RESUMO
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.
Assuntos
Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Linfócitos T CD8-Positivos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de SinaisRESUMO
OM-85 is a bacterial lysate used in clinical practice to reduce duration and frequency of recurrent respiratory tract infections. Whereas knowledge of its regulatory effects in vivo has substantially advanced, the mechanisms of OM-85 sensing remain inadequately addressed. Here, we show that the immune response to OM-85 in the mouse is largely mediated by myeloid immune cells through Toll-like receptor (TLR) 4 in vitro and in vivo. Instead, in human immune cells, TLR2 and TLR4 orchestrate the response to OM-85, which binds to both receptors as shown by surface plasmon resonance assay. Ribonucleic acid-sequencing analyses of human monocyte-derived dendritic cells reveal that OM-85 triggers a pro-inflammatory signature and a unique gene set, which is not induced by canonical agonists of TLR2 or TLR4 and comprises tolerogenic genes. A largely overlapping TLR2/4-dependent gene signature was observed in individual subsets of primary human airway myeloid cells, highlighting the robust effects of OM-85. Collectively, our results suggest caution should be taken when relating murine studies on bacterial lysates to humans. Furthermore, our data shed light on how a standardized bacterial lysate shapes the response through TLR2 and TLR4, which are crucial for immune response, trained immunity, and tolerance.
Assuntos
Imunomodulação , Células Mieloides , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Humanos , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Camundongos , Animais , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Células Mieloides/imunologia , Células Mieloides/metabolismo , Células Dendríticas/imunologia , Transcriptoma , Células Cultivadas , Camundongos Knockout , Regulação da Expressão Gênica , Lisados BacterianosRESUMO
MYC is a pleiotropic transcription factor involved in cancer, cell proliferation, and metabolism. Its regulation and function in NK cells, which are innate cytotoxic lymphocytes important to control viral infections and cancer, remain poorly defined. Here, we show that mice deficient for Myc in NK cells presented a severe reduction in these lymphocytes. Myc was required for NK cell development and expansion in response to the key cytokine IL-15, which induced Myc through transcriptional and posttranslational mechanisms. Mechanistically, Myc ablation in vivo largely impacted NK cells' ribosomagenesis, reducing their translation and expansion capacities. Similar results were obtained by inhibiting MYC in human NK cells. Impairing translation by pharmacological intervention phenocopied the consequences of deleting or blocking MYC in vitro. Notably, mice lacking Myc in NK cells exhibited defective anticancer immunity, which reflected their decreased numbers of mature NK cells exerting suboptimal cytotoxic functions. These results indicate that MYC is a central node in NK cells, connecting IL-15 to translational fitness, expansion, and anticancer immunity.
Assuntos
Interleucina-15 , Células Matadoras Naturais , Animais , Humanos , Camundongos , Citocinas/metabolismo , Regulação da Expressão Gênica , Interleucina-15/genética , Interleucina-15/metabolismo , Transdução de SinaisRESUMO
BTN3A molecules-BTN3A1 in particular-emerged as important mediators of Vγ9Vδ2 T cell activation by phosphoantigens. These metabolites can originate from infections, e.g. with Mycobacterium tuberculosis, or by alterations in cellular metabolism. Despite the growing interest in the BTN3A genes and their high expression in immune cells and various cancers, little is known about their transcriptional regulation. Here we show that these genes are induced by NLRC5, a regulator of MHC class I gene transcription, through an atypical regulatory motif found in their promoters. Accordingly, a robust correlation between NLRC5 and BTN3A gene expression was found in healthy, in M. tuberculosis-infected donors' blood cells, and in primary tumors. Moreover, forcing NLRC5 expression promoted Vγ9Vδ2 T-cell-mediated killing of tumor cells in a BTN3A-dependent manner. Altogether, these findings indicate that NLRC5 regulates the expression of BTN3A genes and hence open opportunities to modulate antimicrobial and anticancer immunity.
RESUMO
The development of new approaches for organ transplantation has become crucial in the last years. In particular, organ engineering, involving the preparation of acellular matrices that provide a natural habitat for reseeding with an appropriate population of cells, is an attractive although technically demanding approach. We here describe a method that allows for the derivation of functional in vitro hepatic organoids and that does not require a previous selection of all the parenchymal hepatocytes and non-parenchymal cells, namely, Kupffer cells, liver endothelial cells, and hepatic stellate cells. The procedure also replaces the costly standard collagenase perfusion step with a trypsin-based enzymatic digestion that results in high-yield decellularization. A combination of physical and chemical treatments through deep immersion and intraluminal infusion of two different consecutive solutions is used: (1) deionized water (DI) and (2) DI + Triton X 1% + ammonium hydroxide (NH4OH) 0.1%. This ensures the isolation of the hepatic constructs that reliably maintain original architecture and ECM components while completely removing cellular DNA and RNA. The procedure is fast, simple, and cheap and warrants an optimal organoid functionality that may find applications in both toxicological and transplantation studies.
