Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.     

Characterization of murine cytolytic-helper hybrid T cell clones

L3T4, Lyt-2 and the T-cell receptor for antigen are cell-surface molecules involved in antigen specific T cell activation. We have constructed functional murine cytolytic-helper T-cell hybrid clones to study the link between expression of cell-surface molecules and specific cell function. Three of the clones express two antigen receptors and both Lyt-2 and L3T4, normally expressed on mutually exclusive subsets of mature T lymphocytes. The pattern of lymphokines produced by the hybrid cells in response to antigen was not controlled by the specific antigen receptor; both T-cell growth factor, produced only by the helper T-cell partner, and gamma-interferon, produced only by the cytolytic T-cell partner, were secreted when either antigen receptor was stimulated. However, cytolytic activity appeared to be restricted to the recognition of antigen by the T-cell receptor of the cytolytic partner. Thus cytolysis appears to be rightly linked to the antigen receptor of the cytolytic parent but lymphokine release is not tightly linked.     

Thymocyte subpopulation enriched for progenitors with an unrearranged T-cell receptor beta-chain gene

The development of T cells within the thymus is not well understood. It is known that thymocytes are derived from a progenitor cell in the bone marrow, the prothymocyte, and that cells in the subcapsular area of the thymus can give rise to progeny in both the cortex and the medulla. However, it is not clear whether all medullary thymocytes are necessarily derived from cortical cells. In particular, it has been difficult to distinguish intrathymic progenitor cells. Recently, however, Lesley et al. have defined a thymocyte subpopulation which can be isolated by treatment of the thymus with cytotoxic anti-Thy-1 antibodies and that seems to be enriched for thymocyte progenitors as measured first by its ability to repopulate transiently the thymus of an irradiated host, and second, by its high content of cells bearing Pgp-1 (refs 10, 11), a cell-surface glycoprotein of relative molecular mass 95,000 that is present on most or all prothymocytes of the bone marrow and on fetal thymocytes, but on only a few per cent of cells in the adult thymus. We show here that the gene encoding the beta-chain of the T-cell receptor for antigen, which is rearranged during T-cell ontogeny, is predominantly in the germline configuration in these cells.     

The role of clonal selection and somatic mutation in autoimmunity

Polyclonal activation has been proposed as the reason that autoantibodies are produced during autoimmune disease. This model denies a role for specific antigen selection of B cells and predicts instead a multiclonal population of unmutated or randomly mutated autoantibodies. We have found that the genetic features and clonal composition of spontaneously derived immunoglobulin G (IgG) antiself-IgG (rheumatoid factor (RF] autoantibodies derived from the autoimmune MRL/lpr mouse strain are inconsistent with both the predictions of this model and the actual outcome of experimental polyclonal activation. Instead we have found that MRL/lpr RFs are oligoclonal or even monoclonal in origin. They harbour numerous somatic mutations which are distributed in a way that suggests immunoglobulin-receptor-dependent selection of these mutations. In this sense, the MRL/lpr RFs resemble antibodies elicited by exogenous antigens after secondary immunization. The parallels suggest that, like secondary immune responses, antigen stimulation is important in the generation of MRL/lpr RFs.     

Second messenger role for Mg2+ revealed by human T-cell immunodeficiency

The magnesium ion, Mg(2+), is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca(2+) does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg(2+) influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg(2+) influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca(2+) influx in T cells but not B cells. These observations reveal a role for Mg(2+) as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.     

Molecular and biological characterization of a murine ligand for CD40.

The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.     

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.     

Importance of IL-2 receptors in intra-thymic generation of cells expressing T-cell receptors

During development, lymphoid stem cells migrate into the thymic rudiment where they proliferate, rearrange their antigen receptor genes and become differentiated into functionally mature T cells. At present, the regulation of these processes is poorly understood, although recent studies have shown that early fetal and adult immature thymocytes express receptors for the T-cell growth factor, interleukin-2 (IL-2). We now present direct evidence that IL-2 receptors have a function in intra-thymic development by demonstrating that proliferation and the generation of cells expressing the T-cell antigen receptor (alpha beta TCR), which is responsible for the recognition of antigens in the context of MHC, are inhibited when antibodies to IL-2 receptors are added to fetal thymus organ cultures. The inhibition is specific in that it does not affect pre-thymic stem cells and can be partially reversed by addition of exogenous recombinant IL-2.     

