RESUMEN
The inducible costimulator (ICOS) is a T cell costimulatory receptor that plays crucial roles in T cell differentiation and function. So far, ICOS has been shown to activate three signaling components: phosphoinositide 3-kinase (PI3K), intracellular calcium mobilization, and TANK binding kinase 1 (TBK1). By generating a knock-in strain of mice in which the ICOS gene is modified such that the ICOS-mediated PI3K pathway is selectively abrogated while the capacity of ICOS to mobilize intracellular calcium remains intact, we have shown that ICOS-mediated PI3K activation is required for some but not all T cell responses. This suggests that the ICOS-calcium signaling axis may explain some of the PI3K-independent ICOS functions. Further, a recent in vivo imaging study indicated that ICOS-dependent intracellular calcium flux facilitates cognate T cell-B cell interactions within the germinal center. However, how ICOS promotes TCR-mediated calcium flux has not been clear. Here we identified a membrane proximal motif in the cytoplasmic tail of ICOS that is essential for ICOS-assisted calcium signaling and demonstrate that ICOS can induce calcium flux independently of other signaling motifs. We also provide evidence that ICOS potentiates phospholipase Cγ1 (PLCγ1) activation to enhance calcium release from the intracellular pool. In parallel, acute ligation of ICOS without TCR co-engagement leads to activation of small GTPases, RhoA and Cdc42, consistent with the capacity of ICOS to induce actin remodeling. Importantly, interruption of actin dynamics during acute TCR or TCR-ICOS co-ligation severely impairs calcium flux in T cells even in the presence of activated PLCγ1. Thus, ICOS potentiates TCR-induced calcium flux by enhancing PLCγ1 activation and actin remodeling in a coordinated manner.
Asunto(s)
Actinas/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Señalización del Calcio/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Fosfolipasa C gamma/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Activación Enzimática/inmunología , Técnicas de Sustitución del Gen , Humanos , Inmunoprecipitación , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Células Jurkat , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Fosfolipasa C gamma/inmunología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
The inducible costimulator (ICOS) is highly expressed in follicular helper T (Tfh) cells, a subset of CD4 T cells that migrate into the B cell zone and facilitate germinal center reactions. Although ICOS is known to play a critical role in forming the Tfh cell population during immune responses, its contribution to the effector functions of Tfh cells remains unclear. Using activated mouse splenic CD4 T cells we demonstrate that ICOS assists TCR-mediated signal transduction by potentiating the PI3K-AKT-mTOR signaling cascade that leads to hyper-phosphorylation of p70S6K and 4E-BP1, events that are known to augment cap-dependent mRNA translation. Consequently, ICOS costimulation promotes the formation of polysomes on IL-4 mRNA in a PI3K-dependent manner. Furthermore, we show that the supply of IL-4 becomes a limiting factor for T-dependent B cell activation during in vitro co-culture when the ICOS-PI3K signaling axis is disrupted in T cells. This ICOS costimulation-dependent translational control may ensure targeted delivery of IL-4 to cognate B cells during T-B collaborations in the germinal center.
Asunto(s)
Linfocitos B/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Interleucina-4/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Técnicas de Cocultivo , Factores Eucarióticos de Iniciación , Citometría de Flujo , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Immunoblotting , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas 70-kDa/inmunología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Serina-Treonina Quinasas TOR/inmunología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
We and others have previously shown that ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models of allogeneic bone marrow transplantation. ICOS potentiates TCR-mediated PI3K activation and intracellular calcium mobilization. However, ICOS signal transduction pathways involved in GVHD remain unknown. In this study, we examined the contribution of ICOS-PI3K signaling in the pathogenic potential of T cells using a knock-in mouse strain, ICOS-YF, which selectively lost the ability to activate PI3K. We found that when total T cells were used as alloreactive T cells, ICOS-YF T cells caused less severe GVHD compared with ICOS wild-type T cells, but they induced much more aggressive disease than ICOS knockout T cells. This intermediate level of pathogenic capacity of ICOS-YF T cells was correlated with similar levels of IFN-γ-producing CD8 T cells that developed in the recipients of ICOS-WT or ICOS-YF T cells. We further evaluated the role of ICOS-PI3K signaling in CD4 versus CD8 T cell compartment using GVHD models that are exclusively driven by CD4 or CD8 T cells. Remarkably, ICOS-YF CD8 T cells caused disease similar to ICOS wild-type CD8 T cells, whereas ICOS-YF CD4 T cells behaved very similarly to their ICOS knockout counterparts. Consistent with their in vivo pathogenic potential, CD8 T cells responded to ICOS ligation in vitro by PI3K-independent calcium flux, T cell activation, and proliferation. Thus, in acute GVHD in mice, CD4 T cells heavily rely on ICOS-PI3K signaling pathways; in contrast, CD8 T cells can use PI3K-independent ICOS signaling pathways, possibly through calcium.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Proteína Coestimuladora de Linfocitos T Inducibles/fisiología , Activación de Linfocitos/inmunología , Fosfatidilinositol 3-Quinasa/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Enfermedad Aguda , Animales , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Enfermedad Injerto contra Huésped/enzimología , Proteína Coestimuladora de Linfocitos T Inducibles/deficiencia , Activación de Linfocitos/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal/genética , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/metabolismoRESUMEN
We previously showed that mice deficient in the Inducible Costimulator ligand (ICOSL-KO) develop more severe disease and lung pathology with delayed bacterial clearance upon respiratory infection of Chlamydia muridarum. Importantly, the exacerbation of disease in ICOSL-KO mice was seen despite heightened IFN-γ/Th1 responses, the major defense mechanisms against Chlamydia. To gain insight into the mechanism of ICOS function in this model, we presently analyzed anti-Chlamydia immune responses in mice lacking the entire ICOS (ICOS-KO) versus knock-in mice expressing a mutant ICOS (ICOS-Y181F) that has selectively lost the ability to activate phosphoinositide 3-kinase (PI3K). Like ICOSL-KO mice, ICOS-KO mice showed worse disease with elevated IFN-γ/Th1 responses compared to wild-type (WT) mice. ICOS-Y181F mice developed much milder disease compared to ICOS-KO mice, yet they were still not fully protected to the WT level. This partial protection in ICOS-Y181F mice could not be explained by the magnitude of IFN-γ/Th1 responses since these mice developed a similar level of IFN-γ response compared to WT mice. It was rather IL-17/Th17 responses that reflected disease severity: IL-17/Th17 response was partially impaired in ICOS-Y181F mice compared to WT, but was substantially stronger than that of ICOS-KO mice. Consistently, we found that both polarization and expansion of Th17 cells were partially impaired in ICOS-Y181F CD4 T cells, and was further reduced in ICOS-KO CD4 T cells in vitro. Our results indicate that once the IFN-γ/Th1 response is above a threshold level, the IL-17/Th17 response becomes a limiting factor in controlling Chlamydia lung infection, and that ICOS plays an important role in promoting Th17 responses in part through the activation of PI3K.