OKT3 resistant rejection in liver transplant patients.
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Adult↗
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Results for “CD3 Complex”
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CD3 antigen modulation in T-lymphocytes during OKT3 treatment.
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CD3 Complex↗
Lymphocyte antigen receptors: a common design?
A recent flurry of papers has provided detailed information on the components of the T- and B-cell antigen receptors. Although these individual elements, and the subunits they form, clearly differ, Frits Koning argues that the T- and B-cell receptor complexes may share a common underlying design.
Animals↗
The role of CD45 in T-cell activation--resolving the paradoxes?
CD45 is one of the most abundant transmembrane glycoproteins expressed on the surface of haematopoietic cells. Despite the striking advances of the past few years in the understanding of the actions of CD45 at a molecular level, several paradoxes remain. This article argues that these paradoxes are more apparent than real, and suggests that CD45 plays a key role on the T-cell surface in regulating coupling of the antigen-receptor complex to intracellular signalling pathways.
CD3 Complex↗
New COP1-binding motifs involved in ER retrieval.
Coatomer-mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins. For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER). Making use of the two-hybrid system, we characterized a new sequence (deltaL) which interacts specifically with the delta-COP subunit of the COP1 complex. Transfer of deltaL to the cytoplasmic domain of a reporter membrane protein resulted in its localization in the ER, in yeast and mammalian cells. This was due to continuous retrieval of tagged proteins from the Golgi back to the ER, in a manner similar to the ER retrieval of KKXX-tagged proteins. Extensive mutagenesis of deltaL identified an aromatic residue as a critical determinant of the interaction with COP1. Similar COP1-binding motifs containing an essential aromatic residue were identified in the cytoplasmic domain of an ER-resident protein, Sec71p, and in an ER retention motif previously characterized in the CD3epsilon chain of the T-cell receptor. These results emphasize the role of the COP1 complex in retrograde Golgi-to-ER transport and highlight its functional similarity with clathrin-adaptor complexes.
Amino Acid Sequence↗
Signaling via the CD2 receptor enhances HTLV-1 replication in T lymphocytes.
Human T lymphotropic virus type 1 (HTLV-1) is considered the etiologic agent of adult T cell leukemia/lymphoma and several chronic progressive immune-mediated diseases. Approximately 1-4% of infected individuals develop disease, generally decades following infection. Increased proviral transcription, mediated by the viral 40-kDa trans-activating protein, Tax, has been implicated in the pathogenesis of HTLV-1-associated diseases. Since the HTLV-1 promoter contains sequences responsive to cyclic AMP and protein kinase C, we hypothesized that lymphocyte activation signals initiated through the TCR/CD3 complex or CD2 receptor promote viral replication in HTLV-1-infected lymphocytes. We demonstrate that mAbs directed against the CD2, but not the CD3 receptor increase viral p24 capsid protein 1.5- to 5.7-fold in CD2/CD3+ HTLV-1-infected cell culture supernatants. Northern blot analysis demonstrated a 2.5- to 4-fold increase in all species of viral mRNA following CD2 cross-linking of OSP2/4 cells, an immortalized HTLV-1 cell line. Consistent with transcriptional regulation, reporter gene activity increased approximately 11-fold in CD2-stimulated Jurkat T cells cotransfected with a Tax-expressing plasmid and a CAT reporter gene construct under control of the HTLV-1 promoter. These data suggest a possible physiologic mechanism, whereby CD2-mediated cell adhesion and lymphocyte activation may promote viral transcription in infected lymphocytes.
CD2 Antigens↗
Delta is the Cx-gene product in the gamma/delta antigen receptor of dendritic epidermal cells.
