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1.
Faraday Discuss ; 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34545865

RESUMO

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during the immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and the negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity.

2.
Nat Commun ; 12(1): 4270, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257311

RESUMO

The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.


Assuntos
Mutação , SARS-CoV-2/fisiologia , Replicação Viral/fisiologia , Animais , Antivirais/farmacologia , COVID-19/tratamento farmacológico , COVID-19/virologia , Sistemas CRISPR-Cas , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desenvolvimento de Medicamentos , Genoma Viral , Células HEK293 , Humanos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Replicação Viral/genética
3.
Nat Immunol ; 22(7): 851-864, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099918

RESUMO

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.


Assuntos
Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Interleucina-33/farmacologia , Linfócitos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo
4.
Nat Commun ; 12(1): 2782, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986293

RESUMO

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Anergia Clonal/imunologia , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Linfopoese/fisiologia , Animais , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteína 2 de Resposta de Crescimento Precoce/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
6.
Sci Adv ; 7(8)2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33608275

RESUMO

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

7.
Sci Adv ; 7(9)2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33637530

RESUMO

CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies.

8.
FEBS J ; 288(1): 81-90, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32248616

RESUMO

Cancer tissue is not homogenous, and individual metastases at different anatomical locations can differ from the primary tumor and from one another in both their morphology and cellular composition, even within an individual patient. Tumors are composed of cancer cells and a range of other cell types, which, together with a variety of secreted molecules, collectively comprise the tumor microenvironment (TME). Cells of the TME can communicate with each other and with distant tissues in a form of molecular cross-talk to influence their growth and function. Cross-talk between cancer cells and local immune cells is well described and can lead to the induction of local immunosuppression. Recently, it has become apparent that tumors located remotely from each other, can engage in cross-talk that can influence their responsiveness to various therapies, including immunotherapy. In this article, we review studies that describe how tumors systemically communicate with distant tissues through motile cells, extracellular vesicles, and secreted molecules that can affect their function. In addition, we summarize evidence from mouse studies and the clinic that indicate an ability of some tumors to influence the progression and therapeutic responses of other tumors in different anatomical locations.


Assuntos
Vesículas Extracelulares/imunologia , Proteínas de Neoplasias/genética , Neoplasias/imunologia , Células Neoplásicas Circulantes/imunologia , Células-Tronco Neoplásicas/imunologia , Animais , Comunicação Celular , Vesículas Extracelulares/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Imunoterapia/métodos , Camundongos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/patologia , Metástase Neoplásica , Proteínas de Neoplasias/imunologia , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
9.
FEBS J ; 288(6): 1734-1741, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33034118

RESUMO

For most researchers, the time they spend as a postdoc stands out as one of challenge, but also enormous personal and professional growth. This Words of Advice is intended to guide the choice of postdoctoral position to help make the venture a success and to launch the first chapter of a happy and fulfilling professional life.

10.
Haematologica ; Online ahead of print2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33147937

RESUMO

This study explored the novel immune checkpoint poliovirus receptor-related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced NK cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK cell PVRIG expression levels were not increased. To understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumour cell recognition, cytokines (IL-2 and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum (ER) and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti- PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIGPVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.


Assuntos
Proteínas de Checkpoint Imunológico , Células Matadoras Naturais , Leucemia Mieloide Aguda , Receptores de Superfície Celular , Humanos , Imunoterapia , Ativação Linfocitária , Receptores de Células Matadoras Naturais
11.
Front Immunol ; 11: 589641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33072137

RESUMO

Immunotherapy has revolutionized the treatment of cancer. Nevertheless, the majority of patients do not respond to therapy, meaning a deeper understanding of tumor immune evasion strategies is required to boost treatment efficacy. The vast majority of immunotherapy studies have focused on how treatment reinvigorates exhausted CD8+ T cells within the tumor. In contrast, how therapies influence regulatory processes within the draining lymph node is less well studied. In particular, relatively little has been done to examine how tumors may exploit peripheral CD8+ T cell tolerance, an under-studied immune checkpoint that under normal circumstances prevents detrimental autoimmune disease by blocking the initiation of T cell responses. Here we review the therapeutic potential of blocking peripheral CD8+ T cell tolerance for the treatment of cancer. We first comprehensively review what has been learnt about the regulation of CD8+ T cell peripheral tolerance from the non-tumor models in which peripheral tolerance was first defined. We next consider how the tolerant state differs from other states of negative regulation, such as T cell exhaustion and senescence. Finally, we describe how tumors hijack the peripheral tolerance immune checkpoint to prevent anti-tumor immune responses, and argue that disruption of peripheral tolerance may contribute to both the anti-cancer efficacy and autoimmune side-effects of immunotherapy. Overall, we propose that a deeper understanding of peripheral tolerance will ultimately enable the development of more targeted and refined cancer immunotherapy approaches.

