CD3 Gamma Chains Research Articles

T cell receptor alpha-chain tail is required for protein kinase C-mediated down-regulation, but not for signaling.

Antigen stimulation through the T cell receptor (TCR) induces phosphorylation of the associated CD3 gamma delta epsilon- and zeta-chain cytoplasmic tails. These events lead to the induction of the intracellular signaling pathways with concomitant receptor down-regulation. The TCR is down-regulated from the cell surface by the activation of protein kinase, C (PKC) and subsequent serine phosphorylation of the CD3 gamma-chain. We report here that the TCR alpha-chain cytoplasmic tail is also necessary for PKC-mediated internalization of the TCR complex. The requirement for the TCR alpha-chain cytoplasmic tail is specific for internalization of the TCR complex, since down-regulation of CD4 is still intact in hybridoma cells expressing a tailless TCR alpha-chain. The absence of TCR internalization directly correlates with defective PKC-mediated phosphorylation of the CD3 gamma-chain. Despite deficient PKC-mediated TCR down-regulation, the tailless alpha beta TCR still transduces antigenic signals resulting in the production of interleukin-2. Although the TCR tails are not obviously required for signal transduction, the TCR alpha-tail may serve as a targeting domain for PKC-mediated down-regulation of the TCR complex.

Interferon-gamma confers resistance to experimental allergic encephalomyelitis.

In experimental allergic encephalomyelitis (EAE), T cells infiltrate the central nervous system (CNS) and induce inflammation. These CD4+ T cells secrete interferon (IFN)-gamma, levels of which correlate with disease severity, and which is proposed to play a key role in disease induction. Many strains of mice are resistant to EAE. We have studied the effect of deletion of IFN-gamma on the ability to induce EAE in resistant BALB/c-backcrossed mice. As expected, only 0-6% of BALB/c or BALB/c-backcrossed mice developed EAE when immunized with myelin basic protein in adjuvant. Strikingly, abrogation of IFN-gamma expression by targeted disruption of the IFN-gamma gene (GKO mice) converted them to a susceptible phenotype. As many as 71% of these IFN-gamma-deficient mice developed EAE, a frequency comparable to that seen with the susceptible SJL/J strain. In addition, EAE was of unusually high severity in mice lacking IFN-gamma. Immunological characteristics of disease in IFN-gamma-deficient mice were comparable to those seen in susceptible (SJL/J) mice with EAE, including perivascular infiltration in the CNS and order-of-magnitude increases for both CD3 gamma chain and TNF-alpha mRNA levels in the spinal cord. We thus demonstrate that lack of IFN-gamma converts an otherwise EAE-resistant mouse strain to become susceptible to disease. Therefore, in BALB/c mice, IFN-gamma confers resistance to EAE.

Modulation of CD3-gamma gene expression after HIV type 1 infection of the WE17/10 T cell line is progressive and occurs in concert with decreased production of viral p24 antigen.

HIV-1 infection of WE17/10, an IL-2-dependent CD4+ human T cell line, abrogates T cell receptor (TCR)/CD3 expression due to a transcription level defect in the CD3-gamma chain gene. Kinetic examination of surface receptor density reveals that these complexes are progressively reduced early after HIV-1 infection as the cells transition from TCR/CD3hi-->TCR/CD3lo-->TCR/CD3-. The passage from TCR/CD3hi reversible TCR/CD3lo is characterized by a steady decrease in receptor density from 100 to 50% of control values with similar kinetic for all of the viral variants tested. This first phase in TCR/CD3 downmodulation was found to occur in concert with a decrease in viral p24 antigen production. The switch from TCR/CD3- is distinguished by the conversion of individual cells to the receptor negative phenotype. Although broad kinetic differences in this second phase were observed between viral variants, its onset was consistently accompanied by a further reduction in virus production. In some of the HIV-1-infected WE17/10 cell lines, surface receptor expression was spontaneously upregulated during the second phase of infection, reversing the progression from TCR/CD3(-)-->TCR/CD3lo-->TCR/CD3hi. Thus, in HIV-1-infected WE17/10 cells, changes in CD3-gamma gene transcription are accompanied by altered viral p24 antigen production and the resulting modulation of surface receptor expression can be summarized by the formula: TCR/CD3hi reversible TCR/CD3lo reversible TCR/CD3-.

