Presence of T-cell receptor mRNA in functionally distinct T cells and elevation during intrathymic differentiation

Understanding the differentiation of functionally distinct subsets of T lymphocytes is essential to unravel their crucial role in the immune response and awaits knowledge of the assembly and expression of genes encoding the T-cell receptor. Recently, we have cloned and sequenced complementary DNA that may specify part of the human T-cell receptor. The deduced protein sequence showed extensive similarity to the entire length of mammalian immunoglobulin light chains. In addition, sequences corresponding to this message undergo somatic rearrangements and are assembled from non-contiguous genomic sequences into a single mRNA molecule, a mechanism similar to those found in the generation of immunoglobulin messages. A related molecule from the mouse was also isolated independently by Hedrick et al. Here we show that the putative T-cell receptor mRNA is expressed at a relatively high level during intrathymic differentiation before decreasing about 10-20-fold in normal, mature peripheral blood T cells and that it can also be detected in T-cell clones with helper and cytotoxic functions, as well as in at least one clone with suppressor properties.     

A malaria T-cell epitope recognized in association with most mouse and human MHC class II molecules

An ideal vaccine should elicit a long lasting immune response against the natural parasite, both at the T- and B-cell level. The immune response should occur in all individuals and be directed against determinants that do not vary in the natural parasite population. A major problem in designing synthetic peptide vaccines is that T cells generally recognize peptide antigens only in association with one or a few of the many variants of major histocompatibility complex (MHC) antigens. During the characterization of epitopes of the malaria parasite Plasmodium falciparum that are recognized by human T cells, we analysed a sequence of the circumsporozoite protein, and found that synthetic peptides corresponding to this sequence are recognized by T cells in association with many different MHC class II molecules, both in mouse and in man. This region of the circumsporozoite protein is invariant in different parasite isolates. Peptides derived from this region should be capable of inducing T-cell responses in individuals of most HLA-DR types, and may represent good candidates for inclusion in an effective anti-malaria peptide vaccine.     

细粒棘球绦虫AgB1抗原表位的生物信息学预测

 以细粒棘球绦虫AgB1基因序列为基础,采用PredictProtein软件预测其编码蛋白的二级结构;应用在线预测软件Bcepred、Abcpred、IEDB及SYFPEITHI等对细粒棘球绦虫AgB1的B细胞表位和T细胞表位进行预测。结果提示,AgB1抗原蛋白存在可以构成抗原表位的区域,经软件分析,分值高的B细胞表位区域:2~9、15~20、22~35和41~52氨基酸序列;T细胞表位区域:3~12、26~33、34~44和52~61氨基酸序列。研究运用生物信息学确定AgB1抗原的4个B细胞优势表位和4个T细胞优势表位,对进一步研究AgB1的抗原性和研发更有价值的免疫诊断方法具有重要意义。     

弧菌外膜蛋白OmpK免疫交叉反应性的分析

本研究旨在研究弧菌外膜蛋白(outer membrane protein,OMP)OmpK免疫交叉反应的特征,探讨OmpK免疫交叉反应性的分子机制.利用克隆得到5株弧菌的ompK基因,通过比对10种(22株)弧菌的OmpK基因序列发现,ompK基因在弧菌中高度保守,其核苷酸序列的相似性达77%⁹3%.对副溶血弧菌(V.parahaemolyticus)VPL4-90的ompK基因进行原核表达,利用纯化的OmpK重组蛋白,制备了兔抗OmpK血清.通过western blot分析发现,OmpK抗血清能够与16种不同的弧菌发生交叉识别反应,其中V.proteolyticus、V.furnissii、V.damsela和V.metschnikovii这4种弧菌OmpK的免疫交叉反应为首次报道,并且证实弧菌OmpK的免疫交叉反应具有种属特异性.通过抗原表位预测和比对发现,弧菌的OmpK具有8个保守的抗原表位,包括7个T细胞表位以及1个T细胞和B细胞的共同表位.流式细胞术分析显示,OmpK抗体能够识别这些相同或相似的抗原表位,提示这些抗原表位可能位于细菌表面.结果表明,这些保守的且位于细胞表面的OmpK抗原表位可能是弧菌OmpK具有免疫交叉反应性的分子基础.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

T细胞功能学与结构免疫学结合方法鉴定HLA-A2限制性的细胞毒性T淋巴细胞表位富集区

 T细胞表位在抗病毒的T细胞免疫中发挥核心作用,目前已在多种病原微生物的蛋白序列上发现存在T细胞表位的聚集现象。本文建立了一套功能学与结构学结合的策略鉴定病原体上细胞毒性T细胞(Cytotoxic T Lymphocyte,CTL)表位富集区的方法,并以严重急性呼吸道综合征(SARS)相关冠状病毒(SARS-CoV)的M蛋白为例,成功地鉴定了一个HLA-A2限制性的表位富集区。首先通过生物信息学的方法预测并合成M蛋白跨膜区的HLA-A2潜在结合多肽,通过体外复性实验和T2细胞结合实验验证多肽与HLA-A2的结合力;然后在HLA-A2.1/Kb转基因小鼠中检测这些多肽的免疫原性;最后通过X射线衍射技术,成功解析了其中一条多肽与HLA-A^*0201的复合物结构,其结构显示该多肽具有典型的HLA-A^*0201表位的结构特点,但却呈现出与以往鉴定多肽不同的构象和锚定残基。本文对于理解机体对SARS-CoV等病原体产生的T细胞免疫反应,以及为更广泛的人群设计T细胞疫苗具有重要意义。     

