RESUMEN
Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8+ T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches.
Asunto(s)
Linfocitos T CD8-positivos , Receptor de Muerte Celular Programada 1 , Linfocitos T CD8-positivos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Transducción de SeñalRESUMEN
Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.
Asunto(s)
Receptores de Trombopoyetina/metabolismo , Trombopoyetina/metabolismo , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Epítopos/inmunología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ligandos , Megacariocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Trombopoyetina/inmunología , Receptores de Trombopoyetina/fisiología , Transducción de Señal/fisiología , Trombocitemia Esencial/metabolismo , Trombopoyetina/fisiologíaRESUMEN
Cytokine and growth-factor ligands typically signal through homo- or hetero-dimeric cell surface receptors via Janus Kinase (JAK/TYK), or Receptor Tyrosine Kinase (RTK)-mediated trans-phosphorylation. However, the number of receptor dimer pairings occurring in nature is limited to those driven by natural ligands encoded within our genome. We have engineered synthethic cytokines (synthekines) that drive formation of cytokine receptor dimer pairings that are not formed by endogenous cytokines and that are not found in nature, and which activate distinct signaling programs. We show that a wide range of non-natural cytokine receptor hetero-dimers are competent to elicit a signaling output. We engineered synthekine ligands that assembled IL-2Rß/IL-4Rα or IL-4Rα/IFNAR2 receptor heterodimers, that do not occur naturally, triggering signaling and functional responses distinct from those activated by the endogenous cytokines IL-2, IL-4, and IFN. Furthermore, hybrid synthekine ligands that dimerized a JAK/STAT cytokine receptor with a receptor tyrosine kinase (RTK) also elicited a signaling response. Synthekines represent a new family of synthetic ligands with pre-defined receptors, but 'orphan' functions, that enable the full combinatorial scope of dimeric signaling receptors encoded within the human genome to be exploited for basic research and drug discovery.
Asunto(s)
Citocinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Línea Celular , Citocinas/genética , Humanos , Proteínas Recombinantes/genéticaRESUMEN
Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.
Asunto(s)
Anafilaxia/metabolismo , Células Madre Hematopoyéticas/inmunología , Mastocitos/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal , Factor de Células Madre/metabolismo , Anafilaxia/inmunología , Animales , Dimerización , Humanos , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas c-kit/agonistas , Proteínas Proto-Oncogénicas c-kit/química , Factor de Células Madre/química , Factor de Células Madre/genéticaRESUMEN
The zebrafish epithalamus is part of the diencephalon and encompasses three major components: the pineal, the parapineal and the habenular nuclei. Using sox2 knockdown, we show here that this key transcriptional regulator has pleiotropic effects during the development of these structures. Sox2 negatively regulates pineal neurogenesis. Also, Sox2 is identified as the unknown factor responsible for pineal photoreceptor prepatterning and performs this function independently of the BMP signaling. The correct levels of sox2 are critical for the functionally important asymmetrical positioning of the parapineal organ and for the migration of parapineal cells as a coherent structure. Deviations from this strict control result in defects associated with abnormal habenular laterality, which we have documented and quantified in sox2 morphants.
