Structural mutations of the T cell receptor zeta chain and its role in T cell activation

T cell hybridomas that express zeta zeta, but not zeta eta, dimers in their T cell receptors (TCRs) produce interleukin-2 (IL-2) and undergo an inhibition of spontaneous growth when activated by antigen, antibodies to the receptor, or antibodies to Thy-1. Hybridomas without zeta and eta were reconstituted with mutated zeta chains. Cytoplasmic truncations of up to 40% of the zeta molecule reconstituted normal surface assembly of TCRs, but antigen-induced IL-2 secretion and growth inhibition were lost. In contrast, cross-linking antibodies to the TCR activated these cells. A point mutation conferred the same signaling phenotype as did the truncations and caused defective antigen-induced tyrosine kinase activation. Thus zeta allows the binding of antigen/major histocompatibility complex (MHC) to alpha beta to effect TCR signaling.     

Selection of amino acid sequences in the beta chain of the T cell antigen receptor

The induction of an immune response in mammals is initiated by specifically reactive T lymphocytes. The specificity of the reaction is mediated by a complex receptor, part of which is highly variable in sequence and analogous to immunoglobulin heavy- and light-chain variable domains. The functional specificity of the T cell antigen receptor is, however, markedly different from immunoglobulins in that it mediates cell-cell interactions via the simultaneous recognition of foreign antigens and major histocompatibility complex-encoded molecules expressed on the surface of various lymphoid and nonlymphoid cells. The relation between the structure of the receptor and its functional specificity was investigated by analyzing the primary sequences of the receptors expressed by a series of T lymphocyte clones specific for a model antigen, pigeon cytochrome c. Within this set of T lymphocyte clones there was a striking selection for amino acid sequences in the receptor beta-chain in the region analogous to the third complementarity-determining region of immunoglobulins. Thus, despite the functional differences between T cell antigen receptors and immunoglobulin molecules, analogous regions appear to be important in determining ligand specificity.     

Activation-driven programmed cell death and T cell receptor zeta eta expression

Activation of spontaneously dividing T cell hybridomas induces interleukin-2 (IL-2) production, a cell cycle block, and programmed cell death. T cell hybridomas that express the T cell antigen receptor (TCR) zeta homodimer (zeta 2), but not the TCR zeta eta heterodimer, were studied. The zeta eta- cells produced little or no inositol phosphates (IP) when stimulated with antigen. In most cases the hydrolysis of phosphoinositides was also impaired after stimulation with antibody to CD3, although one zeta eta- cell produced normal concentrations of IP. The zeta eta- cells slowed their growth and secreted IL-2 in response to both stimuli. However, the zeta eta- cells did not die after activation with antigen. Since activated thymocytes also undergo programmed cell death, these results may have important implications for the role of the zeta eta.TCR in negative selection.     

Molecular cloning of the chicken progesterone receptor

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.     

Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain

In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5' portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3' end of the target fragment. It was used to analyze TCR delta chain mRNA's from human peripheral blood gamma delta T cells. Most of these cells had a V delta gene segment not previously described (V delta 3), and the delta chain junctional sequences formed a discrete subpopulation compared with those previously reported.     

CAR-T细胞免疫疗法在肿瘤治疗中的研究进展

嵌合抗原受体T细胞免疫疗法(chimeric antigen receptor therapy,CAR-T)作为一种肿瘤免疫治疗方法,具有良好的治疗前景.该文总结了CAR-T细胞治疗的发展历程与临床研究进展,阐述了CAR-T细胞治疗耐药机制,并提出了优化CAR-T细胞治疗的策略.     

Early signal transduction by the antigen receptor without commitment to T cell activation

The T lymphocyte antigen-receptor complex mediates antigen-specific cell activation, at least in part, through the production of inositolphospholipid-derived second messengers. Little is known about how second messenger events, typically measured within minutes of ligand binding, eventually lead to distal biologic responses such as expression of lymphokine genes. Several monoclonal antibodies directed against the receptor complex were tested for their ability to elicit transmembrane signaling in the parental Jurkat line and in a somatic mutant (J.CaM1) with a deficient receptor function. One antibody elicited substantial early Ca2+ mobilization responses in both cells but was unable to promote expression of the interleukin-2 gene in J.CaM1. In J.CaM1 there was a diminished production of phosphatidylinositol second messengers, and the elevation in intracellular free Ca2+ was transient. Thus, short-term Ca2+ mobilization does not always indicate complete signal transmission and lead to a full cellular response.     

猪T细胞受体β链基因的克隆及生物信息学分析

以GenBank上登载的猪的T细胞受体β(pTCR-β)基因为参考序列,用RT-PCR法从猪的外周血淋巴细胞中克隆了TCR-β链基因,并进行生物信息学分析.结果表明:pTCR-β基因含有一个完整的开放阅读框架(ORF),大小为849bp,编码283个氨基酸,且含有一段27个氨基酸的信号肽序列,与参考序列相比,在核苷酸序列上的同源性为80.4%,在氨基酸序列上的同源性为70.3%;生物信息学结构预测发现2个结构域,一个为IG结构域,由第32-138位共107个氨基酸残基组成;另一个为IG-LIKE结构域,由第164-211位共48个氨基酸残基组成.     