Computer-based characterization of epidermal growth factor precursor

The cDNA sequence of the precursor of mouse epidermal growth factor (EGFP) has recently been reported by two groups, both of whom noted the presence of repeated similar segments, each about 40 residues long. One of these repeat units overlaps with the sequence of epidermal growth factor itself. The sequence of epidermal growth factor has been reported to be similar to that of pancreatic secretory trypsin inhibitor (PSTI) and a somewhat better match has been found with part of the sequence of bovine factor X, one of the blood coagulating factors. We report here that there is an even stronger similarity between the sequences of some of the repeat units of epidermal growth factor precursor and certain segments in factor X. This sequence similarity is also apparent in comparisons with other blood coagulation factors. On the basis of these sequence comparisons we suggest a scheme for the evolution of the epidermal growth factor precursor. We have also identified certain structural features in the precursor sequence that bear on the way in which the mature epidermal growth factor is generated.     

Two variant surface glycoproteins of Trypanosoma brucei of different sequence classes have similar 6 A resolution X-ray structures

Antigenic variation in the African trypanosome is mediated through changes in the composition of the surface coat. By controlling expression of the major surface protein, the variant surface glycoprotein (VSG), from a repertoire of perhaps 1,000 different genes the organisms exhibit a series of antigenically distinct coats and evade the host's immune system. We have determined the 6 A resolution structure of a T. brucei variant surface glycoprotein, ILTat 1.24, using X-ray crystallography. The crystallized protein consists of the N-terminal two-thirds of the intact VSG which has a relative molecular mass (Mr) of 60,000 (60K). The structure, which includes a 90 A long alpha-helical bundle, is strikingly similar to that of the N-terminal fragment of VSG MITat 1.2 (ref. 4). Although most known VSG sequences show little similarity of primary sequence in the N-terminal domain, the similarity between the structure of a Class I (ILTat 1.24) and a Class II (MITat 1.2) VSG antigen suggests that VSGs may share a common tertiary structure.     

A potential donor gene for the bm1 gene conversion event in the C57BL mouse

The mammalian major histocompatibility complex (MHC; H-2 complex in mouse) is a large multigene complex which encodes cell-surface antigens involved in the cellular immune response to foreign antigens. Class I polypeptides expressed at the H-2K and H-2D loci of numerous mouse strains exhibit an unusually high degree of genetic polymorphism, which is assumed to be related to their function as primary recognition elements in the immune response. We suggested that this H-2 polymorphism may arise by gene conversion-like events between non-allelic class I genes. This is supported by our recent comparison of the DNA sequences of the normal H-2Kb gene sequence, from the C57BL/10 mouse, and a mutant form of this gene called H-2Kbm1: the mutant allele differs from the H-2Kb gene in seven bases out of a region of 13 bases in exon 3 of the class I gene (which encodes alpha 2 (C1) the second highly polymorphic protein domain), suggesting that this region of new sequence had been introduced into the H-2Kb sequence following unequal pairing of two class I genes in the genome of the C57BL mouse. Schulze et al. have obtained similar results. Here we report work identifying a potential donor gene in our library of 26 class I genes cloned from the C57BL/10 mouse.     

Isolation and characterization of a cDNA clone encoding the murine homologue of the human 20K T3/T-cell receptor glycoprotein

The antigen receptor on the surface of human T lymphocytes, which consists of a heterodimer of relative molecular mass (Mr) 90,000 (90K) (alpha- and beta-chains), is associated with the T3 antigen (gamma = 25K, delta = 20K and epsilon = 20K). A working model for the mode of action of the T3/T-cell receptor complex is that the clonotypic alpha- and beta-chains are involved in the recognition and binding of antigen in the context of polymorphic major histocompatibility complex (MHC) gene products on the surface of target cells. Antigen binding by the clonotypic receptor probably results in conformational changes in this structure which are recognized by and subsequently trigger the associated T3 complex to transmit signals into the cell, resulting in a proliferative response. The similarity in structure between murine and human clonotypic antigen receptors suggests that such a mechanism of recognition and activation also exists in mouse T lymphocytes, but so far there has been no evidence for the existence of a murine T3 complex. Here we demonstrate the existence of a T3 delta-chain mRNA in murine T lymphocytes. Our sequence data strongly suggest that this mouse mRNA codes for a complete T3 delta polypeptide chain and reveal some interesting properties of the protein.     

Three Candidate Epitope—Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV—1

HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines. So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies. Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HW-1. According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated El, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2:C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits. After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies. An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide. Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine. Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response. This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 ~tg. Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.     

