Zinc is essential for binding of p56(lck) to CD4 and CD8alpha

Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8alpha is necessary for T-lymphocyte development and activation. Association of p56(lck) with CD4 requires two conserved cysteine residues in the cytosolic domain of CD4 and two in the amino terminus of p56(lck), consistent with the notion that these four residues coordinate a single metal atom (1-5). Here we demonstrate that Zn2+ is essential for complex formation. In an in vitro binding reaction, Zn2+ mediates p56(lck) association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of CD4 or CD8alpha; no other metals tested support binding. Treatment of preformed GST-CD4.p56(lck) dimers with the Zn2+ chelators 1,10-O-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid results in dissociation of GST-CD4 from p56(lck), consistent with the finding of Huse et al. (5) that Zn2+ is contained within similar complexes. Furthermore, we show that, within live cells, CD4.p56(lck) and CD8alpha.p56(lck) interactions occur in a zinc-dependent fashion. Specifically, pretreatment of the human Jurkat T-cell line with membrane permeable zinc chelators disrupts CD4.p56(lck) complexes, and treatment of COS cells co-expressing CD8alpha and p56(lck) with such chelators likewise leads to dissociation of CD8alpha.p56(lck) complexes. CD4. p56(lck) and CD8alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization.     

Lineage-specific control of superantigen-induced cell death by the protein tyrosine kinase p56(lck)

Cell fate decisions in developing T cells depend on signal transduction via the Ag-specific TCR. Although the same TCR can signal for survival or cell death, specific signals that lead to cellular activation or death have not been identified. To study the role of the src tyrosine kinase p56(lck) in cell death of developing T cells, we introduced endogenous mouse mammary tumor retroviruses encoding superantigens (SAG) into p56(lck)-deficient mice. We show that clonal deletion of SAG-reactive CD4+ T cells does occur in p56(lck) -/- mice. Clonal deletion was also evident in CD4+ cells expressing TCRVbeta7, which has low affinity for Mls-1a. However, clonal deletion did not occur in SAG-reactive CD8+ T cells from p56(lck) -/- mice. Deletion of cells expressing SAG-reactive TCRVbeta chains was apparent in CD4+ single-positive but not in CD8+ single-positive thymocytes. Both CD4+ and CD8+ peripheral T cells from Mls-1b p56(lck) -/- mice responded to Mls-1a in vitro. However, CD8+ T cells from Mls-1a p56(lck) -/- mice that did not undergo deletion could not respond to Mls-1a, indicating that these cells are functionally unresponsive. These data show that p56(lck) is not required for clonal deletion of SAG-reactive CD4+ lymphocytes, including CD4+ cell expressing TCRs with low affinity for the SAG. However, p56(lck) appears to be an important signal transduction molecule involved in deletion of SAG-reactive CD8+ T cells.     

A noncomitogenic CD2R monoclonal antibody induces apoptosis of activated T cells by a CD95/CD95-L-dependent pathway

Clonal expansion of activated T and B cells is controlled by homeostatic mechanisms resulting in apoptosis of a large proportion of activated cells, mostly through interaction between CD95 (Fas or Apo-1) receptor and its ligand CD95-L. CD2, which is considered as a CD3/TCR alternative pathway of T cell activation, may trigger activation-induced cell death, but the role of CD95/CD95-L interaction in CD2-mediated apoptosis remains controversial. We show here that the CD2R mAb YTH 655.5, which does not induce comitogenic signals when associated with another CD2 mAb, triggers CD95-L expression by preactivated but not resting T cells, resulting in CD95/CD95-L-mediated apoptosis. The critical role of CD95/CD95-L interaction was supported by complete inhibition in the presence of the antagonist CD95 mAb ZB4 and by blocking CD95-L synthesis and surface expression by cycloheximide, cyclosporin A, EGTA, or cytochalasin B. YTH 655.5 was shown to stimulate p56lck phosphorylation and enzymatic activity. However, p56lck activation is not sufficient to trigger apoptosis, because other CD2R and CD4 mAbs that activate p56lck do not induce apoptosis. In conclusion, CD2 can mediate nonmitogenic signals, resulting in CD95-L expression and apoptosis of CD95+ cells.     

