RESUMEN
KRAS inhibitors have demonstrated exciting preclinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KrasG12D, p53-mutant, murine pancreatic ductal adenocarcinoma-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intratumoral CD8+ T cells without durable responses. Single-cell RNA sequencing revealed an increase in inflammatory cancer-associated fibroblasts (iCAF), M2 macrophages, and a decreased dendritic cell (DC) quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen-presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor-free mice with established immune memory. Our data suggest that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong antitumor effects. SIGNIFICANCE: Combination of SOS1 and MEK inhibitors increase T cell infiltration while blunting pro-immune myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects.
Asunto(s)
Carcinoma Ductal Pancreático , Inmunoterapia , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Proteína SOS1 , Microambiente Tumoral , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Proteína SOS1/genética , Proteína SOS1/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Inmunoterapia/métodos , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , FemeninoRESUMEN
Anti-PD-1 treatment has shown unprecedented clinical success in the treatment of non-small-cell lung cancer (NSCLC), but the underlying mechanisms remain incompletely understood. Here, we performed temporal single-cell RNA and paired T-cell receptor sequencing on 47 tumor biopsies from 36 patients with NSCLC following PD-1-based therapies. We observed increased levels of precursor exhausted T (Texp) cells in responsive tumors after treatment, characterized by low expression of coinhibitory molecules and high expression of GZMK. By contrast, nonresponsive tumors failed to accumulate Texp cells. Our data suggested that Texp cells were unlikely to be derived from the reinvigoration of terminally exhausted cells; instead, they were accumulated by (1) local expansion and (2) replenishment by peripheral T cells with both new and pre-existing clonotypes, a phenomenon we named clonal revival. Our study provides insights into mechanisms underlying PD-1-based therapies, implicating clonal revival and expansion of Texp cells as steps to improve NSCLC treatment.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Receptor de Muerte Celular Programada 1 , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/metabolismoRESUMEN
Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos Inmunológicos/farmacología , Neoplasias Colorrectales , Resistencia a Antineoplásicos/efectos de los fármacos , Inmunoterapia , Neovascularización Patológica , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/inmunología , Angiopoyetina 2/genética , Angiopoyetina 2/inmunología , Animales , Línea Celular , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/terapia , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/inmunología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/inmunología , Ratones , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/terapia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+ T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígenos CD40/agonistas , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Angiopoyetina 2/antagonistas & inhibidores , Angiopoyetina 2/metabolismo , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígenos CD40/inmunología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Neoplasias/irrigación sanguínea , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses toward antibody production via Tfh cells or inflammation by Teff cells. Tfh-Teff cell fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6-Blimp-1 counter-antagonism. We found that the TCR-signal-induced transcription factor Irf4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised Irf4 amounts and promoted Teff cell fates at the expense of Tfh ones. Importantly, orthogonal induction of Irf4 expression redirected Tfh cell fate trajectories toward those of Teff. Mechanistically, we linked greater Irf4 abundance with its recruitment toward low-affinity binding sites within Teff cell cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the "reader" of TCR signal strength, and in turn, concentration-dependent activity of Irf4 "writes" T helper fate choice.
Asunto(s)
Regulación de la Expresión Génica , Redes Reguladoras de Genes , Factores Reguladores del Interferón/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Antígenos/inmunología , Sitios de Unión , Diferenciación Celular/inmunología , Línea Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunización , Factores Reguladores del Interferón/genética , Interleucina-2/metabolismo , Masculino , Ratones , Ratones Noqueados , Motivos de Nucleótidos , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo, we used a unique self-antigen system comprising seven human melanoma gp100(209-217)-specific TCRs spanning physiological affinities (1-100 µM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 µM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.