Most T cells bear an antigen receptor that is a protein of a disulphide-linked heterodimer composed of an alpha chain and a beta chain associated with the non-polymorphic CD3 (T3) complex. A small subpopulation of thymic and peripheral T cells, as well as Thy-1+dendritic epidermal cells (dEC), express an alternative CD3-associated dimeric receptor composed of the product of the T-cell antigen receptor (TCR) gamma gene and a fourth chain, designated delta. Recently a new murine TCR constant-region gene, designated Cx, has been cloned and proposed as a candidate for the C delta gene. We have previously demonstrated that murine Thy-1+ dEC cell lines express a CD3-associated disulphide-linked heterodimer composed of a relative molecular mass Mr 41,000 (41K) gamma chain and a 50K delta chain. We have further analysed the receptor of one of these cloned dEC lines, 7-17.1, by endoglycosidase treatment of the isolated gamma and delta chains. The gamma chain was found to contain two N-linked oligosaccharide residues, consistent with the expression of a chain encoded by the V gamma 3 and C gamma 1 gene segments. The delta chain contains at least three N-linked oligosaccharides and has a core size of 38K. Northern blot analysis indicated the presence of abundant Cx messenger RNA in 7-17.1 cells. Immunoprecipitation with two antisera to peptides comprising distinct regions of the Cx sequence indicates that the delta chain is encoded by the Cx gene.
Amino Acid Sequence↗
Dendritic cells capable of stimulating T cells in germinal centres.
The B cells within the germinal centres of lymphoid organs undergo affinity maturation of their antigen receptors, a critical event for antibody memory. Follicular dendritic cells within the germinal centres retain immune complexes that select the developing B cells for which they have a higher affinity. We have now identified a subset of CD4+ CD11c+ CD3- dendritic cells in the germinal centres. These are strong antigen-presenting cells for T cells, but do not co-stimulate CD40-activated B cells. These dendritic cells probably stimulate germinal centre T cells and aid the complicated processes that are required for the generation of memory B cells.
Antigen Presentation↗
Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.
Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.
Animals↗
Phorbol ester synergizes with Ca2+ ionophore in activation of protein kinase C (PKC)alpha and PKC beta isoenzymes in human T cells and in induction of related cellular functions.
Studies described herein were designed to examine the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA), and a Ca2+ ionophore (ionomycin), singly or in combination, on the activation and expression of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes (alpha, beta and gamma) at the protein and messenger RNA (mRNA) levels in T cells. These two agents induce the activation and proliferation of T lymphocytes by mimicking the action of inositol phospholipid-derived second messengers normally generated by triggering of the antigen-specific T-cell receptor (TcR)/CD3 complex. TPA-induced T-cell proliferation, expression of interleukin-2 receptor-alpha subunit (IL-2R alpha) and transferrin receptor, CD3 down-regulation and, lastly, the cytosol-to-membrane PKC translocation (determined by an enzymatic assay or by immunoblotting with a cross-reactive anti-PKC peptide antibody) were all facilitated by ionomycin. Immunoblots with isoenzyme-specific anti-PKC monoclonal antibodies demonstrated expression of immunoreactive PKC alpha, PKC beta and PKC gamma proteins that were translocated to the membrane upon TPA plus ionomycin stimulation. Resting T cells expressed abundant levels of mRNA for PKC alpha and PKC beta, but very low levels (relative to brain) of PKC gamma. TPA increased by two- to threefold the expression of PKC beta, but not of PKC alpha or PKC gamma, mRNA within 12 hr of stimulation. Ionomycin synergized with TPA in increasing the expression of PKC alpha and PKC beta mRNA. The two agents also synergized in inducing expression of additional activation/growth-associated genes, namely the c-myc protooncogene, ornithine decarboxylase (ODC) and IL-2R alpha. Ionomycin alone was inactive (or marginally active) in all of these assays. The translocation of distinct Ca(2+)-dependent PKC isoenzymes to the membrane and the up-regulation of PKC alpha and beta mRNA suggest that at least these two isoenzymes are involved in discrete steps of the pathway leading to T-cell activation and proliferation. Moreover, the combined effects of TPA and ionomycin on T-cell function and cell-surface antigen expression appear to be due, at least in part, to their synergistic activation of distinct PKC isoenzyme(s).