多粒度时空事件序列相似度算法研究

现有研究集中于不带有时间空间信息或带有固定时间空间信息的活动序列相似度计算,没有从不同层次来度量用户行为序列的相似性,为了实现对用户行为多粒度多视角的动态认知,提出一种基于序列比对算法Needleman-Wunsch的多粒度时空序列比对算法（multi-granular spatiotemporal sequences alignment,MGSSA）,扩展了NW算法的得分函数以结合时间、空间信息,通过粒度调控实现了从不同的粒度来计算时空事件序列的相似度.实验证明,多粒度时空序列比对算法MGSSA是有效且可行的.      

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.     

An embryo protein induced by SV40 virus transformation of mouse cells

A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.     

T gamma protein is expressed on murine fetal thymocytes as a disulphide-linked heterodimer

During the search for genes coding for the mouse alpha and beta subunits of the antigen-specific receptor of mouse T cells we encountered a third gene, subsequently designated gamma. This gene has many properties in common with the alpha and beta genes, somatic assembly from gene segments that resemble the gene segments for immunoglobulin variable (V), joining (J) and constant (C) regions; rearrangement and expression in T cells and not in B cells; low but distinct sequence homology to immunoglobulin V, J and C regions; other sequences that are reminiscent of the transmembrane and intracytoplasmic regions of integral membrane proteins; and a cysteine residue at the position expected for a disulphide bond linking two subunits of a dimeric membrane protein. Despite these similarities the gamma gene also shows some interesting unique features. These include a relatively limited repertoire of the germ-line gene segments, more pronounced expression at the RNA level in immature T cells such as fetal thymocytes and an apparent absence of in-frame RNA in some functional, alpha beta heterodimer-bearing T cells or cultured T clones and hybridomas. To understand the function of the putative gamma protein it is essential to define the cell population that expresses this protein. To this end we produced a fusion protein composed of Escherichia coli beta-galactosidase and the gamma-chain (hereafter referred to a beta-gal-gamma) using the phage expression vector lambda gt11 and raised rabbit antisera against the gamma determinants. Using the purified anti-gamma antibody we detected a polypeptide chain of relative molecular mass 35,000 (Mr 35K) on the surface of 16-day old fetal thymocytes. The gamma-chain is linked by a disulphide bridge to another component of 45K. No such heterodimer was detected on the surface of a cytotoxic T lymphocyte (CTL) clone 2C from which an in-phase gamma cDNA clone was originally isolated.     

A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products

SV40 large T antigen contains a small region of amino acid sequence, conserved among the papovaviruses, that shows considerable similarity to conserved domain 2 of the adenovirus E1A oncogene, a domain which plays an important role in the E1A transforming functions. To learn whether the analogous SV40 T antigen sequences could substitute functionally for E1A domain 2, a chimaeric gene was constructed, coding for T antigen amino acid residues 101 to 118 in place of E1A domain 2. The resulting product showed much of the activity of the wild-type E1A products. It induced proliferation of primary BRK cells and cooperated with the ras oncogene to transform these cells fully. In addition, the chimaeric protein coprecipitated two cellular proteins whose specific binding to the E1A products depends on the presence of domain 2. The activity of the chimaeric product suggests that a similar functional unit exists in the transforming proteins of both SV40 and adenovirus, and that these proteins may exert their cell growth regulating effects through similar mechanisms.     

基于Cp-曲线的蛋白质序列相似性分析

蛋白质是生物体内行使重要功能的生物大分子.蛋白质的功能是由其空间结构决定的,而蛋白质的空间结构取决于其一级序列.因此,研究蛋白质的氨基酸序列就成为蛋白质结构预测的前提和基础,并已经成为生物学家关注的主要研究内容之一.主要介绍基于20种氨基酸的一种5-L模型,将蛋白质序列粗粒化为5-L序列,进而给出蛋白质序列的一种3-D图形表示,称之为Cp曲线.将Cp曲线转化为容易比较的序列不变量,并在此基础上对11个物种β-球蛋白质序列进行相似性分析.     

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.     

CpG DNA enhances the immune responses elicited by the DNA vaccine against foot-and-mouth disease virus in guinea pigs

CpG DNA is DNA sequence that has immune stimulatory effects. Several lines of investigation over the past few years indicate that CpG DNA plays an important role in the induction of immune responses to DNA vaccines. In this study, CpG DNA-containing synthetic oligodeoxynucleotide (CpG-ODN) was cloned into the eukaryotic expression plasmid encoding a fusion protein containing b- galactosidase from E. coli and immunogenic epitopes of foot- and-mouth disease virus (FMDV) type O, and the immune responses induced by the plasmid were assayed. The results showed that guinea pigs immunized with the recombinant plasmid containing CpG-ODN generated a higher level of FMDV-neutralizing antibody and a stronger T cell proliferative response and protection against viral challenge than those receiving the plasmid containing no CpG-ODN. Our study demonstrated that it is an effective route to enhance the efficacy of DNA vaccines by inserting exogenous CpG DNA into the plasmids, and the DNA vaccine developed here is a promising candidate to prevent FMDV infection.