Expression and function of interleukin-2 receptors on immature thymocytes

T-cell differentiation represents a unique system for studying mechanisms of lymphoid development because it occurs in a segregated site, the thymus, in which distinct subpopulations of thymocytes at various stages of differentiation can be defined on the basis of the differential expression of T-cell surface antigens as well as topography. There is particular interest in thymocyte differentiation because the genotype of radioresistant thymus cells influences the specificity repertoire of the pool of T cells that mature therein: that is, the major histocompatibility complex (MHC) antigens expressed by thymus cells bias the pool of maturing T cells towards recognition of antigens in the 'context' of the products of that MHC haplotype ('thymus education'; refs 1-3). Immature T cells with affinity for thymus MHC antigens are generally thought to undergo a stage of positive selection in the thymus. Here we report that 30% of cells in the least mature adult thymocyte subpopulation yet defined, as well as 50% of immature fetal thymocytes, express receptors for interleukin-2 (IL-2, the T-cell growth factor) without in vitro induction, and will proliferate vigorously in an IL-2-dependent fashion if provided with co-stimulating mitogen.     

Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma

Proto-oncogenes may be important in the cellular processes central for the growth and differentiation of normal cells. N-myc is a DNA sequence which shares limited homology to the proto-oncogene c-myc and has been found to be amplified in both primary tissue and cell lines from neuroblastoma, a childhood tumour of neuroectodermal origin. Differentiation of this embryonal tumour is of clinical importance, since occasional tumours have been noted to differentiate in vivo to benign ganglioneuroma. In vitro, many human neuroblastoma cell lines can be induced to differentiate morphologically and biochemically by a variety of agents. Retinoic acid (RA), an analogue of vitamin A, has been shown to inhibit neuroblastoma cell growth and clonability in soft agar, and to induce extensive neurite outgrowth. Therefore we examined the relationship of N-myc expression to the in vitro differentiation of these cells. We report here that in the case of RA-induced differentiation, a decreased level of expression is detected within 6 h of treatment and precedes both cell-cycle changes and morphological differentiation.     

Engagement of the CD4 molecule influences cell surface expression of the T-cell receptor on thymocytes

The intrathymic differentiation process by which precursor cells derived from the bone marrow develop into immuno-competent T lymphocytes is poorly understood. Most thymocytes express both CD4 and CD8 accessory molecules, yet little is known about either the function of these molecules or the responsiveness of the CD4+8+ double positive thymocytes that bear them. Here, we address the possibility that CD4 engagement influences T-cell receptor (TCR) expression on developing thymocytes. We engaged CD4 molecules on murine thymocytes by in vivo injection of an anti-CD4 monoclonal antibody, which reduced the surface expression of CD4 on CD4+ thymocytes. More importantly, CD4 engagement also affected TCR expression on CD4+ thymocytes, but the effect on CD4+8+ double positive and CD4+8- single positive thymocytes was very different. CD4+8+ thymocytes responded to CD4 engagement by dramatically increasing surface expression of TCR, whereas CD4+8- thymocytes decreased surface expression of TCR. These results demonstrate that the effect of CD4 engagement on TCR expression is dependent upon the developmental state of the responding thymocyte, and, most interestingly, results in increased TCR expression by double positive thymocytes.     

Intrathymic signalling in immature CD4+CD8+ thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain

Thymic selection of the developing T-cell repertoire occurs in immature CD4+CD8+ double-positive thymocytes and is thought to be mediated by signals transduced by T-cell antigen receptor (TCR) molecules and possibly by CD4 and CD8 accessory molecules as well. It is not known, however, which signal-transduction mechanisms function in immature CD4+CD8+ thymocytes on engagement of TCR, CD4 or CD8 molecules. In mature T cells, CD4 and CD8 molecules are each associated with the src-like protein tyrosine kinase p56 lck and signals transduced by TCR and CD4 activate tyrosine kinases that phosphorylate TCR-zeta chains and other intracellular substrates. Consequently, we examined whether tyrosine kinases could be similarly activated in immature CD4+CD8+ thymocytes. Unexpectedly, we found that TCR-zeta chains from CD4+CD8+ thymocytes were already phosphorylated in vivo, and that dephosphorylation of this TCR subunit occurred on removal of CD4+CD8+ cells from their intrathymic environment. Rephosphorylation of TCR-zeta in cultured CD4+CD8+ thymocytes occurred rapidly in vitro, either in response to cross-linking of TCR, CD4 or CD8 by specific monoclonal antibodies, or on cell-cell contact. These observations indicate that tyrosine kinases are activated in vivo in immature CD4+CD8+ thymocytes undergoing thymic differentiation and selection. They also indicate that TCR, CD4 and CD8 molecules can function in CD4+CD8+ thymocytes as signalling molecules to activate tyrosine kinases and that phosphorylated TCR-zeta serves as a marker of these signalling events.     

