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PubMed1999

Differential CD3 zeta phosphorylation is not required for the induction of T cell antagonism by altered peptide ligands.

T cells recognize foreign Ags in the form of short peptides bound to MHC molecules. Ligation of the TCR:CD3 complex gives rise to the generation of two tyrosine-phosphorylated forms of the CD3 zeta-chain, pp21 and pp23. Replacement of residues in MHC-bound peptides that alter its recognition by the TCR can generate altered peptide ligands (APL) that antagonize T cell responses to the original agonist peptide, leading to altered T cell function and anergy. This biological process has been linked to differential CD3zeta phosphorylation and generation of only the pp21 phospho-species. Here, we show that T cells expressing CD3zeta mutants, which cannot be phosphorylated, exhibit a 5-fold reduction in IL-2 production and a 30-fold reduction in sensitivity following stimulation with an agonist peptide. However, these T cells are still strongly antagonized by APL. These data demonstrate that: 1) the threshold required for an APL to block a response is much lower than for an agonist peptide to induce a response, 2) CD3zeta is required for full agonist but not antagonist responses, and 3) differential CD3zeta phosphorylation is not a prerequisite for T cell antagonism.

Animals↗
PubMed1988

Alpha/beta heterodimeric T-cell receptor expression early in thymocyte differentiation.

The differentiation of T lymphocytes inside the thymus results in the acquisition of MHC-restricted specific functions mediated by clonally distributed alpha/beta heterodimeric T-cell receptors (TcR). Genes encoding the alpha and beta subunits of the clonotypic receptor (Ti) are rearranged during thymic ontogeny and expressed in association with the monomorphic CD3 complex. The regulation of the expression of functional TcR along T-cell development is thus crucial to establish the ontogenic events involved in the acquisition and selection of T-cell repertoires. Current views support that CD3-alpha/beta heterodimers are acquired late in ontogeny on developing thymocytes already expressing CD4 and/or CD8 surface molecules, whereas CD4- CD8- early precursors, representing the major population in the embryonic thymus, do not yet express the alpha/beta TcR. However, a novel CD3-associated gamma/delta heterodimer has been recently identified on the surface of this "double negative" subset both in thymocytes and in MHC-unrestricted peripheral T cells, suggesting that alpha/beta and gamma/delta heterodimeric receptors are independently expressed on the surface of distinct thymic subpopulations during T-cell development. In contrast to these results, we report here that a major proportion of CD3+1-4-8- adult human thymocytes, included within the early "double negative" subset, express alpha/beta heterodimeric receptors, as assessed by flow cytometric analysis using a frame-work monoclonal antibody (WT.31) against the alpha/beta TcR complex. These and previous data showing that CD3+1-4-8- "double negative" thymocytes constitute a functional intermediate ontogenic stage in the differentiation of CD3+1-4+8-/CD3+1-4-8+ mature T cells from CD3-1-4-8- early prothymocytes further support the relevance of the CD3+1-4-8- transitional subset as immediate intrathymic precursors of alpha/beta TcR-bearing mature T cells. Therefore, developmental regulation of alpha/beta TcR expression was analyzed at the DNA, RNA, and protein levels in those different thymic subpopulations, defined by both functional and phenotypic criteria. Our results demonstrate that multiple Ti beta gene rearrangements and beta RNA messages are already evident at the early prothymocyte stage. Moreover, expression of relative levels of both Ti alpha and Ti beta functional RNA transcripts, similar to those observed in mature thymic cells, were also present in CD3+1-4-8- thymocytes. According with these data, immunoprecipitation analysis using a specific anti-Ti alpha antisera revealed that both alpha and beta molecules are expressed on CD3+ "double negative" and mature thymocytes, but not in prothymocytes

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

At least two non-antigen-binding molecules are required for signal transduction by the T-cell antigen receptor.

In the T-cell somatic mutant J.CaM1, the T-cell antigen receptor complex is poorly coupled to the inositolphospholipid second messenger system; some antibodies against the invariant CD3 subunit of the receptor retain their agonist function in J.CaM1. Here we show by a combination of complementation assays that the mutation in J.CaM1 affects a molecule other than the antigen-binding Ti subunit, suggesting that Ti is coupled indirectly to the signal transduction apparatus through a pathway involving the CD3 complex. We also describe another mutant, J.CaM2, in which the receptor complex is completely uncoupled from inositolphospholipid hydrolysis. J.CaM2 defines an additional complementation group, suggesting that signal transduction by the antigen receptor depends on at least two molecules distinct from Ti.