Characterization of T cell receptor gamma chain expression in a subset of murine thymocytes

While much information exists about the structure and function of the clonally distributed T cell receptor (TCR) alpha beta heterodimer, little is known about the gamma protein, the product of a third rearranging TCR gene. An antiserum to a carboxyl-terminal peptide common to several of the murine gamma chain constant regions and a monoclonal antibody to the murine T3 complex were used to identify products of this TCR gene family in a subpopulation of Lyt2-, L3T4- thymocytes. This subpopulation does not express TCR alpha or full-length TCR beta messenger RNA. The gamma chain is a 35-kilodalton (kD) protein that is disulfide-bonded to a 45-kD partner and is associated with the T3 complex. Analysis of the glycosylation pattern of this thymic gamma chain revealed that the major variable region gamma (V gamma) gene transcribed in activated peripheral T cells is absent from this subpopulation. The cells that bear this second T cell receptor may therefore represent a distinct lineage differentiating within the thymus.     

The T cell receptor

The primary structure of T cell receptor proteins and genes is well understood. Immunologists are now trying to understand the properties of these interesting molecules. Evidence suggests that T cell alpha beta receptors recognize a complex of an antigen-derived peptide bound to one of the cell-surface products of the major histocompatibility complex (MHC) genes. It is likely that alpha beta receptors and MHC proteins have coevolved to have some affinity for each other. During T cell development in the thymus, cells bearing self-reactive receptors are deleted by the mechanisms of tolerance, and cells are preferentially allowed to mature if they bear receptors that will be able to recognize antigen plus self-MHC after they have become full-fledged T cells. Some explanations for these phenomena have been tested, but no satisfactory theory can yet be proposed to account for them.     

Genes for beta chain of human T-cell antigen receptor map to regions of chromosomal rearrangement in T cells

The T-cell antigen receptor is a cell-surface molecule that participates in the immune response. In the present experiments the genes encoding the beta chain of the T-cell receptor were found to reside on the long arm of human chromosome 7 at or near band q32. Related sequences were found on the short arm of chromosome 7 in bands p15-21 in some experiments. Chromosomal rearrangements in T-cells from normal individuals and patients with ataxia telangiectasia have previously been observed at and near these map assignments for the beta-chain genes.     

Molecular cloning and functional expression of glutamate receptor subunit genes

Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate. The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.     

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.     

Differential clustering of CD4 and CD3zeta during T cell recognition

Whereas T helper cells recognize peptide-major histocompatibility complex (MHC) class II complexes through their T cell receptors (TCRs), CD4 binds to an antigen-independent region of the MHC. Using green fluorescent protein-tagged chimeras and three-dimensional video microscopy, we show that CD4 and TCR-associated CD3zeta cluster in the interface coincident with increases in intracellular calcium. Signaling-, costimulation-, and cytoskeleton-dependent processes then stabilize CD3zeta in a single cluster at the center of the interface, while CD4 moves to the periphery. Thus, the CD4 coreceptor may serve primarily to "boost" recognition of ligand by the TCR and may not be required once activation has been initiated.     

欧洲鳗鲡免疫球蛋白轻链基因的克隆及表达分析

通过RT-PCR技术克隆欧洲鳗鲡(Anguilla anguilla)Ig轻链基因cDNA全长序列,命名为AaIgL,其全长为1 016bp,开放阅读框为714bp,编码238个氨基酸。将该基因片段与pET-his载体连接构建原核表达载体,在大肠杆菌BL21中诱导表达,其表达产物经SDS-PAGE和Western blotting分析,结果表明新增的27ku蛋白条带与预期值相符,且与兔抗欧洲鳗鲡IgM血清发生特异性显色反应,证实了AaIgL基因能够在大肠杆菌中以包涵体形式高效表达;实时荧光定量PCR检测结果发现:AaIgL在欧洲鳗鲡各组织中均有表达,其中脾脏的表达量最高,肾脏和心脏中也有较高的表达水平,而肝脏、肌肉、鳃以及肠中的表达量较低;欧洲鳗鲡经山羊IgG肌肉注射后脾脏和肾脏的AaIgL表达水平明显上升,其峰值分别为第7天和第14天,AaIgL在脾脏的表达量显著高于肾脏,但是第21天后均恢复至正常水平。以上结果表明,AaIgL在欧洲鳗鲡机体免疫防御中发挥重要作用,脾脏是AaIgL基因的主要表达器官。     

鸽TLR7基因的克隆、鉴定及表达分析

【目的】克隆鸽Toll样受体(Toll-like receptor 7,TLR7)全基因,预测其主要功能区域并分析在鸽的各种组织中的表达情况.【方法】通过RT-PCR、RACE、相对荧光定量PCR、生物信息学软件分析方法进行研究.【结果和结论】研究发现鸽TLR7基因cDNA全长3 516 bp,ORF全长3 175 bp,编码1 048个氨基酸.其蛋白结构主要由胞外的富含亮氨酸的结构域(LRRs)、跨膜域(TM)和胞内的Toll/白介素-1受体结构域(TIR)3部分构成.鸽TLR7基因编码的氨基酸序列与鸿雁Anser cygnoides、绿头鸭Anas platyrhynchos、鸡Gallus gallus和麻雀Taeniopygia guttata的相似性均高于78%,与哺乳动物的相似性约为60%,与鱼类的相似性低于55%.鸽TLR7基因在小肠、脾脏、肾脏、肝脏中表达量较高,而在大脑、肺脏、气管、心脏、胰腺、肌肉、皮肤中表达量相对较低.该研究克隆了鸽TLR7全基因,并预测其主要功能区域.