CD26: a multifunctional integral membrane and secreted protein of activated lymphocytes

CD26 has proved interesting in the fields of immunology, endocrinology, cancer biology and nutrition owing to its ubiquitous and unusual enzyme activity. This dipeptidyl aminopeptidase (DPP IV) activity generally inactivates but sometimes alters or enhances the biological activities of its peptide substrates, which include several chemokines. CD26 costimulates both the CD3 and the CD2 dependent T-cell activation and tyrosine phosphorylation of TCR/CD3 signal transduction pathway proteins. CD26 in vivo has integral membrane protein and soluble forms. Soluble CD26 is at significant levels in serum, these levels alter in many diseases and soluble CD26 can modulate in vitro T-cell proliferation. CD26, being an adenosine deaminase binding protein (ADAbp), functions as a receptor for ADA on lymphocytes. The focus of this review is the structure and function of CD26 and the influence of its ligand binding activity on T-cell proliferation and the T cell costimulatory activity of CD26.     

CD4-independent signal transduction through the T-cell receptor (TCR/CD3).

The membrane-bound CD4 glycoprotein has been proposed to act like a co-receptor along with the T-cell antigen receptor (TCR/CD3) during ligand recognition and cell activation. Due to its association with the protein tyrosine kinase (PTK) p56lck, CD4 is believed to transduce a signal and support CD3 activation of T cells. In this study we have shown that CD3 ligation on murine T-cell hybridomas induces tyrosine phosphorylation of proteins, including phospholipase C-gamma 1 (PLC gamma 1), both in the presence as well as in the absence of CD4-linked p56lck. Furthermore, using HPB clones deficient in CD3/PTK association, it has been found that the presence of CD4/p56lck does not overcome the defect in signalling. Not even co-aggregation of CD4 with CD3 triggers tyrosine phosphorylation of proteins in these cells. Together, the present results indicate that CD3-linked PTK(s) plays a primary role in the induction of signalling through TCR/CD3, and the presence of CD4/p56lck is neither necessary nor sufficient to elicit these events. In the light of these results a possible role for CD4 in antigen presentation has been proposed.     

Multiple signal transduction pathways activated through the T cell receptor for antigen.

The T cell receptor for antigen (TCR) is a multichain complex on the surface of T lymphocytes which binds peptide antigen and transduces a transmembrane signal leading to IL-2 secretion. Engagement of the TCR leads to activation of a tyrosine phosphorylation pathway and a phospholipase C (PLC) pathway leading to activation of protein kinase C (PCK). Currently available data suggest that the primary event in signal transduction is tyrosine kinase activation, since when this pathway is inhibited, PLC activation is blocked and there is no production of IL-2. The nature of the tyrosine kinase which initiates the signaling cascade is currently unknown. The CD4/CD8 associated kinase p56lck clearly plays a role in tyrosine phosphorylation, but it is clearly not the only tyrosine kinase involved. Studies demonstrating physical association of p59lyn with the TCR implicate fyn as an important candidate for the TCR tyrosine kinase. The protein tyrosine phosphatase CD45 also plays a critical early role in signal transduction since in cells where it is deficient, neither tyrosine kinase activation nor later signaling events are seen. The importance of the PLC/PKC pathway is illustrated by the fact that activation of this pathway alone may lead to IL-2 production. However, there may also be other mechanisms which can generate an IL-2 response. Two proteins known to be involved in growth regulation--p21ras and c-raf--have now been shown to be downstream targets of the PLC/PKC pathway.     

Activation of mitogen-activated protein kinase/ERK-2 in phytohaemagglutin in blasts by recombinant interleukin-2: contrasting features with CD3 activation.