Asunto(s)
Autoinmunidad/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Varianza , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática , Fluorescencia , Humanos , Inmunohistoquímica , Transducción Genética , Antígeno gp100 del Melanoma/inmunologíaRESUMEN
Inflammation of the normally tolerant liver microenvironment precedes the development of chronic liver disease. Study of the pathogenesis of autoimmune liver diseases, such as autoimmune hepatitis (AIH), has been hampered by a lack of autochthonous chronic animal models. Through our studies of T cell costimulation, we generated transgenic mice expressing a ligand specific for the CD28 receptor, which normally shares ligands with the related inhibitory receptor CTLA-4. The mice spontaneously develop chronic inflammatory liver disease with several pathologies found in AIH, including elevated serum aminotransferases in the context of normal alkaline phosphatase and bilirubin levels, lymphocytic inflammation, focal necrosis, oval cell hyperplasia, and fibrosis. The prevalence of IFN-γ-producing CD8(+) T cells in the livers of transgenic mice suggests a role for autoimmune cytotoxicity in the chronic disease state. The CD28 ligand-specific transgenic mice will facilitate evaluation of CD8(+) T cell function in liver disease pathologies found in AIH.
Asunto(s)
Antígenos CD28/inmunología , Hepatitis Autoinmune/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Hepatitis Autoinmune/genética , Hepatitis Autoinmune/patología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ligandos , Masculino , Ratones , Ratones Transgénicos , Unión Proteica/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
CTLA-4 is thought to inhibit effector T cells both intrinsically, by competing with CD28 for B7 ligands, and extrinsically, through the action of regulatory T cells (Tregs). We studied in vivo responses of normal and CTLA-4-deficient Ag-specific murine effector CD4(+) T cells. We directly demonstrate that effector T cell-restricted CTLA-4 inhibits T cell responses in a cell-extrinsic manner. Cotransfer experiments show that CTLA-4 on normal effector CD4(+) T cells completely abrogates the dramatically increased expansion normally experienced by their CTLA-4-deficient counterparts. Neither the wild-type nor the CTLA-4-deficient T cells express the Treg transcription factor Foxp3 when transferred alone or together. Thus, cell-extrinsic inhibition of T cell responses by CTLA-4 is not limited to Tregs but is also a function of effector T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígeno CTLA-4/fisiología , Diferenciación Celular/inmunología , Inhibidores de Crecimiento/fisiología , Tolerancia Inmunológica/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Antígeno CTLA-4/deficiencia , Antígeno CTLA-4/genética , Diferenciación Celular/genética , Epítopos de Linfocito T/inmunología , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/deficiencia , Inhibidores de Crecimiento/deficiencia , Inhibidores de Crecimiento/genética , Inmunidad Celular/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Ratones , Ratones Noqueados , Ratones Transgénicos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplanteRESUMEN
The strength of T-cell receptor (TCR) stimulation and subsequent T-cell response depend on a combination of peptide-major histocompatibility complex (pMHC) density and potency. By comparing two different pMHC at doses yielding similar proliferation in vivo, we have highlighted unexpected differences in the qualitative and quantitative effects of TCR ligand. Measurements of cytokine sensitivity and two-photon imaging of T cell-dendritic cell (T-DC) interactions reveal discrimination between comparably weak stimuli resulting from either decreased pMHC potency or pMHC density. In addition, TCR-induced genes in broad gene expression profiles segregate into two groups: one that responds to cumulative TCR signal and another that responds to pMHC quality, independent of quantity. These observations suggest that models of TCR ligand discrimination must account for disparate sensitivity of downstream responses to specific influences of pMHC potency.
Asunto(s)
Antígenos de Histocompatibilidad/inmunología , Péptidos/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Comunicación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-2/farmacología , Masculino , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Interleucina-2/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacosRESUMEN
The transcription factor Helios has been reported to be a marker of regulatory T cells (Treg) of thymic origin, distinguishing them from Treg induced in the periphery (iTreg). In this study, we demonstrate Helios expression in Foxp3(+) iTreg, both in vitro and in vivo. Following i.v. peptide injection, in vivo Helios expression in adoptively transferred TCR transgenic T cells was more rapid than Foxp3 induction but less stable at later time points without a second injection of peptide. Our in vitro data suggest that APC influence Helios expression in a manner distinct from stimuli required for Foxp3 induction. Thus, Helios expression in iTreg may reflect the context of stimulation during Foxp3 induction. In summary, the robust Helios expression we observe in iTreg precludes its use as a marker of thymic Treg.
