CD2 and TCR synergize for the activation of phospholipase Cgamma1/calcium pathway at the immunological synapse

Upon conjugation with cognate antigen-presenting cells (APCs), T lymphocytes undergo a sustained [Ca(2+)](i) increase resulting from the engagement of TCR and of accessory molecules with ligands expressed on the surface of APCs. We investigated the contribution of the accessory molecule CD2 to the activation of phospholipase Cgamma1 (PLCgamma1)/calcium pathway in antigen-stimulated T cells. We show that CD2 binding with its ligand CD58 expressed on the surface of APCs augments and sustains antigen-induced [Ca(2+)](i) increase in individual T cells interacting with APCs. We also show that in conditions in which CD2-CD58 interaction is impeded, the recruitment of PLCgamma1 to the immunological synapse (IS) is reduced. Interestingly, in these conditions PLCgamma1 phosphorylation in the regulatory tyrosine 783 is also defective. Our results indicate that TCR- and CD2-derived signals converge for the recruitment and activation of PLCgamma1 at the IS and shed new light on the accessory function of CD2 in T cell activation by specific antigen.     

Lipid rafts and T cell receptor signaling: a critical re-evaluation

The current model suggesting that raft integrity is required for T cell activation is mostly (but not exclusively) based on the use of drugs, such as methyl-beta-cyclodextrin (M beta CD), that disorganize rafts and inhibit T cell receptor (TCR)-induced Ca2+ influx. Here we show that conditions that disrupt lipid raft integrity do not inhibit TCR triggering in Jurkat cells and normal T lymphocytes. Indeed, we found that the reported inhibition of TCR-induced Ca2+ influx by M beta CD treatment is mainly due to (a) nonspecific depletion of intracellular Ca2+ stores and (b) plasma membrane depolarization of T cells. When these side-effects are taken into account, raft disorganization does not alter TCR-dependent Ca2+ signaling. In line with these results, also TCR-induced tyrosine phosphorylation is not inhibited by dispersion of lipid rafts. By contrast, in the same conditions, Ca2+ signaling via the glycosylphosphatidylinositol (GPI)-anchored protein CD59 is totally abolished. These results indicate that, while signaling through GPI-anchored proteins requires lipid raft integrity, CD3-dependent TCR activation occurs independently of cholesterol extraction.     

TNFR1-induced sphingomyelinase activation modulates TCR signaling by impairing store-operated Ca2+ influx

Tumor necrosis factor alpha (TNF-alpha) is a potent, pleiotrophic cytokine, which is proinflammatory but can also suppress T lymphocyte function. In chronic inflammatory disease such as rheumatoid arthritis, exposure of T cells to TNF-alpha alters their ability to mount a response by modulating the T cell receptor (TCR) signaling pathway, but the mechanisms involved remain obscure. Here, we investigated the specific role of TNF receptor 1 (TNFR1) signaling in the modulation of the TCR signaling pathway. We observed a down-regulation of the intracellular calcium ([Ca(2+)](i)) signal in Jurkat T cells after just 30 min exposure to TNF-alpha, and maximum suppression was reached after 3 h. This effect was transient, and signals returned to normal after 12 h. This depression of [Ca(2+)](i) was also observed in human CD4+ T lymphocytes. The change in Ca(2+) signal was related to a decrease in the plasma membrane Ca(2+) influx, which was apparent even when the TCR signal was bypassed using thapsigargin to induce a Ca(2+) influx. The role of TNF-alpha-induced activation of the sphingolipid cascade in this pathway was examined. The engagement of TNFR1 by TNF-alpha led to a time-dependent increase in acid sphingomyelinase (SMase; ASM) activity, corresponding with a decrease in cellular sphingomyelin. In parallel, there was an increase in cellular ceramide, which correlated directly with the decrease in the magnitude of the Ca(2+) response to phytohemagglutinin. Exogenous addition of SMase or ceramide mimicked the effects of TNFR1 signals on Ca(2+) responses in Jurkat T cells. Direct evidence for the activation of ASM in this pathway was provided by complete abrogation of the TNF-alpha-induced inhibition of the Ca(2+) influx in an ASM-deficient murine T cell line (OT-II(+/+)ASM(-/-)). This potent ability of TNF-alpha to rapidly modulate the TCR Ca(2+) signal via TNFR1-induced ASM activation can explain its suppressive effect on T cell function. This TNFR1 signaling pathway may play a role as an important regulator of T cell responses.     

CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation

CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.     

CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling

The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR zeta-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.     

Actin polymerization regulates recruitment of Nck to CD3ε upon T-cell receptor triggering

Following T-cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T-cell activation. The non-catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR-induced actin polymerization. Further, Nck is recruited to the CD3ε subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3ε was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3ε phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3ε.     

CD28 as a molecular amplifier extending TCR ligation and signaling capabilities.

Evidence has gathered that CD28 costimulation facilitates T cell activation by potentiating TCR intrinsic-signaling. However, the underlying molecular mechanism is largely unknown. Here we show that, by enhancing T cell/APC close contacts, CD28 facilitates TCR signal transduction. Moreover, the signal supplied by CD28 does not lead to increased Zap-70 and Lat phosphorylation, but amplifies PLCgamma1 activation and Ca(2+) response. We provide evidence that the PTK Itk controls the latter function. Our data suggest that CD28 binding to B7 contributes to setting the level of TCR-induced phosphorylated Lat for recruiting signaling complexes, whereas the CD28 signal boosts multiple pathways by facilitating PLCgamma1 activation. These results should provide a conceptual framework for understanding quantitative and qualitative aspects of CD28-mediated costimulation.     

Ca2+ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca2+ stores. A study with the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin.

Thapsigargin (TG), a sesquiterpene lactone and non-phorbol 12-myristate 13-acetate tumor promoter, stimulates a rapid increase in intracellular free Ca2+ [( Ca2+]i) in human T lymphocytes clone P28. The [Ca2+]i response to TG is sustained in the presence of 1 mM extracellular Ca2+, while it becomes transient in Ca2(+)-free medium suggesting that TG activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from extracellular spaces. TG-induced Ca2+ influx is completely abolished after cell depolarization caused by increased extracellular concentrations of K+. The rise in [Ca2+]i stimulated by TG occurs in the absence of detectable production of inositol phosphates. Moreover, TG does not alter the early biochemical events of T cell activation triggered through the CD2 or the CD3 T cell antigens. Indeed, both inositol phosphate production and intracellular pH increase induced by specific monoclonal antibodies (mAb) remain unchanged after TG treatment. These data suggest that in human T lymphocytes TG releases Ca2+ from an intracellular pool by a mechanism which is independent of the phospholipase C metabolic pathway. Preincubation with TG of T cell clone P28 empties both the CD2 and the CD3-sensitive intracellular Ca2+ pool(s). Conversely, prestimulation of T cell clone P28 by CD3 or CD2-specific mAb inhibits the Ca2(+)-mobilizing effect of TG. Thus it appears that TG and CD2- or CD3-specific mAb mobilize Ca2+ from common Ca2+ pool(s). Taken together, these results demonstrate that Ca2+ influx in human T cells may be linked to mobilization of intracellular Ca2+ pools and by a mechanism independent of phosphoinositide hydrolysis. They further indicate that the release of intracellular Ca2+ pool(s) may play a major role in the opening of cell membrane Ca2+ channels observed during the CD2- or CD3-induced stimulation of human T lymphocytes.     

Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells

C-terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)-mediated signaling. For the first time, we here report the effects of acute elimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70-85% knockdown of Csk was achieved within 48 h. No alterations in surface expression of CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4-5-fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2-3-fold higher Lck activity. In Jurkat cells, basal levels of zeta chain phosphorylation were elevated, and spontaneous NFAT-AP-1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and zeta chains compared to control. TCR-induced activation of NFAT-AP-1 and TCR/CD28-stimulated IL-2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR-stimulated responses.     

IL-4 is able to reverse the CD2-mediated negative apoptotic signal to CD4-CD8- alpha beta and/or gamma delta T lymphocytes.

Activation of immature thymocytes or transformed T lymphocytes via T-cell receptor (TCR)/CD3 signalling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TCR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral blood T lymphocytes. Here we report that triggering of resting CD4-CD8-TCR alpha beta+ and/or TCR gamma delta+ via the alternative CD2-dependent activation pathway is able to induce programmed cell death. A pair of mitogenic anti-CD2 mAb provoked a dramatic rise in [Ca2+]i that was almost entirely sustained by extracellular fluxes, and the inhibition of membrane [Ca2+/Mg2+] ATPase. The resulting endonuclease activation was able to induce DNA fragmentation, as revealed by propidium iodide staining and gel electrophoresis. Induction of apoptosis was prevented by the presence of interleukin-4 (IL-4) as well as by endonuclease inactivation with 100 microM ZnCl2, but enhanced by the contemporary block of protein kinase C. Thus it seems that in resting T lymphocytes the strong calcium signal delivered by the alternative CD2 activation pathway may act as a negative apoptotic signal in both alpha beta and gamma delta T cells with low (non-major histocompatibility complex restricted) antigenic affinity, so limiting the extension of polyclonal T-cell growth.     

