CD3 Zeta Research Articles

Phospholipase C-gamma 1 association with CD3 structure in T cells.

Recently, we and others have reported tyrosine phosphorylation of phospholipase C-gamma 1 (PLC gamma 1) enzyme after CD3 activation of T cells, and have proposed that PLC gamma 1 mediates signal transduction through the T cell receptor (TCR/CD3). Here, using immunoblotting and immune complex PLC assays, we show that CD3 stimulation of Jurkat cells induces the association of PLC gamma 1 enzyme with CD3 complex. PLC activity is also found to co-precipitate with the CD3 zeta chain from activated cells. In addition, in vitro PLC assays show that CD3 activation leads to about 10-fold stimulation of PLC gamma 1 activity. These results, along with the observation that Jurkat cells preferentially express PLC gamma 1, indicate that PLC gamma 1 participates in CD3 signaling.

Surface expression of alternative forms of the TCR/CD3 complex.

T-cell antigen receptor (TCR) heterodimers of both the alpha beta and gamma delta types are expressed at the surface of T cells only in association with a complex of invariant chains called CD3. The requirement for individual CD3 components to achieve TCR surface expression was examined by cotransfection of a non-T-cell line with TCR alpha and beta, as well as CD3 delta, epsilon, gamma, and zeta, cDNAs. Both transient and stable transfectants expressing TCR and CD3 epitopes at the cell surface were generated. By transfection of TCR and CD3 components in different combinations, the TCR chains, as well as the CD3 epsilon and zeta chains, were each shown to be essential for reconstituting surface expression. On the other hand, CD3 delta and gamma chains could be used alternatively, providing evidence for two different types of TCR/CD3 complexes.

Involvement of a metalloprotease in spontaneous and phorbol ester-induced release of natural killer cell-associated Fc gamma RIII (CD16-II).

Two genes encode the CD16 low affinity IgG FcR. CD16-I (Fc gamma RIII-1) is expressed on PMN as a phosphatidylinositol-glycan anchored glycoprotein. CD16-II (Fc gamma RIII-2) is expressed on NK cells and macrophages as a transmembrane glycoprotein associated with CD3 zeta or Fc epsilon RI-gamma. NK cells spontaneously release soluble CD16-II from the cell surface and this is enhanced by activation with phorbol ester. In this study, we demonstrate that a metalloprotease is involved in the spontaneous and PMA-induced release of CD16-II from NK cells. 1,10-phenanthroline, an inhibitor of Zn(2+)-dependent metalloproteases, efficiently inhibits CD16-II release. 1,7-phenanthroline, an inactive analogue that doesn't chelate Zn2+ or other divalent metal cations, and inhibitors of serine proteases do not affect spontaneous or PMA-induced release of CD16-II. Murine P815 mastocytoma cells transfected with human CD16-II cDNA shed membrane CD16, and 1,10-phenanthroline inhibits this process. P815 transfectants expressing CD16-II molecules with truncated cytoplasmic domains also release soluble receptors, indicating that the cytoplasmic segment of CD16-II is not required for interaction with the protease or the cytoskeleton. By contrast, 1,10-phenanthroline does not inhibit PMA-induced release of CD16-I glycoprotein from PMN, indicating a different mechanism of release for this phosphatidylinositol-glycan anchored molecule. Prior studies have demonstrated that NK cells are activated via the inositol phosphate pathway after engagement of CD16-II by immune complexes or Ig-coated tumor cell targets. A membrane metalloprotease with substrate specificity for CD16-II that is activated by PKC stimulation may provide a mechanism for releasing the immune complex or target from the effector cells and halting signal transduction.

Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes.

In the present report, we demonstrated that modulation of CD26 from T cell surface induced by antiCD26 (1F7) led to enhanced phosphorylation of CD3 zeta tyrosine residues and increased CD4 associated p56lck tyrosine kinase activity. We further showed that CD26 was comodulated on the T cell surface with CD45, a known membrane-linked protein tyrosine phosphatase and that anti-CD26 was capable of precipitating CD45 from T cell lysates. These findings strongly suggest that CD26 may be closely associated with the CD45 protein tyrosine phosphatase on T cell surface and further support the notion that the interaction of CD26 with CD45 results in enhanced tyrosine kinase activity, zeta chain phosphorylation, and T cell activation.

