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PubMed1987

Tissue distribution and biochemical and functional properties of Tp55 (CD27), a novel T cell differentiation antigen.

Two monoclonal antibodies (CLB-CD 27/1 and CLB-CD 27/2) were raised against a novel determinant on human T lymphocytes. One of these antibodies, CLB-CD 27/1 (clone 9F4), was grouped by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens together with three other monoclonal antibodies (VIT 14, OKT 18A, and S152) in the new cluster CD27. In this paper we show that antibodies belonging to this cluster recognize an antigen present on a large subset of peripheral T lymphocytes and most medullary thymocytes. At least two different nonoverlapping epitopes were identified with directly labeled monoclonal antibodies. Immunoprecipitation studies indicate that the target antigen of CD27 antibodies is a polypeptide of 55 kDa, which appears in the form of a disulfide-linked homodimer on the T lymphocyte membrane (Tp55). Stimulation of T cells via the T3/T cell antigen-receptor complex, with either phytohemagglutinin or CD3 monoclonal antibodies, resulted in a fivefold increase in the membrane expression of Tp55, whereas activation by phorbol myristate acetate caused a marked down-regulation. Moreover, an additional molecule of 32 kDa was precipitated from the membrane of activated but not of resting T cells. Addition of CD27 antibodies to cultures stimulated with either phytohemagglutinin or CD3 monoclonal antibody led to enhanced proliferation, whereas no effect was observed in phorbol myristate acetate or interleukin 2-stimulated cultures. The possible role of the Tp55 antigen in T cell activation is discussed.

Antibodies, Monoclonal↗

PubMed1999

Molecular interactions between extracellular components of the T-cell receptor signaling complex.

The structural and biochemical basis of antigen recognition by the T-cell receptor (TCR)-CD3 signaling complex has been illuminated greatly over the past few years. Structural biology has contributed enormously to this understanding through the determination of crystal structures of many of the individual components of this complex, and some of the complexes. A number of general principles can be derived for the structure of the alpha beta TCR and its interaction with peptide-major histocompatibility complex (pMHC) in class I systems, as well as interaction of the CD8 co-receptor with MHC. Large buried surface areas within the protein-protein interfaces, and varying degrees of shape complementarity appear critical for modulating the stability of the multicomponent, low-affinity macromolecular complexes consisting of TCR, pMHC, CD8 or CD4, and CD3 gamma, delta, epsilon and zeta. Significant structural alterations in TCR and pMHC, upon complex formation, hint at an as yet unclear role for conformational change in both recognition and activation. Subtle chemical alterations in key peptide residues which contact the TCR can have dramatic agonist or antagonist effects on receptor activation, which correlate only loosely with the TCR/pMHC complex affinity, implying an ability of the signaling complex to "sense" fine differences in the interface. The stoichiometry of an activated TCR signaling complex is still an unresolved issue, as is the structure and disposition of the CD3 components. However, functional experiments are bridging this gap and providing us with preliminary working models of the multimeric assemblies.

Animals↗

PubMed2005

An orderly inactivation of intracellular retention signals controls surface expression of the T cell antigen receptor.

Exit from the endoplasmic reticulum (ER) is an important checkpoint for proper assembly of multimeric plasma membrane receptors. The six subunits of the T cell receptor (TCR; TCRalpha, TCRbeta, CD3gamma, CD3delta, CD3epsilon, and CD3zeta) are each endowed with ER retention/retrieval signals, and regulation of its targeting to the plasma membrane is therefore especially intriguing. We have studied the importance of the distinct ER retention signals at different stages of TCR intracellular assembly. To this end, we have characterized first the presence of ER retention signals in CD3gamma. Despite the presence of multiple ER retention signals in CD3gamma, epsilongamma dimers reach the cell surface when the single CD3epsilon ER retention signal is deleted. Furthermore, inclusion of this CD3epsilon mutant promoted plasma membrane expression of incomplete alphabetagammaepsilon and alphabetadeltaepsilon complexes without CD3zeta. It therefore appears that the CD3epsilon ER retention signal is dominant and that it is only overridden upon the incorporation of CD3zeta. We propose that the stepwise assembly of the TCR complex guarantees that all assembly intermediates have at least one functional ER retention signal and that only a full signaling-competent TCR complex is expressed on the cell surface.