The protein tyrosine kinase p56lck regulates cell adhesion mediated by CD4 and major histocompatibility complex class II proteins

The CD4 protein is expressed on a subset of human T lymphocytes that recognize antigen in the context of major histocompatibility complex (MHC) class II molecules. Using Chinese hamster ovary (CHO) cells expressing human CD4, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In T lymphocytes, CD4 can also function as a signaling molecule, presumably through its intracellular association with p56lck, a member of the src family of protein tyrosine kinases. In the present report, we show that p56lck can affect cell adhesion mediated by CD4 and MHC class II molecules. The expression of wild-type p56lck in CHO-CD4 cells augments the binding of MHC class II+ B cells, whereas the expression of a mutant p56lck protein with elevated tyrosine kinase activity results in decreased binding of MHC class II+ B cells. Using site-specific mutants of p56lck, we demonstrate that the both the enzymatic activity of p56lck and its association with CD4 are required for this effect on CD4/MHC class II adhesion. Further, the binding of MHC class II+ B cells induces CD4 at the cell surface to become organized into structures resembling adhesions-type junctions. Both wild-type and mutant forms of p56lck influence CD4-mediated adhesion by regulating the formation of these structures. The wild-type lck protein enhances CD4/MHC class II adhesion by augmenting the formation of CD4-associated adherens junctions whereas the elevated tyrosine kinase activity of the mutant p56lck decreases CD4-mediated cell adhesion by preventing the formation of these structures.     

Binding of CD4 ligands induces tyrosine phosphorylation of phosphatidylinositol-3 kinase p110 subunit

We have previously reported that different putative CD4 ligands (anti-CD4 antibody, gp160 from HIV, synthetic peptides analogous to the residues 35-46 of HLA class II beta1 chain and residues 134-148 of HLA class II beta2 chain) down-regulate LFA-1-dependent adhesion between CD4+ T cells and HLA class II+ B cells, and also activate p56lck and the phosphatidylinositol-3 kinase (PI3-kinase) associated with the CD4-p56lck complex. It was demonstrated that the latter activation was dependent on the CD4-p56lck association. Since these results suggest a relationship between p56lck and PI3-kinase, we investigated whether PI3-kinase was tyrosine phosphorylated after CD4 binding and whether this phosphorylation was also dependent on the CD4-p56lck association. We show herein that CD4 binding increased tyrosine phosphorylation of the catalytic subunit p110 of PI3-kinase but not of the p85 subunit. Association between p56lck and PI3-kinase was constitutive, and was not modified after CD4 binding. In contrast, p110 tyrosine phosphorylation was inducible, transient and dependent on the CD4-p56lck association. The role of the tyrosine phosphorylation of p110-PI3-kinase following ligand binding to CD4 is unknown. We speculate that this event could link the activation of p56lck and of PI3-kinase after CD4 binding.     

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.     

Phosphatidylinositol 3-kinase participates in p56(lck)/CD4-dependent down-regulation of LFA-1-mediated T cell adhesion

The mechanism inducing cell detachment in Ag-independent adhesion between lymphocytes is poorly understood. Different putative CD4 ligands, anti-CD4 Ab, a DR35-46 peptide mimicking residues 35 to 46 of HLA class II beta1, and a DR134-148 peptide mimicking residues 134 to 148 of HLA class II beta2, were previously found to down-regulate LFA-1-dependent adhesion between CD4+ T cells and HLA class II+ B cells. This down-regulation was shown to be p56(lck) dependent. Here we show that binding of these ligands to CD4 induced the activation of the tyrosine kinase p56(lck) associated with CD4 and also the lipid kinase phosphatidylinositol-3 kinase (PI3-kinase) associated with the CD4-p56(lck) complex in the HUT78 cell line. These events were not detected when p56(lck) was dissociated from CD4 in cell lines expressing mutated forms of CD4. It was also shown, using different inhibitors of the PI3-kinase (wortmannin, Ly294002, and antisense oligonucleotides), that this lipid kinase was necessary for the down-regulation of LFA-1-mediated adhesion induced by CD4 binding. These results strongly suggest that CD4-induced PI3-kinase activation, in the absence of concomitant TCR/CD3 triggering, leads to down-regulation of LFA-1-mediated T cell adhesion to B cells. The mechanism by which PI3-kinase could exert its effect remains unknown. Since PI3-kinase has previously been found to participate in the regulation of cytoskeleton structure, we propose that p56(lck)-associated PI3-kinase activation leads to a cytoskeleton organization unfavorable for LFA-1 function.     

