RESUMEN
Programmed cell death-1 (PD-1) is one of the most famous coinhibitory receptors that are expressed on effector T cells to regulate their function. The PD-1 ligands, PD-L1 and PD-L2, are expressed by various cells throughout the body at steady state and their expression was further regulated within different pathological conditions such as tumor-bearing and chronic inflammatory diseases. In recent years, immune checkpoint inhibitor (ICI) therapies with anti-PD-1 or anti-PD-L1 has become a standard treatment for various malignancies and has shown remarkable antitumor effects. Since the discovery of PD-1 in 1992, a huge number of studies have been conducted to elucidate the function of PD-1. Herein, this paper provides an overview of PD-1 biological findings and sheds some light on the current technology for molecular imaging of PD-1.
Asunto(s)
Neoplasias , Receptor de Muerte Celular Programada 1 , Humanos , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias/metabolismo , Linfocitos T/metabolismo , Antígeno B7-H1/metabolismo , Inmunoterapia/métodos , Imagen MolecularRESUMEN
With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a standard method to evaluate them. Here we establish an advanced imaging system to visualize human PD-1 microclusters, in which a minimal T cell receptor (TCR) signaling unit co-localizes with the inhibitory co-receptor PD-1 in vitro. In these microclusters PD-1 dephosphorylates both the TCR/CD3 complex and its downstream signaling molecules via the recruitment of a phosphatase, SHP2, upon stimulation with the ligand hPD-L1. In this system, blocking antibodies for hPD-1-hPD-L1 binding inhibits hPD-1 microcluster formation, and each therapeutic antibody (pembrolizumab, nivolumab, durvalumab and atezolizumab) is characterized by a proprietary optimal concentration and combinatorial efficiency enhancement. We propose that our imaging system could digitally evaluate PD-1-mediated T cell suppression to evaluate their clinical usefulness and to develop the most suitable combinations among ICIs or between ICIs and conventional cancer treatments.
Asunto(s)
Neoplasias , Receptor de Muerte Celular Programada 1 , Humanos , Imagen Individual de Molécula , Nivolumab/farmacología , Nivolumab/uso terapéutico , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T , Antígeno B7-H1/metabolismo , Inmunoterapia/métodosRESUMEN
The coinhibitory receptor, PD-1, is of major importance for the suppression of T cell activation in various types of immune responses. A high-resolution imaging study showed that PD-1 forms a coinhibitory signalosome, "PD-1 microcluster", with the phosphatase, SHP2, to dephosphorylate the TCR/CD3 complex and its downstream signaling molecules. Such a consecutive reaction entirely depended on PD-1-PD-L1/2 binding. PD-L2 is expressed on professional antigen-presenting cells and also on some tumor cells, which possibly explains the discrepant efficacy of immune checkpoint therapy for PD-L1-negative tumors. Here, we performed precise imaging analysis of PD-L2 forming PD-1-PD-L2 clusters associating with SHP2. PD-L2 could compete with PD-L1 for binding to PD-1, occupying the same space at TCR microclusters. The PD-1 microcluster formation was inhibited by certain mAbs with functional consequences. Thus, PD-1 microcluster formation provides a visible index for the effectiveness of anti-PD-1- or anti-PD-L1/2-mediated T cell suppression. PD-L2 may exert immune suppressive responses cooperatively with PD-L1 on the microcluster scale.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Activación de Linfocitos/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Transducción de SeñalRESUMEN
T cell activation is initiated by signaling molecules downstream of the T cell receptor (TCR) that are organized by adaptor proteins. CIN85 (Cbl-interacting protein of 85 kDa) is one such adaptor protein. Here, we showed that CIN85 limited T cell responses to TCR stimulation. Compared to activated wild-type (WT) T cells, those that lacked CIN85 produced more IL-2 and exhibited greater proliferation. After stimulation of WT T cells with their cognate antigen, CIN85 was recruited to the TCR signaling complex. Early TCR signaling events, such as phosphorylation of ζ-chain-associated protein kinase 70 (Zap70), Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP76), and extracellular signal-regulated kinase (Erk), were enhanced in CIN85-deficient T cells. The inhibitory function of CIN85 required the SH3 and PR regions of the adaptor, which associated with the phosphatase suppressor of TCR signaling-2 (Sts-2) after TCR stimulation. Together, our data suggest that CIN85 is recruited to the TCR signaling complex and mediates inhibition of T cell activation through its association with Sts-2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Receptores de Antígenos de Linfocitos T/genética , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Interleukin (IL)-25, which is a member of the IL-17 family of cytokines, induces production of such Th2 cytokines as IL-4, IL-5, IL-9 and/or IL-13 by various types of cells, including Th2 cells, Th9 cells and group 2 innate lymphoid cells (ILC2). On the other hand, IL-25 can suppress Th1- and Th17-associated immune responses by enhancing Th2-type immune responses. Supporting this, IL-25 is known to suppress development of experimental autoimmune encephalitis, which is an IL-17-mediated autoimmune disease in mice. However, the role of IL-25 in development of IL-17-mediated arthritis is not fully understood. Therefore, we investigated this using IL-1 receptor antagonist-deficient (IL-1Ra-/-) mice, which spontaneously develop IL-17-dependent arthritis. However, development of spontaneous arthritis (incidence rate, disease severity, proliferation of synovial cells, infiltration of PMNs, and bone erosion in joints) and differentiation of Th17 cells in draining lymph nodes in IL-25-/- IL-1Ra-/- mice were similar to in control IL-25+/+ IL-1Ra-/- mice. These observations indicate that IL-25 does not exert any inhibitory and/or pathogenic effect on development of IL-17-mediated spontaneous arthritis in IL-1Ra-/- mice.