Assuntos
Hepatócitos/citologia , Fígado/química , Fígado/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Hidróxido de Amônia/química , Animais , Células Cultivadas , Feminino , Fígado/anatomia & histologia , Octoxinol/química , Organoides/citologia , Perfusão/métodos , CoelhosRESUMO
Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.
Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Epigênese Genética , Fibroblastos/citologia , Células Secretoras de Insulina/citologia , Adulto , Linhagem da Célula , Humanos , Cultura Primária de Células/métodos , Pele/citologia , Pele/crescimento & desenvolvimentoRESUMO
The potential of cell therapy in regenerative medicine has greatly expanded thanks to the availability of sources of pluripotent cells. In particular, induced pluripotent stem cells (iPS) have dominated the scenario in the last years for their ability to proliferate and differentiate into specific cell types. Nevertheless, the concerns inherent to the cell reprogramming process, limit iPS use in therapy and pose questions on the long-term behavior of these cells. In particular, despite the development of virus-free methods for their obtainment, a major and persisting drawback, is related to the acquisition of a stable pluripotent state, that is un-physiological and may lead to cell instability. The increased understanding of epigenetic mechanisms has paved the way to the use of "small molecules" and "epigenetic modifiers" that allow the fine tuning of cell genotype and phenotype. In particular, it was demonstrated that an adult mature cell could be directly converted into a different cell type with the use of these chemicals, obtaining a new patient-specific cell, suitable for cell therapy. This approach is simple and direct and may represent a very promising tool for the regenerative medicine of several and diverse degenerative diseases.
Assuntos
Reprogramação Celular/genética , Epigênese Genética/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Diferenciação Celular/genética , Terapia Baseada em Transplante de Células e Tecidos/métodos , HumanosRESUMO
All the somatic cells composing a mammalian organism are genetically identical and contain the same DNA sequence. Nevertheless, they are able to adopt a distinct commitment, differentiate in a tissue specific way and respond to developmental cues, acquiring a terminal phenotype. At the end of the differentiation process, each cell is highly specialized and committed to a distinct determined fate. This is possible thanks to tissue-specific gene expression, timely regulated by epigenetic modifications, that gradually limit cell potency to a more restricted phenotype-related expression pattern. Complex chemical modifications of DNA, RNA and associated proteins, that determine activation or silencing of certain genes are responsible for the epigenetic control that triggers the restriction of cell pluripotency, with the acquisition of the phenotypic definition and the preservation of its stability during subsequent cell divisions. The process is however reversible and may be modified by biochemical and biological manipulation, leading to the reactivation of hypermethylated pluripotency genes and inducing cells to transit from a terminally committed state to a higher plasticity one.These epigenetic regulatory mechanisms play a key role in embryonic development since they drive phenotype definition and tissue differentiation. At the same time, they are crucial for a better understanding of pluripotency regulation and restriction, stem cell biology and tissue repair process.
Assuntos
Epigênese Genética/fisiologia , Epigênese Genética/genética , Plasticidade Celular , Células Híbridas , Sequência de BasesRESUMO
All the somatic cells composing a mammalian organism are genetically identical and contain the same DNA sequence. Nevertheless, they are able to adopt a distinct commitment, differentiate in a tissue specific way and respond to developmental cues, acquiring a terminal phenotype. At the end of the differentiation process, each cell is highly specialized and committed to a distinct determined fate. This is possible thanks to tissue-specific gene expression, timely regulated by epigenetic modifications, that gradually limit cell potency to a more restricted phenotype-related expression pattern. Complex chemical modifications of DNA, RNA and associated proteins, that determine activation or silencing of certain genes are responsible for the epigenetic control that triggers the restriction of cell pluripotency, with the acquisition of the phenotypic definition and the preservation of its stability during subsequent cell divisions. The process is however reversible and may be modified by biochemical and biological manipulation, leading to the reactivation of hypermethylated pluripotency genes and inducing cells to transit from a terminally committed state to a higher plasticity one.These epigenetic regulatory mechanisms play a key role in embryonic development since they drive phenotype definition and tissue differentiation. At the same time, they are crucial for a better understanding of pluripotency regulation and restriction, stem cell biology and tissue repair process.(AU)