Abnormal differentiation of thymocytes in mice treated with cyclosporin A

Cyclosporin A (CsA) acts as a powerful immunosuppressive agent, and also, when given in repeated doses, can cause T-cell-dependent graft-versus-host disease and organ-specific autoimmune disease in rodents. This suggests that CsA interferes with the processes governing self-tolerance, either by nullifying the activity of T suppressor cells or by preventing the deletion of autoreactive T cells during ontogeny in the thymus. We report here that irradiated mice given repeated injections of CsA show striking dysfunction of the thymus. There are two different effects, the first of which is that CsA seems to block the differentiation of immature CD4+CD8+ thymocytes into mature CD4+CD8- and CD4-CD8+ cells expressing a high density of T-cell receptors and CD3 molecules. Second, CsA-treated mice show incomplete deletion of T cells expressing T-cell receptor molecules reactive to self H-2 I-E molecules.     

A functional T3 molecule associated with a novel heterodimer on the surface of immature human thymocytes

The known T-cell receptors (TCRs) involved in the recognition of antigen and major histocompatibility complex (MHC) molecules are glycoproteins comprised of polymorphic disulphide-linked alpha- and beta-chains. The genes encoding these chains are homologous to immunoglobulin genes and consist of V (variable), J (joining) and C (constant) regions that rearrange during development. TCRs are expressed relatively late in thymocyte development and only in association with an invariant molecular complex of proteins termed T3. Immature thymocytes do not express the TCR-T3 complex but do express messenger RNA encoding a third rearranging T-cell receptor-like gene, termed T gamma. Here we report a clone of normal immature T4-T8- human thymocytes, designated CII, which does not express mature mRNA for T alpha or T beta genes, but does express high levels of T gamma mRNA. This clone also expresses high levels of surface T3, and antibodies to T3 induce immunologically relevant functions in CII cells. Immunoprecipitation of CII surface-labelled proteins with anti-T3 co-precipitates a T3 molecular complex together with two additional and novel peptides of relative molecular mass (Mr), 44,000 (44K) and 62,000 (62K).     

Expression of genes of the T-cell antigen receptor complex in precursor thymocytes

The antigen receptor on T lymphocytes has recently been characterized as a heterodimeric, transmembrane glycoprotein consisting of disulphide-linked alpha (acidic) and beta (basic) subunits of relative molecular mass (Mr) 40,000-45,000 each. The genes encoding these proteins have been cloned and shown to resemble immunoglobulin genes in both overall structure and the requirement for DNA rearrangement before expression. In humans, three additional proteins, termed the T3 complex, are found associated with the clonotypic receptor, and a role for T3 in receptor expression has been proposed. Despite these recent advances in characterizing the antigen receptor complex, there is as yet little understanding of T-cell maturation, particularly the stage of T-cell ontogeny at which the genes encoding the antigen receptor and its associated structures are expressed and assembled. In the adult, stem cells destined to differentiate into T cells arise in the bone marrow and migrate to the thymus, where T-cell precursors proliferate, develop a preference for recognizing antigens in the context of self MHC molecules and are released to the periphery. Recently, cells that have the properties of immature murine thymocytes have been isolated and described. We have now analysed these cells with a series of molecular probes and we describe three distinct patterns of T-cell antigen receptor gene rearrangements in developing thymocytes.     

Interleukins 4 and 5 control expression of IL-2 receptor on murine B cells through independent induction of its two chains

Resting B cells express few, if any, receptors for interleukin-2 (IL-2), whereas activated B cells can express receptors for and respond to IL-2. IL-2 receptors can exist on the cell surface in three different forms; the complete high-affinity receptor, a heterodimer consisting of a chain of relative molecular mass (Mr) 70-75,000 (70-75K) and a chain of Mr 55K; the 70-75K chain alone, with intermediate affinity for IL-2; or the 55K chain alone, with low affinity for IL-2. We have previously reported that IL-5-stimulated B cells are induced to express the 55K chain. We report here evidence for the differential regulation of the expression of the two chains, namely that IL-4 and IL-5 can independently induce expression of the 70-75K and 55K chains respectively on murine B cells. As expected, cells stimulated to express the 55K chain alone are unresponsive to IL-2, whereas cells stimulated to express either the 70-75K chain or the 70-75/55K heterodimer respond to IL-2, at a high and low ligand concentration respectively, with a marked increase in proliferation. This orchestration of receptor expression and factor responsiveness may represent a novel activation pathway for B cells, where the two chains of a compound receptor are shown to be independently regulated.     

Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells

One mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing alpha beta T-cell receptors. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes (which die by apoptosis) but deletion at less and more mature developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.     

Molecular cloning and expression of cDNAs for the human interleukin-2 receptor

We have purified the human T-cell growth factor (interleukin-2) receptor and have cloned, sequenced and expressed cDNAs corresponding to this receptor. We identify one gene, but two interleukin-2 receptor mRNAs which differ in their polyadenylation signals. We have isolated an additional cDNA that may correspond to an alternatively spliced mRNA that lacks a 216 base segment and appears to encode an altered membrane protein which cannot bind interleukin-2.     

Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking

T cells develop from prothymocytes which express no detectable antigen receptors to immature thymocytes with few receptors, eventually becoming mature thymocytes and peripheral T cells with 20,000-40,000 receptors per cell. Recent studies suggest that immature thymocytes are immunologically unresponsive. We have suggested that an early step in signal transduction following engagement of the T cell receptor might differ in immature and mature T cells. Here we examine anti-receptor antibody mediated induction of calcium mobilization in immature and mature T cells. Results indicate that antigen receptors on both immature and mature receptor-positive T cells transduce signals via calcium mobilization. Significant differences were observed, however, between these populations in the magnitude of influx of extracellular Ca2+ following binding of antireceptor antibody. Specifically immature cells show a much reduced Ca2+ influx response compared to mature cells which could result from a low Ca2+ channel frequency in the plasma membranes of immature T cells, or from less efficient activation of existing channels.     

Alternative splicing directs the expression of two D2 dopamine receptor isoforms

Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological properties and the intracellular responses they mediate. The cerebral D2 dopamine receptor is the target of drugs used to alleviate the main symptoms of schizophrenia. Although it is considered to be a single molecular entity, there is evidence that multiple D2-receptor subtypes exist. A complementary DNA encoding a D2 receptor has recently been cloned and the deduced 415-amino-acid sequence indicates that it belongs to the large superfamily of receptors coupled to G proteins, and that its topology consists of seven transmembrane domains. In this family, the genes are frequently without introns and each is believed to encode a unique polypeptide product. Here we show that the gene for the D2 receptor produces two receptor isoforms by alternative messenger RNA splicing, providing a route to receptor diversity in this family. One isoform corresponds to the D2(415) receptor, but the second contains an additional sequence encoding a 29-amino-acid fragment, defining a novel D2(444) receptor isoform. Expression of the two isoforms is tissue-specific, and both are regulated by guanyl nucleotides. As the extra sequence is located within a putative cytoplasmic loop that binds to G proteins, the two isoforms might interact with different G proteins and thereby initiate distinct intracellular signals.     

Prevention of vaccinia virus infection in immunodeficient mice by vector-directed IL-2 expression

Recombinant vaccinia viruses have been proposed as live vaccines against a variety of infectious diseases, including AIDS (acquired immune deficiency syndrome). Objections have been concerned primarily with side effects of the vaccinia virus vector itself. Recently it has been shown that inactivation of the vaccinia virus thymidine kinase gene or deletion of certain other non-essential genes is associated with a marked reduction in pathogenicity. Nevertheless, the ability of vaccinia virus to produce a progressive infection in immunodeficient individuals remains a most serious problem. Indeed, an incident of this type in a vaccinated man seropositive for human immunodeficiency virus was recently reported. We have used immunodeficient athymic nude mice to establish a model of disseminated vaccinia virus infection, and to demonstrate a novel approach to virus attenuation which involves insertion of a gene encoding human interleukin-2 into the genome of vaccinia virus vectors.     