Animals↗

PubMed1991

Differential staining of human alpha beta and gamma delta T cells by the fluorescein conjugate of an anti-CD3 monoclonal antibody.

The enumeration of total T cells, an important function of the clinical immunology laboratory, utilizes antibodies to CD3, the macromolecular complex associated with the antigen-specific receptors of T cells. We compared the ability of some commonly employed commercial anti-CD3 reagents to stain human peripheral blood lymphocytes. Surprisingly, the fluorescein isothiocyanate (FITC) conjugate of Coulter clone T3 (FITC-T3) stained most T cells brightly, but selectively stained gamma delta T cells very dimly or not at all. In contrast, the other anti-CD3 reagents studied (FITC-Leu 4, PE-T3, PE-Leu 4, and indirectly labelled T3 and Leu 4) stained all T cells equivalently. Dual-colour flow cytometric analysis with FITC-T3 and PE-Leu 4 readily demonstrated a FITC-T3-/PE-Leu 4+ population of T cells. This unique population stained dimly or not at all with a combination of anti-CD4 and anti-CD8 monoclonal antibodies and positively with the pan-gamma delta T cell antibody TCR delta 1. Moreover, an excellent correlation was found between the number of FITC-T3-/PE-Leu 4+ cells and the number of TCR delta 1+ cells in 32 normal individuals. Thus, the FITC-T3-/PE-Leu 4+ phenotype accurately marks all gamma delta T cells. In contrast to FITC-T3, both PE-conjugated and unconjugated T3 stained gamma delta T cells brightly. Therefore, T3 binds to an epitope present on all T cells, but fluoresceinylation specifically attenuates this antibody's ability to bind to gamma delta T cells. These findings indicate that the use of FITC-T3 can result in a significant and variable underestimation of peripheral blood T cell number and demonstrate further that the CD3 complexes of human alpha beta and gamma delta T cells are significantly different.

Adult↗

PubMed2002

Cyclosporin-A differentially affects apoptosis during in vivo rat thymocyte maturation.

Maturation arrest and interference with selection are two well-documented effects of cyclosporin-A (CsA) on the thymus. We recently hypothesized that these effects are related and owing to the reduced T-cell receptor (TCR)-CD3 complex-mediated signal transduction in thymocytes upon CsA treatment. In this hypothesis, the maturation arrest is the result of the additional depletion of thymocytes that normally survive by positive selection, whereas the impaired self-tolerance induction is caused by an increased survival of thymocytes that normally undergo negative selection. In this view, it is anticipated that CsA differentially affects thymocyte apoptosis during in vivo thymocyte maturation. Indeed, we report in this study a strong increase in apoptotic cells in the thymic cortex on in situ analysis. Simultaneously, the number of apoptotic cells had decreased at the cortico-medullary zone which is held to be the site for negative selection. Rapamycin (Rapa) also interferes with thymocyte maturation by inhibiting cytokine-driven proliferation. Hence, Rapa preferentially affects the early maturational stages of thymocyte development and is considered not to alter thymocyte selection and subsequent apoptotic events. Indeed, the number of apoptotic events appears not to be altered. However, possibly owing to the decrease in cortical macrophages, the apoptotic cells revealed an atypical enumeration around blood vessels. Taken together, our results favour the hypothesis that the dominant effect of CsA on the thymus is the reduction of the TCR-CD3 complex-mediated signal transduction in thymocytes upon interaction with stromal cells. Furthermore, the preferential localization of apoptotic cells next to blood vessels upon Rapa administration may indicate that endothelial cells are a back-up system for the removal of apoptotic cells.

Animals↗

PubMed1988

CD3-associated alpha/beta and gamma/delta heterodimeric receptors are expressed by distinct populations of CD4- CD8- thymocytes.

