine anti-human xenogeneic mixed lymphocyte reactions (MLR), we studied T cell proliferative responses against various human lymphoid cells by immunization of mice either with cellular or purified HLA-DR antigens. Data presented here indicated that small amounts of soluble HLA-DR antigen were able to prime mice, and that the xenogeneic MLR depends on the expression of HLA class II antigens on the stimulating cells. Experiments using a mutant cell line clearly showed that HLA-DP molecules were also sufficient in eliciting a primary or a secondary xenogeneic MLR while no secondary proliferative response was obtained with cells expressing only HLA class I molecules. Using a large panel of human cells with various haplotypes, our results also showed that (a) nonpolymorphic determinants of HLA class II antigens trigger dominantly the murine T cells and (b) the xenogeneic response required I-E and L3T4 accessory molecules and was not inhibited with anti I-A and monomorphic anti-HLA class II antigen monoclonal antibodies. Altogether these results suggest that HLA class II antigens act as nominal antigens in triggering a murine anti-human proliferative response.     

The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8(+) T lymphocytes in IFN-gamma ELISPOT assays.

ELISPOT assays are increasingly used for a direct detection and quantification of single antigen-specific T cells in freshly isolated peripheral blood mononuclear cells (PBMC). They are particularly attractive for the monitoring of specific T lymphocyte responses in clinical trials assessing antigen-specific immunizations in patients with cancer or chronic viral infections. However, one major limitation for the broad clinical implementation of ELISPOT assays is the lack of an inexhaustible source of suitable HLA-matched antigen-presenting cells (APC). Currently available allogeneic or xenogeneic APC (such as the human lymphoid hybrid T2 or HLA-transfected insect cells) can either lead to strong background spot production by APC-reactive T lymphocytes or have a low antigen presentation capability. Both phenomena can prevent the detection of low frequency T cell responses in PBMC. In search of alternative APC for ELISPOT assays, the human chronic myelogenous leukemia cell line K562 that per se does not express HLA class I and class II molecules on the cell surface was transfected with the HLA-A*0201 gene. Clonal HLA-A*0201-expressing K562 (K562/A*0201) cells were able to process and present endogenously expressed and exogenously loaded melanoma peptide antigens to HLA-A*0201-restricted cytolytic T lymphocyte clones in cytotoxicity and IFN-gamma ELISPOT assays. K562/A*0201 cells were then used as APC in IFN-gamma spot assays to detect ex vivo CD8(+) T lymphocytes responsive to known HLA-A*0201-binding peptide epitopes derived from cytomegalovirus, Epstein-Barr virus, influenza virus and melanoma in PBMC from HLA-A*0201-positive donors. In the majority of cases, peptide-pulsed K562/A*0201 cells were similarly efficient in the ability to visualize single antigen-specific CD8(+) T lymphocytes when compared to T2 cells. However, in contrast to T2, background reactivity of CD8(+) T cells responsive to unpulsed K562/A*0201 was regularly found to be negligible, thereby enhancing the sensitivity of the ELISPOT assay, particularly in donors with strong anti-T2 reactivity. K562 cells transfected with HLA-A*0201 or other HLA genes can serve as standard APC for monitoring T lymphocyte responses against tumor and viral peptide antigens.     

Induction of antigen-specific CD8+ cytolytic T cells by the exogenous bacterial antigen streptolysin O in rhesus monkeys.

We have characterized the T cell responses induced by streptolysin O (SLO), a sulfhydryl-activated hemolysin secreted by streptococci, by applying long-term in vitro culture and cloning rhesus monkey (Macaca mulatta) T cells. T cell lines specific for SLO were obtained from three rhesus monkeys. These T cell lines required autologous antigen-presenting cells (APC) to proliferate in response to SLO and did not respond to purified protein derivative. Phenotypic analysis showed that the cells from two of three SLO-specific T cell lines were more than 85% CD3+CD4-CD8+ after prolonged in vitro culture. The rh 1842 CD8+ T cell line proliferative response to SLO was inhibited by the addition of anti-major histocompatibility complex (MHC) class I and anti-CD8 but not of anti-MHC class II and anti-CD4 monoclonal antibody (mAb). This cell line was able to lyse P815 target cells in the presence of anti-CD3 mAb and did not show natural killer activity. Moreover, specific lysis of autologous but not allogeneic non-rosetting E- cell targets pulsed with SLO was observed. Such lysis was inhibited by the addition of anti-MHC class I mAb. In the attempt to identify the restriction elements involved in SLO presentation APC from six unrelated rhesus monkeys and three humans were used. A CD4+ rh 1842 T cell clone responded when SLO was presented by one of six, and a CD8+ rh 1842 T cell clone by four of six rhesus monkeys APC. Both CD4+ and CD8+ T cell clones did not respond when SLO was presented by human APC. However, both clones responded when APC from all donors were used in conjunction with anti-CD3 mb. Furthermore, SLO required active processing to be presented to CD4+ and CD8+ T cell clones as glutaraldehyde fixation of APC before but not after antigen pulsing inhibited T cell proliferation. The SLO-specific CD8+ cytolytic T cells described here could play a role in the regulation of the immune response occurring during streptococcal infections and/or could participate in the pathogenesis of poststreptococcal nonsuppurative sequelae.     