Antigens, Differentiation, T-Lymphocyte↗
Activation of the CD2 pathway in lamina propria T cells up-regulates functionally active AP-1 binding to the IL-2 promoter, resulting in messenger RNA transcription and IL-2 secretion.
The aim of this study was to identify molecular mechanisms involved in transcriptional regulation of IL-2 expression following CD2 and CD3 activation in lamina propria (LP) T cells. Studies used T cells from normal, ulcerative colitis, and Crohn's disease mucosa and freshly isolated PBMC, PBMC stimulated with IL-2 alone, and PBMC stimulated with IL-2 and cocultured with B cell lines (LP-like T cells). Electrophoretic mobility shift assays were performed with nuclear extracts from cells activated with either anti-CD2 or anti-CD3 Abs. CD2 signaling in LPMC and LP-like T cells led to a pattern of sustained up-regulation of AP-1-binding complexes, whereas CD3 activation resulted in only transient up-regulation. While the pattern of regulation of AP-1 binding observed in normal, uninflamed, or inflamed Crohn's disease LPMC is similar, differences in intensity of AP-1 binding were observed. Activation of LP-like T cells mimics the up-regulation of AP-1 with a kinetic profile similar to that observed with freshly isolated LPMC from Crohn's disease-inflamed tissue. The AP-1 complex formed following CD2 activation is composed of jun/fos heterodimers. The CD2-enhanced responsiveness is reflected in functional analysis experiments utilizing transfection of both multimeric-TRE or IL-2 promoter-luciferase constructs directly into normal, ulcerative colitis, or Crohn's disease LPMC. Our data suggest that activation of LP T cells from normal, ulcerative colitis, or Crohn's disease mucosa through the CD2 pathway leads to induction of AP-1 complexes that bind to the IL-2 promoter, and may play a pivotal role in modulating IL-2 production in the gut.
Animals↗
Short-term treatment with anti-CD3 antibody reduces the development and progression of atherosclerosis in mice.
BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the large arteries that is the primary cause of heart disease and stroke. Anti-CD3-specific antibodies suppress immune responses by antigenic modulation of the CD3 antibody/T-cell receptor complex. Their unique capacity to restore self-tolerance in a mouse model of diabetes and, importantly, in patients with recent-onset type 1 diabetes involves transforming growth factor-beta-dependent mechanisms via expansion and/or activation of regulatory T cells. We hypothesized that treatment with anti-CD3-specific antibodies might inhibit atherosclerosis development and progression in mice. METHODS AND RESULTS: Low-density lipoprotein receptor-deficient mice were fed a high-cholesterol diet for 13 or 24 weeks. Anti-CD3 antibody was administered on 5 consecutive days beginning 1 week before or 13 weeks after the high-cholesterol diet was initiated, respectively. Control mice were injected in parallel with phosphate-buffered saline. Anti-CD3 antibody therapy reduced plaque development when administered before a high-cholesterol diet and markedly decreased lesion progression in mice with already established atherosclerosis. We found increased production of the antiinflammatory cytokine transforming growth factor-beta in concanavalin A-stimulated lymph node cells and enhanced expression of the regulatory T-cell marker Foxp3 in spleens of anti-CD3 antibody-treated mice. A higher percentage of apoptotic cells within the plaques of anti-CD3 antibody-treated mice was also observed. CONCLUSIONS: Altered disease progression, combined with the emergence of this particular cytokine pattern, indicates that short-term treatment with an anti-CD3 antibody induces a regulatory T-cell phenotype that restores self-tolerance in a mouse model of atherosclerosis.
Animals↗
In vivo association of CD5 with tyrosine-phosphorylated ZAP-70 and p21 phospho-zeta molecules in human CD3+ thymocytes.