Immature double negative (DN) CD4-8- thymocytes expressing the CD3 molecular complex can be subdivided into two distinct subsets based on expression of the B2A2 antigen. Thus, B2A2+ CD3+ DN thymocytes express CD3-associated 35 kDa and 45 kDa polypeptide chains characteristic of a gamma/delta T cell receptor, whereas B2A2- DN thymocytes express predominantly 38 to 43 kDa CD3-associated structures typical of alpha/beta TCR. At the mRNA level, B2A2+ DN thymocytes express full length gamma and delta transcripts but no alpha message and only short (1 kb) B transcripts. In contrast, the B2A2- DN subset expresses full length alpha and beta transcripts and minimal levels of delta transcripts. Interestingly, B2A2- DN thymocytes, unlike mature T cells, also express abundant levels of full length TCR gamma mRNA, perhaps suggesting that these cells represent an early stage after divergence of the gamma/delta and alpha/beta T cell lineages.

Animals↗

PubMed1987

Acute leukemia expressing the gamma gene product of the putative second T cell receptor.

Early thymus-derived lymphocytes bearing the T gamma gene product in association with the CD3(T3) complex have recently been described. We report a unique case of human acute lymphoblastic leukemia with a CD2+, CD3+, CD4-, CD5+, CD7+, CD8-, WT31- phenotype. These cells were found to have T gamma gene rearrangement and T gamma transcripts in absence of T alpha or T beta rearrangement or transcripts. Immunoprecipitation studies with anti-CD3 antibodies showed a 43-kD protein associated with T3; this 43-kD protein is also precipitated with antiserum raised against synthetic peptides representing the constant region of the putative T gamma protein.

Antigens, Surface↗

PubMed1994

Differential activation of p21ras in CD45RA+ and CD45RO+ human T lymphocytes.

CD45RA+ and CD45RO+ human T cells differ in their functional responses to different forms of mitogenic stimulation. However, little is known about the intracellular signaling events related to the functional differences in these two subsets. In the present study, we examined the ability of different Abs to the TCR/CD3 complex to activate the p21ras proto-oncogene product in CD45RA+ and CD45RO+ T cells. Stimulation with soluble OKT3 anti-CD3 mAb induced activation of p21ras in CD45RA+ cells but not in CD45RO+ cells, although soluble anti-CD3 was able to induce both activation of CD3-associated kinases and IL-2 responsiveness in CD45RO+ cells. Similar results were obtained by using UCHT1, a different anti-CD3 Ab. In contrast, cross-linked anti-CD3 or the combination of phorbol ester and ionomycin were found to activate p21ras equally in both T cell subsets. Our results demonstrate a selective inability of CD45RO+ human T cells to activate p21ras in response to soluble anti-CD3 mAb.

Antibodies, Monoclonal↗

PubMed1997

Pre-TCR/CD3 and TCR/CD3 complexes: decamers with differential signalling properties?

Currently, the favoured model for the structure of the T-cell receptor (TCR)/CD3 complex proposes a monovalent TCR alpha beta/CD3 gamma epsilon delta epsilon zeta zeta octameric complex. However, other biochemical, structural and functional data point to a bivalent (TCR alpha beta)2/CD3 gamma epsilon delta epsilon zeta zeta decameric organization, which has a favourable neutral transmembrane net charge within the complex. Here, Heinz Jacobs proposes a model for the organization of the pre-TCR and discusses its function in signal transduction.

CD3 Complex↗

PubMed1993

Stimulus-dependent modulation of human B cell function by T cell clones.

T cells exert both positive and negative regulatory effects on B cell function. To determine whether the nature of the stimulus could alter the immunoregulatory effects of T cells, the capacity of a battery of human T cell clones to modulate B cell function after stimulation with either pokeweed mitogen or a mAb to the CD3 molecular complex was examined. It was observed that most clones induced B cell differentiation when stimulated with immobilized mAb to CD3 (64.1) regardless of their phenotype. Moreover, the majority of clones (42 of 51) augmented the generation of immunoglobulin-secreting cells (ISC) supported by anti-CD3-stimulated blood CD4+ T cells. By contrast, none of the clones induced B cell differentiation when stimulated with PWM and 48 of 51 clones suppressed the generation of ISC induced by blood CD4+ T cells. This suppression could not be accounted for by the depletion of essential molecules or factors or by secretion of suppressive factors. Suppressive activity of clones did not correlate with the CD4 or CD8 phenotype and was not overcome by the addition of supernatants generated from mitogen-stimulated T cells or recombinant IL-2. Suppression by most clones, however, was abrogated when the clones were treated with mitomycin C or irradiated. A number of suppressive mechanisms by individual PWM-stimulated clones was identified, including direct inhibition of B cell function by cytotoxic and non-lytic means and suppression of helper T cell function. The failure of anti-CD3-stimulated clones to suppress the differentiation of B cells appeared to reflect the capacity of this stimulus to induce apoptosis by the clones after initial activation. These results indicate that T cell clones can provide help for B cell differentiation or can suppress B cell function by a variety of mechanisms depending upon the mode of stimulation.

