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PubMed1999

TCRzeta is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly.

It is generally assumed that TCR assembly occurs in the endoplasmic reticulum (ER), and ER retention/degradation signals have been identified in several of the TCR chains. These signals are probably responsible for retention of incompletely assembled TCR complexes and free TCR chains in the ER. This study focused on the intracellular localization and transport of partially assembled TCR complexes as determined by confocal microscopy analyses. We found that none of the TCR chains except for TCRzeta were allowed to exit the ER in T cell variants in which the hexameric CD3gammaepsilonTi alphabetaCD3 deltaepsilon complex was not formed. Interestingly, TCRzeta was exported from the ER independently of other TCR chains and was predominantly located in a compartment identified as the Golgi apparatus. Furthermore, in the TCRzeta-negative cell line MA5.8, the hexameric CD3gammaepsilonTi alphabetaCD3 deltaepsilon complex was allowed to exit the ER and was also predominantly located in the Golgi apparatus. However, neither hexameric TCR complexes nor TCRzeta chains were efficiently expressed at the cell surface without the other. The observations that TCRzeta and hexameric TCR complexes are transported from the ER to the Golgi apparatus independently of each other and that these partial TCR complexes are unable to be efficiently expressed at the cell surface suggest that final TCR assembly occurs in the Golgi apparatus.

Biological Transport↗
PubMed1997

Human intestinal epithelial cell lines produce factor(s) that inhibit CD3-mediated T-lymphocyte proliferation.

Peripheral blood T lymphocytes (PBT) proliferate more to anti-CD3 stimulation than to anti-CD2 stimulation. On the other hand, fresh, but not cultivated, intestinal intraepithelial lymphocytes (iIEL) exhibit a lower response to CD3 stimulation in comparison to CD2. The goal of this study was to show that the anti-CD3 T-cell response depends on the microenvironment and is independent of the origin of the lymphocytes. Cultured T-cell lines were stimulated with either an anti-CD3 mAb or an anti-CD2 mAb. Either conditioned supernatant from intestinal epithelial cell (IEC) lines or non- conditioned medium (negative control) was added. After 2 days cytokine production and proliferation were measured. Conditioned supernatant decreased the proliferative response of small and large bowel iIEL compared to controls (P = 0.04). In the same experiments, the cytokine production was non-significantly decreased. Immortalized iIEL, that are not regularly stimulated by their CD3 pathway, showed a similar decrease in proliferation (P < 0.001) and cytokine production (P = 0.01) when incubated with conditioned supernatant. Similar results were also obtained with a non-immortalized and an immortalized PBT line (P < 0.001). In a small bowel iIEL cell line, that exhibited a significant response to anti-CD2 stimulation, the proliferative response to anti-CD2 stimulation was preserved. Active conditioned supernatant could be generated from three independent IEC lines and a liver derived epithelial cell line, but not from a non-epithelial control cell line or two extraintestinal epithelial cell lines. We conclude that supernatants of cultured IEC contain soluble factor(s) that cause cultured iIEL and extraintestinal lymphocytes to behave like fresh iIEL. These results, therefore, support and extend the studies of others which suggest that the intestinal microenvironment mucosalizes lymphocytes.

CD2 Antigens↗
PubMed2002

Induction of IL-15 by TCR/CD3 aggregation depends on IFN-gamma and protects against apoptosis of immature thymocytes in vivo.

TCR/CD3 aggregation by injection of anti-CD3 Ab produces T cell activation, release of cytokines such as IFN-gamma, and apoptosis in the cortical region of the thymus. We show that anti-CD3 Ab induces IL-15 mRNA in spleens of wild-type but not IFN-gamma receptor-knock-out (IFN-gammaR KO) mice. The loss of IL-15 mRNA induction in IFN-gammaR KO mice was associated with increased thymocyte apoptosis. Pretreatment of wild-type mice with neutralizing anti-IL-15 Ab increased the anti-CD3-triggered thymocyte apoptosis, thus mimicking the sensitive phenotype of IFN-gammaR KO mice. Inversely, anti-CD3-induced apoptosis in IFN-gammaR KO mice was suppressed by administration of recombinant IL-15. In IFN-gammaR KO mice and in wild-type mice that were treated with anti-IL-15, augmented apoptosis affected mainly CD4+CD8+ immature thymocytes. IL-15 as well as IL-15Ralpha mRNA expression in thymocytes was not increased by anti-CD3. These data demonstrate that systemic IL-15 exerts anti-apoptotic activity on immature T cells and establish a regulatory mechanism whereby TCR/CD3 engagement induces IL-15 expression via an IFN-gamma-dependent pathway. The self-amplifying nature of this IFN-gamma/IL-15 connection may constitute a regulatory pathway in central tolerance to self.