CD5 is a 67-kDa T cell surface Ag that can be found physically associated with the CD3-TCR molecular complex. In different experimental models it has been shown to act as a costimulatory receptor for T cell activation. Unexpectedly, studies in CD5-deficient mice suggested a negative role for the CD5 Ag in CD3-TCR signaling in the thymus. In this report we assessed the constitutive interactions of CD5 in freshly isolated human thymocytes with signaling elements of the CD3-TCR complex. We determined that the ZAP-70 protein tyrosine kinase was present in CD5 immunoprecipitates. The two molecules were constitutively tyrosine phosphorylated in a complex also associating with unphosphorylated as well as phosphorylated zeta-chains. Although both p21 and p23 tyrosine-phosphorylated forms of zeta as well as phospho-CD3 epsilon molecules were constitutively present in human thymocytes and could be immunoprecipitated with ZAP-70- or CD3 epsilon-specific Abs, the p21 species of zeta was predominant in CD5 immune complexes. The interaction between CD5 and ZAP-70 was not observed in CD3-negative thymocytes, where the constitutive tyrosine phosphorylation of ZAP-70 was very low. We conclude that CD5 may affect in vivo the signaling capacity of TCRs expressed by human thymocytes by altering the phosphorylation state of CD3 and/or by retaining ZAP-70 with the p21 species of zeta.
Biotinylation↗
Non FcR-binding murine antihuman CD3 monoclonal antibody is capable of productive TCR signalling and induces proliferation in the presence of costimulation.
CLB T3/4.A is a non FcR-binding CD3 mAb of the murine IgA isotype, which may be used as an alternative for the mitogenic OKT3 mAb in the treatment of acute cellular rejection after organ transplantation. We studied TCR signalling and T cell activation in response to T3/4.A in normal human PBMC in vitro. T3/4.A induced a rapid rise in free cytoplasmic Ca(2+), not different from the response to mitogenic CD3 mAb. However, protein tyrosine phosphorylation and, particularly, MAPK activation, were reduced as compared to mitogenic CD3 mAb. T3/4.A enhanced expression of both CD69 and CD25, but proliferation and detectable cytokine production did not occur. Addition of either CD28 mAb or IL-2 induced a strong proliferative response, which was accompanied by cytokine production. At higher mAb concentrations, T cell activation decreased, which correlated with TCR downmodulation. To exclude the possibility that activation by T3/4.A depends on interaction of murine IgA Fc with as yet unknown FcR, we showed that also with CD3 mAb F(ab')2 fragments upregulation of activation molecules occurred, as well as proliferation in the presence of costimulation. We conclude that the non FcR-binding murine IgA mAb T3/4.A acts as a partial agonist and leads to proliferation and cytokine production only in the presence of appropriate costimuli. These findings may explain the mitigated cytokine release syndrome observed in vivo with some nonmitogenic CD3 mAbs.
Animals↗
Different activation states of human lymphocytes after antibody-mediated stimulation via CD3 and the alpha/beta T-cell receptor.
BMA031 is an IgG2b antibody directed towards the human alpha/beta T-cell receptor that is able to induce proliferation of peripheral blood mononuclear cells independent of antibody crosslinking. The proliferative response to BMA031 during the first 3 days of culture is usually of similar magnitude to that induced by the IgG2a CD3 antibody OKT3 but decreases quickly afterwards. Stimulation by BMA031 induces no measurable IL-2 release, very low expression of the IL-2 receptor, and does not trigger cytotoxic effector function. However, cross-linking of the antibody or addition of IL-2 leads to enhanced and prolonged proliferation, strong IL-2 receptor expression, and cytotoxic activity, features that are usually found after stimulation by the IgG2a CD3 antibody OKT3 in soluble form. The stimulatory effect of BMA031 cannot be diminished by IL-2 receptor blocking, whereas stimulation by OKT3 is strongly reduced. Moreover, proliferation induced by BMA031 has lower sensitivity to inhibition by ciclosporin than OKT3. From these results two major conclusions can be drawn: (1) an IL-2-independent way of activation may be important for the short-term proliferation of the T cells stimulated by BMA031 and (2) after stimulation by BMA031, cells reach a state of activation that is different from that induced by OKT3. These differences are most likely related to the different specificities of the antibodies, alpha/beta TcR versus CD3, suggesting that different activation signals are triggered via CD3 and via the alpha/beta TcR.