Animals↗

PubMed1989

A case of CD3+, CD4-, CD8-, WT31- acute T-cell leukemia.

The article reports a case of acute T-cell leukemia (T-ALL) with the unusual CD3+, WT31 phenotype. On surface marker analysis, the blast cells were found to be CD7+, CD2-, CD5-, CD1-, CD4-, CD8-, CD3+ and negative for B-lymphoid and myeloid lineage. The cells had both TCR beta and TCR gamma gene rearrangement but had negative results with the monoclonal antibody WT31, which reacts with a framework epitope on the T alpha/beta heterodimer (Ti) of the conventional T-cell receptor (TCR). This suggests an association of the CD3 molecular complex with a different polypeptide (the alternate TCR gamma). In two similar reported cases of T-ALL (WT31-, CD3+, CD4-, CD8-), the CD3 molecular complex was found to be associated with a product of the T gamma gene.

Antigens, Differentiation, T-Lymphocyte↗

PubMedDate not supplied

Antibodies to the T-cell receptor (CD3) induce LFA-1/Mac-1-dependent mononuclear cell adhesion to human arterial endothelium.

Monoclonal antibodies to the CD3 determinant on T lymphocytes have been used extensively as immunosuppressive agents. The T-cell receptor forms a complex with CD3 so that antibodies to CD3 mimic T-cell receptor cross-linking by human leukocyte antigens. Both CD3 and T-cell receptor cross-linking have been associated with lymphocyte activation and expression of the LFA-1 adhesion ligand, which leads to adhesion to intercellular adhesion molecule 1 immobilized on artificial surfaces. We sought to determine if anti-CD3 antibodies would increase adhesion of heart transplant recipient peripheral blood mononuclear cells to confluent human aortic endothelial cells by a similar mechanism. Peripheral blood mononuclear cells were incubated for 30 minutes with monoclonal CD3 or CD3 + CD18 antibody, before a 15-minute incubation with unstimulated human aortic endothelial cells. Mononuclear cell adhesion doubled after anti-CD3 antibody preincubation from 15.1 +/- 2.1 to 29.3 +/- 6.7 cells/high-power field (p = 0.002). This increase was abolished by coincubation with anti-CD18 (7.1 +/- 3 cells/high-power field; p = 0.0002). The major increase was the result of increased lymphocyte adhesion (3.4 +/- 0.6 to 14.8 +/- 6.7 cells/high-power field; p = 0.02); however, monocyte adhesion also increased from 11.7 +/- 1.5 to 14.5 +/- 1.6 cells/high-power field (p = 0.04). Anti-CD18 antibodies markedly reduced binding of both cell types. Cross-linking of the T-cell receptor by antibodies to CD3 causes acute adhesion of mononuclear cells (particularly lymphocytes) to arterial endothelium. Increased adhesion is mediated through CD18 (LFA-1/Mac-1).

Antibodies, Monoclonal↗

PubMed1993

Inhibition of anti-CD3 monoclonal antibody-induced T-cell proliferation and interleukin-2 secretion by physiologic combinations of dexamethasone and prostaglandin E2.

