RESUMO
The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.
Assuntos
Antígenos de Neoplasias , Linfócitos T CD8-Positivos , Antígenos HLA-E , Animais , Humanos , Masculino , Fosfatase Ácida , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citomegalovirus/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Macaca mulatta , MesotelinaRESUMO
The commonly used antibodies 3D12 and 4D12 recognise the human leukocyte antigen E (HLA-E) protein. These antibodies bind distinct epitopes on HLA-E and differ in their ability to bind alleles of the major histocompatibility complex E (MHC-E) proteins of rhesus and cynomolgus macaques. We confirmed that neither antibody cross-reacts with classical HLA alleles, and used hybrids of different MHC-E alleles to map the regions that are critical for their binding. 3D12 recognises a region on the alpha 3 domain, with its specificity for HLA-E resulting from the amino acids present at three key positions (219, 223 and 224) that are unique to HLA-E, while 4D12 binds to the start of the alpha 2 domain, adjacent to the C terminus of the presented peptide. 3D12 staining is increased by incubation of cells at 27°C, and by addition of the canonical signal sequence peptide presented by HLA-E peptide (VL9, VMAPRTLVL). This suggests that 3D12 may bind peptide-free forms of HLA-E, which would be expected to accumulate at the cell surface when cells are incubated at lower temperatures, as well as HLA-E with peptide. Therefore, additional studies are required to determine exactly what forms of HLA-E can be recognised by 3D12. In contrast, while staining with 4D12 was also increased when cells were incubated at 27°C, it was decreased when the VL9 peptide was added. We conclude that 4D12 preferentially binds to peptide-free HLA-E, and, although not suitable for measuring the total cell surface levels of MHC-E, may putatively identify peptide-receptive forms.
Assuntos
Antígenos HLA-E , Antígenos de Histocompatibilidade Classe I , Humanos , Epitopos , Antígenos HLA , Peptídeos , Antígenos de Histocompatibilidade Classe II , Anticorpos MonoclonaisRESUMO
Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro , but their role in infection and pathogenesis is unknown. To examine the in vivo function of vFcγRs in animal hosts closely related to humans, we identified and characterized vFcγRs encoded by rhesus CMV (RhCMV). We demonstrate that Rh05, Rh152/151 and Rh173 represent the complete set of RhCMV vFcγRs, each displaying functional similarities to their respective HCMV orthologs with respect to antagonizing host FcγR activation in vitro . When RhCMV-naïve rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma viremia levels and anti-RhCMV antibody responses were comparable to wildtype infections. However, the duration of plasma viremia was significantly shortened in immunocompetent, but not in CD4+ T cell-depleted animals. Since vFcγRs were not required for superinfection, we conclude that vFcγRs delay control by virus-specific adaptive immune responses, particularly antibodies, during primary infection.
RESUMO
Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8+ T cells recognizing epitopes presented by major histocompatibility complex II (MHC-II) and MHC-E but not MHC-Ia. These immune responses mediate replication arrest of SIV in 50 to 60% of monkeys. We show that the peptide VMAPRTLLL (VL9) embedded within the RhCMV protein Rh67 promotes intracellular MHC-E transport and recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8+ T cells. Deletion or mutation of viral VL9 abrogated MHC-E-restricted CD8+ T cell priming, resulting in CD8+ T cell responses exclusively targeting MHC-II-restricted epitopes. These responses were comparable in magnitude and differentiation to responses elicited by 68-1 vectors but did not protect against SIV. Thus, Rh67-enabled direct priming of MHC-E-restricted T cells is crucial for RhCMV/SIV vaccine efficacy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/metabolismo , Vetores Genéticos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/metabolismo , Vacinas contra a SAIDS/imunologia , Animais , Linhagem Celular , Citomegalovirus/genética , Epitopos de Linfócito T/imunologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/genética , Ligantes , Macaca mulatta , Fragmentos de Peptídeos/genética , Transporte Proteico , Vírus da Imunodeficiência Símia , Antígenos HLA-ERESUMO
Increasing evidence suggests that cancer cells highjack developmental programs for disease initiation and progression. Melanoma arises from melanocytes that originate during development from neural crest stem cells (NCSCs). Here, we identified the transcription factor Yin Yang 1 (Yy1) as an NCSCs regulator. Conditional deletion of Yy1 in NCSCs resulted in stage-dependent hypoplasia of all major neural crest derivatives due to decreased proliferation and increased cell death. Moreover, conditional ablation of one Yy1 allele in a melanoma mouse model prevented tumorigenesis, indicating a particular susceptibility of melanoma cells to reduced Yy1 levels. Combined RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and untargeted metabolomics demonstrated that YY1 governs multiple metabolic pathways and protein synthesis in both NCSCs and melanoma. In addition to directly regulating a metabolic gene set, YY1 can act upstream of MITF/c-MYC as part of a gene regulatory network controlling metabolism. Thus, both NCSC development and melanoma formation depend on an intricate YY1-controlled metabolic program.
