RESUMEN
Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.
Asunto(s)
Inmunoterapia Adoptiva , Linfocitos T , Humanos , Inmunoterapia Adoptiva/métodos , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Toxic shock syndrome toxin-1 (TSST-1) is a superantigen (SAg) produced by Staphylococcus aureus thought to be responsible for essentially all cases of menstrual-associated toxic shock syndrome (TSS). As a potent exotoxin, it is not surprising that S. aureus has evolved multiple systems to control expression of TSST-1. Although the accessory gene regulator (Agr) system is recognized to enhance TSST-1 expression, how Agr regulates TSST-1 is unclear. Using an agr-null mutant, complementation experiments demonstrated that Agr controls TSST-1 expression through the activity of the RNAIII effector molecule. RNAIII can repress translation of the repressor of toxins (Rot) regulator, and deletion of rot increased expression of TSST-1 during the exponential phase of growth. Deletion of agr did not affect rot transcription, but did result in overexpression of the Rot protein, and Rot was also shown to bind and positively regulate the rot promoter. Overexpression of Rot dramatically repressed TSST-1, and Rot bound directly to the TSST-1 promoter. Deletion of both agr and rot in S. aureus returned TSST-1 expression to wild-type levels. This work demonstrates that Agr, although widely considered to be an inducer of TSST-1, has evolved in combination with Rot, to restrict the expression of this potent SAg.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Enterotoxinas/genética , Choque Séptico/genética , Superantígenos/genética , Transactivadores/metabolismo , Proteínas Bacterianas/genética , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Enterotoxinas/inmunología , Enterotoxinas/metabolismo , Exotoxinas/inmunología , Regulación Bacteriana de la Expresión Génica/genética , Genes Reguladores/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Choque Séptico/metabolismo , Infecciones Estafilocócicas/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Superantígenos/inmunología , Superantígenos/metabolismo , Transactivadores/genéticaRESUMEN
UNLABELLED: Toxic shock syndrome toxin 1 (TSST-1) is a Staphylococcus aureus superantigen that has been implicated in both menstrual and nonmenstrual toxic shock syndrome (TSS). Despite the important role of TSST-1 in severe human disease, a comprehensive understanding of staphylococcal regulatory factors that control TSST-1 expression remains incomplete. The S. aureus exotoxin expression (Sae) operon contains a well-characterized two-component system that regulates a number of important exotoxins in S. aureus, although regulation of TSST-1 by the Sae system has not been investigated. We generated a defined deletion mutant of the Sae histidine kinase sensor (saeS) in the prototypic menstrual TSS strain S. aureus MN8. Mutation of saeS resulted in a complete loss of TSST-1 expression. Using both luciferase reporter experiments and quantitative real-time PCR, we demonstrate that the Sae system is an important transcriptional activator of TSST-1 expression. Recombinant SaeR was able to bind directly to the tst promoter to a region containing two SaeR consensus binding sites. Although the stand-alone SarA transcriptional regulator has been shown to be both a positive and a negative regulator of TSST-1, deletion of sarA in S. aureus MN8 resulted in a dramatic overexpression of TSST-1. As expected, mutation of agr also reduced TSST-1 expression, but this phenotype appeared to be independent of Sae. A double mutation of saeS and sarA resulted in the loss of TSST-1 expression. This work indicates that the Sae system is a dominant and direct transcriptional activator that is required for expression of TSST-1. IMPORTANCE: The TSST-1 superantigen is an exotoxin, produced by some strains of S. aureus, that has a clear role in both menstrual and nonmenstrual TSS. Although the well-characterized agr quorum sensing system is a known positive regulator of TSST-1, the molecular mechanisms that directly control TSST-1 expression are only partially understood. Our studies demonstrate that the Sae two-component regulatory system is a positive transcriptional regulator that binds directly to the TSST-1 promoter, and furthermore, our data suggest that Sae is required for expression of TSST-1. This work highlights how major regulatory circuits can converge to fine-tune exotoxin expression and suggests that the Sae regulatory system may be an important target for antivirulence strategies.
