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1.
STAR Protoc ; 4(1): 102082, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36861834

RESUMO

Recognition of Cas9 and other proteins encoded in delivery vectors has limited CRISPR technology in vivo. Here, we present a protocol for genome engineering using selective CRISPR antigen removal (SCAR) lentiviral vectors in Renca mouse model. This protocol describes how to conduct an in vivo genetic screen with a sgRNA library and SCAR vectors that can be applied to different cell lines and contexts. For complete details on the use and execution of this protocol, please refer to Dubrot et al. (2021).1.


Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Camundongos , Animais , Sistemas CRISPR-Cas/genética , Biblioteca Gênica , Genoma , Linhagem Celular
2.
Nat Immunol ; 23(10): 1495-1506, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36151395

RESUMO

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Inibidores de Checkpoint Imunológico , Evasão da Resposta Imune , Interferon gama/genética , Interferon gama/metabolismo , Subfamília C de Receptores Semelhantes a Lectina de Células NK
3.
Immunity ; 54(3): 571-585.e6, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33497609

RESUMO

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.


Assuntos
Carcinoma de Células Renais/imunologia , Testes Genéticos/métodos , Vetores Genéticos/genética , Imunoterapia/métodos , Neoplasias Renais/imunologia , Células Matadoras Naturais/imunologia , Lentivirus/genética , Animais , Apresentação de Antígeno , Autofagia , Carcinoma de Células Renais/terapia , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Renais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular
4.
Cell Rep ; 21(1): 208-221, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978474

RESUMO

Interleukin 21 (IL-21) plays key roles in humoral immunity and autoimmune diseases. It is known to function in mature CD4+ T follicular B cell helper (TFH) cells, but its potential involvement in early T cell ontogeny is unclear. Here, we find that a significant population of newly activated thymic and peripheral CD4+ T cells functionally expresses IL-21 soon after birth. This naturally occurring population, termed natural (n)TH21 cells, exhibits considerable similarity to mature TFH cells. nTH21 cells originating and activated in the thymus are strictly dependent on autoimmune regulator (AIRE) and express high levels of NUR77, consistent with a bias toward self-reactivity. Their activation/expansion in the periphery requires gut microbiota and is held in check by FoxP3+ TREG cells. nTH21 cells are the major thymic and peripheral populations of IL-21+ cells to expand in an IL-21-dependent humoral autoimmune disease. These studies link IL-21 to T cell ontogeny, self-reactivity, and humoral autoimmunity.


Assuntos
Artrite/genética , Autoimunidade/genética , Interleucinas/genética , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Artrite/imunologia , Artrite/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Microbioma Gastrointestinal/imunologia , Regulação da Expressão Gênica , Imunidade Humoral , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/patologia , Linfócitos T Reguladores/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Proteína AIRE
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