Calcium↗
PubMed1989

Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors.

Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.

Animals↗
PubMed2005

Type II phosphatidylinositol 4-kinase beta associates with TCR-CD3 zeta chain in Jurkat cells.

Phosphatidylinositol lipid signaling cascades are integral part of TCR-CD3 signaling. The mechanisms by which phosphatidylinositol kinases are coupled to TCR-CD3 complex remain elusive. Here we report an association of type II PtdIns 4-kinase with TCR-CD3 zeta chain upon cross-linking. Mapping studies have revealed that the C-terminal ITAM is critical for docking of the enzyme on the zeta chain. The association is shown to be tyrosyl phosphorylation dependent as mutation of Y-151 and Y-142 on the C-terminal ITAM disrupts interaction of the two proteins. Identification of the associated type II PtdIns 4-kinase revealed that the beta isoform of the enzyme interacts with the zeta chain in vivo.

1-Phosphatidylinositol 4-Kinase↗
PubMed2005

pH-specific aqueous synthetic chemistry in the binary cadmium(II)-citrate system. Gaining insight into cadmium(II)-citrate speciation with relevance to cadmium toxicity.

The involvement of Cd(II) in toxic manifestations and pathological aberrations in lower and higher organisms entails interactions with low and high molecular mass biological targets. To understand the relevant chemistry in aqueous media, we have launched pH-dependent synthetic efforts targeting Cd(II) with the physiological ligand citric acid. Reactions of Cd(II) with citric acid upon the addition of NaOH at pH 2.5 and pyridine at pH 3 and the addition of ammonia at pH approximately 7 led to the new complexes [Cd3(C6H5O7)2(H2O)5] x H2O (1) and (NH4)[Cd(C6H5O7)(H2O)] x H2O (2), respectively. Complexes 1 and 2 were characterized by elemental analysis, spectroscopy (FT-IR and NMR), and X-ray crystallography. Complex 1 crystallizes in the monoclinic space group P2(1)/n, with a = 18.035(6) A, b = 10.279(4) A, c = 12.565(4) A, beta = 109.02(1) degrees, V = 2202(2) A3, and Z = 4. Complex 2 crystallizes in the monoclinic space group P2(1), with a = 9.686(4) A, b = 8.484(4) A, c = 7.035(3) A, beta = 110.28(1) degrees, V = 542.3(4) A3, and Z = 2. Complex 1 is a trinuclear assembly with the citrate ligand securing a stable metallacyclic ring around one Cd(II), with the terminal carboxylates spanning into the coordination sphere of two nearby Cd(II) ions. Complex 2 contains mononuclear units of Cd(II) bound by citrate in an overall coordination number of 8. In both 1 and 2, the participating citrates exhibit three different modes of coordination, thus projecting a distinct yet variable aqueous structural chemistry of Cd(II) with physiological substrates. The pH-dependent chemistry and its apparent structural diversity validate past solution speciation studies, projecting the existence of mononuclear species such as the one in the anion of 2. The spectroscopic and structural properties of 2 emphasize the significance of the information emerging from synthetic studies that otherwise would not have been revealed through conventional solution studies, while concurrently shedding light onto the linkage of the requisite chemistry with the potential biological toxicity of Cd(II).

Cadmium↗
PubMed1989

The CD4/CD8:p56lck complex in T lymphocytes: a potential mechanism to regulate T-cell growth.

The CD4 and CD8 antigens on the surface of T cells appear to bind to major histocompatibility complex (MHC) class II and I antigens, respectively. These receptors have also been found to regulate T cell growth in a manner independent of MHC recognition. In this report, we describe recent work showing that the CD4 and CD8 receptors are coupled to a protein-tyrosine kinase, p56lck, from T lymphocytes. The p56lck protein is a member of the src family, which plays a crucial role in the activation and transformation of various mammalian cells. The CD4/CD8:p56lck complex is catalytically active as shown by its ability to phosphorylate at 55-60 kDa. Two-dimensional, nonequilibrium gel electrophoresis demonstrated the similarity of p56lck associated with the CD4 and CD8 antigens. Detergents were found to vary in their ability to solubilize the CD4:p56lck complex in a catalytically active form. We further demonstrated by in vitro phosphorylation that members of the CD3 complex including the gamma, delta, and epsilon chains, as well as a putative zeta subunit can be phosphorylated at tyrosyl residues by the CD4/CD8:p56lck complex. Thus, this interaction may play an important role in the activation of T cells, and may mediate the cooperative interaction between the CD4/CD8 antigens and the Ti(TcR)/CD3 complex. This interaction also represents a possible precedent by which other members of the src family (c-src, c-yes, c-fgr, etc.) may be found to interact with mammalian growth receptors.