Asunto(s)
Factores de Transcripción Forkhead , Factor de Transcripción Ikaros/biosíntesis , Animales , Células Presentadoras de Antígenos , Ratones , Ratones Transgénicos , Péptidos/administración & dosificación , Linfocitos T Reguladores/metabolismo , Distribución TisularRESUMEN
The TCR can detect subtle differences in the strength of interaction with peptide/MHC ligand and transmit this information to influence downstream events in T cell responses. Manipulation of the factor commonly referred to as TCR signal strength can be achieved by changing the amount or quality of peptide/MHC ligand. Recent work has enhanced our understanding of the many variables that contribute to the apparent cumulative strength of TCR stimulation during immunogenic and tolerogenic T cell responses. In this review, we consider data from in vitro studies in the context of in vivo immune responses and discuss in vivo consequences of manipulation of strength of TCR stimulation, including influences on T cell-APC interactions, the magnitude and quality of the T cell response, and the types of fate decisions made by peripheral T cells.
Asunto(s)
Antígenos de Histocompatibilidad/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Humanos , Péptidos/inmunologíaRESUMEN
αß T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4(+) T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Ligandos , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Transducción de SeñalRESUMEN
T cell receptor (TCR) ligation is required for the extrathymic differentiation of forkhead box p3(+) (Foxp3(+)) regulatory T cells. Several lines of evidence indicate that weak TCR stimulation favors induction of Foxp3 in the periphery; however, it remains to be determined how TCR ligand potency influences this process. We characterized the density and affinity of TCR ligand favorable for Foxp3 induction and found that a low dose of a strong agonist resulted in maximal induction of Foxp3 in vivo. Initial Foxp3 induction by weak agonist peptide could be enhanced by disruption of TCR-peptide major histocompatibility complex (pMHC) interactions or alteration of peptide dose. However, time course experiments revealed that Foxp3-positive cells induced by weak agonist stimulation are deleted, along with their Foxp3-negative counterparts, whereas Foxp3-positive cells induced by low doses of the strong agonist persist. Our results suggest that, together, pMHC ligand potency, density, and duration of TCR interactions define a cumulative quantity of TCR stimulation that determines initial peripheral Foxp3 induction. However, in the persistence of induced Foxp3(+) T cells, TCR ligand potency and density are noninterchangeable factors that influence the route to peripheral tolerance.
Asunto(s)
Diferenciación Celular/inmunología , Epítopos de Linfocito T/inmunología , Factores de Transcripción Forkhead/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Apoptosis/inmunología , Recuento de Células , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Citocromos c/inmunología , Proteínas de Unión al ADN/genética , Epítopos de Linfocito T/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Interleucina-2/farmacología , Antígeno Ki-67/metabolismo , Ligandos , Ganglios Linfáticos/citología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fosforilación , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Bazo/citología , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/efectos de los fármacos , Linfocitos T/trasplante , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
SUMMARY: The generation of productive adaptive immune responses depends on the antigen-specific activation of T and B cells. The outcome of T-cell receptor engagement is influenced by signals from both positive and negative regulatory molecules that can either activate or inhibit T-cell function. CD28 and cytotoxic T-lymphocyte antigen-4 are the prototypical members of an immunoglobulin domain-containing protein family that play important roles in the control of T-cell responses against infection, cancer, and in autoimmune disease. Although the precise molecular details of their functions are still under active investigation, tumors and chronic pathogens seem to have exploited these pathways to achieve immune evasion. Furthermore, malfunction of the inhibitory arm of the immune response appears responsible for the development of multiple autoimmune pathologies. As a result, the negative regulators of T-cell activation have become attractive targets for therapeutic intervention in cancer, chronic infection, and autoimmune disease. The application of findings from basic research has provided insight into the manipulation of these pathways in the clinic and offers promising strategies for the treatment of disease.