Animals↗
PubMed2003

Differential expression of TcR-CD3 zeta as evidence for altered immunoregulation in preeclamptic versus normotensive women.

OBJECTIVE: The study was undertaken to exhibit and quantify the difference in modulation of CD3-zeta protein (an integral component of the T-cell receptor) in preeclamptic and normotensive women. STUDY DESIGN: Serum was collected from 10 preeclamptic and 10 normotensive women at >or=37 weeks' gestation on admission. Jurkat E-61 cells were incubated with the sera (20% volume to volume) and analyzed with Western immunoblot using mouse monoclonal CD3-zeta antibody. Enhanced chemiluminescence and densitometry were used to qualitatively measure zeta expression of the cells. A de novo flow cytometry assay was developed to quantify the difference in CD3-zeta expression of these cells. Comparisons were performed by t test (P<.05 was significant). RESULTS: Preeclamptic patient sera produced a 2.4-fold increase in CD3-zeta expression than normotensive patients on Western blot (P<.01). Flow cytometry showed that preeclamptic sera had a 1.4-fold higher expression of CD3-zeta compared with normotensive patients (P<.0003). CONCLUSION: TcR/CD3-zeta expression is normally suppressed in pregnancy. Loss of this suppression occurs in preeclamptic patients, implying increased T-cell function.

Adult↗
PubMed1989

Immobilized anti-CD3 monoclonal antibodies induce accessory cell-independent lymphokine production, proliferation and helper activity in human T lymphocytes.

Monoclonal antibodies (mAb) directed against the human CD3 molecular complex are able, when immobilized on the plastic of microtitre wells, to induce accessory cell-independent T-cell proliferation. In this study, we show that the anti-CD3 mAb CLB-T3/3 induces strong T-cell stimulation that is proportional to the density of the immobilized antibody. T cells, optimally stimulated with plastic-immobilized CLB-T3/3, showed a five-fold higher proliferation compared to cells that were stimulated with soluble anti-CD3 in the presence of accessory cells. The difference in magnitude of proliferation was found to be correlated with the expression of the CD25 (TAC) antigen and the production of interleukin (IL)-2, but not with the number of high-avidity IL-2 receptors expressed on the surface of these differentially activated cells. In addition, immobilized CLB-T3/3 initiated the production of interferon-gamma (IFN-gamma), but not of IL-4, in purified T lymphocytes. Coated anti-CD3 mAb induced helper activity in T cells for IgM and IgG production by B lymphocytes. Whereas addition of IL-1 or IL-2 had only a moderate effect on T-cell proliferation induced by immobilized anti-CD3 mAb, helper activity was strongly enhanced in the presence of these factors. This T-cell activation system may prove useful for a standardized analysis of both activation requirements and immunoregulatory capacities of human T cells.

Antibodies, Monoclonal↗
PubMed1991

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.

Animals↗
PubMed2001

Molecular modelling and endoplasmic reticulum retention of mutated TCR/CD3 complexes.

T cell receptor (TCR)/CD3 complex assembly takes place in the endoplasmic reticulum (ER). Normal TCR/CD3 complexes egress from the ER to the cis-Golgi, where the interaction with zeta2 homodimers occurs. This interaction leads to further uncontrolled transport of TCR/CD3/zeta molecules to the cell surface. The purpose of the present experiments was to determine firstly the basis for the impact of the Phe195/216 --> Val mutations on TCR/CD3 expression in Jurkat cells, and secondly why mutated J79-cell TCRalphabeta/CD3 hexamers are prevented from interacting with zeta2 homodimers. We found that Phe --> Val mutations cause serious perturbations in a so far undefined hydrophobic area formed by the two Phe195/216 on beta-strand F and aromatic/large hydrophobic amino acids on neighboring beta-strands B and A in Calpha and Cbeta domains, respectively. In addition, TCR/CD3 hexamers and zeta2 homodimers colocalize in normal Jurkat T cells, in revertant J79r58 cells, and in J79 cells transfected with wild-type TCRalpha cDNA but not in J79 mutant cells (confocal microscopy). Furthermore, mutated TCR/CD3 complexes seem to be actively retained in the ER in J79 cells but not in revertant J79r58 cells by a nondominant mechanism. We propose that a hitherto undefined ER-retention molecule controls both the protein structure and egress of TCR/CD3 complexes from the ER of alphabeta and gammadelta T cells.

