RESUMO
A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.
Assuntos
Proteína Forkhead Box O1 , Memória Imunológica , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/genética , Proteína Forkhead Box O1/metabolismo , Edição de Genes , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/citologiaRESUMO
Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3-7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.
RESUMO
Engagement of platelet endothelial cell adhesion molecule 1 (PECAM, PECAM-1, CD31) on the leukocyte pseudopod with PECAM at the endothelial cell border initiates transendothelial migration (TEM, diapedesis). We show, using fluorescence lifetime imaging microscopy (FLIM), that physical traction on endothelial PECAM during TEM initiated the endothelial signaling pathway. In this role, endothelial PECAM acted as part of a mechanotransduction complex with VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2), and this predicted that VEGFR2 was required for efficient TEM. We show that TEM required both VEGFR2 and the ability of its Y1175 to be phosphorylated, but not VEGF or VEGFR2 endogenous kinase activity. Using inducible endothelial-specific VEGFR2-deficient mice, we show in three mouse models of inflammation that the absence of endothelial VEGFR2 significantly (by ≥75%) reduced neutrophil extravasation by selectively blocking diapedesis. These findings provide a more complete understanding of the process of transmigration and identify several potential anti-inflammatory targets.
Assuntos
Migração Transendotelial e Transepitelial , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Animais , Camundongos , Adesão Celular , Movimento Celular , Endotélio Vascular , Mecanotransdução Celular , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.
Assuntos
Engenharia Celular , Imunoterapia Adotiva , Lógica , Neoplasias , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos , Transdução de Sinais , Linfócitos T , Humanos , Engenharia Celular/métodos , Imunoterapia Adotiva/efeitos adversos , Leucemia de Células B , Linfoma de Células B , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To "benchmark" exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8+ T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive in vitro but were nonfunctional in vivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation-driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.
Assuntos
Doença Enxerto-Hospedeiro , Receptores de Antígenos Quiméricos , Humanos , Linfócitos T Reguladores , Exaustão das Células T , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC, components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12-deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.
Assuntos
Ciclina C , Complexo Mediador , Neoplasias , Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Complexo Mediador/genética , Linfócitos T/imunologia , Fatores de Transcrição/genética , Estudo de Associação Genômica Ampla , Ciclina C/genética , Testes Genéticos , Imunoterapia Adotiva , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Peptídeo Hidrolases , Receptores de Antígenos de Linfócitos T , Linfócitos T/patologiaRESUMO
Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.
Assuntos
Cromatina/metabolismo , Neoplasias/terapia , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos Quiméricos , Linfócitos T/fisiologia , Animais , Antígenos CD19 , Linhagem Celular , Cromatina/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Receptor de Morte Celular Programada 1/genéticaRESUMO
T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.
Assuntos
Dasatinibe/farmacologia , Epigênese Genética , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigenoma , Feminino , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Memória Imunológica , Ativação Linfocitária , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Neoplasias Experimentais/terapia , Domínios Proteicos , Estabilidade Proteica , Receptores de Antígenos Quiméricos/química , Receptores de Antígenos Quiméricos/imunologia , Transdução de Sinais , Linfócitos T/metabolismo , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cell therapies present an entirely new paradigm in drug development. Within this class, immune cell therapies are among the most advanced, having already demonstrated definitive evidence of clinical benefits in cancer and infectious disease. Numerous features distinguish these "living therapies" from traditional medicines, including their ability to expand and contract in proportion to need and to mediate therapeutic benefits for months or years following a single application. Continued advances in fundamental immunology, genetic engineering, gene editing, and synthetic biology exponentially expand opportunities to enhance the sophistication of immune cell therapies, increasing potency and safety and broadening their potential for treatment of disease. This perspective will summarize the current status of immune cell therapies for cancer, infectious disease, and autoimmunity, and discuss advances in cellular engineering to overcome barriers to progress.
Assuntos
Doenças Autoimunes/terapia , Terapia Baseada em Transplante de Células e Tecidos , Imunoterapia/métodos , Neoplasias/terapia , Viroses/terapia , Engenharia Celular , Edição de Genes , Engenharia Genética , Humanos , Biologia SintéticaRESUMO
Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. SIGNIFICANCE: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence.This article is highlighted in the In This Issue feature, p. 627.
Assuntos
Receptores de Antígenos Quiméricos/metabolismo , Animais , Humanos , Camundongos , Transdução de SinaisRESUMO
Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.
Assuntos
Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Receptores de Antígenos de Linfócitos T/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologia , Transcrição GênicaAssuntos
Dasatinibe/farmacologia , Receptores de Antígenos Quiméricos , Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD19/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Xenoenxertos , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T/imunologiaRESUMO
Transient receptor potential (TRP) channels are a ubiquitously expressed multi-family group of cation channels that are critical to signaling events in many tissues. Their roles have been documented in many physiologic and pathologic conditions. Nevertheless, direct studies of their roles in maintain barrier function in endothelial and epithelia are relatively infrequent. This seems somewhat surprising considering that calcium ion concentrations are known to regulate barrier function. This short review provides an introduction to TRP channels and reviews some of the work in which investigators directly studied the role of TRP channels in endothelial permeability to electric current, solute, or leukocytes during the inflammatory response.