Characterization of functional GTP binding proteins in Jurkat T cell mutants lacking either CD3-Ti or CD2 surface receptors

G proteins are membrane-bound molecules involved in coupling of surface receptors with signal transduction effector systems in multiple cell types including T lymphocytes. Given that mature T cells which lack antigen receptors (CDl-Ti) are refractory to stimulation through CD2 or other accessory molecules, T cell receptor components likely play a critical role in coupling surface receptors with signal transduction effectors. It has recently been proposed that modulation of T cell receptor components with MAbs results in a physical loss or functional inactivation of G protein(s). In view of the importance of the T cell activation process, we herein examined G proteins in untreated or antibody-modulated Jurkat T cells as well as in genetic variants lacking either CD3-Ti or CD2 surface receptors. 43- and 41-kDa G protein alpha chains are ADP ribosylated with cholera (CTX) and pertussis (PTX) toxins, respectively, in wild type and receptor minus cell populations. In the wild type Jurkat cell line as well as in CD3- and CD2- variants, AlF4- can activate the G protein(s) presumably associated with phospholipase C to generate polyphosphoinositide turnover as well as an increase in cytoplasmic free calcium ions. Furthermore, G protein(s) linked to adenylylcyclase, a pathway which inhibits T lymphocyte activation, can be directly activated with CTX in the absence of CD3-Ti or CD2 on the membrane. Importantly, AlF4- can also induce polyphosphoinositide turnover in Jurkat cells whose T cell receptor proteins have been modulated with anti-CD3 MAb. These data provide functional and biochemical evidence that at least certain G proteins are intact in the absence of surface expression of CD3-Ti or CD2 molecules and imply that CD3-Ti desensitization is not singularly due to G protein loss.     

The lymphocyte receptor CD6 interacts with syntenin-1, a scaffolding protein containing PDZ domains

CD6 is a type I membrane glycoprotein expressed on thymocytes, mature T and B1a lymphocytes, and CNS cells. CD6 binds to activated leukocyte cell adhesion molecule (CD166), and is considered as a costimulatory molecule involved in lymphocyte activation and thymocyte development. Accordingly, CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse (IS) on T lymphocytes. However, the signaling pathway used by CD6 is still mostly unknown. The yeast two-hybrid system has allowed us the identification of syntenin-1 as an interacting protein with the cytoplasmic tail of CD6. Syntenin-1 is a PDZ (postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1) domain-containing protein, which functions as an adaptor protein able to bind cytoskeletal proteins and signal transduction effectors. Mutational analyses showed that certain amino acids of the most C-terminal sequence of CD6 (-YDDISAA) and the two postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1 domains of syntenin-1 are relevant to the interaction. Further confirmation of the CD6-syntenin-1 interaction was obtained from pull-down and co-immunoprecipitation assays in mammalian cells. Image analyses also showed that syntenin-1 accumulates at CD6 caps and at the IS. Therefore, we propose that syntenin-1 may function as a scaffolding protein coupling CD6 and most likely other lymphocyte receptors to cytoskeleton and/or signaling effectors during IS maturation.     

Differences in signaling molecule organization between naive and memory CD4+ T lymphocytes

The immunological synapse is a highly organized complex formed at the junction between Ag-specific T cells and APCs as a prelude to cell activation. Although its exact role in modulating T cell signaling is unknown, it is commonly believed that the immunological synapse is the site of cross-talk between the T cell and APC (or target). We have examined the synapses formed by naive and memory CD4 cells during Ag-specific cognate interactions with APCs. We show that the mature immunological synapse forms more quickly during memory T cell activation. We further show that the composition of the synapse found in naive or memory cell conjugates with APCs is distinct with the tyrosine phosphatase, CD45, being a more integral component of the mature synapses formed by memory cells. Finally, we show that signaling molecules, including CD45, are preassociated in discrete, lipid-raft microdomains in resting memory cells but not in naive cells. Thus, enhanced memory cell responses may be due to intrinsic properties of signaling molecule organization.     

CD97 stabilises the immunological synapse between dendritic cells and T cells and is targeted for degradation by the Salmonella effector SteD

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.     

Relevance of CD6-mediated interactions in T cell activation and proliferation

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.     

