RESUMO
The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.
Assuntos
Linfócitos B , Melanoma , Animais , Camundongos , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ativação Linfocitária , Melanoma/imunologia , Melanoma/patologia , Melanoma/prevenção & controle , Linfócitos T/citologia , Linfócitos T/imunologia , Citometria de Fluxo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Linfonodos/citologia , Linfonodos/imunologia , Apresentação de Antígeno , Receptores de Antígenos de Linfócitos B/genética , Análise da Expressão Gênica de Célula Única , Carga Tumoral , Interferon Tipo IRESUMO
Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cell's digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9Δ yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.
RESUMO
Examining host-pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host-parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host-parasite interaction.
RESUMO
Successful infection strategies must balance pathogen amplification and persistence. In the obligate intracellular parasite Toxoplasma gondii this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. In this study we examine five factors that are transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc-finger family, which we name bradyzoite formation deficient 2 (BFD2). Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript under stress, and deletion of BFD2 reduces BFD1 protein levels but not messenger RNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces the chronic-stage gene-expression programme. Thus, our findings help explain how parasites both initiate and commit to chronic differentiation. This work provides new mechanistic insight into the regulation of T. gondii persistence, and can be exploited in the design of strategies to prevent and treat these key reservoirs of human infection.
Assuntos
Toxoplasma , Camundongos , Animais , Humanos , Toxoplasma/metabolismo , Retroalimentação , Regulação da Expressão Gênica , Fatores de Transcrição/genéticaRESUMO
Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.
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CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.
Assuntos
Sistemas CRISPR-Cas , Células Clonais , Técnicas de Inativação de Genes/métodos , Animais , Linhagem Celular , Células HEK293 , Humanos , Mamíferos , Plasmídeos , RNA Guia de Cinetoplastídeos , TransfecçãoRESUMO
Ninety-seven percent of drug-indication pairs that are tested in clinical trials in oncology never advance to receive U.S. Food and Drug Administration approval. While lack of efficacy and dose-limiting toxicities are the most common causes of trial failure, the reason(s) why so many new drugs encounter these problems is not well understood. Using CRISPR-Cas9 mutagenesis, we investigated a set of cancer drugs and drug targets in various stages of clinical testing. We show that-contrary to previous reports obtained predominantly with RNA interference and small-molecule inhibitors-the proteins ostensibly targeted by these drugs are nonessential for cancer cell proliferation. Moreover, the efficacy of each drug that we tested was unaffected by the loss of its putative target, indicating that these compounds kill cells via off-target effects. By applying a genetic target-deconvolution strategy, we found that the mischaracterized anticancer agent OTS964 is actually a potent inhibitor of the cyclin-dependent kinase CDK11 and that multiple cancer types are addicted to CDK11 expression. We suggest that stringent genetic validation of the mechanism of action of cancer drugs in the preclinical setting may decrease the number of therapies tested in human patients that fail to provide any clinical benefit.
Assuntos
Antineoplásicos/toxicidade , Ensaios Clínicos como Assunto , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Células Clonais , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Técnicas de Inativação de Genes , Genoma Humano , Humanos , Terapia de Alvo Molecular , Quinolonas/farmacologia , Interferência de RNA/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆9-tetrahydrocannabinol (THC), are metabolized and inactivated by cytochrome P450 enzymes primarily within the liver. The lipophilic nature of cannabinoids necessitates mechanism(s) to facilitate their intracellular transport to metabolic enzymes. Here, we test the central hypothesis that liver-type fatty acid binding protein (FABP1) mediates phytocannabinoid transport and subsequent inactivation. Using X-ray crystallography, molecular modeling, and in vitro binding approaches we demonstrate that FABP1 accommodates one molecule of THC within its ligand binding pocket. Consistent with its role as a THC carrier, biotransformation of THC was reduced in primary hepatocytes obtained from FABP1-knockout (FABP1-KO) mice. Compared to their wild-type littermates, administration of THC to male and female FABP1-KO mice potentiated the physiological and behavioral effects of THC. The stark pharmacodynamic differences were confirmed upon pharmacokinetic analyses which revealed that FABP1-KO mice exhibit reduced rates of THC biotransformation. Collectively, these data position FABP1 as a hepatic THC transport protein and a critical mediator of cannabinoid inactivation. Since commonly used medications bind to FABP1 with comparable affinities to THC, our results further suggest that FABP1 could serve a previously unrecognized site of drug-drug interactions.
Assuntos
Dronabinol/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Animais , Sítios de Ligação , Biotransformação , Células Cultivadas , Cristalografia por Raios X , Dronabinol/administração & dosagem , Proteínas de Ligação a Ácido Graxo/química , Feminino , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos MolecularesRESUMO
The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Previously, we established that triple-negative breast cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK proliferate at wild-type levels in vitro (
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Carcinogênese , Expressão Gênica , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Inativação de Genes , Humanos , Camundongos NusRESUMO
The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.
Assuntos
Marcação de Genes , Mutagênese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Inativação de Genes , HumanosRESUMO
We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo-electron microscopy structure reveals how the adenosine triphosphatases (ATPases) form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring adenosine triphosphate hydrolysis cycle that results in stepwise substrate translocation. A hingelike linker accommodates the large-scale nucleotide-driven motions of the ATPase spiral relative to the planar proteolytic base. The translocation mechanism is likely conserved for other AAA+ ATPases.