Asunto(s)
Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Quinasas/metabolismo , Staphylococcus aureus/metabolismo , Superantígenos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Enterotoxinas/genética , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Staphylococcus aureus/genética , Superantígenos/genéticaRESUMEN
Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as 'trademark' virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to â¼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC -II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Exotoxinas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Proteínas de la Membrana/metabolismo , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Superantígenos/inmunología , Enfermedad Aguda , Animales , Proteínas Bacterianas/inmunología , Exotoxinas/inmunología , Humanos , Proteínas de la Membrana/inmunología , Ratones Endogámicos C57BL , Nasofaringe/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Infecciones Estreptocócicas/prevención & control , Streptococcus pyogenes/genética , Superantígenos/genética , Linfocitos T/inmunologíaRESUMEN
Staphylococcus aureus is a versatile bacterial pathogen that produces T cell-activating toxins known as superantigens (SAgs). Although excessive immune activation by SAgs can induce a dysregulated cytokine storm as a component of what is known as toxic shock syndrome (TSS), the contribution of SAgs to the staphylococcal infection process is not well defined. Here, we evaluated the role of the bacterial superantigen staphylococcal enterotoxin A (SEA) in a bacteremia model using humanized transgenic mice expressing SAg-responsive HLA-DR4 molecules. Infection with S. aureus Newman induced SEA-dependent Vß skewing of T cells and enhanced bacterial survival in the liver compared with infection by sea knockout strain. SEA-induced gamma interferon, interleukin-12, and chemokine responses resulted in increased infiltration of CD11b(+) Ly6G(+) neutrophils into the liver, promoting the formation of abscesses that contained large numbers of viable staphylococci. Hepatic abscesses occurred significantly more frequently in S. aureus Newman-infected livers than in livers infected with the Newman sea knockout strain, promoting the survival of S. aureus in vivo. This represents a novel mechanism during infection whereby S. aureus utilizes SAgs to form a specialized niche and manipulate the immune system.
Asunto(s)
Absceso/inmunología , Neutrófilos/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Absceso/microbiología , Animales , Antígenos Ly/inmunología , Antígeno CD11b/inmunología , Enterotoxinas/inmunología , Antígeno HLA-DR4/inmunología , Interferón gamma/inmunología , Interleucina-12/inmunología , Hígado/inmunología , Hígado/microbiología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Transgénicos , Neutrófilos/microbiología , Infecciones Estafilocócicas/microbiología , Linfocitos T/inmunología , Linfocitos T/microbiologíaRESUMEN
Despite the success of CAR-T cell cancer immunotherapy, challenges in efficacy and safety remain. Investigators have begun to enhance CAR-T cells with the expression of accessory molecules to address these challenges. Current systems rely on constitutive transgene expression or multiple viral vectors, resulting in unregulated response and product heterogeneity. Here, we develop a genetic platform that combines autonomous antigen-induced production of an accessory molecule with constitutive CAR expression in a single lentiviral vector called Uni-Vect. The broad therapeutic application of Uni-Vect is demonstrated in vivo by activation-dependent expression of (1) an immunostimulatory cytokine that improves efficacy, (2) an antibody that ameliorates cytokine-release syndrome, and (3) transcription factors that modulate T cell biology. Uni-Vect is also implemented as a platform to characterize immune receptors. Overall, we demonstrate that Uni-Vect provides a foundation for a more clinically actionable next-generation cellular immunotherapy.