The high affinity Fc epsilon receptor gamma subunit (Fc epsilon RI gamma) facilitates T cell receptor expression and antigen/major histocompatibility complex-driven signaling in the absence of CD3 zeta and CD3 eta

The T cell receptor (TCR) is a molecular complex formed by at least seven transmembrane proteins: the antigen/major histocompatibility complex recognition unit (Ti alpha-beta heterodimer) and the invariant CD3 chains (gamma, delta, epsilon, zeta, and eta). In addition to targeting partially assembled Ti alpha-beta CD3 gamma delta epsilon TCR complexes to the cell surface, CD3 zeta appears to be essential for interleukin-2 production after TCR stimulation with antigen/major histocompatibility complex. The gamma chain of the high affinity Fc receptor for IgE (Fc epsilon RI gamma) has significant structural homology to CD3 zeta and the related CD3 eta subunit. To identify the functional significance of sequence homologies between CD3 zeta and Fc epsilon RI gamma in T cells, we have transfected a Fc epsilon RI gamma cDNA into a T cell hybridoma lacking CD3 zeta and CD3 eta proteins. Herein we show that a Fc epsilon RI gamma-gamma homodimer associates with TCR components to up-regulate TCR surface expression. A TCR composed of Ti alpha-beta CD3 gamma delta epsilon Fc epsilon RI gamma-gamma is sufficient to restore the coupling of TCR antigen recognition to the interleukin-2 induction pathway, demonstrating the functional significance of structural homology between the above receptor subunits. These results, in conjunction with the recent finding that CD3 zeta, CD3 eta, and Fc epsilon RI gamma are coexpressed in certain T cells as subunits of an unusual TCR isoform, suggest that Fc epsilon RI gamma is likely to play a role in T cell lineage function.

Biochemical identification of a direct physical interaction between the CD4: p56lck and Ti(TcR)/CD3 complexes

The CD4 and CD8 antigens function in synergy with the TcR/CD3 complex in the generation of intracellular signals leading to T cell proliferation. The association of the protein-tyrosine kinase p56lck with CD4 and CD8 provides a potential mechanism in the generation of intracellular signals. Several studies have shown that CD4 can co-modulate with TcR/CD3 suggesting that these receptor complexes may associated on the surface of the T cell. Nevertheless, it has proven difficult to formally demonstrate a direct physical interaction between the CD4 and TcR/CD3 complexes using biochemical techniques. In this study, we have used the sensitivity of the in vitro kinase assay to show a direct physical linkage between the CD4:p56lck complex and various CD3 subunits. Immunoprecipitation of CD4 from cell lysates derived from the T lymphoblastoid line HPB-ALL results in the co-purification of p56lck with an additional polypeptide at 20 kDa. Re-precipitation analysis and isoelectric focusing demonstrated that this band corresponds to the CD3 epsilon chain. An alternative approach which involves the labeling of microsomal membranes with [gamma-32P]ATP revealed the presence of CD3 epsilon and zeta chains in anti-CD4 immunoprecipitates. By contrast, we were unable to demonstrate the association of the CD4:p56lck and TcR/CD3 complex in resting peripheral blood lymphocytes. These data indicate that the CD4:p56lck and TcR/CD3 complexes have the ability to form stable complexes on the surface of certain T cell lines.

CD3 eta and CD3 zeta are alternatively spliced products of a common genetic locus and are transcriptionally and/or post-transcriptionally regulated during T-cell development.

The CD3 eta subunit of the T-cell receptor is thought to subserve an important role in signal transduction and possibly T-cell development. Herein we characterize the organization of the mouse CD3 eta gene and show that it is part of one gene locus that also encodes CD3 zeta on chromosome 1. The NH2-terminal sequence of CD3 zeta and CD3 eta, which share the same leader peptide and are identical through amino acid 122 of each mature protein, is encoded by exons 1-7. However, exons 8 and 9 are differentially spliced to give rise to CD3 zeta and CD3 eta: exons 1-8 encode CD3 zeta and exons 1-7 plus 9 encode CD3 eta. RNase protection analysis with RNA from a variety of fetal, neonatal, and adult cell types indicates that expression of both gene products is T-lineage-restricted. Importantly, expression of CD3 zeta and CD3 eta mRNA appears before or on day 16 of fetal gestation. Expression is apparently coordinate since no cell types tested express CD3 zeta or CD3 eta alone. The steady-state level of CD3 zeta mRNA is greater than or equal to 40-60 times that of CD3 eta mRNA. In immature CD4+CD8+CD3low double-positive thymocytes and CD4+CD8-CD3high or CD4-CD8+CD3high single-positive thymocytes, the respective steady-state CD3 zeta and CD3 eta mRNA levels are equivalent, whereas the amount of receptor-associated CD3 zeta and CD3 eta proteins in double-positive thymocytes is approximately 10 times less than in single-positive thymocytes. Nevertheless, the CD3 zeta/CD3 eta protein ratio remains constant in all populations (40-60:1). Furthermore, discordance between mRNA and protein levels for CD3 zeta and CD3 eta is also observed in splenic T cells. Thus, posttranscriptional and/or transcriptional regulatory mechanisms control CD3 zeta and CD3 eta expression during T-cell development.