Qualitative and quantitative contributions of the T cell receptor zeta chain to mature T cell apoptosis.

Engagement of the T cell receptor (TCR) of mature T lymphocytes can lead either to activation/proliferation responses or programmed cell death. To understand the molecular regulation of these two fundamentally different outcomes of TCR signaling, we investigated the participation of various components of the TCR-CD3 complex. We found that the TCR-zeta chain, while not absolutely required, was especially effective at promoting mature T cell apoptosis compared with the CD3 epsilon, gamma, or delta chains. We also carried out mutagenesis to address the role of the immunoreceptor tyrosine-based activation motifs (ITAMs) that are the principal signaling components found three times in the TCR-zeta chain and once in each of the CD3 epsilon, gamma, or delta chains. We found that the ability of the TCR-zeta chain to promote apoptosis results both from a quantitative effect of the presence of multiple ITAMs as well as qualitatively different contributions made by individual ITAMs. Apoptosis induced by single chain chimeras revealed that the first zeta ITAM stimulated greater apoptosis than the third zeta ITAM, and the second zeta ITAM was unable to trigger apoptosis. Because microheterogeneity in the amino acid sequence of the various ITAM motifs found in the TCR-zeta and CD3 chains predicts interactions with distinct src-homology-2-domain signaling proteins, our results suggest the possibility that individual ITAM motifs might play unique roles in TCR responses by engaging specific signaling pathways.

The T cell antigen receptor beta chain interacts with the extracellular domain of CD3-gamma.

Selective pairwise interactions between a number of CD3 chains and the clonotypic T cell antigen receptor (TCR-alpha, -beta) chains have recently been reported. What still remains unanswered is the site of interaction between TCR-beta and CD3-gamma chains. To examine the region of interaction between TCR-beta and CD3-gamma chains, a variety of genetically altered TCR-beta and CD3-gamma chains were constructed using recombinant cDNA techniques. Non-T cells (COS-7) were transfected with cDNA constructs, metabolically labelled, and immunoprecipitates were analysed for assembly using non-equilibrium pH gel electrophoresis (NEPHGE)/SDS-PAGE. The results demonstrated that assembly between TCR-beta and CD3-gamma chains was localized to their extracellular domain. These findings, when coupled with the information on pairwise interactions and formation of higher order subcomplexes, extend our model of the structure of the TCR complex.

T cell receptor expression by dendritic epidermal T cells.

Dendritic epidermal T cells (DETC) in murine epidermis express the gamma delta T cell receptor (TCR). The major population of DETC utilize V gamma 5 and V delta 1 without any junctional diversity, corresponding to the earliest fetal thymocytes which express TCR gamma delta. Using PCR, we recently found another population of DETC which express V gamma 1-V delta 6 with junctional diversity in addition to V gamma 5-V delta 1, although they exist in small numbers in normal mice. In athymic nude mice, V gamma 1+ cells also exist. Therefore, this subset of gamma delta T cells is the product of an extrathymic pathway. These V gamma 1+ cells may recognize mycobacterium antigen or heat shock protein (HSP), thus playing an important role in the first defense of the skin. In contrast to normal mice, in nude mice, we could not detect any DETC using anti CD3 epsilon antibody (2C11). In order to solve this puzzle, we examined the components of TCR complex utilizing immunoprecipitation and northern blot analysis. TCR is composed of either alpha beta or gamma delta chains associated with CD3 gamma, delta, epsilon and zeta-zeta chains. By immunoprecipitation of 125I labelled DETC cell lines using anti-CD3 epsilon antibody, we detected gamma delta chains and CD3 gamma and CD3 epsilon chains, but not CD3 delta or CD3 zeta chains. Northern blot analysis showed that these cells express CD3 gamma, epsilon, and zeta chains, but not the CD3 delta chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of Raf with the CD3 delta and gamma chains of the T cell receptor-CD3 complex.