Abnormal tyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders

The study of human autoimmune diseases has benefited greatly from analysis of animal models. Mice that are homozygous for either the lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mutant genes develop a disease characterized by massive lymphadenopathy and autoantibody formation. With age, the lymphoid organs in these mice are replaced with a greatly expanded population of abnormal lymphocytes. Recent work has shown that these cells are likely to be in the T-cell lineage. They rearrange and transcribe the genes for the alpha and beta subunits of the T-cell receptor (TCR) and a third, T-cell receptor-like gene, T gamma. As determined by immunofluorescence with anti-receptor antibodies the cells also express TCR on the cell surface. The murine T-cell receptor consists of the alpha and beta chains, derived from the rearranged alpha and beta genes, in non-covalent association with seven other chains; the delta chain, of relative molecular mass (Mr) 26,000 (26K), the epsilon chain (25K), a glycosylated 21K chain (gp21) which is probably the homologue of the gamma chain of T3 (CD3), a 16K homodimer (zeta) and a 21K dimer (p21). This multichain complex is thought to be the murine analogue of the human T3 complex. After activation of normal T cells by antigen or lectin, p21 is phosphorylated on tyrosine residues and gp21 is phosphorylated on serine residues. In contrast, in the gld and lpr cells, p21 is phosphorylated even in the absence of antigen or lectin, whereas gp21 is not phosphorylated.     

Spermatogenic failure in male mice lacking H-Y antigen

The mammalian Y chromosome carries a factor that initiates male sexual development by directing the fetal gonads to form testes. Wachtel and his colleagues proposed that this testis-determining function of the Y is mediated by the male-specific cell-surface antigen H-Y, originally defined by skin grafting. This attractive hypothesis, which has been widely accepted, was based on the assumption that serological tests using antisera raised against male cells were recognizing H-Y antigen. Although disputed this assumption is supported by some recent studies. However, mice have been described which develop testes but lack the cell-surface H-Y antigen as defined by T-cell-mediated transplantation tests. Thus, although it remains possible that a serologically detected male-specific antigen is responsible for testis determination, it seems that H-Y, as originally defined, is not. We show here that H-Y negative male mice, in losing the genetic information that encodes H-Y, have also lost genetic information required for spermatogenesis. This result identifies a gene on the mouse Y, distinct from the testis-determining gene, which is necessary for spermatogenesis, and raises the intriguing possibility that the product of this 'spermatogenesis gene' is H-Y antigen.     

Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) gamma-gene has recently been found to be expressed on a subset of both peripheral cells and thymocytes. As an initial approach to understanding the role of this gamma-chain of TCR (TCR gamma) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a gamma-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR delta). This TCR gamma delta is first detected approximately two days before the appearance of cell-surface TCR alpha beta heterodimers. We report that N-glycosidase digestions reveal that all of the gamma-protein expressed on fetal thymocytes, as in adult CD4-8-(L3T4-, Lyt2-) thymocytes, bear N-linked carbohydrate side chains. The major gamma-gene transcribed in mature, alpha beta-bearing T cells (V gamma 1.2C gamma 2)encodes no N-linked glycosylation site so these results suggest that the fetal gamma delta receptor defines a distinct T-cell lineage whose development in the thymus precedes classical alpha beta-bearing cells.     

T-cell recognition of antigen and the Ia molecule as a ternary complex

T-lymphocyte co-recognition of antigen and major histocompatibility complex (MHC)-encoded molecules (such as murine Ia molecules) is thought to be mediated by a single cell-surface receptor, although the molecular mechanism by which this occurs is controversial (reviewed in ref. 1). One possibility is that the antigen molecule and the Ia molecule interact physically, either before or after encountering the T-cell antigen-specific receptor. Alternatively, both molecules could bind to the receptor independently of one another, accounting for the dual specificity of the receptor without postulating a physical interaction between a limited number of Ia molecules present in any given animal and the myriad antigens to which T cells can respond. Here, we used a recently described approach for analysing the relative avidity of the T-cell receptor for different ligands to address these two possibilities. We describe a T-cell clone whose response to a single antigen, presented in the context of two different Ia molecules, strongly suggests that the antigen and the Ia molecule interact physically.     

Generation of cytotoxic T-lymphocyte precursor cells in T-cell colonies grown in vitro

The role of the thymus in T-lymphocyte differentiation remains unclear. The demonstration that the thymus can restrict the T-lymphocyte specificity repertoire suggests that T cells acquire specificity within the thymus. However, the demonstrations of immunocompetent helper T cells and cytotoxic T-lymphocyte precursor cells (CLPs) in athymic nude mice suggest that the acquisition of some T-cell reactivity may occur without the thymus. We have been using T-cell colonies grown in vitro as a model system for studying various aspects of T-cell differentiation in both mouse and man. In one study we showed that CLPs can be found in T-cell colonies grown from spleen cells of normal mice, each colony containing CLPs of several different specificities. The colonies containing CLPs are not clonal, appearing to have a colony-forming unit (CFU-T) of two (perhaps three) cells. Here we provide direct evidence that the CLPs are spontaneously produced in the colonies. In addition, the cells of the CFU-T were characterized with antisera directed against the cell-surface marker Thy-1, which is present on all murine T cells, and the cell-surface markers Lyt-1 and Lyt-2, which are differentially distributed on different T-cell subclasses. We found that the CFU-T contains both a Thy-1+ and a Thy-1- cell, neither of which seems to carry either Lyt-1 or Lyt-2 surface markers.     