We investigated activation of mitogen-activated protein (MAP) kinase, also known as microtubule associated protein-2 kinase (MAP-2K), by recombinant interleukin-2 (rIL-2) in phytohaemagglutinin (PHA)-induced peripheral blood lymphoblasts (PBL). MAP-kinase activation has been implicated in growth of lymphocytes and other cell types. Enzyme activity was purified from cell lysates by ion-exchange chromatography and activity measured by the ability to phosphorylate the substrates MAP-2 and myelin basic protein peptide (APRTPGGRR) in vitro. Recombinant IL-2 stimulated a variable (two-to 10-fold) and evanescent MAP-2K response which was dose dependent over the range 0-50 U/ml. In contrast to MAP-kinase activation by the CD3 receptor, activation by the IL-2 receptor (IL-2R) proceeded independently from protein kinase C (PKC) and extracellular-free Ca2+. MAP-kinase activation by CD3 involves an activation cascade which depends on Ca2+ influx and PKC activation. These events culminate in tyrosine phosphorylation and activation of MAP kinase. Recombinant IL-2 induced tyrosine phosphorylation of several intracellular proteins, including a 40,000 MW substrate which co-electrophoresed with ERK-2 on SDS-PAGE. The ERK-2 gene encodes a 41,000 MW MAP-2K and is subject to regulation by a variety of mitogens and growth factors in lymphocytes and non-lymphoid cells. MAP-kinase activation by rIL-2 was abrogated when PHA blasts were pretreated with the tyrosine protein kinase (TPK) inhibitor, methyl-2,5-dihydroxy-cinnamate. Although the TPK, p56lck, has been implicated in the activation of MAP kinase and the function of IL-2R, we found no mobility shift from a 56,000 to a 60,000 MW position as seen during PKC activation. Together these data suggest that tyrosine phosphorylation is critical to IL-2-mediated signal transduction and that MAP kinase is one of the cellular intermediates involved in this pathway.     

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4(+) Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphorylation of ZAP-70, zeta, or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAP kinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10 secretion. ERK or JNKinhibitors partially inhibited IL-4 and IL-10 secretion, while PKC or p38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.     

CD28 signal transduction: tyrosine phosphorylation and receptor association of phosphoinositide-3 kinase correlate with Ca2+-independent costimulatory activity

The interaction of CD28 with its counter-receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen-dependent interleukin-2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross-linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28-induced, p56^lck kinase-independent pathway causes tyrosine-phosphorylation of a 110-kDa substrate while recruitment of p56^lck kinase activity is apparently required for CD28-induced tyrosine-phosphorylation of 97- and 68-kDa substrates as well as CD28-induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium-independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)-3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI-3 kinase. PI-3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was “activated” by cross-linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI-3 kinase with the “activated” CD28 receptor was rapid, occurring within 30 s of receptor cross-linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI-3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate-treated Jurkat B2.7 cells, but not untreated cells. Pervanadate-induced tyrosine phosphorylation of CD28 correlated with receptor association of PI-3 kinase in the absence of CD28 cross-linking, suggesting that CD28 association with PI-3 kinase uses a tyrosine phosphorylation-dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI-3 kinase in mediating the CD28 calcium-independent, cyclosporin A-insensitive costimulatory signal.     

Cyclic AMP Sensitive Signalling by the CD28 Marker Requires Concomitant Stimulation by the T-Cell Antigen Receptor (TCR/CD3) Complex

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RIα₂-Cβ₂) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anli-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP)     

Lack of evidence for aggregation-dependent enhancement of p56lck in the signal transduction upon major histocompatibility complex recognition by mature T cells

The kinase activity of lymphocyte-specific tyrosine kinase p56lck (Lck) upon physiological major histocompatibility complex (MHC) recognition by normal mature T cells was examined. Recognition of the target MHC molecules by T cells induced phosphorylation of the zeta-chain without obvious enhancement of the background Lck activity. There was no sign of enhancement of Lck through putative T-cell receptor (TCR)-independent class II MHC/CD4 interactions either. As has been reported, cross-linking of CD4 molecules by antibodies induced a marked enhancement of Lck activity. However, it did not have an immediate relevance to TCR-mediated signal transduction, as judged from the lack of detectable de novo phosphorylation of zeta-chain and the absence of functional responses of T cells. These results strongly favour the model in which TCR-mediated signal transduction does not involve aggregation-dependent enhancement of Lck, suggesting that the signal can be triggered simply by the recruitment of already active Lck with basal kinase activity through the formation of a TCR/MHC/CD4 ternary complex.     

Alternative p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases

Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.     