Protein kinases and phosphatases play an important role in signal transduction. In the T cell, activation via the T cell receptor-CD3 complex results in rapid tyrosine phosphorylation of proteins, as well as subsequent increases in serine/threonine phosphorylation. The Raf serine/threonine kinase has been implicated in many receptor signaling pathways, including those of platelet-derived growth factor, epidermal growth factor, insulin, and interleukin-2 receptors. We show here that Raf is associated with the T cell receptor-CD3 complex in unstimulated murine T cells. Using a COS cell expression system, we show that a hypophosphorylated form of Raf specifically associates with the CD3 gamma and delta chains but not with the CD3 epsilon or zeta chains. These results suggest that Raf mediates signal transduction from the gamma and delta chains of the T cell receptor-CD3 complex, analogous to the role played by ZAP-70 in signal transduction mediated by the zeta chain.

CD3 epsilon-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR beta chain expression.

Recent studies have shown that TCR beta chain expression can effect the differentiation of CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) thymocytes. The TCR beta chain is expressed on the surface of DP thymocytes in association with CD3 gamma, delta and epsilon chains, suggesting a potential role for CD3 components in this signaling process. We now report detection of a very low level of surface expression of CD3 epsilon on adult DN RAG-2-/- thymocytes. This surface CD3 epsilon was associated with CD3 gamma and delta chains, as detected by anti-CD3 epsilon immunoprecipitation analyses. Significantly, injection of anti-CD3 epsilon mAb into RAG-2-/- mice led to the accumulation of an IL-2R alpha- CD2+ DP cell population and a nearly 100-fold increase in thymic cellularity to essentially normal levels. Together, these data strongly indicate that TCR beta chain-mediated developmental signals are transduced by CD3 components and provide potential insights into mechanisms by which TCR beta chain expression may effect this process.

Specificity of signal transduction through CD16, TCR-CD3 and BCR receptor chains containing the tyrosine-associated activation motif.

FcR gamma III (CD16) is a member of a family of receptor proteins that bind to Fc regions of IgG molecules. Ligation of CD16 transduces signal and alters tyrosine phosphorylation of proteins in natural killer, B and T cells. However, the identity of protein tyrosine kinases (PTKs) involved in CD16 signaling and the mode of their interaction with the receptor are not completely understood. Here, we show that the transmembrane form of CD16 transfected in a CD3- Jurkat cell line associates with p56lck and ZAP-70 PTKs. Using chimeric proteins consisting of the extracellular domain of CD16 and the cytoplasmic portions of the zeta chain, we find that these PTKs interact with the 17-residue activation motif present in the zeta chain. Substitution of tyrosine residues in the motif with phenylalanine blocks the interaction of PTKs and abrogates signal transduction. Comparative studies demonstrate that similar to zeta, MB1 protein of the IgM receptor also binds to p56lck and ZAP-70 in T cells and induces strong activation responses, whereas CD3 gamma, delta and epsilon chains do not interact with ZAP-70 and transduce weak signals. Together, these results demonstrate that: (i) PTKs bound to CD16 mediate signal transduction, (ii) ZAP-70 plays a crucial role in signaling and (iii) apart from tyrosine residues in the activation motif, other structural constraints also regulate the interaction of PTKs with the receptor molecule.

Induction of CD3δεω) by phorbol 12‐myristate 13‐acetate

The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3 omega chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Ti alpha, CD3 gamma and CG3 zeta chains two- to threefold and the synthesis of Ti beta and CD3 delta epsilon omega complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount of total assembled TcR complexes increased approximately threefold and the maturation of the TcR was not affected as determined by analysis of oligosaccharide side chain processing in the Golgi apparatus. Activation of Jurkat cells with anti-CD3 monoclonal antibody, calcium ionophore, or mitogenic lectins did not affect the synthesis of the TcR components. In other cells studied (the human leukemic T cell line CEM, a panel of variants of the Jurkat T cell line and peripheral blood mononuclear cells) PMA also increased the synthesis of the TcR components. However, for all cell lines studied the amount of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3 omega in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3 delta epsilon omega complexes was immunoprecipitated as compared to the amount obtained from untreated Jurkat cells, and these observations indicate that the CEM cell line may be a qualified candidate for purification of CD3 omega.

CD3 and CD2 antigen-mediated CD3 gamma-chain phosphorylation in permeabilized human T cells. Regulation by cytosolic phosphatases.