Assuntos
Melanoma/metabolismo , Melanoma/patologia , Crista Neural/citologia , Crista Neural/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fator de Transcrição YY1/deficiênciaRESUMO
Human cytomegalovirus (HCMV) depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC), membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM) cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-ß-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.
Assuntos
Membrana Celular/metabolismo , Citomegalovirus/fisiologia , Fibroblastos/virologia , Proteômica/métodos , Tetraspaninas/metabolismo , Internalização do Vírus , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Integrinas/química , Integrinas/metabolismo , Marcação por Isótopo , Domínios Proteicos/efeitos dos fármacos , Tetraspaninas/química , Tetraspaninas/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
The neural crest (NC) is an embryonic stem/progenitor cell population that generates a diverse array of cell lineages, including peripheral neurons, myelinating Schwann cells, and melanocytes, among others. However, there is a long-standing controversy as to whether this broad developmental perspective reflects in vivo multipotency of individual NC cells or whether the NC is comprised of a heterogeneous mixture of lineage-restricted progenitors. Here, we resolve this controversy by performing in vivo fate mapping of single trunk NC cells both at premigratory and migratory stages using the R26R-Confetti mouse model. By combining quantitative clonal analyses with definitive markers of differentiation, we demonstrate that the vast majority of individual NC cells are multipotent, with only few clones contributing to single derivatives. Intriguingly, multipotency is maintained in migratory NC cells. Thus, our findings provide definitive evidence for the in vivo multipotency of both premigratory and migrating NC cells in the mouse.
Assuntos
Antígenos de Diferenciação/metabolismo , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Células-Tronco Multipotentes/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Crista Neural/citologiaRESUMO
Schwann cells, the myelinating glia of the peripheral nervous system (PNS), originate from multipotent neural crest cells that also give rise to other cells, including neurons, melanocytes, chondrocytes, and smooth muscle cells. The transcription factor Sox10 is required for peripheral glia specification. However, all neural crest cells express Sox10 and the mechanisms directing neural crest cells into a specific lineage are poorly understood. We show here that histone deacetylases 1 and 2 (HDAC1/2) are essential for the specification of neural crest cells into Schwann cell precursors and satellite glia, which express the early determinants of their lineage myelin protein zero (P0) and/or fatty acid binding protein 7 (Fabp7). In neural crest cells, HDAC1/2 induced expression of the transcription factor Pax3 by binding and activating the Pax3 promoter. In turn, Pax3 was required to maintain high Sox10 levels and to trigger expression of Fabp7. In addition, HDAC1/2 were bound to the P0 promoter and activated P0 transcription. Consistently, in vivo genetic deletion of HDAC1/2 in mouse neural crest cells led to strongly decreased Sox10 expression, no detectable Pax3, virtually no satellite glia, and no Schwann cell precursors in dorsal root ganglia and peripheral nerves. Similarly, in vivo ablation of Pax3 in the mouse neural crest resulted in strongly reduced expression of Sox10 and Fabp7. Therefore, by controlling the expression of Pax3 and the concerted action of Pax3 and Sox10 on their target genes, HDAC1/2 direct the specification of neural crest cells into peripheral glia.