Asunto(s)
Neurogénesis/fisiología , Glándula Pineal/embriología , Factores de Transcripción SOX/metabolismo , Transducción de Señal/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Técnicas de Silenciamiento del Gen , Metaloproteinasas de la Matriz Secretadas/genética , Metaloproteinasas de la Matriz Secretadas/metabolismo , Glándula Pineal/citología , Factores de Transcripción SOX/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Tyk2 belongs to the Janus protein tyrosine kinase family and is involved in signaling of immunoregulatory cytokines (type I and III IFNs, IL-6, IL-10, and IL-12 families) via its interaction with shared receptor subunits. Depending on the receptor complex, Tyk2 is coactivated with either Jak1 or Jak2, but a detailed molecular characterization of the interplay between the two enzymes is missing. In human populations, the Tyk2 gene presents high levels of genetic diversity with >100 nonsynonymous variants being detected. In this study, we characterized two rare Tyk2 variants, I684S and P1104A, which have been associated with susceptibility to autoimmune disease. Specifically, we measured their in vitro catalytic activity and their ability to mediate Stat activation in fibroblasts and genotyped B cell lines. Both variants were found to be catalytically impaired but rescued signaling in response to IFN-α/ß, IL-6, and IL-10. These data, coupled with functional study of an engineered Jak1 P1084A, support a model of nonhierarchical activation of Janus kinases in which one catalytically competent Jak is sufficient for signaling provided that its partner behaves as proper scaffold, even if inactive. Through the analysis of IFN-α and IFN-γ signaling in cells with different Jak1 P1084A levels, we also illustrate a context in which a hypomorphic Jak can hamper signaling in a cytokine-specific manner. Given the multitude of Tyk2-activating cytokines, the cell context-dependent requirement for Tyk2 and the catalytic defect of the two disease-associated variants studied in this paper, we predict that these alleles are functionally significant in complex immune disorders.
Asunto(s)
Enfermedades Autoinmunes/genética , Linfocitos B/metabolismo , Janus Quinasa 1/genética , Polimorfismo de Nucleótido Simple , Transducción de Señal/inmunología , TYK2 Quinasa/genética , Alelos , Secuencia de Aminoácidos , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Línea Celular Transformada , Expresión Génica , Predisposición Genética a la Enfermedad , Vectores Genéticos , Herpesvirus Humano 4/genética , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/farmacología , Interleucina-10/inmunología , Interleucina-10/farmacología , Interleucina-6/inmunología , Interleucina-6/farmacología , Janus Quinasa 1/inmunología , Janus Quinasa 1/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos , TYK2 Quinasa/inmunología , TYK2 Quinasa/metabolismo , TransfecciónRESUMEN
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) protein cause one of the most common and severe forms of inherited retinal dystrophy. In spite of numerous studies, the precise function of RPGR remains unclear, as is the mechanism by which RPGR mutations cause retinal degeneration. We have analysed the function of RPGR by RNA interference-mediated translational suppression [knockdown (KD)] using a model cellular system for studying the formation, maintenance and function of primary cilia (human telomerase-immortalized retinal pigmented epithelium 1 cells). We observed that RPGR-deficient cells exhibited reduced numbers of cilia, slower cell cycle progression and impaired attachment to fibronectin, but showed no migration defects in a wound-healing assay. RPGR KD cells showed stronger actin filaments, associated with basal dysregulation of the Akt, Erk1/2, focal adhesion kinase and Src signalling pathways, as well as a 20% reduction in ß1-integrin receptors at the cell surface and impaired fibronectin-induced signalling. Stronger actin filaments and impairment of the above signalling pathways suggest a common underlying mechanism for all of the cellular phenotypes observed in RPGR KD cells. Our data underline a novel function for RPGR in cilia formation and in the regulation of actin stress filaments, suggesting that, in the retina, it may regulate nascent photoreceptor disc formation by regulating actin-mediated membrane extension.
Asunto(s)
Actinas/metabolismo , Cilios/metabolismo , Proteínas del Ojo/metabolismo , Organogénesis , Citoesqueleto de Actina/metabolismo , Animales , Ciclo Celular , División Celular , Línea Celular , Movimiento Celular , Forma de la Célula , Células Epiteliales/citología , Células Epiteliales/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal , Adhesiones Focales/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Integrina beta1/metabolismo , Ratones , Epitelio Pigmentado de la Retina/citología , Transducción de SeñalRESUMEN
PURPOSE: The authors investigated the expression and function of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene. METHODS: Zebrafish RP2 (ZFRP2) cDNA was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was examined by RT-PCR. The deduced peptide sequence was aligned with RP2 orthologues from different species. Translational suppression (knockdown) of zebrafish RP2 was carried out by antisense morpholino-injection. The phenotype of ZFRP2 knockdown morphants was characterized by immunohistology and histology. Human wild-type and mutant RP2 mRNAs were coinjected with ZFRP2 morpholinos to test whether human RP2 mRNA could rescue ZFRP2 knockdown phenotypes. RESULTS: ZFRP2 encodes a protein of 376 amino acids containing an N-terminal tubulin folding cofactor C-like domain and a C-terminal nucleoside diphosphate kinase-like domain. It shares 63% to 65% amino acid identity with human, mouse and bovine RP2. RP2 is expressed at the earliest stages of zebrafish development and persists into adulthood. Knockdown of RP2 in zebrafish causes a curved body axis and small eye phenotype, associated with increased cell death throughout the retina. Human wild-type RP2 mRNA could rescue the body curvature phenotype of ZFRP2 morphants, and the eye size of the resultant morphants was significantly increased over that of morphants in which ZFRP2 had been depleted. CONCLUSIONS: Zebrafish RP2 is widely expressed throughout development. ZFRP2 knockdown caused retinal degeneration in zebrafish. Human RP2 could partially rescue the small eye phenotype of ZFRP2 morphants.