Rat CD16 is defined by a family of class III Fc gamma receptors requiring co-expression of heteroprotein subunits.

Rat CD16 is defined here by a family of highly homologous class III Fc gamma receptor isoforms. Northern blot and polymerase chain reaction analysis indicate that multiple rtFc gamma RIII alpha isoforms are expressed by rat NK and macrophages contrasting the expression of a single class III receptor isoform by human and mouse NK and macrophages. Analysis of genomic Southern blots and splice variants identified by polymerase chain reaction suggests the existence of several homologous rat Fc gamma RIII alpha genes organized similar to human and mouse class III receptor genes. All rat Fc gamma RIII alpha isoform protein sequences have conventional transmembrane insertion sequences containing the unique LFAVDTGL motif conserved in all other class III Fc gamma and Fc epsilon RI alpha receptor sequences. A model is proposed for the protein-protein interactions between this sequence and the transmembrane sequences of two heteroprotein subunits, Fc epsilon RI gamma and CD3 zeta, known to interact with human and mouse class III receptors. Rat NK, monocytes, and neutrophils were all found to express transcripts for both of these subunits, whereas rat macrophages express only Fc epsilon RI gamma subunit transcripts. Furthermore, the Fc epsilon RI gamma subunit was found to promote the cell surface expression of rtFc gamma RIII alpha isoforms in transfected COS cells.

Interleukin 1‐induced maturation of progenitor thymocytes

Regulation of thymocyte development was assessed by culturing purified CD4-CD8- thymocytes with cytokines. Sorted CD3-CD4-CD8- adult thymocytes responded to the combination of interleukin (IL) 1 plus IL 2 without additional mitogens or co-mitogens with both cellular proliferation and cell surface expression of the T cell receptor (TcR)/CD3 complex. IL2 alone induced neither proliferation nor cell surface TcR/CD3 expression. IL1 alone was sufficient to induce cell surface TcR/CD3 without proliferation. Prior to stimulation with cytokines, the progenitor CD4-CD8- thymocytes accumulated TcR beta and CD3 gamma, delta, epsilon and zeta mRNA but TcR alpha mRNA was not detectable. Stimulation with IL 1 led to a dramatic induction of TcR alpha mRNA without an increase in the other transcripts. These studies suggest that IL 1 regulates the differentiation status of immature adult thymocytes. Nuclear run-on studies suggested that the increase in TcR alpha mRNA accumulation induced by cytokines might result from post-transcriptional accumulation.

Differential signal transduction via T-cell receptor CD3 zeta 2, CD3 zeta-eta, and CD3 eta 2 isoforms.

The T-cell antigen receptor (TCR) consists of an antigen-binding heterodimer, termed Ti, which is noncovalently associated with the invariant CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3 zeta and -eta subunits form either homodimeric or heterodimeric structures in turn associated with the other components of the TCR complex. This feature increases the structural complexity of TCRs by creating "isoforms." Both CD3 zeta and -eta are thought to play an important role in signal transduction triggered by antigen/major histocompatibility complex. To compare signaling functions of TCR isoforms, MA5.8, a CD3 zeta-eta- variant of the cytochrome c-specific, I-Ek-restricted T-cell hybridoma 2B4.11, was stably transfected with cDNAs encoding CD3 zeta and/or CD3 eta, and resulting clones were characterized. The findings indicate that signals inducing Ca2+ mobilization, phosphatidylinositol turnover, and interleukin 2 production are each transmitted by the above TCR isoforms. In contrast, tyrosine phosphorylation of the CD3 zeta subunit but not the CD3 eta subunit follows TCR stimulation. Given the general importance of tyrosine phosphorylation for receptor signaling, it is likely that this difference between TCR isoforms plays a regulatory role in T-lineage function by qualitatively or quantitatively altering signaling events.