1. The purpose of these studies was to characterize further previous observations from our laboratory indicating that physiologic concentrations of dexamethasone (DEX) and prostaglandin E2 (PGE2) added together result in synergistic inhibition of the proliferative response of T cells stimulated via the T-cell receptor CD3 signaling complex (TCR/CD3). 2. Various physiologic concentrations of DEX and PGE2 were added to T cells stimulated with immobilized anti-CD3 monoclonal antibody (mAb) and cultured at optimal and suboptimal cell densities. The results demonstrate that the proliferative response of anti-CD3 mAb-stimulated T cells cultured at a suboptimal cell density is more suppressed than that of T cells cultured at optimal concentrations. 3. The proliferative response of CD4+ T cells to immobilized anti-CD3 mAb was also determined in the presence of PGE2 and DEX. The data indicate that the CD4+ subset of T cells is more sensitive to the synergistic antiproliferative effects of DEX and PGE2 compared to whole T-cell populations. 4. Various concentrations of DEX and/or PGE2 were added to T cells stimulated with anti-CD3 mAb and the secretion of interleukin-2 (IL-2) was determined. The results demonstrate that concentrations of DEX and PGE2 which individually do not significantly suppress IL-2 synthesis act together to inhibit the synthesis of IL-2 synergistically. 5. The addition of an exogenous source of recombinant IL-2 (rIL-2) to T cells stimulated in the presence of DEX and PGE2 completely reversed the synergistic antiproliferative effect of these two compounds. This reversal was even more pronounced with T cells cultured at a suboptimal cell density. Additionally, PGE2 and DEX did not affect expression of the IL-2 receptor (IL-2R), as measured by upregulation of the alpha chain, on anti-CD3 mAb stimulated T cells. 6. Collectively these data indicate that physiologic concentrations of PGE2 and DEX, which alone have no effect on anti-CD3 mAb-induced T-cell proliferation, act synergistically to inhibit T-cell proliferation as well as IL-2 synthesis.

Antibodies, Monoclonal↗

PubMed1998

Activation-induced apoptosis of mature T cells is dependent upon the level of surface TCR but not on the presence of the CD3 zeta ITAM.

Activation-induced cell death (AICD) occurs primarily in recently activated T cells after a second TCR triggering. Since a threshold in the activation status may be critical for AICD, it is likely that the CD3 ITAM, docking sites for tyrosine kinases, regulate AICD. A 'threshold model' for AICD was tested by using two targeted mutant mouse strains lacking either the zeta chain (CD3zeta-/-) or the ITAM of the zeta chain (CD3zeta-/-:Tgzetadelta67-150). Although the T cells from the CD3zeta-/- mice express extremely low levels of surface TCR, a subpopulation (approximately 18%) of activated T cells could be induced to express TCR/FcepsilonRI gamma by using a powerful polyclonal activation protocol. These activated TCR/FcRI gamma T cells were capable of undergoing AICD, but its induction required 10 times as much anti-CD3epsilon mAb as that required for AICD of wild-type T cells. Thus, the intensity of AICD correlated with the level of CD3 expression and was less efficient with activated, CD3zeta(-/-)-derived T cells. By contrast, AICD of T cells from the CD3zeta-/-:Tgzetadelta67-150 mice could be induced with low doses of anti-CD3epsilon mAb and the extent of AICD was comparable to T cells from wild-type mice. The AICD induced in T cells from CD3-/-, CD3zeta-/-:Tgzetadelta67-150 and normal controls was specifically inhibited by Fas-Ig fusion proteins. Our data support the 'threshold model' of AICD by demonstrating that AICD is controlled by the strength of T cell activation.

Animals↗

PubMed1998

Activation of T cells via CD55: recruitment of early components of the CD3-TCR pathway is required for IL-2 secretion.

It was previously reported that the glycosylphosphatidylinositol (GPI)-anchored CD55 molecule provides a co-stimulatory signal for T lymphocytes and is constitutively associated with the Src-related kinase p56lck. The present studies were undertaken to clarify the mechanism of action of CD55 in T cells. We describe the failure of cross-linking of CD55 alone to induce both the elevation of the intracellular calcium concentration and the tyrosine phosphorylation of PLC-gamma in CD3+ Jurkat cells. By contrast, it is sufficient to induce the phosphorylation of tyrosine residues on p56lck, the TCR-zeta chain as well as ZAP-70. Surprisingly, the observed TCR-zeta and ZAP-70 tyrosine phosphorylations appear delayed compared to stimulation via CD3. Calcium ionophore A23187 in combination with cross-linked CD55 mAb initially caused an acceleration in the kinetic of these two phosphorylation events, followed by IL-2 secretion. Furthermore, transfection of the cytoplasmic domain of TCR-zeta in CD3- Jurkat cells, using a CD16-zeta chimera, demonstrates that CD55-mediated T-cell activation depends on the expression of this chain of the CD3-TCR complex.