CD80 cytoplasmic domain controls localization of CD28, CTLA-4, and protein kinase Ctheta in the immunological synapse

The binding of costimulatory ligand CD80 to CD28 or CTLA-4 on T cells plays an important role in the regulation of the T cell response. We have examined the role of the cytoplasmic domain of CD80 in murine T cell costimulation and its organization in the immunological synapse (IS). Removal of CD80 cytoplasmic tail decreased its effectiveness in costimulating T cell proliferative response and early IL-2 production in response to agonist MHC-peptide complexes. Immunofluorescent study showed a decreased tailless CD80 accumulation in the IS of naive T cells. The two forms of CD80 accumulated differently at the IS; the tailless CD80 was colocalized with the TCR whereas the full-length CD80 was segregated from the TCR. In addition, we showed that CD80, CD28, and protein kinase Ctheta colocalized in the presence or absence of the CD80 cytoplasmic tail. Thus, the cytoplasmic tail of CD80 regulates its spatial localization at the IS and that of its receptors and T cell signaling molecules such as protein kinase Ctheta, and thereby facilitates full T cell activation.     

Immunological synapse formation licenses CD40-CD40L accumulations at T-APC contact sites

The maintenance of tolerance is likely to rely on the ability of a T cell to polarize surface molecules providing "help" to only specific APCs. The formation of a mature immunological synapse leads to concentration of the TCR at the APC interface. In this study, we show that the CD40-CD154 receptor-ligand pair is also highly concentrated into a central region of the synapse on mouse lymphocytes only after the formation of the TCR/CD3 c-SMAC. Concentration of this ligand was strictly dependent on TCR recognition, the binding of ICAM-1 to T cell integrins and the presence of an intact cytoskeleton in the T cells. This may provide a novel explanation for the specificity of T cell help directing the help signal to the site of Ag receptor signal. It may also serve as a site for these molecular aggregates to coassociate and/or internalize alongside other signaling receptors.     

CD5 inhibits signaling at the immunological synapse without impairing its formation

Physiologically, Ag detection by T cells occurs at the immunological synapse (IS) formed at the interface with an APC. CD5 is considered as an inhibitory molecule for Ag receptor-mediated signals in T cells. However, the influence of CD5 at the IS on synapse formation and functioning has not yet been reported. We demonstrate here that CD5 is recruited and tightly colocalized with CD3 in different human and murine IS. Following transfection in a CD5-negative T cell line of CD5 fused to the green fluorescent protein, we show that CD5 recruitment includes a fast Ag-independent and a slower Ag-dependent component. In video-imaging recordings of doubly transfected cells, the movements of CD3 and CD5 show similar kinetics, and the amount of CD3 recruited to the synapse is unaffected by CD5 expression. Moreover, APC-T cell adhesion is unchanged in CD5-expressing cells. Despite this, the extent of tyrosine phosphorylation at the synapse and the amplitude of calcium responses induced by Ag recognition are both decreased by CD5. These inhibitions increase with CD5 membrane levels. They also requires the pseudo-immunoreceptor tyrosine-based activation motif expressed in the cytoplasmic domain of the molecule. Thus, CD5 is rapidly recruited at the IS and lowers the T cell response elicited by Ag presentation by targeting downstream signaling events without affecting IS formation.     

CD4 raft association and signaling regulate molecular clustering at the immunological synapse site

T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) theta clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKCtheta raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKCtheta clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.     

CD28 signals in the immature immunological synapse

T cell recognition of peptide-MHC complexes on APCs results in the aggregation of TCRs at a central supramolecular activation complex (c-SMAC) within a mature immunological synapse. T cells require a second "costimulatory" signal for activation, the most important of which, for naive T cells, is from CD28. However the time at which CD28-derived signals are induced relative to c-SMAC formation is not well understood. In this study, we have assessed the kinetics of CD28 localization and function relative to well-established aspects of c-SMAC formation. CD28 accumulates at the immature synapse alongside the TCR and is likewise enriched at the synapse at the onset of the calcium signal. In addition, using CD28 deficient or reconstituted murine cells in a single-cell recording approach shows that CD28 regulates this signal within seconds of a TCR-mediated rise in intracellular calcium levels. Finally, CD28 exerts effects on both the initiation and stabilization of the synapse in parallel with its effects on the downstream proliferation of T cells. Together, the data show that CD28 functions in the immunological synapse before the formation of the c-SMAC.     

Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement

T cell polarization and redistribution of cellular components are critical to processes such as activation, migration, and potentially HIV infection. Here, we investigate the effects of CD4 engagement on the redistribution and localization of chemokine receptors, CXCR4 and CCR5, adhesion molecules, and lipid raft components including cholesterol, GM1, and glycosyl-phosphatidylinositol (GPI)-anchored proteins. We demonstrate that anti-CD4-coated beads (alpha CD4-B) rapidly induce co-capping of chemokine receptors as well as GPI-anchored proteins and adhesion molecules with membrane cholesterol and lipid rafts on human T cell lines and primary T cells to the area of bead-cell contact. This process was dependent on the presence of cellular cholesterol, cytoskeletal reorganization, and lck signaling. Lck-deficient JCaM 1.6 cells failed to cap CXCR4 or lipid rafts to alpha CD4-B. Biochemical analysis reveals that CXCR4 and LFA-1 are recruited to lipid rafts upon CD4 but not CD45 engagement. Furthermore, we also demonstrate T cell capping of both lipid rafts and chemokine receptors at sites of contact with HIV-infected cells, despite the binding of an HIV inhibitory mAb to CXCR4. We conclude that cell surface rearrangements in response to CD4 engagement may serve as a means to enhance cell-to-cell signaling at the immunological synapse and modulate chemokine responsiveness, as well as facilitate HIV entry and expansion by synaptic transmission.     

Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected "dominant-negative" TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-kappaB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.     

Modulation of CD6 function through interaction with Galectin-1 and -3

CD6 is a lymphocyte glycoprotein receptor that physically associates with the antigen-specific receptor complex at the center of the immunological synapse, where it interacts with its ligand CD166/ALCAM. The present work reports the carbohydrate-dependent interaction of CD6 and CD166/ALCAM with Galectin-1 and -3, two well-known soluble mammalian lectins. Both galectins interfered with superantigen-induced T cell proliferation and cell adhesion phenomena mediated by the CD6-CD166/ALCAM pair, while CD6 expression protected cells from galectin-induced apoptosis. The results suggest that interaction of Galectin-1 and -3 with CD6 and CD166/ALCAM might modulate some relevant aspects of T cell physiology.     

Polar redistribution of the sialoglycoprotein CD43: implications for T cell function

Contact between T cells and APCs results in the orchestrated segregation of molecules at the cell-cell interface and formation of a specialized structure termed the immunological synapse. This model predicts the topological seclusion of large molecules such as CD43 from the site of closest contact between the T cell and APC, allowing for the close apposition of cell membranes and effective TCR engagement. Similarly, during T cell migration segregation of CD43 to the uropod is thought to aid integrin adhesion at the leading edge of the cell by removing steric hindrance. We show in this work that CD43 distribution on T cells is regulated by a membrane proximal ezrin binding site and that failure to displace CD43 from the immunological synapse has no inhibitory effects on primary T cell activation. We also report that CD43 expression at the contact zone between T cells and matrix does not negatively regulate motility but may regulate LFA-1 de-adhesion. These results suggest that the steric barrier model of CD43 is inadequate and that alternative mechanisms account for the negative regulatory properties of CD43.     

Live-cell dynamics and the role of costimulation in immunological synapse formation

Using transfected fibroblasts expressing both wild-type I-E(k) and green fluorescent protein-tagged I-E(k) with covalently attached antigenic peptide, we have monitored movement of specific MHC:peptide complexes during CD4(+) T cell-APC interactions by live-cell video microscopy. Ag recognition occurs within 30 s of T cell-APC contact, as shown by a sharp increase in cytoplasmic calcium ion concentration. Within 1 min, small MHC:peptide clusters form in the contact zone that coalesce into an immunological synapse over 3-20 min. When T cells conjugated to APC move across the APC surface, they appear to drag the synapse with them. This system was used to examine the role of costimulation in the formation of the immunological synapse. Blocking CD80/CD28 or ICAM-1/LFA-1 interactions alters synapse morphology and reduces the area and density of accumulated complexes. These reductions correlate with reduced T cell proliferation, while CD69 and CD25 expression and TCR down-modulation remain unaffected. Thus, costimulation is essential for normal mature immunological synapse formation.     

Impaired induction of CD27 and CD28 predicts naive CD4 T cell proliferation defects in HIV disease

Many immunological defects have been described in HIV disease, including a diminished capacity of naive CD4+ T cells to expand after TCR stimulation. The mechanisms underlying impaired naive CD4+ T cell expansion in HIV disease are not well described. Using a rigorous phenotypic definition of naive T cells, we found that cell cycle entry after TCR engagement was restricted to cells that increased surface expression of costimulatory molecules CD27 and CD28. Induction of these receptors, however, was not sufficient to result in cell cycle entry among the CD4+CD31- naive T cell subset. Analyses of cells from HIV-infected persons indicated that naive CD4+CD31+ T cells from these subjects were impaired in their ability to enter the cell cycle after stimulation and this impairment was predicted by the relatively poor induction of costimulatory molecules on these cells. Thus, failure to increase surface expression of costimulatory molecules may contribute to the naive T cell expansion failure that characterizes HIV infection.     