Platelet‐induced expression of FcγRIII (CD16) on human monocytes

Monocytes constitutively express Fc receptor (FcR) for IgG type I (CD64) and type II (CD32), but not type III (CD16). Prior studies have indicated that in vitro culture of monocytes results in spontaneous induction of CD16, but this phenomenon has been variable and the mechanism unexplained. Here, we demonstrate that activated platelets are responsible for induction of CD16 on monocytes, as a consequence of TGF-beta release. Local release of TGF-beta by activated platelets at sites of tissue damage may induce CD16 on infiltrating or resident monocytes that in turn facilitate the function of these effector cells. The FcR on these CD16+ monocytes is functionally competent, and enables the monocytes to kill murine anti-CD16 hybridoma cells. CD16 expressed on platelet-activated monocytes is structurally similar to the transmembrane-anchored CD16-II polypeptide expressed on natural killer (NK) cells. However, whereas CD16-II on NK cells is co-associated with CD3 zeta, we were unable to detect CD3 zeta transcript or protein in monocytes. Transcripts for the gamma subunit of the high-affinity IgE FcR (Fc epsilon RI-gamma) were detected in monocytes, and presumably gamma proteins are co-associated with CD16-II in these cells. Nucleotide sequence analysis of Fc epsilon RI-gamma cDNA derived from both NK cells and cultured monocytes indicated identity with the structure previously cloned from basophils. Since CD16+ monocytes can kill anti-CD16 hybridoma cell targets in the absence of CD3 zeta, these results indicate that CD3 zeta is not essential for signal transduction in CD16-II-mediated cytotoxicity.

T cell receptor-independent CD2 signal transduction in FcR+ cells.

CD2 subserves both adhesion and signal transduction functions in T cells, thymocytes, and natural killer (NK) cells. In mature T lymphocytes, CD2-mediated signaling function apparently requires surface expression of T cell receptors (TCRs). In contrast, in CD2+ CD3- NK cells and thymocytes, signal transduction through CD2 is TCR independent. To resolve this paradox and characterize TCR-independent triggering mechanisms, we transfected a human CD2 cDNA into a murine mast cell line, C1.MC/57 (Fc epsilon RI+, Fc gamma RII+, Fc gamma RIII+), which is known to produce interleukin 6 (IL-6) as well as release histamine in response to crosslinking of Fc epsilon RI. In the CD2 transfectant, a combination of anti-T11(2) + anti-T11(3) monoclonal antibodies (mAbs) induced a rise in intracellular free calcium [( Ca2+]i), IL-6 production, and histamine release. As expected, no activation was mediated by the same mAbs in C1.MC/57. F(ab)'s fragments of the activatory combination of anti-T11(2) + anti-T11(3) mAbs induced IL-6 in the CD2-transfected mast cells, demonstrating an Fc gamma receptor ectodomain-independent triggering mechanism. In addition, either intact anti-T11(2) or anti-T11(3) IgG alone, which failed to induce [Ca2+]i mobilization in the transfectant, was able to induce IL-6 production. A mAb directed against both Fc gamma RII (previously denoted as Fc gamma RIIb) and Fc gamma RIII (previously denoted as Fc gamma RIIa) inhibits this induction. These results indicate that: (a) Ca2+ mobilization is not essential for IL-6 production; and (b) crosslinking of CD2 and Fc gamma receptors via intact anti-CD2 IgG stimulates IL-6 production. Thus, CD2-mediated IL-6 production occurs by both Fc receptor ectodomain-independent as well as Fc receptor ectodomain-dependent mechanisms in these nonlymphoid cells. Northern blot analysis demonstrates that although the mast cells do not express CD3 zeta or CD3 eta mRNA, they express Fc epsilon RI gamma mRNA. The latter is a known component of Fc gamma RIII as well as Fc epsilon RI, has significant homology to CD3 zeta/eta, and is thought to have a signal transduction function. In these mast cells, CD2 signaling machinery does not require CD3 zeta/eta and may be linked to the Fc epsilon RI gamma subunit. We predict that this subunit or a related structure may confer a TCR-independent signal transduction pathway upon CD2 in CD3- NK cells, thymocytes, and certain B lymphocytes.