Fyn and Lck tyrosine kinases regulate tyrosine phosphorylation of p105CasL, a member of the p130Cas docking protein family, in T-cell receptor-mediated signalling

We have previously shown that engagement of the T-cell receptor (TCR)/CD3 complex with anti-CD3 antibody induces tyrosine phosphorylation of p105CasL (CasL), a member of the p130Cas docking protein family. In the present work, we attempted to determine which protein tyrosine kinases (PTKs) regulate TCR-mediated phosphorylation of CasL. We show here that an association between CasL and two types of Src family PTKs, Fyn and Lck, is induced by anti-CD3 cross-linking of human H9 T cells. In contrast, ZAP-70, another PTK that also plays a critical role in the TCR signalling, failed to bind CasL, even after anti-CD3 stimulation. In vitro kinase assays revealed that Fyn and Lck, but not ZAP-70, were capable of phosphorylating CasL. Moreover, we found that CasL was constitutively hyperphosphorylated in vivo in splenocytes of MRL-MP-lpr/lpr mice, in which overproduction and excessive activation of Fyn and Lck have previously been shown to occur. Constitutive in vivo binding of CasL to both kinases was also demonstrated in lpr splenocytes. These results strongly suggest that CasL is a substrate for Fyn and Lck PTKs in TCR signal transduction.     

A novel 90-kDa tyrosine-phosphorylated protein associated with TCR complex in thymocytes

Ligation of the TCR-CD3 complex initiates a cascade of tyrosine phosphorylation that results in T cell activation. Initial activation of tyrosine kinases depends on the phosphorylation of activation motifs on CD3 chains. We previously found that a 90-kDa protein was tyrosine phosphorylated upon TCR cross-linking and the induction of the phosphorylation was dependent on the structure of the CD3 complex. In this study, we further characterized p90 phosphorylation. Phosphorylation of p90 was induced only by stimulation through the TCR-CD3 complex but not by other kinds of stimulation including CD28- or hydrogen peroxide-mediated activation and was dynamically regulated. Phosphorylated p90 was associated with the TCR-CD3 complex upon T cell activation. In a normal T cell population, thymocytes but not splenic T cells induced the tyrosine phosphorylation of p90 upon TCR cross-linking. These results suggest that p90 is a novel phosphoprotein associated with the TCR-CD3 complex and may play a role in TCR signaling during thymocyte differentiation.     

Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorylations of zeta-chains and p59fyn/p56lck in human T cells

The expression and catalytic activity of the protein tyrosine kinase (PTK) ZAP-70 are needed for normal intracellular signaling through the T-cell receptor (TCR)/CD3 complex. However, the possible effect of aging on the catalytic activity of ZAP-70 in human peripheral blood T cells stimulated via the TCR/CD3 complex is unknown. The current studies show that T cells from a substantial proportion of elderly humans (12) exhibit significant reductions in the catalytic activity, but not expression of ZAP-70 when stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. In addition, the reduced catalytic activity of ZAP-70 in T cells from elderly subjects was not restored to the normal levels in response to ligation of CD4 receptors, suggesting defects in PTKs linked to both CD3 and CD4 receptors. Other experiments demonstrated that the age-related impairments of ZAP-70 activation in anti-CD3-stimulated T cells were accompanied by decreased tyrosine phosphorylations of zeta-chains and autophosphorylations of the PTKs p561ck/p59fyn. Moreover, the age-related defects in these early TCR/CD3-mediated phosphorylation events were readily detectable in both CD45RO+ memory and CD45RA+ naive T cells. Thus, these results suggest that defects in early TCR/CD3-mediated phosphorylation events among CD45RO+ memory and CD45RA+ naive T cells from certain elderly humans may con tribute to impaired induction of ZAP-70 catalytic activity.     

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.     

Obligatory cross-talk with the tyrosine kinases assembled with the TCR/CD3 complex in CD4 signal transduction.