Asunto(s)
Enfermedades Autoinmunes/terapia , Inmunoterapia , Infecciones/terapia , Activación de Linfocitos , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/inmunología , Antígenos CD28/inmunología , Antígeno CTLA-4 , HumanosRESUMEN
Coronavirus budding at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) requires accumulation of the viral envelope proteins at this point in the secretory pathway. Here we demonstrate that the spike (S) protein from the group 3 coronavirus infectious bronchitis virus (IBV) contains a canonical dilysine endoplasmic reticulum retrieval signal (-KKXX-COOH) in its cytoplasmic tail. This signal can retain a chimeric reporter protein in the ERGIC and when mutated allows transport of the full-length S protein as well as the chimera to the plasma membrane. Interestingly, the IBV S protein also contains a tyrosine-based endocytosis signal in its cytoplasmic tail, suggesting that any S protein that escapes the ERGIC will be rapidly endocytosed when it reaches the plasma membrane. We also identified a novel dibasic motif (-KXHXX-COOH) in the cytoplasmic tails of S proteins from group 1 coronaviruses and from the newly identified coronavirus implicated in severe acute respiratory syndrome. This dibasic motif also retained a reporter protein in the ERGIC, similar to the dilysine motif in IBV S. The cytoplasmic tails of S proteins from group 2 coronaviruses lack an intracellular localization signal. The inherent differences in S-protein trafficking could point to interesting variations in pathogenesis of coronaviruses, since increased levels of surface S protein could promote syncytium formation and direct cell-to-cell spread of the infection.
Asunto(s)
Retículo Endoplásmico/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas del Envoltorio Viral/fisiología , Ensamble de Virus , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dipéptidos , Aparato de Golgi/fisiología , Células HeLa , Humanos , Virus de la Bronquitis Infecciosa/fisiología , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Transporte de Proteínas , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/químicaRESUMEN
Virus-like particle (VLP) formation by the coronavirus E and M proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. We studied interactions between the infectious bronchitis virus (IBV) E and M proteins using in vivo crosslinking and VLP assembly assays. We show that IBV E and M can be crosslinked to each other in IBV-infected and transfected cells, indicating that they interact. The cytoplasmic tails of both proteins are important for this interaction. We also examined the ability of the mutant and chimeric E and M proteins to form VLPs. IBV M proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support VLP formation, regardless of their ability to be crosslinked to IBV E. Interactions between the E and M proteins and the membrane bilayer are likely to play an important role in VLP formation and virus budding.
Asunto(s)
Virus de la Bronquitis Infecciosa/fisiología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus , Animales , Línea Celular , Aparato de Golgi/metabolismo , Virus de la Bronquitis Infecciosa/genética , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/genética , Virión/metabolismoRESUMEN
We have previously shown that the E protein of the coronavirus infectious bronchitis virus (IBV) is localized to the Golgi complex when expressed exogenously from cDNA. Here, we report that neither the transmembrane domain nor the short lumenal domain of IBV E is required for Golgi targeting. However, an N-terminal truncation containing only the cytoplasmic domain (CTE) was efficiently localized to the Golgi complex, and this domain could retain a reporter protein in the Golgi. Thus, the cytoplasmic tail of the E protein is necessary and sufficient for Golgi targeting. The IBV E protein is palmitoylated on one or two cysteine residues adjacent to its transmembrane domain, but palmitoylation was not required for proper Golgi targeting. Using C-terminal truncations, we determined that the IBV E Golgi targeting information is present between tail amino acids 13 and 63. Upon treatment with brefeldin A, both the E and CTE proteins redistributed to punctate structures that colocalized with the Golgi matrix proteins GM130 and p115 instead of being localized to the endoplasmic reticulum like Golgi glycosylation enzymes. This suggests that IBV E is associated with the Golgi matrix through interactions of its cytoplasmic tail and may have interesting implications for coronavirus assembly in early Golgi compartments.