Brefeldin A↗
PubMed2001

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Amino Acid Sequence↗
PubMed1998

Loss of original antigenic specificity in T cell hybridomas transduced with a chimeric receptor containing single-chain Fv of an anti-collagen antibody and Fc epsilonRI-signaling gamma subunit.

T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted with a chimeric cell surface receptor, scC2Fv/gamma, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling gamma subunit of the Fc epsilonRI. When transduced into MD.45 cells, scC2Fv/gamma or its mutated form lacking immunoreceptor tyrosine-based activation motif (ITAM), scC2Fv/gammaIC-, formed mainly homodimers. A small proportion of these molecules formed heterodimers with endogenous CD3zeta in these hybridoma cells. By contrast, in HCQ6 cells, the majority of scC2Fv/gamma and scC2Fv/gammaIC- molecules formed heterodimers with CD3zeta, and only a small proportion of them was expressed as homodimers. Stimulation with plastic-immobilized collagen induced IL-2 production in scC2Fv/gamma-transduced MD.45 cells, but not in MD.45 cells transduced with the ITAM-less chimera scC2Fv/gammaIC-. HCQ6 cells transduced with scC2Fv/gamma responded to plastic-bound collagen. Due to the high content of CD3zeta-associated chimeras, HCQ6 cells transduced with the ITAM-less scC2Fv/gammaIC- chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced with scC2Fv/gamma produced IL-2, whereas transduced HCQ6 cells were unresponsive, hence suggesting that the ability of cells transduced with scC2Fv chimeras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/gamma homodimers, but not monovalent scC2Fv/gamma-CD3zeta or scC2Fv/gammaIC(-)-CD3zeta heterodimers. Of interest, expression of CD3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resulting in decreased response to cognate Ags.

Animals↗
PubMed1993

CD4, CD8 and the role of CD45 in T-cell activation.

CD4, CD8 and CD45 regulate the coupling of the T-cell receptor complex (CD3-TCR) to tyrosine kinase activation and phosphorylation of key substrates such as phospholipase C gamma 1. CD4 and CD8 contribute to activation signals through their cytoplasmic association with p56lck. Expression of the zeta-chain is required for functional synergy of the T-cell receptor with CD4 in the activation of phospholipase C gamma 1, which probably reflects an interaction between p56lck and zeta-associated kinase ZAP-70. CD45 expression is required for CD3-TCR signaling. CD45 may positively regulate signaling by dephosphorylating the carboxyl-terminal tyrosine of p56lck and p59fyn, and negatively regulate signaling by dephosphorylation of other TCR-associated substrates directly. One ligand for CD45 receptor has been identified as the B cell CD22 molecule. The positive and negative effects of CD45 are sensitive to the composition of CD45 in receptor complexes, and may be regulated by specific associations of CD45 isoforms with other receptors such as CD3-TCR, CD2 and CD4.

Animals↗
PubMed1991

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.

Antigens, Differentiation, T-Lymphocyte↗
PubMedDate not supplied

In vivo T cell activation properties of anti-T cell monoclonal antibodies.

Murine monoclonal antibodies specifically directed at the CD3 molecular complex are widely used in clinical transplantation. They are potent inhibitors of immune function and may be administered in association with conventional immunosuppressive drugs. However, their use has been complicated by the severe clinical syndrome that invariably follows the first injections of the antibody. We have shown that this reaction relates to transient T cell activation, with production of a cohort cytokines. This in vivo activating effect is unique to anti-CD3 MoAbs. Furthermore, this property is highly monocyte-dependent, engaging Fc receptors, and thus varies according to the isotype of the administered MoAb. Another anti-T cell MoAb such as the BMA031 directed towards monomorphic determinants of the T cell receptor (TCR) does not promote such massive in vivo activation.