Asunto(s)
Proteínas del Ojo/genética , Silenciador del Gen/fisiología , Degeneración Retiniana/genética , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Bovinos , Clonación Molecular , Proteínas del Ojo/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/genética , Retina/embriología , Retina/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Mutations in the human RPGR gene cause one of the most common and severe forms of inherited retinal dystrophy, but the function of its protein product remains unclear. We have identified two genes resembling human RPGR (ZFRPGR1, ZFRPGR2) in zebrafish (Danio rerio), both of which are expressed within the nascent and adult eye as well as more widely during development. ZFRPGR2 appears to be functionally orthologous to human RPGR, because it encodes similar protein isoforms (ZFRPGR2(ORF15), ZFRPGR2(ex1-17)) and causes developmental defects similar to other ciliary proteins, affecting gastrulation, tail and head development after morpholino-induced knockdown (translation suppression). These defects are consistent with a ciliary function and were rescued by human RPGR but not by RPGR mutants causing retinal dystrophy. Unlike mammals, RPGR knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. Developmental abnormalities in the eye included lamination defects, failure to develop photoreceptor outer segments and a small eye phenotype, associated with increased cell death throughout the retina. These defects could be rescued by expression of wild-type but not mutant forms of human RPGR. ZFRPGR2 knockdown also resulted in an intracellular transport defect affecting retrograde but not anterograde transport of organelles. ZFRPGR2 is therefore necessary both for the normal differentiation and lamination of the retina and to prevent apoptotic retinal cell death, which may relate to its proposed role in dynein-based retrograde transport processes.
Asunto(s)
Dineínas/metabolismo , Proteínas del Ojo/metabolismo , Retina/crecimiento & desarrollo , Enfermedades de la Retina/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Apoptosis , Muerte Celular , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Técnicas de Silenciamiento del Gen , Humanos , Transporte de Proteínas , Retina/anomalías , Retina/citología , Retina/metabolismo , Enfermedades de la Retina/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Tyk2 is a Jak family member involved in cytokine signaling through heterodimeric-type receptors. Here, we analyzed the impact of the Val(678)-to-Phe substitution on Tyk2 functioning. This mutation is homologous to the Jak2 Val(617)-to-Phe mutation, implicated in myeloproliferative disorders. We studied ligand-independent and ligand-dependent Jak/Stat signaling in cells expressing Tyk2 V678F. Moreover, the effect of Tyk2 V678F was monitored in the context of the native heterodimeric interferon alpha receptor and in the context of a homodimeric receptor chimera, EpoR/R1, containing the ectodomain of the erythropoietin receptor. We show that Tyk2 V678F has increased catalytic potential in vivo and in vitro and more so when it is anchored to the homodimeric receptor. Tyk2 V678F leads to constitutive Stat3 phosphorylation but has no notable effect on the canonical interferon alpha-induced signaling. However, if anchored to the homodimeric EpoR/R1, the mutant confers to the cell increased sensitivity to erythropoietin. Thus, despite the catalytic gain of function of Tyk2 V678F, the effect on ligand-induced signaling is manifest only when two mutant enzymes are juxtaposed via the homodimeric receptor.