RESUMEN
Programmed cell death 1 (PD-1) is highly expressed on exhausted T cells and inhibits T cell activation. Antibodies that block the interaction between PD-1 and its ligand prevent this inhibitory signal and reverse T cell dysfunction, providing beneficial anti-tumor responses in a substantial number of patients. Mechanisms for the induction and maintenance of high PD-1 expression on exhausted T cells have not been fully understood. Utilizing a genome-wide loss-of-function screening method based on the CRISPR-Cas9 system, we identified genes involved in the core fucosylation pathway as positive regulators of cell-surface PD-1 expression. Inhibition of Fut8, a core fucosyltransferase, by genetic ablation or pharmacologic inhibition reduced cell-surface expression of PD-1 and enhanced T cell activation, leading to more efficient tumor eradication. Taken together, our findings suggest that blocking core fucosylation of PD-1 can be a promising strategy for improving anti-tumor immune responses.
Asunto(s)
Fucosiltransferasas , Regulación Neoplásica de la Expresión Génica/inmunología , Inmunidad Celular , Proteínas de Neoplasias , Neoplasias Experimentales , Receptor de Muerte Celular Programada 1 , Linfocitos T/inmunología , Animales , Sistemas CRISPR-Cas , Fucosiltransferasas/genética , Fucosiltransferasas/inmunología , Estudio de Asociación del Genoma Completo , Glicosilación , Humanos , Activación de Linfocitos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/patologíaRESUMEN
T cell antigen receptor (TCR) stimulation induces recruitment and accumulation of various types of signaling molecules and forms signaling microclusters. The dynamics of the microclusters are important for regulating the quality and quantity of T cell activation. We describe here our protocols for analysis of signaling microclusters by using supported planar bilayers.
Asunto(s)
Estructuras de la Membrana Celular/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Estructuras de la Membrana Celular/genética , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/genéticaRESUMEN
The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.
Asunto(s)
Activación de Linfocitos/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/inmunología , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Paxillin/genética , Paxillin/inmunología , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citologíaRESUMEN
Protein arginine methyltransferase 1 (PRMT1), a major PRMT in mammalian cells, has been shown to play a crucial role in multiple biological functions in vitro. To explore the role of PRMT1 in B cells in vivo, we generated B cell-specific PRMT1-deficient (Prmt1(-/-) ) mice using a Cre-loxP system. Prmt1(-/-) mice showed a defect in B-cell development with diminished levels of serum antibodies. Antibody responses in Prmt1(-/-) mice were absent after stimulation with the type 2 T cell-independent antigen NP-Ficoll but intact after stimulation with the T cell-dependent antigen NP-OVA. Our findings comprise the first evidence showing that PRMT1 is necessary for lymphocyte functions in vivo.