The insulin-like growth factor-I receptor is regulated by CD28 and protects activated T cells from apoptosis

Co-stimulatory signals through the CD28 receptor enhance the survival of T cells that have their antigen receptor (TCR) engaged. Here we show that stimulation through the CD28 receptor in the absence of TCR engagement with either an anti-CD28 cross-linking antibody or the CD80 ligand transiently increases expression of the insulin-like growth factor-I receptor (IGF-IR) on T cells. Antibodies that block signaling through the IGF-IR decrease the survival of T cells activated through the TCR and CD28 in the presence of IL-2 by more than 50%, and also enhance susceptibility to Fas-induced apoptosis. CD28 stimulation increases IGF-IR expression on Jurkat cells, and exogenously added IGF-I can protect these cells from Fas-induced apoptosis. We conclude that CD28-mediated enhancement of IGF-IR expression provides activated T cells with essential survival signals that are independent of survival mediated by IL-2 and Bcl-xl.     

Role of cAMP phosphodiesterase 4 in regulation of T-cell function

Ligation of both the T-cell receptor (TCR) and the CD28 receptor is required for full T-cell activation to occur. Engagement of the TCR in primary T cells is followed by rapid cAMP production in lipid rafts resulting in raft-associated protein kinase A (PKA) activation and inhibition of proximal T-cell signaling. However, upon TCR and CD28 cross-ligation, beta-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) is recruited to lipid rafts, thus downregulating cAMP levels. Consequently, the activities of both PKA and PDE4 seem to be important for the regulation of TCR-induced signaling and T-cell function. We, therefore, propose a novel role for TCR and CD28 co-stimulation in the downmodulation of TCR-induced cAMP-mediated inhibitory signals through the recruitment of beta-arrestin and PDE4 to lipid rafts, thus allowing a full T-cell response to occur.     

Intracellular concentrations of Ca(2+) modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4(+) T lymphocytes

The costimulatory receptors CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 and their ligands, CD80 and CD86, are expressed on T lymphocytes; however, their functional roles during T cell-T cell interactions are not well known. The consequences of blocking CTLA-4-CD80/CD86 interactions on purified mouse CD4(+) T cells were studied in the context of the strength of signal (SOS). CD4(+) T cells were activated with phorbol 12-myristate 13-acetate (PMA) and different concentrations of a Ca(2+) ionophore, Ionomycin (I), or a sarcoplasmic Ca(2+) ATPase inhibitor, Thapsigargin (TG). Increasing concentrations of I or TG increased the amount of interleukin (IL)-2, reflecting the conversion of a low to a high SOS. During activation with PMA and low amounts of I, intracellular concentrations of calcium ([Ca(2+)](i)) were greatly reduced upon CTLA-4-CD80/CD86 blockade. Further experiments demonstrated that CTLA-4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of I or TG (high SOS) but the opposite occurred with PMA and low amounts of I or TG (low SOS). These results were confirmed by surface T-cell receptor (TCR)-CD3 signalling using a low SOS, for example soluble anti-CD3, or a high SOS, for example plate-bound anti-CD3. Also, CTLA-4-CD80/CD86 interactions enhanced the generation of reactive oxygen species (ROS). Studies with catalase revealed that H(2)O(2) was required for IL-2 production and cell cycle progression during activation with a low SOS. However, the high amounts of ROS produced during activation with a high SOS reduced cell cycle progression. Taken together, these results indicate that [Ca(2+)](i) and ROS play important roles in the modulation of T-cell responses by CTLA-4-CD80/CD86 interactions.     

