Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation

Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.     

Kinetics of T-cell receptor binding by bivalent HLA-DR. Peptide complexes that activate antigen-specific human T-cells

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at >100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.     

Post-thymic selection of peripheral CD4+ T-lymphocytes on class II major histocompatibility antigen-bearing cells.

Following positive and negative selection in the thymus, mature CD4+ T-cells emigrate into peripheral lymphoid organs. Whether resting T-cells require periodic stimulation to remain viable in the absence of antigen is important for understanding peripheral T-cell homeostasis. A prerequisite for T-cell receptor (TCR)-mediated signals in maintaining peripheral CD4+ T-cell longevity has been demonstrated. Here, we show in mice expressing a mutant I-Abeta transgene on an I-Abeta knockout background that na?ve CD4+ T-cells also require engagement of their CD4 coreceptors by peripheral, class II MHC-bearing cells for their survival. The transgene's product combines with endogenous Aalpha, but this mutant AalphaAbeta heterodimer cannot interact with CD4 molecules, although it efficiently presents antigens to TCRs. Resting CD4+ T-lymphocytes from mutant Abeta transgenic mice die by apoptosis at a much higher rate than do CD4+ T-cells from normal mice. Apoptosis of CD4+ T-cells in mutant Abeta transgenic mice is partially mediated by Fas. Adoptive transfer experiments revealed that the increase in apoptosis is due to a lack of interactions with mutant MHC class II rather than to an intrinsic defect in the CD4+ T-cells selected on mutant Abeta-expressing thymic epithelial cells. Thus, interactions between CD4 and MHC class II molecules contribute to the regulation of homeostasis in the peripheral immune system. Our results further suggest that thymic emigrant cells are continuously retested in the periphery for appropriate coreceptor interactions. Peripheral selection may be important in eliminating potentially autoreactive T-cells.     

Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav

The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cgamma1 (PLCgamma1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y(132), Y(171), Y(191), and Y(226) have been shown previously to be critical for binding to Gads, Grb2, and PLCgamma1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y(171) and potentially Y(226) are docking sites for the Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.     

Disruption of Mekk2 in mice reveals an unexpected role for MEKK2 in modulating T-cell receptor signal transduction

MEKK2 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase gene family involved in regulating multiple MAPK signaling pathways. To elucidate the in vivo function of MEKK2, we generated mice carrying a targeted mutation in the Mekk2 locus. Mekk2(-/-) mice are viable and fertile. Major subsets of thymic and spleen T cells in Mekk2-deficient mice were indistinguishable from those in wild-type mice. B-cell development appeared to proceed similarly in the bone marrow of Mekk2-deficient and wild-type mice. However, Mekk2(-/-) T-cell proliferation was augmented in response to anti-CD3 monoclonal antibody (MAb) stimulation, and these T cells produced more interleukin 2 and gamma interferon than did the wild-type T cells, suggesting that MEKK2 may be involved in controlling the strength of T-cell receptor (TCR) signaling. Consistently, Mekk2(-/-) thymocytes were more susceptible than wild-type thymocytes to anti-CD3 MAb-induced cell death. Furthermore, TCR-mediated c-Jun N-terminal kinase activation was not blocked but moderately enhanced in Mekk2(-/-) T cells. Neither extracellular signal-regulated kinase nor p38 MAPK activation was affected in Mekk2(-/-) T cells. In conclusion, we found that MEKK2 may be required for controlling the strength of TCR/CD3 signaling.     

Protein kinase D orchestrates the activation of DRAK2 in response to TCR-induced Ca2+ influx and mitochondrial reactive oxygen generation

DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca(2+) influx through the Ca(2+) release-activated Ca(2+) channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor G?6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca(2+) influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca(2+) influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.     

Immunocytochemical localization of phospholipase C isozymes in cord blood and adult T-lymphocytes.

The response of T-cells to peptide antigen plus major histocompatibility complex (MHC) consists of a series of cellular events collectively called T-cell activation. An essential component of this pathway is phospholipase C (PLC)gamma1, whose hydrolytic activity increases rapidly after binding of ligands to the T-cell receptor (TCR) and consequent activation of tyrosine kinases. Recent studies also suggest a GTP binding protein-dependent activation of PLCbeta during the early steps of T-cell activation. On the basis of these findings, we first checked the expression of PLC isoforms by Western blotting and by confocal and electron microscopy techniques, and then we looked for the phosphoinositide breakdown induced by CD3 engagement in cord and adult T-lymphocytes. Our results indicated that PLCbeta1 was almost exclusively expressed in cord T-cells, whereas PLCbeta2 was more strongly represented in the adult. The amount of PLCgamma1 was found to be larger in the adult than in cord cells. No significant differences were found in PLCgamma2 and delta2 expression. PLCdelta1 was scarcely detectable. On CD3 stimulation, adult lymphocytes gave rise, as expected, to a dramatic increase in phosphoinositide breakdown, whereas in cord cells this response was scarcely detected. These results indicate that a shift in PLC expression occurs in the postnatal period and that this change is associated with induction of the capability to respond to CD3 engagement with phosphoinositide hydrolysis.     