Asunto(s)
Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Humanos , Inmunoterapia Adoptiva/métodos , Linfocitos T , Vectores Genéticos/genética , Citocinas/metabolismoRESUMEN
OBJECTIVES: With a rapidly growing list of candidate immune-based cancer therapeutics, there is a critical need to generate highly reliable animal models to preclinically evaluate the efficacy of emerging immune-based therapies, facilitating successful clinical translation. Our aim was to design and validate a novel in vivo model (called Xenomimetic or 'X' mouse) that allows monitoring of the ability of human tumor-specific T cells to suppress tumor growth following their entry into the tumor. METHODS: Tumor xenografts are established rapidly in the greater omentum of globally immunodeficient NOD-scid IL2Rγnull (NSG) mice following an intraperitoneal injection of melanoma target cells expressing tumor neoantigen peptides, as well as green fluorescent protein and/or luciferase. Changes in tumor burden, as well as in the number and phenotype of adoptively transferred patient-derived tumor neoantigen-specific T cells in response to immunotherapy, are measured by imaging to detect fluorescence/luminescence and flow cytometry, respectively. RESULTS: The tumors progress rapidly and disseminate in the mice unless patient-derived tumor-specific T cells are introduced. An initial T cell-mediated tumor arrest is later followed by a tumor escape, which correlates with the upregulation of the checkpoint molecules programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (LAG3) on T cells. Treatment with immune-based therapies that target these checkpoints, such as anti-PD-1 antibody (nivolumab) or interleukin-12 (IL-12), prevented or delayed the tumor escape. Furthermore, IL-12 treatment suppressed PD-1 and LAG3 upregulation on T cells. CONCLUSION: Together, these results validate the X-mouse model and establish its potential to preclinically evaluate the therapeutic efficacy of immune-based therapies.
RESUMEN
Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Carcinogénesis/inmunología , Epítopos de Linfocito T/inmunología , Neoplasias Pulmonares/inmunología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Traslado Adoptivo , Alelos , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Mutación , Péptidos/genética , Péptidos/inmunología , Proteómica , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Inmunoconjugados , Activación de Linfocitos , Microdominios de Membrana/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Abatacept , Células Presentadoras de Antígenos/inmunología , Antígenos CD , Antígenos de Diferenciación/genética , Antígeno CTLA-4 , Citometría de Flujo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Interleucina-2/antagonistas & inhibidores , Células Jurkat , Microdominios de Membrana/química , Microscopía Confocal , Datos de Secuencia Molecular , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Linfocitos T/citologíaRESUMEN
Stimulation of T cells through their antigen receptors (TCRs) causes a transient increase in the intracellular concentration of cyclic AMP (cAMP). However, sustained high levels of cAMP inhibit T-cell responses, suggesting that TCR signaling is coordinated with the activation of cyclic nucleotide phosphodiesterases (PDEs). The molecular basis of such a pathway is unknown. Here we show that TCR-dependent signaling activates PDE4B2 and that this enhances interleukin-2 production. Such an effect requires the regulatory N terminus of PDE4B2 and correlates with partitioning within lipid rafts, early targeting of this PDE to the immunological synapse, and subsequent accumulation in the antipodal pole of the T cell as activation proceeds.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Compartimento Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Activación Enzimática , Humanos , Técnicas In Vitro , Interleucina-2/biosíntesis , Células Jurkat , Activación de Linfocitos , Microdominios de Membrana/enzimología , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transducción de SeñalRESUMEN
BACKGROUND: Dendritic cells (DCs) are crucial regulators of immunity and important in inducing and maintaining tolerance. Here, we investigated the potential of a novel DC-immunomodulating agent, soluble CD83 (sCD83), in inducing transplant tolerance. METHODS: We used the C3H-to-C57BL/6 mouse cardiac transplantation model that exhibits a combination of severe cell-mediated rejection and moderate antibody-mediated rejection and investigated whether sCD83 could augment a combination therapy consisting of Rapamycin (Rapa) and anti-CD45RB monoclonal antibody (α-CD45) to prolong allograft survival. RESULTS: Monotherapies consisting of Rapa and α-CD45 were incapable of preventing rejection. However, all treatments involving sCD83 were capable of (1) down-modulating expression of various DC surface molecules, such as major histocompatibility complex class II and costimulatory molecules, (2) reducing the allogeneic stimulatory capacity of the DCs, and (3) significantly inhibiting antidonor antibody responses. Most striking results were observed in the triple therapy-treated group, sCD83Rapaα-CD45, where cell-mediated rejection and antibody-mediated rejection were abrogated for over 100 days. Donor-specific tolerance was achieved in long-term surviving recipients, because donor skin transplants were readily accepted for an additional 100 days, whereas third-party skin grafts were rejected. Success of triple therapy treatment was accompanied by enhancement of tolerogenic-DCs that conferred antigen-specific protection on adoptive transfer to recipients of an allogeneic heart graft. CONCLUSIONS: Our study revealed that sCD83 is capable of attenuating DC maturation and function, and inducing donor-specific allograft tolerance, in the absence of toxicity. Thus, sCD83 seems to be a safe and valuable counterpart to current DC-modulating agents.