Interelationship between CD3 and CD28 pathways in a murine T cell thymoma

It is well known that the CD28 costimulatory signal is important to complement T cell receptor (TCR)/CD3-initiated T cell activation, but the mechanism by which these two distinct signaling pathways are integrated is not clearly understood. In our laboratory, we dispose of a murine T cell hybridoma transfected with human CD28 molecule which is able to produce IL-2 in response to stimulation, suggesting that the signal transduction machinery coupled to the CD28 molecule is capable of triggering effector functions. Nevertheless, the action of three immunosuppressive agents previously shown in our model, suggested an interaction between the CD3 and CD28 pathways. We confirmed here this hypothesis by transfecting the cDNA of the human CD28 molecule in the BW5147 thymoma which lacks CD3 surface expression. Stimulation of the human CD28 did not lead to IL-2 secretion while the restoration of the TCR/CD3 complex re-established the functionality of this costimulatory molecule. These data demonstrate that the IL-2 production induced by the CD28 activation pathway is dependent of the TCR/CD3 complex cell surface expression and suggest the formation of a functional membrane complex between the CD3 and CD28 molecules. The molecular basis of the functional dependence of CD28 signaling on the TCR/CD3 complex is presently unknown. Nonetheless, we showed that some early events induced by CD28 stimulation, such as PI3-kinase association, are independent of the TCR/CD3 complex expression.     

Polycyclic aromatic hydrocarbon diol epoxides increase cytosolic Ca(2+) of airway epithelial cells

Polycyclic aromatic hydrocarbons (PAHs) increase cytosolic Ca(2+) concentration ([Ca(2+)](i)) in lymphocytes and mammary epithelial cells, but little is known regarding their effects on [Ca(2+)](i) in airway epithelium. We hypothesized that benzo[a]pyrene (BP) and/or anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a carcinogenic BP metabolite, increases [Ca(2+)](i) in untransformed human small airway epithelial (SAE) cells and that their effects on [Ca(2+)](i) are directly proportional to carcinogenicity. SAE [Ca(2+)](i) was determined by a ratiometric digital Ca(2+) imaging system. BPDE increased SAE [Ca(2+)](i) within 20 s in media with high (1 mM) and low (10 nM) Ca(2+) at a threshold concentration of 0.2 nM. Elevation of [Ca(2+)](i) persisted longer with high Ca(2+). Neither BP nor solvent altered [Ca(2+)](i). Thapsigargin and inositol 1,4,5- phosphate receptor (InsP(3)R) antagonists inhibited this BPDE action with low Ca(2+). We conclude that BPDE but not BP increases [Ca(2+)](i) partly by mobilizing Ca(2+) from cytosolic stores through an InsP(3)R. The most potent carcinogenic PAH diol epoxide increased in SAE [Ca(2+)](i) at the lowest threshold concentration, suggesting that carcinogenicity is directly proportional to the action of PAHs on SAE [Ca(2+)](i). Short-term exposure to BPDE 36 to 48 h before the study rendered SAE cells less sensitive to BPDE, suggesting that BPDE may also induce persistent changes in Ca(2+) signaling pathways.     

Regulation of Lck activity by CD4 and CD28 in the immunological synapse

Although the Src family tyrosine kinase Lck is essential for T cell receptor (TCR) signaling, whether or how Lck is activated is unknown. Using a phosphospecific antiserum to Lck, we show here that Lck becomes autophosphorylated when T cells are stimulated by antigen-presenting cells (APCs). We found that TCR cross-linking alone could not stimulate Lck autophosphorylation and CD45 was not required for this process. Instead, the T cell accessory molecules CD4 and CD28 cooperated to induce autophosphorylation of Lck. CD4 recruited Lck to the T cell--APC interface, whereas CD28 sustained Lck activation. These data show how the multiple interactions afforded by the immunological synapse drive efficient and highly specific signaling.     

CD28 affects the earliest signaling events generated by TCR engagement

The efficiency and magnitude of T cell responses are influenced by ligation of the co-stimulatory receptor CD28 by B7 molecules expressed on antigen-presenting cells (APC). In contrast to most previous studies in which agonistic anti-TCR/CD3 and anti-CD28 antibodies were employed, here we have investigated the contribution of CD28 to T cell activation under physiological conditions of antigen presentation. Jurkat T cells and primary T cells from TCR-transgenic mice stimulated with superantigen and antigen, respectively, presented by B7-expressing APC were utilized. In both systems we show that inhibiting CD28/B7 interaction resulted in impaired TCR-induced tyrosine phosphorylation of the signal-transducing ζ chain and ZAP-70. Consistent with a blockade of TCR-proximal signaling events, Jurkat cells stimulated in the absence of CD28 ligation were found to have strongly diminished tyrosine phosphorylation of cellular substrates and downstream signaling pathways such as Ca²⁺ /calcineurin, ERK/MAPK and JNK. Our results provide evidence for a role of CD28 in enhancing TCR signaling capacity during the earliest stages of T cell : APC interaction.