T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand

The binding of a T-cell antigen receptor (TCR) to peptide antigen presented by major histocompatibility antigens (pMHC) on antigen-presenting cells (APCs) is a central event in adaptive immune responses. The mechanism by which TCR-pMHC ligation initiates signalling, a process termed TCR triggering, remains controversial. It has been proposed that TCR triggering is promoted by segregation at the T cell-APC interface of cell-surface molecules with small ectodomains (such as TCR-pMHC and accessory receptors) from molecules with large ectodomains (such as the receptor protein tyrosine phosphatases CD45 and CD148). Here we show that increasing the dimensions of the TCR-pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR-pMHC ligation. A similar dependence on receptor-ligand complex dimensions was observed with artificial TCR-ligand systems that span the same dimensions as the TCR-pMHC complex. Interfaces between T cells and APCs expressing elongated pMHC showed an increased intermembrane separation distance and less depletion of CD45. These results show the importance of the small size of the TCR-pMHC complex and support a role for size-based segregation of cell-surface molecules in TCR triggering.     

Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus

A role for proto-oncogenes in the regulation and modulation of cell proliferation has been suggested by the findings that the B-chain of platelet-derived growth factor (PDGF) is encoded by the proto-oncogene sis and that the erb-B oncogene product is a truncated form of the epidermal growth factor (EGF) receptor. Furthermore, the product of the proto-oncogene fms (c-fms) may be related or identical to the receptor for macrophage colony-stimulating factor (CSF-1). v-fms is the transforming gene of the McDonough strain of feline sarcoma virus (SM-FeSV) and belongs to the family of src-related oncogenes which have tyrosine-specific kinase activity. Furthermore, nucleotide sequence analysis of the v-fms gene product revealed topological properties of a cell-surface receptor protein. To elucidate the features involved in the conversion of a normal cell-surface receptor gene into an oncogenic one, we have now determined the complete nucleotide sequence of a human c-fms complementary DNA. The 972-amino-acid c-fms protein has an extracellular domain, a membrane-spanning region, and a cytoplasmic tyrosine protein kinase domain. Comparison of the feline v-fms and human c-fms sequences reveals that the proteins share extensive homology but have different carboxyl termini.     

炭疽毒素及其克隆受体的研究进展

综述炭疽毒素研究的最新进展，炭疽毒素由3种蛋白组成：致死因子（L ethal factor,LF)，水肿因子(edema factor,EF)和保护性抗原（protective antigen,PA)，本文主要讲述了炭疽毒素的关键致病因子－致死因子（LF）的结构和功能，炭疽素受体（anthrax toxin receptor,ATR）的结构及其特征性功能基团，介绍了通过基因互补对ATR进行克隆的方法，并讨论了ATR和ATR的cDNA克隆在炭疽治疗的可能应用。     

Origin of Thy-1+ dendritic epidermal cells of adult mice from fetal thymic precursors

The skin of mice contains dendritic epidermal cells carrying the Thy-1 antigen (Thy-1+ dEC) which express antigen receptors composed of the T-cell antigen receptor (TCR) gamma- and delta-chains. Although the role of the thymus in the generation of most T cells is well established, the involvement of the thymus in the generation of Thy-1+ dEC is not clear. Because bone marrow cells can give rise in Thy-1+ dEC in chimaeric mice and Thy-1+ dEC are detected in the skin of athymic nude nice, it has been proposed that Thy-1+ dEC arise continuously from bone marrow precursors by a thymus-independent mechanism. But it has recently been determined that Thy-1+ dEC in nude mice do not express TCR at the cell surface, and that the gamma- and delta-chain genes are in germ-line configuration, leaving the role of the thymus in the generation of Thy-1+ dEC uncertain. Most Thy-1+ dEC in all normal mouse strains examined express TCR containing the V gamma 3 gene product. This V gene segment is expressed on the first wave of TCR-expressing cells to emerge during fetal development, and in adult mice is detectable only on cells in the epidermis. In addition to use of this 'fetal' V gamma segment, other features of the Thy-1+ dEC TCR genes, including absence or minimal presence of nongerm-line-encoded nucleotides at the junctions and use of a single D element in the rearranged delta-chain gene are typical of rearrangements found in fetal, and not adult, thymus. Here we demonstrate that precursors that are present only in the fetal thymus give rise to Thy-1+ dEC in the skin of adult mice.