The role of cytosolic and membrane-associated phosphatases in regulating dephosphorylation of the CD3 antigen gamma-chain has been investigated using streptolysin-O-permeabilized T lymphoblasts and Jurkat T leukaemia cells. Permeabilization of T cells caused a rapid extrusion of cytosolic type 2A phosphatases, but a membrane-associated phosphorylase phosphatase activity remained inside the cells. This activity had the properties characteristic of type 2A phosphatases, being resistant to inhibition by type 1 phosphatase inhibitors, though it was inhibited in a time-dependent manner by ATP or by non-hydrolysable ATP analogues, but not by GTP, CTP, ITP or PPi. The membrane-associated type 2A phosphatase in permeabilized cells did not dephosphorylate the CD3 antigen gamma-chain, suggesting that cytosolic phosphatases dephosphorylate the gamma-chain in situ. Cross-linking the CD2 and CD3 antigens with a bivalent monoclonal antibody in the absence of cytosolic phosphatases induced marked phosphorylation of the CD3 gamma-chain, immunoprecipitated using a novel gamma-chain peptide analogue directed antiserum (TG1). Phosphorylation was inhibited by a protein kinase C (PKC) pseudosubstrate inhibitor, indicating that CD2/CD3-induced gamma-chain phosphorylation is a PKC-mediated event. Activation of T cells either with phorbol 12,13-dibutyrate or by CD2-CD3 cross-linking caused [32P]Pi incorporation into the same gamma-chain Ser residues. The site-mapping data suggested that PKC in situ may incorporate phosphate at the CD3 gamma-chain Ser-123 and Ser-126 residues, but that phosphate is rapidly lost from Ser-123 by cytosolic phosphatase action. Our findings underline the importance of the dual actions of kinases and phosphatases as potential regulators of T cell antigen-receptor complex function.

Instability of assembled T-cell receptor complex that is associated with rapid degradation of zeta chains in immature CD4+CD8+ thymocytes.

The intracellular fate of newly synthesized T-cell receptor (TCR) chains was compared in CD4+CD8+ (double positive; DP) thymocytes and in CD4+CD8- or CD4-CD8+ (single positive; SP) thymocytes. Purified DP and SP thymocytes from normal adult mice were analyzed by pulse-chase metabolic labeling and immunoprecipitation with specific anti-TCR antibodies. Biosynthesis of invariant chains (CD3 gamma, -delta, -epsilon, and zeta) was comparable between DP and SP thymocytes, whereas DP thymocytes synthesized TCR alpha and TCR beta chains at lower and higher levels than SP thymocytes, respectively. These newly synthesized TCR chains were degraded at different rates in SP thymocytes based on their sensitivities for degradation as previously reported: TCR alpha, TCR beta, CD3 gamma, and CD3 delta chains were rapidly degraded and CD3 epsilon and zeta chains were stable. Although the degradation rates of clonotypic and invariant CD3 chains were similar in DP and SP thymocytes, the zeta subunit was rapidly degraded in DP thymocytes (t1/2, approximately 1.5 hr). Degradation of zeta was inhibited by NH4Cl, implicating lysosomes as the site of degradation. Comparison of TCR subunit assembly in DP and SP thymocytes demonstrated that, despite the same relative rate of formation of TCR complexes in a pulse period (30 min), complete complexes were unstable and degraded during the subsequent 6 hr of chase in DP thymocytes. This contrasted with the stability and a progressive increase in the levels of completely assembled complexes in SP thymocytes. Thus, these results demonstrate that a unique posttranslational regulation operates in the formation of TCR complexes in DP thymocytes and that lack of stability of complete TCR complexes is a crucial mechanism that may account for the limited surface TCR expression on this thymocyte subset.

Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression.

The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex is produced in the endoplasmic reticulum in the absence of CD3-gamma; 2) CD3-zeta does not associate with the Ti alpha beta-CD3 delta epsilon complex; 3) the Ti alpha beta-CD3 delta epsilon complex is not exported from the endoplasmic reticulum to the Golgi apparatus; and 4) CD3-gamma is required for cell surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.

Structural analysis of low TCR-CD3 complex expression in T cells of an immunodeficient patient.