Antibodies, Monoclonal↗

PubMed2004

Crystal structure of a human CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody fragment.

The alpha/beta T cell receptor complex transmits signals from MHC/peptide antigens through a set of constitutively associated signaling molecules, including CD3-epsilon/gamma and CD3-epsilon/delta. We report the crystal structure at 1.9-A resolution of a complex between a human CD3-epsilon/delta ectodomain heterodimer and a single-chain fragment of the UCHT1 antibody. CD3-epsilon/delta and CD3-epsilon/gamma share a conserved interface between the Ig-fold ectodomains, with parallel packing of the two G strands. CD3-delta has a more electronegative surface and a more compact Ig fold than CD3-gamma; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-epsilon opposite the dimer interface, occluding this region from direct interaction with the TCR. This immunodominant epitope may be a uniquely accessible surface in the TCR/CD3 complex, because there is overlap between the binding site of the UCHT1 and OKT3 antibodies. Determination of the CD3-epsilon/delta structure completes the set of TCR/CD3 globular ectodomains and contributes information about exposed CD3 surfaces.

CD3 Complex↗

PubMed1994

The L-selectin (Leu8) molecule is associated with the TcR/CD3 receptor; fluorescence energy transfer measurements on live cells.

Several accessory molecules were shown to play important roles in T cell functions and be in close proximity to the T cell receptor (TcR/CD3). The L-selectin molecule (Leu8, LAM1-1, LECAM1) also plays an important role in lymphocyte homing and proliferation. We were interested in determining the proximity of this molecule to the TcR/CD3 complex on live peripheral human T cells. Using a fluorescence energy transfer method, designed to study individual cells, we could show that L-selectin is within 170 A of the TcR/CD3 complex. Monoclonal antibody directed against the LAM1-1 (Leu8) epitope of the L-selectin molecule suppressed the mitogenic activity of antibodies specific for various CD3 epitopes in vitro. Intracellular Ca2+ mobilization obtained with wt31 followed by cross-linking antibody or with anti-CD3 was not influenced by anti-Leu8 antibody. Also antibody directed against the LAM1-1 epitope did not influence the binding of the mitogenic antibodies, as shown by fluorescence-based flow cytometry. Therefore, we suggest that binding of TcR/CD3 bound mitogenic antibodies to accessory cell Fc receptors may be hindered by antibodies bound to the close proximity L-selectin molecules.

CD3 Complex↗

PubMed1990

Dissociation of signal transduction via Thy-1 and CD3 antigens in murine T cells.

To understand the proliferation/differentiation of immature thymocytes which have not express T cell antigen receptor (TCR), we studied whether Thy-1 has signal-transducing capacity. Thy-1+ CD3-TCR- cells including thymocytes from BALB/c embryos and SCID mice and nude mouse splenic cells did not show proliferative responses in the culture with anti-Thy-1 (G7) plus phorbol myristate acetate (PMA), whereas Thy-1+ CD3+ cells from normal thymus or spleen did show a response to them. Since Thy-1-mediated activation is suggested to require co-expression of the CD3-TCR complex, we compared the T cell proliferative response in mature T cells stimulated with anti-Thy-1 (G7) and anti-CD3-epsilon (2C11). Under the presence of PMA or IL-2, accessory cell-depleted splenic T cells were cultured with G7 or 2C11. PMA augmented the proliferative response of splenic T cells cultured with G7 much more than that with 2C11. IL-2, however, showed reciprocal effect on the proliferation of G7 and 2C11-treated splenic T cells. These data suggest that signals triggered via Thy-1 and CD3-epsilon may provide a distinct intracellular pathway for T cell activation.

Animals↗