Animals↗
PubMed1993

Defective signal transduction via T-cell receptor-CD3 structure in T cells from rheumatoid arthritis patients.

T cells from patients with active RA are known to produce low levels of IL-2 and proliferate poorly in response to various mitogenic stimuli. The present work shows that cross-linking of CD3 antigen on patients' T-cell surface induces two- to threefold lower Ca2+ response than in T cells from age-matched controls. Immunofluorescence studies indicate that the attenuated signal may be due to the suppressed expression of CD3 and/or CD45 molecules on patients' T cells. In the majority of the patients, the level of CD45 expression is reduced by 60%-70% as compared with that in the control T cells. Therefore, the poor mitogenic response of patient cells is apparently due to a defect in early stages of signal transduction through the T-cell receptor (TCR-CD3).

Arthritis, Rheumatoid↗
PubMed1992

Anti-CD3-stimulated T cells induce the production of multiple Ig H chain isotypes by individual human peripheral B lymphocytes.

Polyclonal activation of human B cells is achieved by coculture with T cells stimulated by mAb to the CD3 molecular complex. By formal limiting dilution analysis, approximately 60% of human peripheral blood B cells were found to produce Ig in this system. When individual B cells were cultured in microtiter wells with anti-CD3-activated T cells, more than one-third of cultures producing Ig contained multiple Ig H chain isotypes. Similar results were observed when individual IgM-expressing B cells, selected and dispersed by FACS were cultured with anti-CD3-activated T cells. The clonality of the B cells producing multiple Ig isotypes was supported by L chain analysis of the secreted Ig. Of the wells containing more than one H chain isotype, nearly 85% contained only a single L chain type. Clonality was further examined by polymerase chain reaction amplification of cDNA harvested from cultures originally seeded with individual B cells. In general, only a single VH gene family could be amplified from cultures producing more than one Ig isotype. Three separate VH regions were cloned and sequenced. One, a VHIV-mu was nearly identical to a previously described VH gene VH71.4; as second, a VHIV-gamma was very similar to a previously described VH gene segment V-79, whereas a third, a VHIII-gamma differed by 14% in nucleotide sequence from its closest germline counterpart VH3005. These results indicate that anti-CD3-activated T cells not only stimulate the majority of B cells to secrete Ig, but also induce individual B cells to produce multiple Ig H chain isotypes. Additionally, the procedure described provides a reliable method to sample a large proportion of the human peripheral B cell repertoire.

Amino Acid Sequence↗
PubMed1996

Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes.

Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down-regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses.

Antigen-Presenting Cells↗
PubMed1999

Expression of the T-cell markers CD3, CD4 and CD8 in healthy and atopic children during the first 18 months of life.

BACKGROUND: There is little information available about the development of T-cell immunity in healthy and atopic children. We have studied prospectively the mean fluorescence intensity of the T-cell receptor complex-associated CD3, CD4 and CD8 in relation to atopic family history (AFH) and the development of atopic disease. METHODS: Children with a defined AFH (n = 172) were followed from birth to 18 months and the cumulative history of atopic disease was recorded. Blood samples were obtained at birth and at 18 months, and in a subgroup of 78 children also at 3, 6 and 12 months. Multicolour flow cytometry was used to analyse pan T-cells (CD3+CD45+CD14-), T-helper-(CD3+CD4+) and T-cytotoxic-(CD3+CD8+) cells. RESULTS: At 18 months, 31 children were atopic and 118 non-atopic. Children who developed atopic disease had a higher CD4 expression (mean fluorescence intensity, MFI) on CD4+CD3+ lymphocytes at birth and at 3 months, particularly as compared with non-atopic children without AFH. Furthermore, the CD3 expression on CD3+CD45+CD14- lymphocytes increased more slowly with age in children with double atopic heredity, as compared with children with no or only one atopic family member. CONCLUSIONS: The higher expression of the CD4 receptor in early infancy in children who developed atopic disease compared with non-atopics suggests a delayed expression in T-helper cells. Children with a strong AFH had a slower increase in the expression of CD3, indicating a delayed T-cell maturation.

CD3 Complex↗