13.
Cancer Res ; 80(19): 4129-4144, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32816860

RESUMO

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/imunologia , Cobre/metabolismo , Neuroblastoma/imunologia , Evasão Tumoral/fisiologia , Animais , Antígeno B7-H1/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Quelantes/farmacologia , Transportador de Cobre 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunoterapia/métodos , Células Matadoras Naturais , Linfócitos do Interstício Tumoral/patologia , Camundongos Endogâmicos BALB C , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Trietilenofosforamida/farmacologia , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Clin Transl Immunology ; 9(7): e1157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32704371

RESUMO

Objectives: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells is a form of cancer immunotherapy that has achieved remarkable efficacy in patients with some haematological cancers. However, challenges remain in CAR T-cell treatment of solid tumours because of tumour-mediated immunosuppression. Methods: We have demonstrated that CAR T-cell stimulation through T-cell receptors (TCRs) in vivo can generate durable responses against solid tumours in a variety of murine models. Since Clec9A-targeting tailored nanoemulsion (Clec9A-TNE) vaccine enhances antitumour immune responses through selective activation of Clec9A+ cross-presenting dendritic cells (DCs), we hypothesised that Clec9A-TNE could prime DCs for antigen presentation to CAR T cells through TCRs and thus improve CAR T-cell responses against solid tumours. To test this hypothesis, we used CAR T cells expressing transgenic TCRs specific for ovalbumin (OVA) peptides SIINFEKL (CAROTI) or OVA323-339 (CAROTII). Results: We demonstrated that the Clec9A-TNEs encapsulating full-length recombinant OVA protein (OVA-Clec9A-TNE) improved CAROT T-cell proliferation and inflammatory cytokine secretion in vitro. Combined treatment using the OVA-Clec9A-TNE and CAROT cells resulted in durable responses and some rejections of tumours in immunocompetent mice. Tumour regression was accompanied by enhanced CAROT cell proliferation and infiltration into the tumours. Conclusion: Our study presents Clec9A-TNE as a prospective avenue to enhance CAR T-cell efficacy for solid cancers.

15.
J Clin Invest ; 130(7): 3391-3402, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538896

RESUMO

Pediatric cancers, particularly high-risk solid tumors, urgently need effective and specific therapies. Their outlook has not appreciably improved in decades. Immunotherapies such as immune checkpoint inhibitors offer much promise, but most are only approved for use in adults. Though several hundred clinical trials have tested immune-based approaches in childhood cancers, few have been guided by biomarkers or clinical-grade assays developed to predict patient response and, ultimately, to help select those most likely to benefit. There is extensive evidence in adults to show that immune profiling has substantial predictive value, but few studies focus on childhood tumors, because of the relatively small disease population and restricted use of immune-based therapies. For instance, only one published study has retrospectively examined the immune profiles of pediatric brain tumors after immunotherapy. Furthermore, application and integration of advanced multiplex techniques has been extremely limited. Here, we review the current status of immune profiling of pediatric solid tumors, with emphasis on tumor types that represent enormous unmet clinical need, primarily in the context of immune checkpoint inhibitor therapy. Translating optimized and informative immune profiling into standard practice and access to personalized combination therapy will be critical if childhood cancers are to be treated effectively and affordably.


Assuntos
Neoplasias Encefálicas , Imunoterapia , Adulto , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Criança , Humanos
16.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140457, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32473350

RESUMO

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.


Assuntos
Granzimas/química , Granzimas/metabolismo , Muromegalovirus/imunologia , Peptídeos/metabolismo , Animais , Apoptose , Caspases/metabolismo , Morte Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Granzimas/genética , Infecções por Herpesviridae/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Peptídeos/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Especificidade por Substrato , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Proteína bcl-X/metabolismo
17.
Cancer Immunol Res ; 8(8): 1085-1098, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32444423

RESUMO

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1ß and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.


Assuntos
Transplante de Medula Óssea/efeitos adversos , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Inflamassomos/imunologia , Leucemia/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Apoptose , Caspase 1/metabolismo , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Inflamassomos/metabolismo , Leucemia/imunologia , Leucemia/patologia , Camundongos , Camundongos Endogâmicos BALB C
18.
Nat Immunol ; 21(8): 914-926, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32424363

RESUMO

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.


Assuntos
Células Dendríticas/imunologia , Imunoterapia Adotiva , Proteínas de Membrana/imunologia , Neoplasias Experimentais/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Humanos , Fatores Imunológicos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia
20.
J Immunol ; 204(8): 2308-2315, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32152070

RESUMO

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Animais , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Eletroporação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologia
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