Regulation of CTL by ecto-nictinamide adenine dinucleotide (NAD) involves ADP-ribosylation of a p56lck-associated protein

Receptor-mediated activation of T lymphocytes involves protein phosphorylation by several protein tyrosine kinases, among those the src-related enzymes p56lck and p59fyn. Accumulating evidence supports the notion that these enzymes are regulated by tyrosine phosphorylation and dephosphorylation, but much is yet to be learned about regulation of their activity. Here we demonstrate that p56lck but not p59fyn exists as a complex with a 40-kDa protein, which in its ADP-ribosylated form inhibits p56lck kinase activity. ADP-ribosylation of this protein is mediated by an arginine-specific mono-ADP-ribosyltransferase, which makes use of extracellular nicotinamide adenine dinucleotide (NAD). This enzyme is a glycosyl-phosphatidylinositol-anchored protein releasable from the surface of cytotoxic T cells by glycosyl-phosphatidylinositol-specific phospholipase C. Release of arginine-specific mono-ADP-ribosyltransferase results in failure of extracellular NAD to downmodulate p56lck kinase activity. Concomitant to suppression of the kinase by NAD, CD8 mediated transmembrane signaling and p56lck kinase activation are inhibited.     

The BB1 monoclonal antibody recognizes both cell surface CD74 (MHC class II-associated invariant chain) as well as B7-1 (CD80), resolving the question regarding a third CD28/CTLA-4 counterreceptor

The identification of all CD28/CTLA-4 counterreceptors is critical to our understanding of this pivotal pathway of T cell activation. Clouding our understanding has been the reported discrepancies in expression and function of the B7-1 (CD80) molecule based upon the use of the BB1 vs other anti-B7-1 mAbs. To resolve this issue, we have cloned a BB1-binding molecule from the BB1+B7-1(-) NALM-6 pre-B cell line. Here, we demonstrate that this BB1-binding molecule is identical to the cell surface form of CD74 (MHC class II-associated invariant chain). CD74-transfected cells bound the BB1 mAb but not other anti-CD80 mAbs, CD28-Ig, or CTLA4Ig. Absorption and blocking experiments confirmed the reactivity of BB1 mAb with CD74. A region of weak homology was identified between CD74 and the region of B7-1 encoding the BB1 epitope. Therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Many of the puzzling observations in the literature concerning the expression of human B7-1 are resolved by an understanding that BB1 staining is the summation of CD74 plus B7-1 expression. This observation requires the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.     

Differential requirement for p56lck in fetal and adult thymopoiesis

The protein tyrosine kinase p56lck is critical for the generation of mature thymocytes in adult mice. However its requirement during the maturation of thymocytes from the fetal to the adult stage has not been clearly defined. We analyzed prenatal and postnatal thymocyte maturation in mice deficient for p56lck (lck[-/-]). Before birth, lck appears to play a crucial role in the expansion and proliferation of CD4+CD8+ double positive thymocytes, whereas proliferation and absolute numbers of CD4-CD8- double negative thymocyte precursors remained within the normal range until the end of the second week postnatal. Three weeks after birth, the total numbers of double negative and immature single positive thymocytes underwent a dramatic reduction that correlated with a decrease in the double positive population. This ontogenic defect was associated with a significant decrease in the proliferation rates of thymocyte precursors. Our data suggest that signaling via p56lck kinase is differentially required within a given phenotypically defined thymocyte subpopulation, depending on its stage of thymocyte maturation.     

The CD4-associated tyrosine kinase p56lck is required for lymphocyte chemoattractant factor-induced T lymphocyte migration

Lymphocyte chemoattractant factor (LCF) is a polypeptide cytokine which induces both cell motility and activation of T lymphocytes. These LCF-induced events demonstrate an absolute requirement for the cell surface expression of CD4. Because many CD4-mediated T lymphocyte activation events have been demonstrated to require the association of the src-related tyrosine kinase p56lck with the cytoplasmic domain of CD4, we examined the role of p56lck in LCF-induced lymphocyte migration in a murine T cell hybridoma line expressing transfected human CD4. LCF induces the catalytic activity of CD4 associated p56lck at chemoattractant concentrations of cytokine. Hybridoma cells that express CD4 with cytoplasmic point mutations which uncouple the CD4-lck association lack both lck enzymatic activity and chemotactic responses to LCF. The enzymatic activity of lck however does not appear to be required for CD4-mediated migratory signal. First, the protein tyrosine kinase inhibitor herbimycin A blocked LCF-induced p56lck activation but had no effect on the LCF-induced motile response. Second, T cell hybridomas expressing a chimeric receptor combining the extracellular domain of human CD4 and murine p56lck which lacked the kinase domain had a normal LCF-induced motile response. We conclude from these observations that CD4-lck coupling is essential for LCF-induced T lymphocyte migration but the motile response is independent of the enzymatic activity of CD4-associated p56lck.     

