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1.
Cell ; 184(15): 4016-4031.e22, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34081922

RESUMO

Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8+ T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8+ T cell responses.


Assuntos
Apresentação Cruzada/imunologia , Gelsolina/metabolismo , Imunidade , Lectinas Tipo C/metabolismo , Neoplasias/imunologia , Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Gelsolina/química , Gelsolina/deficiência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mutação/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Análise de Sobrevida
2.
BMC Biol ; 22(1): 26, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38302906

RESUMO

BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.


Assuntos
Sistemas CRISPR-Cas , Dependovirus , Camundongos , Animais , Dependovirus/genética , Reprodutibilidade dos Testes , Zigoto , Marcação de Genes , Técnicas de Introdução de Genes/métodos
3.
Cancer Cell ; 11(4): 311-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17418408

RESUMO

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.


Assuntos
Carcinoma de Células Renais/etiologia , Fumarato Hidratase/genética , Inativação Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Doenças Renais Císticas/etiologia , Neoplasias Renais/etiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Hipóxia Celular , Proliferação de Células , Feminino , Transportador de Glucose Tipo 1/metabolismo , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
bioRxiv ; 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37609190

RESUMO

To increase antibody affinity against pathogens, positively selected GC-B cells initiate cell division in the light zone (LZ) of germinal centres (GCs). Among those, higher-affinity clones migrate to the dark zone (DZ) and vigorously proliferate by relying on oxidative phosphorylation (OXPHOS). However, it remains unknown how positively selected GC-B cells adapt their metabolism for cell division in the glycolysis-dominant, cell cycle arrest-inducing, hypoxic LZ microenvironment. Here, we show that microRNA (miR)-155 mediates metabolic reprogramming during positive selection to protect high-affinity clones. Transcriptome examination and mass spectrometry analysis revealed that miR-155 regulates H3K36me2 levels by directly repressing hypoxia-induced histone lysine demethylase, Kdm2a. This is indispensable for enhancing OXPHOS through optimizing the expression of vital nuclear mitochondrial genes under hypoxia. The miR-155-Kdm2a interaction is crucial to prevent excessive production of reactive oxygen species and apoptosis. Thus, miR-155-mediated epigenetic regulation promotes mitochondrial fitness in high-affinity clones, ensuring their expansion and consequently affinity maturation.

5.
J Exp Med ; 219(4)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35238865

RESUMO

Eosinophils are potent sources of inflammatory and toxic mediators, yet they reside in large numbers in the healthy intestine without causing tissue damage. We show here that intestinal eosinophils were specifically adapted to their environment and underwent substantial transcriptomic changes. Intestinal eosinophils upregulated genes relating to the immune response, cell-cell communication, extracellular matrix remodeling, and the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor with broad functions in intestinal homeostasis. Eosinophils from AHR-deficient mice failed to fully express the intestinal gene expression program, including extracellular matrix organization and cell junction pathways. AHR-deficient eosinophils were functionally impaired in the adhesion to and degradation of extracellular matrix, were more prone to degranulation, and had an extended life span. Lack of AHR in eosinophils had wider effects on the intestinal immune system, affecting the T cell compartment in nave and helminth-infected mice. Our study demonstrates that the response to environmental triggers via AHR partially shapes tissue adaptation of eosinophils in the small intestine.


Assuntos
Eosinófilos , Receptores de Hidrocarboneto Arílico , Animais , Eosinófilos/metabolismo , Homeostase , Intestino Delgado , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/metabolismo
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