RESUMO
CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8+ CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes. The CAR T cell post-infusion changes are further characterized by repression of genes (e.g., TCF7 and LEF1) associated with memory potential and a DNA methylation signature (e.g., demethylation at CX3CR1, BATF, and TOX) demarcating a transition toward exhaustion-progenitor T cells. Thus, CD19-CAR T cells undergo exhaustion-associated DNA methylation programming, indicating that efforts to prevent this process may be an attractive approach to improve CAR T cell efficacy.
Assuntos
Antígenos CD19/imunologia , Linfócitos T CD8-Positivos/imunologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Imunoterapia Adotiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Adulto JovemRESUMO
Chimeric antigen receptor (CAR) T cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies and is now being developed for solid tumors and chronic viral infections. Although clinical trials have demonstrated the curative potential of CAR T cell therapy, a substantial and well-established limitation is the heightened contraction and transient persistence of CAR T cells during prolonged antigen exposure. The underlying mechanism(s) for this dysfunctional state, often termed CAR T cell exhaustion, remains poorly defined. Here, we report that exhaustion of human CAR T cells occurs through an epigenetic repression of the T cell's multipotent developmental potential. Deletion of the de novo DNA methyltransferase 3 alpha (DNMT3A) in T cells expressing first- or second-generation CARs universally preserved the cells' ability to proliferate and mount an antitumor response during prolonged tumor exposure. The increased functionality of the exhaustion-resistant DNMT3A knockout CAR T cells was coupled to an up-regulation of interleukin-10, and genome-wide DNA methylation profiling defined an atlas of genes targeted for epigenetic silencing. This atlas provides a molecular definition of CAR T cell exhaustion, which includes many transcriptional regulators that limit the "stemness" of immune cells, including CD28, CCR7, TCF7, and LEF1. Last, we demonstrate that this epigenetically regulated multipotency program is firmly coupled to the clinical outcome of prior CAR T cell therapies. These data document the critical role epigenetic mechanisms play in limiting the fate potential of human T cells and provide a road map for leveraging this information for improving CAR T cell efficacy.
Assuntos
Imunoterapia Adotiva , Neoplasias , Antígenos CD28 , Epigênese Genética , Humanos , Neoplasias/terapia , Linfócitos T , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression. IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human chimeric antigen receptor T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector DNA demethylation programming in CD8 T cells and serve as proof of concept that cytokines can guide induction of epigenetically regulated traits for T cell-based immunotherapies.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Interferon gama/metabolismo , Interleucina-12/farmacologia , Coriomeningite Linfocítica/enzimologia , Células T de Memória/efeitos dos fármacos , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Modelos Animais de Doenças , Humanos , Memória Imunológica/efeitos dos fármacos , Interferon gama/genética , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Células T de Memória/enzimologia , Células T de Memória/imunologia , Células T de Memória/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estudo de Prova de Conceito , Transdução de SinaisRESUMO
OBJECTIVES: Fine-needle aspiration (FNA) of thyroid lesions may result in infarction and diagnostic difficulties on subsequent thyroidectomy specimens. Next-generation sequencing (NGS) methods for detection of hallmark driver BRAF V600E mutations may help characterize such tumors in which histologic alterations preclude definitive tissue diagnosis. METHODS: Thyroidectomy specimens with both malignant FNA diagnoses and resultant infarction were identified from our institutional database. NGS methods were used to detect BRAF V600E mutations in the infarcted thyroid carcinomas. RESULTS: Nine thyroid carcinomas with infarction were characterized as BRAF-like papillary thyroid carcinoma based on molecular driver categorization and histologic diagnosis. BRAF V600E mutations were detected in the infarcted tissue in four (67%) of six lesions. CONCLUSIONS: We demonstrate detection of hallmark BRAF V600E mutations by NGS within infarcted tissue of thyroid carcinomas after FNA. This suggests a potential ancillary method of characterizing infarcted thyroid carcinomas whose altered histology may be nondiagnostic.