Analysis of Fc gamma RIII (CD16) membrane expression and association with CD3 zeta and Fc epsilon RI-gamma by site-directed mutation.

Two genes encode Fc gamma RIII (CD16), a low affinity FcR for IgG. CD16-I is expressed as a phosphatidylinositol glycan-anchored membrane glycoprotein on neutrophils, whereas CD16-II is a transmembrane-linked glycoprotein on NK cells. Membrane anchoring is determined by codon 203. Site-directed mutation of codon 203 and transient expression of these cDNA in COS-7 cells indicated that Phe, Ile, Leu, and Val permit transmembrane expression, whereas Ser, Thr, Tyr, Asn, Gly, Ala, Asp and Lys enable phosphatidylinositol-glycan attachment. Thus, the involvement of amino acid 203 in membrane anchoring cannot be explained simply on the basis of size, charge, or polarity of the amino acid side groups at this site. Efficient expression of CD16-II in COS-7 cells requires co-transfection with either CD3 zeta or Fc epsilon RI-gamma. Truncation of the cytoplasmic segment of CD16 failed to affect association with CD3 zeta. CD3 zeta and Fc epsilon RI-gamma with truncated cytoplasmic segments were also able to facilitate membrane expression of CD16-II, implicating the transmembrane segments as the interaction site between CD16-II and CD3 zeta or Fc epsilon RI-gamma. Prior studies have suggested that the acidic residue in the CD3 zeta transmembrane segment may be important for the association of CD3 zeta complexes. Although site-directed mutation of CD3 zeta-Asp36 to Glu, Leu, or Val retained the ability to permit membrane expression of CD16-II, quantitatively the wild-type CD3 zeta-Asp36 provided optimal levels of expression, consistent with conservation of this amino acid in mouse and human CD3 zeta.

Genetic reconstitution of the T cell receptor (TcR) α/β heterodimer restores the association of CD3 ζ2 with the TcR/CD3 complex

The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3 zeta. In its absence, a pentameric complex (TcR alpha/beta/CD3 gamma delta epsilon) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3 zeta by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 zeta 2 with the TcR/CD3 complex is dependent on the presence of both the TcR alpha and beta polypeptide chains. Despite wild-type levels of the CD3 zeta protein in a TcR alpha-negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 zeta 2 and consisted of beta gamma delta epsilon and beta 2 gamma delta epsilon. Upon transfection of the mutant with a TcR alpha cDNA, a TcR/CD3 complex which contained CD3 zeta 2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 epsilon-specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 zeta 2 was not interacting with the TcR alpha protein alone, implying that a conformation provided by either the TcR alpha/beta heterodimer or the TcR alpha/beta/CD3 gamma delta epsilon complex was necessary for the association of CD3 zeta 2. Transfection studies performed in a TcR alpha/beta-negative murine T-T hybridoma confirmed the requirement of both the TcR alpha and beta proteins in CD3 zeta 2 binding. We conclude that the TcR alpha and beta chains harbor polypeptide sequences essential for the association of CD3 zeta 2 with the TcR/CD3 complex.

Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells

The T-cell antigen receptor (TCR) consists of a glycoprotein heterodimer (alpha/beta or gamma/delta) which is non-covalently associated with at least four or five invariant polypeptides (CD3 gamma, delta, epsilon, zeta and eta). In T-cell variants lacking TCR alpha, beta or zeta, it has been shown that incomplete TCR/CD3 complexes are retained within the cell. To examine requirements for cell surface expression of TCR/CD3, we transfected COS monkey kidney cells with cDNAs encoding TCR alpha, beta and CD3 gamma, delta, epsilon and zeta. We report that cell surface appearance of TCR/CD3 on COS cells requires coordinate expression of all six proteins. In the absence of the zeta chain, subcomplexes comprising from two to five chains were readily demonstrable in COS cells, but they failed to reach the cell surface or to acquire N-linked oligosaccharide side chains indicating failure to reach the medial Golgi. Pulse-chase metabolic labelling of transfected COS cells showed that three chains (CD3 gamma, CD3 epsilon, and zeta) were stable while three (TCR alpha, TCR beta and CD3 delta) were rapidly degraded. In two- and three-chain co-transfections specific intracellular subcomplexes were formed between TCR alpha and CD3 gamma, TCR alpha and CD3 delta, or TCR beta and CD3 epsilon. Binary subcomplexes having at least one stable chain (CD3 epsilon - TCR beta) were stable while one formed by two unstable chains (TCR alpha - CD3 delta) was still degraded. Assembly of the TCR/CD3 complex in COS cells thus appears centered around the metabolically stable CD3 gamma and CD3 epsilon proteins. Site-specific mutations of the negatively-charged transmembrane amino acid of residues of the CD3 chains to alanines served to either abolish (for TCR alpha - CD3 delta and TCR beta - CD3 epsilon) or diminish (for TCR alpha -CD3 gamma) these TCR-CD3 interactions. These mutations had no effect, however, on CD3-CD3 interactions or upon synthesis, metabolism, or intracellular distributions of the CD3 proteins. The transmembrane domains of CD3 gamma, delta, and epsilon thus appear to play a major role in associations of CD3 with TCR chains.