Asunto(s)
Formación de Anticuerpos/inmunología , Proteína-Arginina N-Metiltransferasas/metabolismo , Linfocitos T/inmunología , Animales , Antígenos/metabolismo , Linfocitos B/inmunología , Femenino , Ficoll/inmunología , Inmunoglobulinas/sangre , Activación de Linfocitos , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Proteína-Arginina N-Metiltransferasas/deficienciaRESUMEN
CARMA1-mediated NF-κB activation controls lymphocyte activation through antigen receptors and survival of some malignant lymphomas. CARMA1 clusters are formed on physiological receptor-mediated activation or by its oncogenic mutation in activated B-cell-diffuse large B-cell lymphomas (ABC-DLBCLs) with constitutive NF-κB activation. However, regulatory mechanisms and relevance of CARMA1 clusters in the NF-κB pathway are unclear. Here we show that SH3 and GUK domain interactions of CARMA1 link CARMA1 clustering to signal activation. SH3 and GUK domains of CARMA1 interact by either intra- or intermolecular mechanisms, which are required for activation-induced assembly of CARMA1. Disruption of these interactions abolishes the formation of CARMA1 microclusters at the immunological synapse, CARMA-regulated signal activation following antigen receptor stimulation as well as spontaneous CARMA1 clustering and NF-κB activation by the oncogenic CARMA1 mutation in ABC-DLBCLs. Thus, the SH3-GUK interactions that regulate CARMA1 cluster formations are promising therapeutic targets for ABC-DLBCLs.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/biosíntesis , Guanilato Ciclasa/biosíntesis , Subunidad p50 de NF-kappa B/metabolismo , Transducción de Señal , Animales , Proteínas Adaptadoras de Señalización CARD/química , Análisis por Conglomerados , Cristalografía por Rayos X , Homólogo 4 de la Proteína Discs Large , Femenino , Guanilato Ciclasa/química , Guanilato-Quinasas/biosíntesis , Guanilato-Quinasas/química , Humanos , Sistema Inmunológico/fisiología , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/química , Células Jurkat , Linfocitos/citología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Mutación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Ratas , Fracciones Subcelulares/metabolismo , Dominios Homologos srcRESUMEN
Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4-PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4-PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.
Asunto(s)
Antígeno CTLA-4/metabolismo , Sinapsis Inmunológicas/metabolismo , Inmunoterapia/tendencias , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Complejos Multiproteicos/metabolismo , Proteína Quinasa C/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Humanos , Tolerancia Inmunológica/genética , Células Jurkat , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteína Quinasa C/genética , Proteómica , Transducción de SeñalRESUMEN
Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/inmunología , Antígenos CD28/inmunología , Proteínas Portadoras/inmunología , Guanilato Ciclasa/inmunología , Proteína Quinasa C/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Citometría de Flujo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Análisis de Secuencia de ADN , Organismos Libres de Patógenos EspecíficosRESUMEN
Programmed cell death 1 (PD-1) is a negative costimulatory receptor critical for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a molecular mechanism of PD-1-mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently associated with the phosphatase SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addition to PD-1 clustering, PD-1-TCR colocalization within microclusters is required for efficient PD-1-mediated suppression. This inhibitory mechanism also functions in PD-1(hi) T cells generated in vivo and can be overridden by a neutralizing anti-PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.
Asunto(s)
Receptor de Muerte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/metabolismo , Activación de Linfocitos , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Fosforilación , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Transducción de Señal , Linfocitos T/metabolismo , Tirosina/metabolismoRESUMEN
Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-θ was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-θ-mediated downstream signaling and the differentiation of T helper type 2 cells (T(H)2 cells) and interleukin 17-producing helper T cells (T(H)17 cells) but not of T helper type 1 cells (T(H)1 cells). Ectopic expression of V3 sequestered PKC-θ from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC-θ and indicate V3-based 'decoys' may be therapeutic modalities for T cell-mediated inflammatory diseases.
Asunto(s)
Antígenos CD28/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Subgrupos de Linfocitos T/metabolismo , Células Th17/metabolismo , Células Th2/metabolismo , Secuencias de Aminoácidos/genética , Animales , Antígenos CD28/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Sinapsis Inmunológicas , Inmunomodulación , Isoenzimas/genética , Isoenzimas/inmunología , Activación de Linfocitos , Ratones , Ratones Noqueados , Dominios Proteicos Ricos en Prolina/genética , Unión Proteica/inmunología , Proteína Quinasa C/genética , Proteína Quinasa C/inmunología , Proteína Quinasa C-theta , Transporte de Proteínas/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Células Th17/inmunología , Células Th17/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
CIN85, an adaptor protein which binds the C-terminal domain of tyrosine phosphorylated Cbl and Cbl-b, has been thought to be involved in the internalization and subsequent degradation of receptors. However, its physiological function remains unclear. To determine its role in B cells, we used Mb1-cre to generate mice with a B cell-specific deletion of CIN85. These mice had impaired T cell-independent type II antibody responses in vivo and diminished IKK-ß activation and cellular responses to B cell receptor (BCR) cross-linking in vitro. Introduction of a constitutively active IKK-ß construct corrected the defective antibody responses as well as cellular responses in the mutant mice. Together, our results suggest that CIN85 links the BCR to IKK-ß activation, thereby contributing to T cell-independent immune responses.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Formación de Anticuerpos , Linfocitos B/citología , Secuencia de Bases , Proliferación Celular , Supervivencia Celular , Cartilla de ADN/genética , Quinasa I-kappa B/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
When T cells recognize a peptide-major histocompatibility complex on antigen-presenting cells (APCs), T cell receptor microclusters (TCR-MCs) are generated and move to the center of the T cell-APC interface to form the central supramolecular activation cluster (cSMAC). cSMAC formation depends on stimulation strength and regulates T cell activation. We demonstrate that the dynein motor complex colocalized and coimmunoprecipitated with the TCR complex and that TCR-MCs moved along microtubules (MTs) toward the center of the immune synapse in a dynein-dependent manner to form cSMAC. MTs are located in close proximity to the plasma membrane at the activation site. TCR-MC velocity and cSMAC formation were impaired by dynein or MT inhibitors or by ablation of dynein expression. T cells with impaired cSMAC formation exhibited enhanced cellular activation including protein phosphorylation and interleukin-2 production. These results indicate that cSMAC formation by TCR-MC movement depends on dynein and MTs, and the movement regulates T cell activation.