Dissection of the CD4 signal transduction pathway has revealed striking similarities with the TCR/CD3 pathway. Furthermore, downstream signaling by CD4 is impaired in cells lacking surface TCR, suggesting a role for the TCR/CD3 complex in CD4 signal transduction. We have investigated the molecular basis for the dependence of CD4 signaling on TCR/CD3 expression. Using the phosphotyrosine binding domains of the Shc adaptor and the Fyn kinase, which both participate in CD4 signaling, as baits, we show that CD4 induces tyrosine phosphorylation of a subset of the proteins phosphorylated in response to TCR/CD3 engagement. The phosphoprotein patterns were dramatically altered in cells defective for TCR/CD3 expression, and were recoverable by reconstitution of correctly assembled TCR, suggesting that CD4 uses TCR/CD3-associated tyrosine kinases to signal. Among the tyrosine kinases associated with the resting TCR/CD3 complex, only Fyn is activated following CD4 engagement. The failure of Fyn to become phosphorylated in cells defective for TCR expression underlines the unique role of TCR/CD3 associated Fyn in CD4 signal transduction. While no calcium mobilization was measurable in cells defective for TCR/CD3 expression in response to CD4 engagement, the Ras/MAP kinase pathway could be partially activated. Thus, CD4 activates at least two signaling pathways, and tyrosine kinases associated with the TCR/CD3 complex are key components of one of these pathways.     

Regulation of the p59fyn protein tyrosine kinase by the CD45 phosphotyrosine phosphatase.

Triggering of the T cell antigen receptor/CD3 (TcR/CD3) complex leads to rapid tyrosine phosphorylation of regulatory proteins that participate in initiating T cell activation and proliferation. This signal transduction event requires the presence of the TcR/CD3-associated protein tyrosine kinase p59fyn. There is also evidence that the CD45 phosphotyrosine phosphatase is involved in TcR/CD3 signalling. We show here by capping experiments using double indirect immunofluorescence techniques that the receptor phosphotyrosine phosphatase CD45 and the intracellular protein tyrosine kinase p59fyn specifically co-distribute in functional T lymphocytes. Furthermore, we provide evidence that isolated p59fyn is a substrate for CD45 as indicated by the rapid dephosphorylation of the regulatory Tyr531 of p59fyn by CD45. This dephosphorylation is accompanied by a severalfold increase in the catalytic activity of p59fyn as measured by its autophosphorylation and phosphorylation of an exogenous substrate. We also demonstrate that CD45-mediated dephosphorylation and activation of p59fyn apparently occurs at a slow basal rate in resting T cells. This represents the first identification of a physiologic regulator of p59fyn and implies a mechanism for the role of CD45 in TcR/CD3 signal transduction.     

T-cell receptor downregulation by ceramide-induced caspase activation and cleavage of the zeta chain

Regulation of T-cell receptor (TCR) cell surface expression levels is probably an important mechanism by which T-cell responsiveness is controlled. Previously, two distinct pathways for TCR downregulation have been described. One is dependent on protein kinase C (PKC) and the leucine-based receptor-sorting motif (L-based motif) of the CD3 gamma chain but independent of tyrosine kinases, whereas the other is dependent on the tyrosine kinase activation but independent of the PKC and the CD3 gamma L-based motif. In this study, we describe a new pathway for TCR downregulation distinct from both the PKC/CD3 gamma L-based motif-dependent and the tyrosine kinase-dependent pathways. This pathway is dependent on ceramide-induced activation of caspases and correlate with caspase-mediated cleavage of the zeta chain. Thus, a 10--15% downregulation of the TCR was induced following the treatment of the T cells with ceramide for 4 h. A close correlation between TCR downregulation, caspase activation, and cleavage of the zeta chain was found. Furthermore, the caspase inhibitors abolished the cleavage of the zeta chain and TCR downregulation in parallel with the inhibition of the caspase activity.     

The phosphatase domains of CD45 are required for ligand induced T-cell receptor downregulation

Down-regulation of the T-cell receptor (TCR) plays an important role in modulating T-cell responses, both during T-cell development and in mature T cells. At least two distinct pathways exist for TCR down-regulation: down-regulation following TCR ligation; and down-regulation following activation of protein kinase C (PKC). Ligand-induced TCR down-regulation is dependent on protein tyrosine kinase (PTK) activity and seems to be closely related to T-cell activation. In addition, previous studies have indicated that ligand-induced TCR down-regulation is dependent on the expression of CD45, a transmembrane protein tyrosine phosphatase. The role of the different domains of CD45 in TCR down-regulation was investigated in this study. We found that the phosphatase domains of CD45 are required for efficient ligand-induced TCR down-regulation. In contrast, the extracellular domain of CD45 is dispensable for ligand-mediated TCR down-regulation. Finally, PKC-mediated TCR down-regulation was found to be independent of both the extra-and intracellular domains of CD45.     

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.