Herpesvirus saimiri transformed T cells and peripheral blood mononuclear cells restimulate identical antigen-specific human T cell clones

Panels of human antigen-specific T cell clones (TCC) have been established by limiting dilution using Herpesvirus saimiri (HVS) subtype C transformed T cells as antigen presenting cells (APC). They showed antigen-specific proliferation when peripheral blood mononuclear cells (PBMC), HVS-transformed T cells and Epstein Barr Virus transformed lymphoblastoid B cell lines (EBV-LCL) were used as APC. All T cell clones were CD4+ and HLA class II restricted. For a detailed analysis, two panels of T cell clones specific for an epitope located in the N-terminus of the Merozoite Surface Protein 1 (MSP-1) of Plasmodium falciparum were established from the same founder T cell line using either PBMC or HVS-transformed T cells as APC. TCR analysis of the two panels of TCC demonstrated that the same founder cells could be propagated in both culture systems. Furthermore, no difference in the cytokine expression pattern or antigen processing and co-stimulatory requirements was observed between TCC established on PBMC or HVS-transformed T cells. Based on the finding that HVS-transformed T cells can replace PBMC as APC for isolation and propagation of antigen-specific TCC, a protocol was developed and successfully executed, which allows to establish and maintain vaccine-specific T cell clones from 20 ml of blood. This method might be particularly significant in clinical trials of immune intervention strategies.     

Human Th1 cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class II molecules

We have used a synthetic-peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT-6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT-6-specific CD4+ T-cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA-DR-typed tuberculosis patients with complete antigen in vitro. The established T-cell lines were then screened for proliferation and interferon-gamma (IFN-gamma) secretion in response to eight overlapping 20-mer peptides covering the ESAT-6 sequence. The response of the T-cell lines to ESAT-6 and peptides from a human leucocyte antigen (HLA)-heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti-HLA class I and class II antibodies suggested that the T-cell lines recognized ESAT-6 in association with HLA-DR and -DQ molecules. Furthermore, testing of selected T-cell lines with ESAT-6 and the peptides in the presence of autologous and allogeneic HLA-DR- and -DQ-typed antigen-presenting cells identified HLA-DR2, -DR52 and -DQ2 amongst the HLA molecules involved in the presentation of ESAT-6 and its peptides to human Th1 cells. In addition, the T-cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT-6 and peptides. In conclusion, the recognition of ESAT-6 by IFN-gamma-secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.     

Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro.

Autoantibodies against the human asialoglycoprotein receptor (ASGPR) occur in the sera of patients with autoimmune liver disorders. Liver-infiltrating T cell clones that specifically recognize the ASGPR have been described in patients with autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with AI-CAH or PBC but not chronic viral hepatitis secreted anti-ASGPR antibodies in vitro. In this study we characterized the influence of liver-infiltrating T cells on the secretion of ASGPR-specific autoantibodies by autologous B cells in cell culture supernatants. T cell clones from liver biopsies of three patients with chronic autoimmune liver disorders (one with AI-CAH, two with PBC) were isolated and investigated for their proliferative response to soluble ASGPR and their helper function provided to autoantibody-secreting B lymphocytes. PBMC from these patients secreted autoantibodies spontaneously in their cell culture supernatants and showed a proliferative response to ASGPR. T cell-depleted PBMC, however, lacked spontaneous antibody secretion. Four CD4+CD8- liver-infiltrating T cell clones showed a proliferative response to ASGPR and also induced spontaneous anti-ASGPR antibody production in cell culture supernatants when added to autologous T cell depleted PBMC. Activated supernatants of these T cell clones failed to induce antibody production. None of seven CD4+CD8- and two CD4-CD8+ T cell clones non-responding to ASGPR provided this help for antibody secretion. Anti-ASGPR secretion in vitro could not be inhibited by the addition of MoAbs raised against monomorphic determinants on HLA class II molecules. The addition of purified ASGPR or polyclonal-activating pokeweed mitogen showed no influence on the production of autoantibodies in these cultures. These data show that B lymphocytes require T cell help for the production of ASGPR-specific antibodies. This help can be provided by ASGPR-responsive T helper cells via cellular interactions.     

HLA class II-restricted recognition of common tumor epitopes on human melanoma cells by CD4+ melanoma-infiltrating lymphocytes

CD4⁺ T cell clones derived from lymphocytes infiltrating four human melanomas specifically recognized melanoma-derived tumor epitopes as shown by secretion of tumor necrosis factor (TNF) in vitro upon interaction with autologous melanoma cells, whereas they did not recognize HLA class II-expressing autologous lymphoblasts or HLA class II mismatched allogeneic melanoma cells. Specificity was further established by demonstrating that TNF responses to tumor cells were inhibited by HLA-DR or HLA-DQ monoclonal antibodies. Most of these clones cross-reacted with allogeneic melanoma cells expressing a potentially restricting HLA allele or a structurally similar one. These data show that shared epitopes of human melanoma cells presented on HLA class II molecules are frequently recognized by autologous CD4⁺ T lymphocytes.     

Individual variation in the frequency of HLA class II-specific cytotoxic T lymphocyte precursors

The frequencies of HLA class II-specific cytotoxic T lymphocyte precursors (CTLp) were studied in number of unrelated individuals using a limiting dilution analysis system optimized for the detection of CD4+ CTLp. Peripheral blood mononuclear cells (PBMC) were enriched for CD4+ T cells by immunomagnetic depletion of CD8+ T cells. In some allogeneic combinations high CTLp frequencies were obtained with no significant difference between PBMC and CD4-enriched PBMC populations. In other combinations CTLp frequencies in CD4-enriched PBMC were found to be at least twentyfold lower than in the starting, unfractionated PBMC, suggesting a predominance in these pairs of CD8+ CTLp. In addition there was variation in CTLp frequencies against the same set of HLA class II gene products between individuals, and variation in CTLp frequencies against different HLA class II gene products within individuals. The HLA class II specificity of the assay system was demonstrated unequivocally with detection of CTLp against HLA-DR1 expressed on a murine L cell transfectant.