Asunto(s)
Dineínas/inmunología , Sinapsis Inmunológicas/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Membrana Celular/inmunología , Membrana Celular/metabolismo , Sinapsis Inmunológicas/ultraestructura , Ratones , Microscopía Electrónica , Unión Proteica , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
TCR-microclusters (MC) are generated upon TCR stimulation prior to the immune synapse formation independently of lipid rafts. TCR-MCs contain receptors, kinases and adaptors, and function as the signaling unit for T cell activation. The TCR complex, but not the signaling molecules, is transported to the center to form cSMAC. The co-stimulation receptor CD28 joins the signaling region of cSMAC and recruits PKCθ and Carma1. CTLA-4 accumulates in the same region and competes with CD28 for negative regulation of T cell activation. T cell activation is therefore mediated by two spatially distinct signaling compartments: TCR signaling by the peripheral TCR-MC and co-stimulation signal by the central signaling cSMAC.
Asunto(s)
Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Humanos , Transducción de SeñalRESUMEN
T cell activation is positively and negatively regulated by a pair of costimulatory receptors, CD28 and CTLA-4, respectively. Because these receptors share common ligands, CD80 and CD86, the expression and behavior of CTLA-4 is critical for T cell costimulation regulation. However, in vivo blocking of CD28-mediated costimulation by CTLA-4 and its mechanisms still remain elusive. Here, we demonstrate the dynamic behavior of CTLA-4 in its real-time competition with CD28 at the central-supramolecular activation cluster (cSMAC), resulting in the dislocalization of protein kinase C-θ and CARMA1 scaffolding protein. CTLA-4 translocation to the T cell receptor microclusters and the cSMAC is tightly regulated by its ectodomain size, and its accumulation at the cSMAC is required for its inhibitory function. The CTLA-4-mediated suppression was demonstrated by the in vitro anergy induction in regulatory T cells constitutively expressing CTLA-4. These results show the dynamic mechanism of CTLA-4-mediated T cell suppression at the cSMAC.
Asunto(s)
Antígenos CD/fisiología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Proteínas Adaptadoras de Señalización CARD/fisiología , Antígenos CD28/fisiología , Complejo CD3/fisiología , Antígeno CTLA-4 , Células Cultivadas , Tolerancia Inmunológica , Isoenzimas/fisiología , Ratones , Proteína Quinasa C/fisiología , Proteína Quinasa C-theta , Linfocitos T Reguladores/fisiologíaRESUMEN
We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3 zeta and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.
Asunto(s)
Activación de Linfocitos/inmunología , Activación de Linfocitos/fisiología , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Complejo CD3/química , Complejo CD3/metabolismo , Endocitosis , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Sustancias Macromoleculares , Microdominios de Membrana/química , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteína Tirosina Quinasa ZAP-70/química , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
T cell activation begins with the interaction between an antigen-specific T cell and an antigen-presenting cell (APC). This interaction results in the formation of the immunological synapse, which had been considered to be responsible for antigen recognition and T cell activation. Recent advances in imaging analysis have provided new insights into T cell activation. The T cell receptor (TCR) microclusters, TCRs, kinases, and adaptors are generated upon antigen recognition at the interfaces between the T cells and the APCs and serve as a fundamental signaling unit for T cell activation. CD28-mediated costimulation is also found to be regulated by the formation of microclusters. Therefore, the dynamic regulations of TCR and CD28 microcluster formation, migration, and interaction are the key events for the initiation of T cell-mediated immune responses. Comprehensive analyses of the composition and characteristics of the TCR microcluster have identified its dynamic features. This review will outline new discoveries of the microclusters and its related concept in T cell activation.