Assuntos
Diferenciação Celular/fisiologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Crista Neural/metabolismo , Células-Tronco Neurais/metabolismo , Oligodendroglia/metabolismo , Células de Schwann/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Camundongos , Crista Neural/citologia , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/citologiaRESUMO
The embryonic head development, including the formation of dental structures, is a complex and delicate process guided by specific genetic programs. Genetic changes and environmental factors can disturb the execution of these programs and result in abnormalities in orofacial and dental structures. Orofacial clefts and hypodontia/ oligodontia are examples of such abnormalities frequently seen in dental clinics. An insight into the mechanisms and genes involved in the formation of orofacial and dental structures has been gradually gained by genetic analysis of families and by the use of experimental vertebrate models such as the mouse and chick models. The development of novel clinical therapies for orofacial and dental pathological conditions depends very much on a detailed knowledge of the molecular and cellular processes that are involved in head formation.
Assuntos
Anodontia/genética , Fissura Palatina/genética , Palato Duro/embriologia , Transdução de Sinais/genética , Crânio/embriologia , Animais , Anodontia/embriologia , Fenda Labial/embriologia , Fenda Labial/genética , Fissura Palatina/embriologia , Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas Hedgehog/fisiologia , Humanos , Fator de Transcrição MSX1/genética , Camundongos , Crista Neural , Fator de Transcrição PAX9/genética , Fatores de Transcrição Box Pareados/genética , Fator de Crescimento Transformador beta/fisiologia , Proteínas Wnt/genéticaRESUMO
During vertebrate development, neural crest stem cells (NCSCs) give rise to neural cells of the peripheral nervous system and to a variety of mesenchymal cell types, including smooth muscle, craniofacial chondrocytes, and osteocytes. Consistently, mesenchymal stem cells (MSCs) have recently been shown to derive in part from the neural crest (NC), although the mechanisms underlying MSC generation remains to be identified. Here, we show that transforming growth factor ß (TGFß)-mediated suppression of the NCSC transcription factor Sox10 induces a switch in neural to mesenchymal potential in NCSCs. In vitro and in vivo, TGFß signal inactivation results in persistent Sox10 expression, decreased cell cycle exit, and perturbed generation of mesenchymal derivatives, which eventually leads to defective morphogenesis. In contrast, TGFß-mediated downregulation of Sox10 or its genetic inactivation suppresses neural potential, confers mesenchymal potential to NC cells in vitro, and promotes cell cycle exit and precocious mesenchymal differentiation in vivo. Thus, negative regulation of Sox10 by TGFß signaling promotes the generation of mesenchymal progenitors from NCSCs. Our study might lay the grounds for future applications demanding defined populations of MSCs for regenerative medicine.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Crista Neural/metabolismo , Fatores de Transcrição SOXE/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese/genética , Crista Neural/citologia , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/embriologia , Sistema Nervoso Periférico/metabolismo , Reação em Cadeia da Polimerase , Fatores de Transcrição SOXE/antagonistas & inibidores , Transdução de Sinais , Fator de Crescimento Transformador beta/genéticaRESUMO
During vertebrate development, neural crest cells are exposed to multiple extracellular cues that drive their differentiation into neural and non-neural cell lineages. Insights into the signals potentially involved in neural crest cell fate decisions in vivo have been gained by cell culture experiments that have allowed the identification of instructive growth factors promoting either proliferation of multipotent neural crest cells or acquisition of specific fates. For instance, members of the TGFbeta factor family induce neurogenesis and smooth muscle cell formation at the expense of other fates in culture. In vivo, conditional ablation of various TGFbeta signaling components resulted in malformations of non-neural derivatives of the neural crest, but it is unclear whether these phenotypes involved aberrant fate decisions. Moreover, it remains to be shown whether neuronal determination indeed requires TGFbeta factor activity in vivo. To address these issues, we conditionally deleted Smad4 in the neural crest, thus inactivating all canonical TGFbeta factor signaling. Surprisingly, neural crest cell fates were not affected in these mutants, with the exception of sensory neurogenesis in trigeminal ganglia. Rather, Smad4 regulates survival of smooth muscle and proliferation of autonomic and ENS neuronal progenitor cells. Thus, Smad signaling plays multiple, lineage-specific roles in vivo, many of which are elicited only after neural crest cell fate decision.