Cloning, sequence and expression of human interleukin-2 receptor

T lymphocytes, essential for the generation of a normal immune response, require the presence of the lymphokine interleukin-2 (IL-2) in order to proliferate. Cells that respond to IL-2 possess a surface receptor glycoprotein specific for this lymphokine. We have recently purified and chemically characterized the IL-2 receptor from both phytohaemagglutinin-activated human T cells and the human T-cell lymphoma HUT-102 (ref. 5). From the NH2-terminal protein sequence obtained in that study, we have now used synthetic oligonucleotides to probe a complementary DNA library, prepared from HUT-102 messenger RNA, for the presence of cDNA clones that might code for the IL-2 receptor. Two cDNA clones were isolated which had closely related DNA sequences. Interestingly, only one coded for an active receptor when transfected into COS-7 cells. This clone contained a 216-base pair (bp) insert that was not present in the other clone. The insert was flanked by an 8-bp direct repeat reminiscent of a transposable element, and appeared to code for a region of marked structural homology to the NH2-terminal region of the receptor molecule.     

MyHC基因在C2C12成肌细胞分化过程中的时序表达

以体外培养的C2C12成肌细胞为研究对象,然后利用荧光定量PCR检测肌球蛋白重链(myosin heavy chains,MyHC)的3种亚型MyHC1、MyHC2x和MyHC2b在成肌诱导分化中(0-8 d)中的时序表达规律.结果表明,MyHC1基因在诱导分化的0-4 d表达呈上升趋势,4-8 d又呈下降趋势,在第4 d和第6 d的表达水平显著高于其他天数(p0.05).MyHC2x基因在诱导分化的0-6 d表达呈上升趋势,但在第8 d又开始下降.MyHC2b在诱导分化的0-8 d中表达一直呈上升趋势,第6 d和第8 d表达水平显著高于其他天数(p0.05).     

Differential expression of calmodulin-binding proteins in B, T lymphocytes and thymocytes

Changes in intracellular free Ca2+ are involved in the transmembrane signalling of different cells, including lymphocytes. Since calmodulin (CaM) is a primary receptor for Ca2+ (ref. 4), it may mediate the activation of crucial enzymes after antigen-induced increases in cytosolic Ca2+. Using a biotinylated-CaM (Bio-CaM) detection procedure to identify such proteins, we found that a peptide of relative molecular mass 59,000 (59K) was the predominant soluble CaM-binding protein (CaM-BP) in T cells and B lymphocytes from murine spleen; immunoblotting experiments identified it as a subunit of the CaM-dependent phosphatase, 'calcineurin' (CN). Smaller amounts of larger CaM-BPs, thought to be cytoskeletal-binding proteins, were also detected. CaM-BPs were expressed differentially, with B lymphocytes having four times more of the CN-like protein than T lymphocytes, while in thymocytes, a 65K polypeptide was the major CaM-BP. However, limited proteolysis analysis suggested that this thymus-specific peptide may be a precursor of CN. These data suggest that Ca2+-stimulated protein dephosphorylation may be an important and highly regulated function in lymphoid cells.     

The adult T-cell receptor delta-chain is diverse and distinct from that of fetal thymocytes

T lymphocytes recognize foreign molecules using the T-cell receptor (TCR), a disulphide-linked heterodimer closely associated with the CD3 polypeptide complex on the cell surface. The TCR alpha beta heterodimers seem largely responsible for the recognition properties of both helper (TH) and cytotoxic (TC) T cells. Recently, a second CD3-associated T-cell receptor heterodimer, gamma delta, has been described. Cells bearing the gamma delta receptor appear before those bearing alpha beta during thymic ontogeny and persist as a minor component (1-10%) of mature peripheral T cells. Their function is unknown. As there are a limited number of functional TCR V gamma gene segments, the size and potential diversity of the V delta repertoire is important for the number of different antigens that may be recognized by gamma delta heterodimers. The delta-chain locus is located 75 kilobases (kb) 5' to the TCR C alpha coding region, raising the possibility that the alpha and delta V-region repertoires may overlap. Also, analysis of rearrangements at the delta-chain locus in developing thymocytes shows distinct fetal and adult patterns indicating that there may be differences between the fetal and adult V delta repertoires. To address these questions, we have characterized a large number of delta-containing complementary DNA clones from adult double-negative thymocytes (CD4-8-), an immature population that is enriched for gamma delta-bearing cells. We find that a limited number of V delta sequences are used, showing little overlap with known adult V alpha s and differing significantly from fetal V delta s. But as two D elements may participate simultaneously in V delta gene assembly, and random nucleotides may be added at any one of three junctional points, the potential number of different delta chains that can be made in the adult thymus is very large (approximately 10(13)).