Asunto(s)
Antígenos CD/farmacología , Células Dendríticas/efectos de los fármacos , Rechazo de Injerto/prevención & control , Trasplante de Corazón/inmunología , Inmunoglobulinas/farmacología , Inmunosupresores/farmacología , Glicoproteínas de Membrana/farmacología , Tolerancia al Trasplante/efectos de los fármacos , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/genética , Antígeno CD11c/inmunología , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Quimioterapia Combinada , Rechazo de Injerto/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Inmunoglobulinas/genética , Inmunofenotipificación , Antígenos Comunes de Leucocito/inmunología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Estructura Terciaria de Proteína , Proteínas Recombinantes/farmacología , Sirolimus/farmacología , Trasplante de Piel , Factores de Tiempo , Trasplante Homólogo , Antígeno CD83RESUMEN
The host determinants of susceptibility to recurrent urinary tract infections (UTI) are poorly understood. We investigated whether the susceptibility is associated with abnormalities in the immunological defense and further explored the linkage to vaginal microbiota. For this purpose, we compared vaginal, urine, and blood samples collected during a disease-free period from 22 women with recurrent UTI and from 17 controls. In UTI-prone women, interleukin-12 (IL-12) production in peripheral monocytes and myeloid dendritic cells (DCs) was significantly (P < 0.05) enhanced whether measured in relative numbers of IL-12-producing cells or in mean IL-12 production per cell. In contrast, no T-cell polarization was observed. Interestingly, it seemed that the cytokine production of DCs and monocytes did not translate into T-cell activation in the UTI-prone group in a manner similar to that seen with the controls. In vaginal mucosa, UTI-prone women had a lower concentration of tissue repair-associated vascular endothelial growth factor (VEGF) (P = 0.006) and less often had detectable amounts of the chief monocyte and DC chemoattractant, monocyte chemotactic protein 1 (P = 0.005), than the controls. The microbiota of UTI-prone women was characterized by a diminished lactobacillus morphotype composition, with an abnormally high (>3) mean Nugent score of 4.6 compared to 1.7 for the controls (P = 0.003). Normal lactobacillus composition was associated with increased IL-17 and VEGF concentrations in vaginal mucosa. In conclusion, immunological defects and a persistently aberrant microbiota, a lack of lactobacilli in particular, may contribute to susceptibility to recurrent UTI. Further studies of antigen-presenting-cell function and T-cell activation in recurrent UTI are called for.
Asunto(s)
Susceptibilidad a Enfermedades/microbiología , Infecciones Urinarias/inmunología , Vagina/inmunología , Vagina/microbiología , Adulto , Anciano , Bacterias/citología , Sangre/inmunología , Quimiocina CCL2/análisis , Células Dendríticas/inmunología , Femenino , Humanos , Interleucina-12/biosíntesis , Lactobacillus/aislamiento & purificación , Activación de Linfocitos , Persona de Mediana Edad , Monocitos/inmunología , Membrana Mucosa/química , Orina/química , Vagina/química , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.