MHC class II-peptide complexes and APC lipid rafts accumulate at the immunological synapse

Activation of CD4(+) Th cells requires their cognate interaction with APCs bearing specific relevant MHC class II-peptide complexes. This cognate interaction culminates in the formation of an immunological synapse that contains the various proteins and lipids required for efficient T cell activation. We now show that APC lipid raft membrane microdomains contain specific class II-peptide complexes and serve as platforms that deliver these raft-associated class II molecules to the immunological synapse. APC rafts are required for T cell:APC conjugate formation and T cell activation at low densities of relevant class II-peptide complexes, a requirement that can be overcome at high class II-peptide density. Analysis of confocal microscopy images revealed that over time APC lipid rafts, raft-associated relevant class II-peptide complexes, and even immunologically irrelevant class II molecules accumulate at the immunological synapse. As the immunological synapse matures, relevant class II-peptide complexes are sorted to a central region of the interface, while irrelevant class II molecules are excluded from this site. We propose that T cell activation is facilitated by recruitment of MHC class II-peptide complexes to the immunological synapse by virtue of their constitutive association with lipid raft microdomains.     

Human CD4+ T cells displaying viral epitopes elicit a functional virus-specific memory CD8+ T cell response

The Ag-specific cellular recall response to herpes virus infections is characterized by a swift recruitment of virus-specific memory T cells. Rapid activation is achieved through formation of the immunological synapse and supramolecular clustering of signal molecules at the site of contact. During the formation of the immunological synapse, epitope-loaded MHC molecules are transferred via trogocytosis from APCs to T cells, enabling the latter to function as Ag-presenting T cells (T-APCs). The contribution of viral epitope expressing T-APCs in the regulation of the herpes virus-specific CD8+ T cell memory response remains unclear. Comparison of CD4+ T-APCs with professional APCs such as Ag-presenting CD40L-activated B cells (CD40B-APCs) demonstrated reduced levels of costimulatory ligands. Despite the observed differences, CD4+ T-APCs are as potent as CD40B-APCs in stimulating herpes virus-specific CD8+ T cells resulting in a greater than 35-fold expansion of CD8+ T cells specific for dominant and subdominant viral epitopes. Virus-specific CD8+ T cells generated by CD4+ T-APCs or CD40B-APCs showed both comparable effector function such as specific lysis of targets and cytokine production and also did not differ in their phenotype after expansion. These results indicate that viral epitope presentation by Ag-specific CD4+ T cells may contribute to the rapid recruitment of virus-specific memory CD8+ T cells during a viral recall response.     

Requirements for modulation of the CD4 molecule in response to phorbol myristate acetate. Role of the cytoplasmic domain

CD4 (T4) is a 60 kD glycoprotein expressed on a subset of T lymphocytes. CD4 augments T cell responses to suboptimal Ag stimulation. In addition, the CD4 molecule is the receptor for HIV-1. CD4 is phosphorylated on serine residues within the cytoplasmic domain and its cell surface expression is decreased in response to PMA, APC bearing the appropriate Ag or HIV infection. The kinetics of CD4 phosphorylation and modulation are similar, suggesting that the two events may be related. L3T4, the murine CD4 equivalent, is not modulated from the surface of mature, peripheral T cells in response to PMA. The difference in the ability to modulate L3T4 and CD4 in response to PMA may be due to differences between the two molecules or to differences between the cells in which they are expressed. To further define the requirements for CD4 modulation, we used retroviral vectors to transfer the cDNA for CD4 and various mutants of CD4 into two murine T cell hybridomas that express L3T4. One of these hybridomas, By155.16, does not modulate L3T4 in response to PMA and the other, 5D5.63, does modulate L3T4 in response to PMA. When expressed by these hybridomas CD4 is not modulated from the surface of By155.16 and is modulated from the surface of 5D5.63 in response to PMA. In both of these hybridomas, CD4 is phosphorylated on serine residues in response to PMA. A mutant form of CD4, CD4 delta, was constructed in which the majority of the cytoplasmic domain was deleted. When expressed in 5D5.63, CD4 delta was not modulated in response to PMA. Replacing the cytoplasmic domain of CD4 with that of the human IL-2 receptor did not reconstitute the ability of CD4 to be modulated. These results suggest that the inability to modulate L3T4 from the surface of murine peripheral T cells is due to features of the cell and not the molecule. Furthermore, the cytoplasmic domain of CD4 is required for its modulation from the cell surface in response to PMA.