In vitro expansion of CD3/TCR- human thymocyte populations that selectively lack CD3 delta gene expression: a phenotypic and functional analysis.

Highly purified CD1-3-4-8- human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3 epsilon chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIL-2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3-TCR-. The remaining cells (5-40%) were represented by CD3+ TCR gamma/delta+ (BB3- A13+) cells. Further removal of CD3+ TCR-gamma/delta+ cells resulted in highly purified CD3- populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR-alpha/beta+ cells could be detected, thus indicating that PMA did not affect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3- thymocytes. Surface marker analysis of cultured CD3- thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent. Importantly, these cells were different from peripheral NK cells, as 80-95% of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the Fc gamma R+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) (but not IL-2) production by CD3- thymocytes. Cloning of fresh CD1-3-4-8-thymocytes in the presence of PMA and rIL-2 resulted in CD3-CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3 zeta, epsilon, and gamma transcripts, while CD3 delta was undetectable. Mature transcripts for both gamma and delta TCR chains could be detected, whereas no TCR-alpha mRNA and only a truncated (1.0 kb) form of TCR-beta mRNA were revealed.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of thymus-derived lymphocytes expressing Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta antigen receptor isoforms: analysis by gene transfection.

To characterize the function of the CD3 eta subunit of the T cell receptor (TCR), we have used cDNAs encoding CD3 zeta, CD3 eta, or both to reconstitute a variant of a cytochrome c-specific, I-Ek-restricted murine T cell hybridoma, termed MA5.8, which lacks CD3 zeta and CD3 eta proteins. We provide direct evidence that assembly and surface expression of TCRs can be mediated by either of these subunits separately or together. However, the level of TCR expression on zeta transfectants is up to one order of magnitude greater than that on eta transfectants, implying that CD3 eta is weakly associated with the pentameric Ti alpha-beta CD3 gamma delta epsilon complex and/or inefficient at salvaging the incomplete TCR from lysosomal degradation. As a component of the TCR, the CD3 eta subunit preferentially forms a heterodimer with CD3 zeta, but is also able to form a CD3 eta-eta homodimer. Crosslinking of Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta, or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta TCR isotypes with anti-CD3 epsilon monoclonal antibody or a cytochrome c peptide epitope on I-Ek antigen-presenting cells mediates signal transduction resulting in reversible cell-cycle arrest of transfected clones. Given the potential for diversity of signals generated by these functional TCR isotypes and the expression of the CD3 eta gene product in the thymus, CD3 eta is likely to play a role in selection and/or activation of thymocytes during development.

CD3 zeta subunit can substitute for the gamma subunit of Fc epsilon receptor type I in assembly and functional expression of the high-affinity IgE receptor: evidence for interreceptor complementation.

The high-affinity receptor for IgE (Fc epsilon RI) is a four-subunit structure consisting of three distinct polypeptides: the IgE-binding alpha chain, the four-fold membrane-spanning beta chain, and the disulfide-linked gamma-gamma homodimer. cDNAs encoding each subunit have previously been isolated. Here we show that microinjection of Xenopus oocytes with a mixture of in vitro transcribed RNAs encoding each subunit results in expression of IgE receptors at the oocyte surface as detected by binding of IgE or anti-Fc epsilon RI alpha subunit monoclonal antibody to intact oocytes. Surface expression of Fc epsilon RI requires injection of all three subunits (alpha, beta, and gamma) RNAs. In particular, omission of Fc epsilon RI gamma RNA from the mixtures abolishes surface binding of either IgE or anti-Fc epsilon RI alpha monoclonal antibody to microinjected oocytes. However, addition of CD3 zeta RNA to Fc epsilon RI alpha and Fc epsilon RI beta RNAs restores IgE receptor surface expression when this combination is microinjected into oocytes. Metabolic labeling and immunoprecipitation of oocyte microinjected with a mixture of CD3 zeta plus Fc epsilon RI alpha and Fc epsilon RI beta RNAs reveals a noncovalent association between the CD3 zeta-zeta disulfide-linked homodimer and Fc epsilon RI alpha-beta. These results provide direct evidence for the functional relatedness of CD3 zeta and Fc epsilon RI gamma.