Antibodies, Monoclonal↗
PubMed1989

Qa-1 restricted recognition of foreign antigen by a gamma delta T-cell hybridoma.

Distinct T-lymphocyte subsets recognize antigens in conjunction with different classes of major histocompatibility complex (MHC) glycoproteins using the T-cell receptor (TCR), a disulphide-linked heterodimer associated with the CD3 complex on the cell surface. In general, class I and class II MHC products provide a context for the recognition of foreign antigens by CD8+ and CD4+ T cells, respectively. This recognition seems to be largely dependent on alpha beta TCR heterodimers, whereas the function of the second gamma delta TCR, present on a minor subpopulation of cells, is still unknown. In the mouse, the existence of six cell-surface MHC class I products (K, D, L, Qa-1, Qa-2 and Tla) has been firmly established by serological, biochemical and genetic evidence. So far, only the most polymorphic of them, K, D and L ('classical' class I) have been reported as restriction elements for T-cell recognition of foreign antigens. The function of the relatively invariant Qa and Tla molecules remains unknown. We have made a T-helper cell hybridoma clone (DGT3) that recognizes synthetic copolymer poly(Glu50Tyr50) in the context of Qa-1 cell surface product, and has a CD4-CD8- phenotype. Our studies indicate that DGT3 cells express the gamma delta TCR on the cell surface, implicating its role in Qa-1-restricted antigen recognition. This is the first evidence that T cells can recognize foreign antigen in association with self Qa product, confirming that Qa molecules not only topologically, but also functionally, belong to the MHC.

Animals↗
PubMed1991

Treatment of mice bearing BCL1 lymphoma with bispecific antibodies.

Bispecific antibodies with specificity for the CD3/TCR complex of CTL and a target cell Ag can bridge both cell types and trigger cellular cytoxicity. We have produced bispecific antibodies, directed against the surface-expressed Id of the mouse BCL1 lymphoma and the mouse CD3 complex, by hybrid-hybridoma fusion. Two recombination Ig were purified to homogeneity: B1 X 7D6F, which is univalent for Id and CD3 binding and B1 X 7D6M, which is univalent for Id binding but has lost the CD3 binding because of association of the anti-CD3 H chain with the inappropriate L chain. In vitro studies indicate that bridging the TCR/CD3 complex of resting T cells with tumor IgM Id and the appropriate bispecific antibody induced proliferation and secretion of IL-2. Furthermore, in cytotoxicity assays using 51Cr-labeled tumor cells, preactivated T cells could be targeted with the bispecific antibody to give complete lysis of the Ag+ tumor. Finally, the activity of the bispecific antibody was confirmed in vivo. Animals treated i.v. with 5 micrograms of bispecific antibody 9 days after receiving BCL1 cells were cured. Furthermore, when these animals were checked at 150 days for dormant or variant tumors, as have been reported after other forms of immunotherapy in this model, none could be found. Immunotherapy experiments comparing a mixture of control antibodies with the bispecific antibody demonstrate that tumor cell-T cell bridging is established in vivo and is required for therapeutic success. These results indicate the importance of bispecific antibodies as a novel form of treatment for cancer.

Animals↗
PubMed1989

Activation of human T lymphocytes by crosslinking of anti-CD3 monoclonal antibodies.

The proliferation of human T lymphocytes induced by anti-CD3 monoclonal antibodies (mAb) is used as a model for antigen-induced activation via the T cell receptor-CD3 complex. Since both systems are accessory cell (AC)-dependent, an understanding of the role of AC in anti-CD3-induced proliferation may provide an understanding of physiological activation via the T cell receptor. Previous work has implicated receptor crosslinking as an important AC function. To determine its necessity in anti-CD3-induced lymphocyte proliferation, we prepared highly purified T lymphocytes and found that these cells did not respond to the anti-CD3 mAb UCHT1, either alone or with interleukin 1 (IL1), interleukin 2 (IL2), or tetradecanoyl phorbol acetate (TPA). However, the response, as measured by appearance of IL2 receptors and proliferation, was restored by crosslinking with immobilized goat anti-mouse antibodies (GAM) and did not require the addition of IL1, IL2, or TPA. Thus, crosslinking of CD3 receptors was a sufficient signal for proliferation of these cells. Cyclosporine A (CsA) inhibited the activation induced by immobilized UCHT1. Since macrophages are the principle targets of CsA-mediated suppression of mitogen-induced proliferation, but macrophages do not participate in the response to immobilized anti-CD3, this may indicate that CsA was inhibiting crosslinking or a signal generated by it.

