Cytotoxic-T-lymphocyte antigen 4 receptor signaling for lymphocyte adhesion is mediated by C3G and Rap1.
ABSTRACT: T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.
Project description:WAVE2 regulates T cell receptor (TCR)-stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.
Project description:Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-? and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-?. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.
Project description:T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.
Project description:C3G, a Guanine nucleotide Exchange Factor (GEF) for Rap1 and R-Ras, has been shown to play important roles in development and cancer. Previous studies determined that C3G regulates cell death through down-regulation of p38? MAPK activity. Here, we found that C3G knock-down in MEFs and HCT116 cells promotes migration and invasion through Rap1-mediated p38? hyper-activation. These effects of C3G were inhibited by Rap1 knock-down or inactivation. The enhanced migration observed in C3G depleted HCT116 cells was associated with reduction in E-cadherin expression, internalization of ZO-1, actin cytoskeleton reorganization and decreased adhesion. We also found that matrix metalloproteases MMP2 and MMP9 are involved in the pro-invasive effect of C3G down-regulation. Additionally, our studies revealed that both C3G and p38? collaborate to promote growth of HCT116 cells in vitro and in vivo, possibly by enhancing cell survival. In fact, knocking-down C3G or p38? individually or together promoted cell death in vitro, although only the double C3G-p38? silencing was able to increase cell death within tumors. Notably, we found that the pro-tumorigenic function of C3G does not depend on p38? or Rap1 activation. Altogether, our studies uncover novel mechanisms by which C3G controls key aspects of tumorigenesis.
Project description:Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecules (ICAMs) facilitates T cell antigen receptor (TCR)-mediated killing. To dissect TCR and LFA-1 contributions, we evaluated cytolytic activity and granule release by cytotoxic T lymphocytes (CTL) as well as intracellular granule redistribution and morphology of CTL stimulated with natural TCR ligand in the presence or absence of LFA-1 engagement. Although other adhesion mechanisms, e.g., CD2-CD58 interaction, could substitute for LFA-1 to trigger CTL degranulation, productive LFA-1 ligation was indispensable for effective target cell lysis by the released granules. LFA-1-mediated adhesion to glass-supported bilayers containing intercellular adhesion molecule-1 was characterized by a much larger junction area, marked by LFA-1 segregation, and a more compact cell shape compared with those observed for CD2-mediated adhesion to bilayers containing CD58. A larger contact induced by intercellular adhesion molecule 1 determined a unique positioning of granules near the interface. These data provide evidence that LFA-1 delivers a distinct signal essential for directing released cytolytic granules to the surface of antigen-bearing target cells to mediate the effective destruction of these cells by CTL.
Project description:Leukocyte trafficking is crucial to facilitate efficient immune responses. Here, we report that the large GTPase dynamin2, which is generally considered to have a key role in endocytosis and membrane remodeling, is an essential regulator of integrin-dependent human T lymphocyte adhesion and migration. Chemical inhibition or knockdown of dynamin2 expression significantly reduced integrin-dependent T cell adhesion in vitro. This phenotype was not observed when T cells were treated with various chemical inhibitors which abrogate endocytosis or actin polymerization. We furthermore detected dynamin2 in signaling complexes and propose that it controls T cell adhesion via FAK/Pyk2- and RapGEF1-mediated Rap1 activation. In addition, the dynamin2 inhibitor-induced reduction of lymphocyte adhesion can be rescued by Rap1a overexpression. We demonstrate that the dynamin2 effect on T cell adhesion does not involve integrin affinity regulation but instead relies on its ability to modulate integrin valency. Taken together, we suggest a previously unidentified role of dynamin2 in the regulation of integrin-mediated lymphocyte adhesion via a Rap1 signaling pathway.
Project description:<h4>Background</h4>Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined.<h4>Methodology/principal findings</h4>By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/-) mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells.<h4>Conclusions/significance</h4>We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer.
Project description:Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P(2)) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2).
Project description:Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3(+) regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9-12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4-13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing ?-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.
Project description:The mechanisms regulating the size of the cerebral cortex are poorly understood. Here, we demonstrate that the Rap1 guanine nucleotide exchange factor, C3G (Grf2, Rapgef1), controls the size of the cerebral precursor population. Mice lacking C3G show overproliferation of the cortical neuroepithelium. C3G-deficient neuroepithelial cells accumulate nuclear beta-catenin and fail to exit the cell cycle in vivo. C3G mutant neural precursor cells fail to activate Rap1, exhibit activation of Akt/PKB, inhibition of the beta-catenin-degrading enzyme, Gsk3beta and accumulation of cytosolic and nuclear beta-catenin when exposed to growth factors, in vitro. Our results show that the size of the cortical neural precursor population is controlled by C3G-mediated inhibition of the Ras signalling pathway.