Human natural killer cells and mature T lymphocytes express identical CD3ζ subunits as defined by cDNA cloning and sequence analysis

In order to characterize the CD3 zeta-related protein found in human natural killer (NK) cells and compare it with CD3 zeta expressed in T lymphocytes, the present study was performed. A polyclonal CD3-CD16+NK population displaying a strong non-major histocompatibility complex-restricted cytotoxic activity against the NK target K-562 was isolated and a product corresponding to CD3 zeta amplified using the polymerase chain reaction method. This 0.6-kb product was present in similar amounts in NK cells and T cells. In contrast, a product corresponding to CD3 delta was amplified from T lymphocytes exclusively. Thus, the CD3 zeta product detected in NK cells did not originate from contaminating T cells. DNA sequence analysis of two independent polymerase chain reaction products from the NK cells demonstrates that human NK cells and mature T cells share a CD3 zeta subunit with an identical primary amino acid sequence. The nucleotide sequence of a third NK-derived cDNA revealed an insertion of a CAG triplet encoding an additional glutamine residue in the cytoplasmic domain. Since this residue is encoded by nucleotides at a putative RNA splice junction, it possibly results from a difference in pre-mRNA splicing. Taken together, these data show that CD3 zeta is not structurally distinct in NK cells and in T lymphocytes.

Assembly and function of the T cell antigen receptor. Requirement of either the lysine or arginine residues in the transmembrane region of the alpha chain.

The T cell receptor (TCR) for antigen consists, on the majority of peripheral lymphocytes, of an immunoglobulin-like, disulfide-linked heterodimeric glycoprotein: the alpha and beta chain. These proteins are noncovalently linked to at least four nonvariant proteins which comprise the CD3 complex: CD3 gamma, delta, epsilon, and zeta. Whereas the TCR alpha and beta proteins have positively charged residues in the transmembrane region, all the CD3 proteins have similarly placed negatively charged amino acid residues. It has been suggested that these basic and acidic amino acid residues may play an important role in TCR.CD3 complex assembly and/or function. In this paper, the structural and functional role of the lysine and arginine residues of the TCR alpha chain was addressed using oligonucleotide mediated site directed mutagenesis. The Arg256 and Lys261 residues of the TCR alpha cDNA of the HPB-ALL cell line were mutated to either Gly256 and/or Ile261. The altered cDNAs were transfected into a TCR alpha negative recipient mutant cell line of REX, clone 20A. Metabolic labeling of the T cell transfectants showed that mutation of either the Arg256 or Lys261 amino acid residues had no effect on the ability of the TCR alpha chain to form either a heterodimer with the TCR beta chain or a complex with the CD3 gamma, delta, and epsilon proteins. Consequently, the Arg256 to Gly256 and Lys261 to Ile261 mutations did not prevent the formation of a mature, functional TCR.CD3 complex on the cell surface as determined by immunofluorescence, cell surface radioiodination, and the ability of the transfectants to mobilize intracellular calcium after stimulation with a mitogenic anti-CD3 epsilon monoclonal antibody. In contrast, a mutant cDNA in which both the Arg256 and Lys261 residues were mutated to Gly256 and Ile261, respectively, failed to reconstitute the cell surface expression of the TCR.CD3 complex and, consequently, the ability to respond to mitogenic stimuli. In the absence of both the Arg256 and Lys261 residues, TCR alpha beta heterodimer formation was not observed. Cotransfection studies in COS cells showed that the failure of assembly of a heterodimer was likely due to an inability of the mutated TCR alpha chain to form a subcomplex with either the CD3 gamma, delta, epsilon, or zeta proteins.(ABSTRACT TRUNCATED AT 400 WORDS)