Antibodies, Monoclonal↗
Phosphorylation of multiple CD3 zeta tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation.
T lymphocyte activation resulting from antigen recognition involves a protein tyrosine kinase pathway which triggers phosphorylation of several cellular substrates including the CD3 zeta subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3 eta, is an alternatively spliced product of the same gene locus as CD3 zeta. CD3 eta lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3 zeta and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phosphorylation studies herein demonstrates that Tyr-132 is required for the formation of pp21. Moreover, the differential phosphorylation of CD3 zeta versus CD3 eta is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59fyn; p59fyn but not p56lck or p62yes is associated with each of the three TCR isoforms containing CD3 zeta 2, or CD3 eta 2, or CD3 zeta-eta. This association occurs through components of the TCR complex distinct from CD3 zeta or CD3 eta. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3 zeta residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3 zeta Tyr----Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3 zeta upon TCR stimulation as reflected by alteration in its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis may affect subsequent events such as receptor desensitization, receptor movement, and/or protein associations.
Amino Acid Sequence↗
Control of T-cell-mediated immune response by HLA class I in human pancreatic carcinoma.
PURPOSE: Cell surface HLA class I molecules present peptides derived from human cellular proteins to T cells. In the present study, we investigated the expression of HLA class I in human pancreatic carcinoma. EXPERIMENTAL DESIGN: The expression of HLA class I antigen and the extent of tumor infiltration by T cells were investigated in 46 primary tumors and in 14 metastases of pancreatic cancer by standard immunohistochemistry. RESULTS: The locus-specific expression of HLA I was reduced in 61% of primary tumors and in 93% of metastases. The total loss of this molecule complex was detected in 6% of primary tumors and in 43% of metastases. Pancreatic carcinoma and peritumoral tissue showed a significantly higher infiltration by CD3+, CD4+, and CD8+ T-cells compared with the tumor-distant pancreatic tissue. The negative expression of HLA class I was uniformly accompanied by a low density of tumor-infiltrating cytotoxic T-cells whereas the HLA class I-positive tumors were characterized by a substantial lymphocyte accumulation. However, the infiltration by cytotoxic T-cells was not correlated with the density of tumor cells. Patients with a high accumulation of cytotoxic cells showed a longer median survival. CONCLUSIONS: Pancreatic carcinoma frequently induces a cellular immune response that results in intratumoral and peritumoral T-cell infiltration. The expression of HLA class I is frequently lost in pancreatic carcinoma, which represents an effective mechanism to escape the tumor infiltration by cytotoxic T-cells. However, the infiltration by cytotoxic cells represents a favorable prognostic sign in pancreatic cancer patients.
Adenocarcinoma↗
Role of cross-linking in stepwise activation of T cells.
Activation of T cells is a complex process which we have hypothesized involves a series of functional intermediates possessing some, but not all, of the characteristics of fully functional effector cells. We have identified two such functional intermediates, the poised T cell (poTc) and the pre-effector T cell (peTc). poTc do not proliferate or mediate cytolytic activity but are responsive to help in the form of interleukin 2 (IL-2); peTc are proliferating cells which are not cytolytic. Here the role of T-cell receptor/CD3 complex cross-linking in generating these functional intermediates is examined and it is shown that cells can be driven to different stages of functional maturation depending upon the sequence of antibody binding to CD3 and cross-linking. Highly purified T cells can be activated to poTc stage if they are first labelled with the anti-CD3 monoclonal antibody OKT3 and then cross-linked with goat anti-mouse IgG-coated beads. If the OKT3 antibody is first bound to the IgG-coated beads and then added to highly purified T cells, peTc are generated. In both cases the intermediates can be driven to become fully functional effector cells by the addition of IL-2. Finally, by removing the OKT3-bound beads during the activation process, were are able to show that poTc and peTc are sequential intermediates along the same pathway of activation.
Antibodies, Monoclonal↗