Adult↗
PubMed1989

Do CD4 and CD8 control T-cell activation via a specific tyrosine protein kinase?

The CD4 and CD8 glycoproteins play an important role in T-cell activation by binding to major histocompatibility complex (MHC) class II or class I molecules, respectively, and stabilizing their interactions with the T-cell receptor-CD3 complex during antigen presentation. Recent evidence suggesting that the cytoplasmic domains of CD4 and CD8 are physically, and perhaps functionally, linked to the T-cell specific tyrosine protein kinase, p56lck, adds a new dimension to our current understanding of their physiological function. Based on these and other recent findings, Tomas Mustelin and Amnon Altman present a working hypothesis that defines a novel role for CD4 or CD8 in regulating T-cell activation, and perhaps other processes, such as thymic repertoire selection and human immunodeficiency virus (HIV)-induced immunosuppression.

Animals↗
PubMed1987

Inhibition of antibodies to CD3 surface antigen and phytohemagglutinin-mediated T cellular responses by inhibiting Ca2+/phospholipid-dependent protein kinase activity with the aid of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride.

Ca2+/phospholipid-dependent kinase activity (C-kinase) plays an important second messenger role in T lymphocyte responses initiated by the cluster of differentiation (CD3) complex and presumably also lectinic receptors. During treatment with submitogenic or mitogenic amounts of phytohemagglutinin, as well as with anti-CD3 monoclonal antibody and 12-O-tetradecanoyl 13-phorbol acetate, the enzyme was intracellularly redistributed between the cytosol and the surface membrane. Submitogenic amounts of lectin and anti-CD3 were ineffective in inducing proliferation unless exogenous interleukin 2 (IL-2) was supplied, implying that even though IL-2 receptors were expressed, additional signals were required for IL-2 production. This would also indicate that there is a direct relationship between activation of C-kinase and expression of IL-2 receptors. The importance of C-kinase was further substantiated by the ability of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H7), a potent inhibitor of this enzyme, to interfere with IL-2 receptor expression and cellular [methyl-3H]thymidine uptake during primary activation. The drug concentration at which these cellular responses were inhibited by 50% was about the same as that which decreased c-kinase activity by 50% in vitro. H7 also prevented anti-CD3-induced translocation in intact cells. This effect may be related to competition with the phosphatidylserine binding site, which is important for membrane attachment. This drug apparently also interferes with the active center of the enzyme as demonstrated by its ability to inhibit Ca2+/phospholipid-independent phosphorylation of protamine sulfate. This additional mode of inhibition may be important in suppressing intact cell responses under circumstances during which the enzyme displacement to the membrane is nonphysiologic in nature, e.g., during treatment with 12-O-tetradecanoyl 13-phorbol acetate.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1986

Distinction of two CD3-monoclonal antibodies in respect to the effectivity to modulate the induction of IgM- and gamma-interferon synthesis.

Monoclonal antibodies which specifically bind to epitopes within the T3-antigen complex (CD3 mAb) are excellent tools to analyse mechanisms of T-cell activation and of the interactions between T-cells, B-cells, accessory cells and intermediating immunefactors. In the present study we could show, that the CD-3 mAbs BMA 030 and BMA 031 are highly effective in modulating immunereactions, similar to antibodies directed to the T4-antigen (CD4-mAb) or the T8-antigen (CD8-mAbs) respectively. The data substantiate that depending on the experimental system BMA 030 and BMA 031 are clearly distinctive in their efficacy to modulate immunereactions. For therapeutical use of CD-3-monoclonal antibodies in organ transplantation those clone-specific differences of mAbs with identical binding specificity might cause for crucial differences in clinical applicability.

Antibodies, Monoclonal↗
PubMedDate not supplied

Identification and sequence of a fourth human T cell antigen receptor chain.