Normal membrane expression of the T cell receptor (TCR) depends on the coordinated synthesis and assembly of all seven proteins composing the complex, i.e. the TCR alpha and beta chains, the CD3 gamma, delta, and epsilon chains, and the zeta-zeta or zeta-eta dimer. In an experimental TCR membrane-defective T cell variant (Sussman, J. L., Bonifacino, J. S., Lippincott-Schwartz, J., Weissman, A. M., Saito, T., Klausner, R. D., and Ashwell, J. D. (1988) Cell 52, 85-95) and in two siblings whose lymphocytes express only a low level of the TCR/CD3 complex (Alarcon, B., Berkhout, B., Breitmeyer, J., and Terhorst, C. (1988) N. Engl. J. Med. 319, 1203-1208), a defect in zeta chain synthesis and/or assembly was thought to account for the defective membrane synthesis of the whole complex. We report on another immunodeficient patient whose T lymphocytes express the T cell receptor at one-tenth of normal fluorescence intensity and are not triggered to proliferate in vitro by anti-CD3 or anti-CD2 antibodies. Biochemical analysis of the patient's surface-iodinated peripheral blood lymphocytes failed to detect TCR alpha and beta, or CD3 gamma, delta, and epsilon proteins but revealed the presence of the zeta homodimer (probably as a result of the high proportion of natural killer cells). In the cytoplasm, TCR alpha and beta proteins and the zeta chain were detected, but, using monoclonal anti-CD3 antibodies, the CD3 gamma, delta, and epsilon chains were not. In addition, the CD3 epsilon chain was not detected with polyclonal antiserum in a very sensitive Western blotting detection system. The zeta chain was shown to be synthesized by the patient's T and natural killer cells. Northern blot analysis revealed normal levels of normal-size TCR beta and CD3 gamma, delta gene-specific mRNAs and decreased levels of TCR alpha mARN; CD3 epsilon gene transcripts were of abnormal size and present in lower than normal amounts. These findings suggest that this defect in T cell receptor-CD3 expression involves a mutation in the CD3 epsilon gene leading to the synthesis of an abnormal and unstable CD3 epsilon subunit.

Identification of a murine monoclonal antibody specific for an allotypic determinant on mouse CD3

A murine monoclonal antibody (mAb; 7D6) that was mitogenic for T cells was derived from 129/Sv animals immunized with a T helper clone from C57BL/6 origin. Fluoresceinated 7D6 labeled T cells from most common mouse strains but not from 129/Sv and LP/J animals, and this labeling was inhibited by the anti-CD3 epsilon mAb 145-2C11. The mitogenicity of 7D6 for T cells had a similar strain specificity. The antibody immunoprecipitated the T cell receptor (TcR) complex from a T cell hybridoma. After dissociation of this immunoprecipitate with detergents, the CD3 gamma and epsilon chains were retained by the 7D6 antibody. Immunoprecipitation data were also obtained with COS cells transfected with the CD3 gamma, delta or epsilon chains alone, in pairs or together. They confirmed that 7D6 bound the CD3 gamma epsilon pair, suggesting that the antibody recognizes a conformational epitope formed by gamma epsilon pairing, whereas 145-2C11 bound both gamma epsilon and delta epsilon pairs. These results, therefore, add to current information about TcR structure and subunit stoichiometry. We have demonstrated that the 7D6 mAb specifically binds to a CD3 dimer comprised of gamma and epsilon chains. We thus provide additional evidence that indicates that two CD3 epsilon chains are found within the receptor, one linked to CD3 gamma and the other to CD3 delta.

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.

Structural comparison of α/β and γ/δ T cell receptor‐CD3 complexes reveals identical subunit interactions but distinct cross‐linking patterns of T cell receptor chains