EphrinB1 is essential in T-cell-T-cell co-operation during T-cell activation

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands are ephrins (EFNs), which are also cell surface molecules. We have very limited knowledge about the expression and function of these kinases and their ligands in the immune system. In this study we investigated the effect of EFNB1 on mouse T-cells. EFNB1 and the Eph kinases it interacts with (collectively called EFNB1 receptors (EFNB1R)) were expressed on T-cells, B cells, and monocytes/macrophages. Some T-cells were double positive for EFNB1 and EFBB1R. Solid phase EFNB1 in the presence of suboptimal TCR ligation augmented T-cell responses in terms interferon-gamma secretion, proliferation, and cytotoxic T lymphocyte activity but not interleukin-2 production. After T-cell receptor (TCR) ligation, EFNB1R congregated to TCR caps, and then both of them translocated to raft caps. This provides a morphological basis for EFNB1R to enhance TCR signaling. Further downstream of the signaling pathway, EFNB1R stimulation led to increased LAT (linker for activation of T-cells) phosphorylation and p44/42 and p38 MAPK activation. Similar to CD28 costimulation, EFNB1R costimulation was insensitive to cyclosporin A inhibition. On the other hand, unlike the former, EFNB1R costimulation failed to activate Akt, which is essential in triggering interleukin-2 production. Our study suggests that EFNB1 is pivotal in T-cell-T-cell costimulation and in reducing T-cell response threshold to antigen stimulation.     

A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse

Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCgamma1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or "antisynapse".     

Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.     

Maturation and function of mouse T-cells with a transgenic TCR positively selected by highly disparate xenogeneic porcine MHC.

Remarkably normal cellular immune function, along with specific T-cell tolerance to highly disparate xenogeneic donors, can be achieved by grafting fetal pig thymus (FP THY) tissue to T and NK cell-depleted, thymectomized (ATX) mice. Porcine MHC can mediate positive selection of mouse CD4+ T-cells with a mouse MHC-restricted TCR in FP THY-grafted, T- and NK cell-depleted, ATX TCR-transgenic "AND" mice. However, functional studies were not performed on transgenic mouse T-cells selected in a FP THY graft. We have now performed further studies to confirm the ability of porcine MHC to mediate the positive selection of mouse T-cells with a mouse MHC-restricted TCR, and to exclude the possibility that the maturation of mouse T-cells with a mouse MHC-restricted TCR in FP THY grafts in ATX "AND" mice is a special case. For this purpose, TCR-transgenic mice with an unrelated transgenic TCR ["3A9", specific for hen egg lysozyme (HEL) peptide 46-61 presented by I-Ak] were employed. Similar to FP THY-grafted ATX "AND" mice, large numbers of mouse CD4 single positive thymocytes expressing the transgenic TCR (Vbeta8.2) and expressing a mature phenotype (Qa-2high and heat stable antigen, HSAlow) were detected in FP THY grafts. Porcine thymus grafting led to a high level of peripheral repopulation with mouse naive-type (CD44low CD45RBhigh CD62Lhigh) CD4+ cells expressing the transgenic TCR in T and NK cell-depleted ATX "3A9" mice, regardless of whether the recipients had a positive selecting or a non-selecting, class II deficient MHC background. The mouse CD4+ T-cells expressing the "3A9" TCR showed efficient primary proliferative responses to the protein antigen (HEL) when it was presented by mouse class II+ antigen presenting cells (APC) in vitro. These results, collectively, support the general conclusion that discordant xenogeneic porcine MHC can mediate positive selection of mouse T-cells with mouse MHC-restricted TCR. This study has implications for the potential clinical use of xenogeneic thymus transplantation to reconstitute cellular immunity in the setting of thymic insufficiency or thymectomy, and hence for its applicability to the induction of xenograft tolerance and in the treatment of immunodeficiency diseases.     

PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes

Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.     

Genetic Evidence of a Role for Lck in T-Cell Receptor Function Independent or Downstream of ZAP-70/Syk Protein Tyrosine Kinases

T-cell antigen receptor (TCR) engagement results in sequential activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the kinase domain of Syk for the homologous region in ZAP-70 (designated as KS for kinase swap). Expression of the KS chimera resulted in its autophosphorylation, the phosphorylation of cellular proteins, the upregulation of T-cell activation markers, and the induction of interleukin-2 gene synthesis in a TCR-independent fashion. The KS chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells. However, expression of the KS chimera in JCaM1.6 cells failed to rescue downstream signaling events, demonstrating a functional role for Lck beyond the activation of the ZAP-70 and Syk PTKs. These results indicate that downstream TCR signaling pathways may be differentially regulated by ZAP-70 and Lck PTKs and provide a mechanism by which effector functions may be selectively activated in response to TCR stimulation.