Thymus-derived lymphocytes (T cells) use clonally distributed antigen receptors to recognize peptide fragments associated with products of the major histocompatibility complex (MHC) (refs 1-4). On most murine and human T cells the T cell receptor (TCR) is composed of disulphide-linked alpha and beta chains (TCR alpha/beta), each of which contains constant and variable domains, and which are associated with the invariant chains of the CD3 complex. It has been demonstrated, however, that a distinct CD3-associated TCR is expressed on a small subset of T cells or immature thymocytes which fail to express either CD4 or CD8 (refs 7-14), the molecules associated with class II or class I MHC antigen recognition. Instead of TCR alpha/beta, these cells express heterodimers of gamma and delta chains (TRC gamma/delta). The genes encoding alpha, beta, and gamma have been isolated and characterized. A new murine T cell receptor (Cx) gene which undergoes rearrangement and expression early during T cell ontogeny has recently been identified 5' of the murine J alpha C alpha gene locus. Here we isolate and sequence the homologous transcript from PEER, a human cell line that expresses a TCR gamma/delta, and show that it encodes a protein with characteristic V, D, J, and C segments. Using probes derived from this transcript, we have shown that both PEER and MOLT-13, another TCR gamma/delta-expressing cell line, rearrange this locus and express two sizes of transcripts differing in the 3' untranslated region. Using a synthetic peptide derived from the deduced C region sequence, we have prepared antisera that precipitates the delta chain of the TCR from both PEER and MOLT-13, thus demonstrating that Cx and its human homologue code for the delta chain of the TCR.

Amino Acid Sequence↗
PubMed1993

Human CD4/CD45RA+ and CD4/CD45RA- T cell subsets express CD4-p56lck complexes, CD4-associated lipid kinases, TCR/CD3-p59fyn complexes, and share similar tyrosine kinase substrates.

T cell activation appears to be regulated by an interplay between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). p56lck and p59fyn have been found to associate with CD4 and TCR-CD3 respectively. The CD45 family of transmembrane PTPases has been shown to be able to regulate the activities of these receptor-associated PTKs in vitro. In man, CD45 contains five different isoforms whose distribution defines subsets of T cells having distinct activation requirements and in vitro functions. Several groups have reported a physical interaction between distinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex. Given the potential regulatory interaction between CD45 and PTKs in CD4+ subsets expressing different CD45 isoforms, we have examined CD4 associated and TCR-CD3- associated PTK activities, associated phosphatidyl inositol (PI) kinases and substrates of tyrosine phosphorylation in CD45RA+ and CD45RA- CD4+ T cell lines derived from peripheral blood. Both subsets express CD4-associated p56lck and TCR-CD3-associated p59fyn kinases which exhibit identical in vitro phosphorylation at the Y-394 and Y-420 autophosphorylation sites respectively. Further, both subsets exhibited PI kinases activity associated with CD4-p56lck. Consistent with these observations, anti-CD3 crosslinking induced the phosphorylation of a similar spectrum of intracellular substrates in these CD45RA+ and CD45RA- CD4+ T cell lines. These observations indicate that despite the possible interaction between CD45 isoforms and CD4 or TCR-CD3, the mere expression of the CD45RA isoform does not in and of itself alter the presence of receptor-associated kinases or their intracellular targets.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

Induction of non-MHC-restricted cytotoxicity in a patient with pure red cell aplasia: functional relevance to antigen-specific cytotoxic T cells.

A functional analysis of an expanded T-cell subpopulation was studied in a patient with pure red cell aplasia who had no evidence of malignancy. In the time of an aggravation of the anemia, an expanded population of large granular lymphocytes (LGL) with T-cell receptor (TCR) alpha beta +CD3+CD8+ phenotype was noted, which reverted to normal with remission. These T cells displayed a polyclonal pattern in DNA analysis. Functionally, the T cells, with suppressor/cytotoxic phenotype exhibited normal capabilities for transducing the signals of the proliferative response, and of interleukin-2R (IL-2R) expression and IL-2 production via the TCR-CD3 complex structure. While they suppressed erythroid colony formation in vitro, neither non-major histocompatibility complex-restricted cytotoxic activity, cytotoxic T-cell activity, nor suppressive activity for immunoglobulin synthesis by B cells was detected. Pretreatment by anti-CD3 or anti-T-cell receptor antibody generated cytotoxicity for FcR+ target cells in the patient cells, but no such augmentation was found with other monoclonal antibodies of FcR- target cells. These findings indicated that the expanded T cells are functionally relevant to antigen-specific cytotoxic T lymphocytes.

Adult↗
PubMed2004

Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.

Amino Acid Sequence↗