Here we compare the subunit interactions within the alpha/beta and gamma/delta T cell receptor-CD3 (TcR-CD3) complexes. By using different detergent solubilization protocols, subunit interactions can be defined within such cell surface receptor complexes. It was found that in both TcR-CD3 complexes analogous subunit interactions can be defined: CD3 gamma and CD3 delta each form a stable complex with separate CD3 epsilon chains. Both the TcR gamma/delta and TcR alpha/beta form stable receptor complexes with these CD3 gamma/epsilon and CD3 delta/epsilon subunit. A zeta homodimer is present in these TcR-CD3 complexes. However, biochemical cross-linking results in a covalent bond between the CD3 gamma chain and the TcR beta chain in alpha/beta TcR-CD3 complexes and between the CD3 gamma chain and the TcR delta in gamma/delta TcR-CD3 complexes. This latter observation was independent of the type of TcR gamma/delta studied (e.g. disulfide or non-disulfide linked). These cross-linking results either indicate that the TcR beta and delta chains are each others structural homolog or that in TcR-CD3 complexes both TcR chains (alpha and beta, gamma and delta) are spatially closely associated with the CD3 gamma chain. The identical subunit interactions in the alpha/beta and gamma/delta TcR-CD3 complexes indicate no large structural differences between these receptor complexes but rather suggest a striking conservation of crucial interaction between subunits in the TcR-CD3 complexes.

A specific defect in CD3 gamma-chain gene transcription results in loss of T-cell receptor/CD3 expression late after human immunodeficiency virus infection of a CD4+ T-cell line.

Sequential effects on cellular protein expression following human immunodeficiency virus (type 1) infection of a CD4+ T-cell line in vitro were investigated. Events in the human interleukin 2-dependent helper T-cell line WE17/10 are similar in several respects to the clinical progression in acquired immunodeficiency syndrome. WE17/10 cell infection is characterized by an extended period during which viral replication occurs without accompanying cytotoxicity and with a maximum 30% decrease in surface CD4. Cellular protein expression generally remains unaffected during this first phase of infection. However, after 2-3 months, a severe defect in the expression of the T-cell receptor/CD3 complex both on the cell surface and inside the cell becomes apparent. Other cell membrane markers, such as CD2 and CD25, remain constant throughout the course of infection; after its initial decrease, CD4 remains at 70% of control values. Lack of surface expression of the TCR/CD3 complex is correlated with a specific defect in transcription of the CD3 gamma-chain gene.

Characterization of the CD3 γ and δ invariant subunits of the sheep T cell antigen receptor

The CD3 gamma and delta chains of the sheep T cell antigen receptor have been characterized by cDNA cloning. In comparison to human and mouse protein sequences, short oligopeptide stretches have been deleted from both the gamma and delta chains. However, apart from further variability in the number and position of potential N-linked glycosylation sites, the general structure of the CD3 gamma and delta proteins is markedly conserved. Particularly remarkable are two motifs consisting of 8 and 32 amino acids, respectively, which locate in the C-terminal half of each protein and which have been precisely conserved in both the CD3 gamma and delta chains of all three species. These motifs probably contribute structural properties essential for the function of the CD3/T cell receptor complex. mRNA of the sheep CD3 gamma (1.5 and 1.2 kb) and CD3 delta (1.5 kb) chains are transcribed from closely linked, single-copy genes, suggesting that common mechanisms for the regulation of CD3 genes are also preserved between species.

Inhibition of protein kinase C and anti-CD3-induced Ca2+ influx in Jurkat T cells by a synthetic peptide with sequence identity to HIV-1 gp41.

We have previously shown that a synthetic peptide containing env residues 581-597 from HIV-1 inhibits lymphoproliferation of human PBMC. We have investigated the molecular mechanism(s) by which this HIV-1-derived peptide inhibits CD3-mediated signal transduction. We show that the peptide containing residues 581-597 from the HIV-1 transmembrane protein gp41 specifically inhibited the intracellular Ca2+ influx in Jurkat cells stimulated by the mAb OKT3 whereas it had no effect on the production of inositol triphosphate. In addition, the peptide inhibited protein kinase C (pkC)-mediated phosphorylation of the CD3 gamma-chain in intact cells and directly inhibited partially purified pkC. The inhibition was noncompetitive with respect to the substrates histone and ATP and independent of the regulatory domain of the enzyme. Furthermore, the peptide required internalization for inhibitory activity because no inhibition of lymphoproliferation was observed when cells were treated with peptide at 4 degrees C. Based on these results obtained with the peptide aa581-597, we postulate that the transmembrane protein gp41 of HIV-1 may inhibit pkC activity and thus block pkC-dependent immune function contributing to the immunosuppression of HIV-1-infected individuals.