Asunto(s)
Antígenos Bacterianos/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Isoenzimas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Superantígenos/inmunología , Fosfolipasas de Tipo C/metabolismo , Antígenos CD4/inmunología , Calcio/metabolismo , Células Cultivadas , Enterotoxinas/inmunología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Humanos , Interleucina-2/biosíntesis , Isoenzimas/genética , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fosfolipasa C beta , Fosfoserina/metabolismo , Proteína Quinasa C/metabolismo , Fosfolipasas de Tipo C/genéticaRESUMEN
Understanding the regulatory events involved in the activation and inactivation of T cells is crucial to develop therapeutic approaches for autoimmune diseases and for organ transplantation. Co-stimulatory signals delivered through the CD28 receptor and inhibitory signals through CTLA-4 are required for the proper modulation of T cell responses and the induction and maintenance of peripheral tolerance. Manipulation of these signals is emerging as a potential strategy to prevent allograft rejection in different animal models. Recent data on the compartmentalization and the structural features of CTLA-4 within T cells provides critical information not only on the molecular basis of T cell inactivation by CTLA-4, but also on the key requirements for the successful development of therapeutic strategies targeting this molecule.
Asunto(s)
Antígenos de Diferenciación/inmunología , Compartimento Celular , Linfocitos T/inmunología , Antígenos CD , Antígenos de Diferenciación/química , Antígenos de Diferenciación/metabolismo , Antígeno CTLA-4 , Humanos , Transducción de SeñalRESUMEN
The catalytic subunit of the serine/threonine phosphatase 2A (PP2A) can interact with the cytoplasmic tail of CTLA-4. However, the molecular basis and the biological significance of this interaction are unknown. In this study, we report that the regulatory subunit of PP2A (PP2AA) also interacts with the cytoplasmic tail of CTLA-4. Interestingly, TCR ligation induces tyrosine phosphorylation of PP2AA and its dissociation from CTLA-4 when coligated. The association between PP2AA and CTLA-4 involves a conserved three-lysine motif in the juxtamembrane portion of the cytoplasmic tail of CTLA-4. Mutations of these lysine residues prevent the binding of PP2AA and enhance the inhibition of IL-2 gene transcription by CTLA-4, indicating that PP2A represses CTLA-4 function. Our data imply that the lysine-rich motif in CTLA-4 may be used to identify small molecules that block its binding to PP2A and act as agonists for CTLA-4 function.
Asunto(s)
Antígenos de Diferenciación/fisiología , Regulación hacia Abajo/inmunología , Inmunoconjugados , Inmunosupresores/antagonistas & inhibidores , Inmunosupresores/farmacología , Fosfoproteínas Fosfatasas/fisiología , Abatacept , Secuencias de Aminoácidos/genética , Animales , Antígenos CD , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación/farmacología , Antígeno CTLA-4 , Línea Celular Transformada , Citoplasma/genética , Citoplasma/inmunología , Citoplasma/metabolismo , Regulación hacia Abajo/genética , Humanos , Inmunosupresores/metabolismo , Células Jurkat , Ligandos , Activación de Linfocitos/genética , Lisina/genética , Lisina/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Unión Proteica/genética , Unión Proteica/inmunología , Proteína Fosfatasa 2 , Estructura Terciaria de Proteína/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunologíaRESUMEN
Human embryonic stem cells (hESCs) are envisioned to be a major source for cell-based therapies. Efforts to overcome rejection of hESCs include nuclear transfer and collection of hESC banks representing the broadest diversity of major histocompatability complex (MHC) polymorphorisms. Surprisingly, immune responses to hESCs have yet to be experimentally evaluated. Here, injection of hESCs into immune-competent mice was unable to induce an immune response. Undifferentiated and differentiated hESCs failed to stimulate proliferation of alloreactive primary human T cells and inhibited third-party allogeneic dendritic cell-mediated T-cell proliferation via cellular mechanisms independent of secreted factors. Upon secondary rechallenge, T cells cocultured with hESCs were still responsive to allogeneic stimulators but failed to proliferate upon re-exposure to hESCs. Our study demonstrates that hESCs possess unique immune-privileged characteristics and provides an unprecedented opportunity to further investigate the mechanisms of immune response to transplantation of hESCs that may avoid immune-mediated rejection.