Vascular density in melanoma xenografts correlates with vascular permeability factor expression but not with metastatic potential

Previous studies have shown that human airway epithelial cells (AEC) can stimulate allogeneic peripheral blood T-lymphocyte (PBT) proliferation. We now sought to determine which AEC surface molecule/T-cell coreceptors are involved in this process. AEC-induced PBT proliferation was inhibited by 25 microM genestein or herbamycin A (0.9 and 1.8 microM), both tyrosine kinase inhibitors. Anti-phosphotyrosine immunoblots performed on PBT lysates after coculture with AEC demonstrated phosphorylation of 56kD and 60kD bands. To determine whether CD3 associated p59fyn, or CD4 and CD8 associated p56lck phosphotyrosine kinases (PTK) were involved, we assayed kinase activity in lymphocyte lysates immunoprecipitated with anti-p56lck and p59fyn mAbs. PBT cells or murine T-cell line transfectants expressing human CD4 (3G4) or human CD8alpha (3G8) were cocultured with AEC or A549, an alveolar-like cell line lacking class II Ag expression. After A549 or AEC coculture, p56lck activity in PB T-cells peaked at 2 min whereas p59fyn kinase activity continued to rise at 8 min. AEC (expressing class II Ags) stimulate PTK activity in both 3G8 and 3G4 cells. A549 stimulated p56lck in 3G8, but not in 3G4 cells. This activation of p56lck was not blocked by preincubation of A549 with anti-class I or anti-CD1d mAbs. An antibody generated in our laboratory, which recognizes an epithelial specific surface molecule (mAb L12) and which blocks AEC driven PBT proliferation, was shown to block PTK activity of peripheral blood T-cell lysates, though not of 3G8 lysates. These studies suggest that AEC are capable of stimulating CD4 and CD8 associated lck and CD3 associated fyn kinases through class II dependent and independent pathways.     

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes

Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin ("caveolae"). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low-density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes. We provide a detailed analysis of the two most highly expressed NRPTK, p53/p56lyn and p56lck, which are involved in the transduction of signals for proliferation and differentiation of monocytes/B lymphocytes and T lymphocytes, respectively. We show that lyn is selectively recovered in LDTI complexes isolated from human leukemic cell lines (promyelocytic [HL-60], erythroid [K562] and B-lymphoid [697]) and from normal human granulocytes, and that lck is recovered from LDTI fractions of leukemic T- and B-lymphoid cell lines (CEM, 697). In LDTI fractions of leukemic cells, lck and lyn are enriched 100-fold as compared with the total cell lysates. Analysis of these fractions by electron microscopy shows the presence of 70- to 200-nm vesicles: lyn and lck are homogenously distributed in the vesicles, as revealed by an immunogold labeling procedure. These novel results propose a role for these vesicles in signal transduction mechanisms of normal and neoplastic hematopoietic cells. In support of this hypothesis, we further observed that molecules participating in B- and T-cell receptor activation cofractionate in the LDTI fractions, CD45/lyn (B cells) and CD45/lck/CD4 (T cells).     

Human 4-1BB regulates CD28 co-stimulation to promote Th1 cell responses

Our present study provides evidence that the 4-1BB signal is critical to CD28 co-stimulation in maintaining T cell activation when CD28 has been down-regulated because of repeated stimulation. The 4-1BB signal synergized with CD28 co-stimulation by lowering the threshold of anti-CD28 required to sustain proliferation and IL-2 production. The 4-1BB signal also modulated CD28-mediated cytokine profiles by markedly enhancing Th1 but suppressing Th2-type cytokine production. The 4-1BB signal generated Th1-type cells, as identified by intracellular IFN-gamma production. IFN-gamma induction was detected preferentially in 4-1BB-expressing cells, but not in those expressing CD30. 4-1BB and CD30 were induced in both CD4+ and CD8+ cells, but the location of the two molecules was mutually exclusive in each T cell subset. Our study suggests that the 4-1BB signal regulates CD28 co-stimulation in the targeted subset cells to favor Th1 development and maintain long-term cell growth.