Assuntos
Biópsia por Agulha Fina/efeitos adversos , Infarto/etiologia , Proteínas Proto-Oncogênicas B-raf/genética , Câncer Papilífero da Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infarto/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adulto JovemRESUMO
In ovo electroporation of chick embryos at ages ≥ E2 is simple to conduct and widely used to manipulate gene function. However, in ovo electroporation at early E1 stages has so far been unsuccessful because of unacceptable levels of tissue damage and embryonic lethality. Early E1 manipulations in the chick have therefore relied on in vitro electroporation, posing problems for morphogenetic studies in which the long-term preservation (>24 h) of three-dimensional tissue organization is critical. This article describes a simple technique for in vivo electroporation of E1 embryos as young as Hamburger-Hamilton stage 4 (HH4). It uses thin microelectrodes and low voltages, which permit precise localization of gene misexpression while causing minimal tissue damage and embryonic lethality. Critically, it does not depend on the presence of a lumen for DNA injections and can easily be adapted for a wide variety of tissues.
Assuntos
Embrião de Galinha , Eletroporação/métodos , Animais , Análise de SobrevidaRESUMO
The floor plate (FP) is a midline signaling center, known to direct ventral cell fates and axon guidance in the neural tube. The recent identification of midbrain FP as a source of dopaminergic neurons has renewed interest in its specification and organization, which remain poorly understood. In this study, we have examined the chick midbrain and spinal FP and show that both can be partitioned into medial (MFP) and lateral (LFP) subdivisions. Although Hedgehog (HH) signaling is necessary and sufficient for LFP specification, it is not sufficient for MFP induction. By contrast, the transcription factor FOXA2 can execute the full midbrain and spinal cord FP program via HH-independent and dependent mechanisms. Interestingly, although HH-independent FOXA2 activity is necessary and sufficient for inducing MFP-specific gene expression (e.g., LMX1B, BMP7), it cannot confer ventral identity to midline cells without also turning on Sonic hedgehog (SHH). We also note that the signaling centers of the midbrain, the FP, roof plate (RP) and the midbrain-hindbrain boundary (MHB) are physically contiguous, with each expressing LMX1B and BMP7. Possibly as a result, SHH or FOXA2 misexpression can transform the MHB into FP and also suppress RP induction. Conversely, HH or FOXA2 knockdown expands the endogenous RP and transforms the MFP into a RP and/or MHB fate. Finally, combined HH blockade and FOXA2 misexpression in ventral midbrain induces LMX1B expression, which triggers the specification of the RP, rather than the MFP. Thus we identify HH-independent and dependent roles for FOXA2 in specifying the FP. In addition, we elucidate for the first time, a novel role for SHH in determining whether a midbrain signaling center will become the FP, MHB or RP.
Assuntos
Proteínas Hedgehog/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Transdução de Sinais/genética , Animais , Padronização Corporal/genética , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/genética , Fator 3-beta Nuclear de Hepatócito/genética , Modelos Biológicos , Coluna Vertebral/metabolismoRESUMO
BACKGROUND: The amenability of the chick embryo to a variety of manipulations has made it an ideal experimental model organism for over 100 years. The ability to manipulate gene function via in ovo electroporations has further revolutionized its value as an experimental model in the last 15 years. Although in ovo electroporations are simple to conduct in embryos ≥ E2, in ovo electroporations at early E1 stages have proven to be technically challenging due to the tissue damage and embryonic lethality such electroporations produce. RESULTS AND CONCLUSIONS: Here we report our success with in vivo microelectroporations of E1 embryos as young as Hamburger-Hamilton Stage 4 (HH4). We provide evidence that such electroporations can be varied in size and can be spatially targeted. They cause minimal disruption of tissue-size, 3-dimensional morphology, cell survival, proliferation, and cell-fate specification. Our paradigm is easily adapted to a variety of experimental conditions since it does not depend upon the presence of a lumen to enclose the DNA solution during electroporation. It is thus compatible with the in vivo examination of E1 morphogenetic events (e.g., neural tube closure) where preservation of 3-dimensional morphology is critical.