RESUMEN
Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has achieved tremendous success treating B cell malignancies; however, some patients fail to respond due to poor autologous T cell fitness. To improve response rates, we investigated whether disruption of the co-inhibitory receptors CTLA4 or PD-1 could restore CART function. CRISPR-Cas9-mediated deletion of CTLA4 in preclinical models of leukemia and myeloma improved CAR T cell proliferation and anti-tumor efficacy. Importantly, this effect was specific to CTLA4 and not seen upon deletion of CTLA4 and/or PDCD1 in CAR T cells. Mechanistically, CTLA4 deficiency permitted unopposed CD28 signaling and maintenance of CAR expression on the T cell surface under conditions of high antigen load. In clinical studies, deletion of CTLA4 rescued the function of T cells from patients with leukemia that previously failed CAR T cell treatment. Thus, selective deletion of CTLA4 reinvigorates dysfunctional chronic lymphocytic leukemia (CLL) patient T cells, providing a strategy for increasing patient responses to CAR T cell therapy.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Receptores Quiméricos de Antígenos , Humanos , Receptores de Antígenos de Linfocitos T/metabolismo , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Linfocitos T , Inmunoterapia Adoptiva , Antígenos CD19RESUMEN
Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/inmunología , Células Th17/inmunología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/inmunología , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Citometría de Flujo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Immunoblotting , Interleucina-17/inmunología , Interleucina-17/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Complejos Multiproteicos/inmunología , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Células Th17/metabolismoRESUMEN
Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.
Asunto(s)
5-Metilcitosina/metabolismo , Antígenos CD19/inmunología , Dioxigenasas/genética , Inmunoterapia/métodos , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Linfocitos T/inmunología , Linfocitos T/trasplante , Traslado Adoptivo , Anciano , Alelos , Diferenciación Celular , Ensayos Clínicos como Asunto , Células Clonales/citología , Células Clonales/inmunología , Dioxigenasas/metabolismo , Epigénesis Genética , Células HEK293 , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Mutación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , TransgenesRESUMEN
T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteínas de Unión al ADN/metabolismo , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Metilación de ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Memoria Inmunológica , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Invariant NKT (iNKT) cells bridge innate and adaptive immunity by rapidly secreting cytokines and lysing targets following TCR recognition of lipid antigens. Based on their ability to secrete IFN-γ, IL-4 and IL-17A, iNKT-cells are classified as NKT-1, NKT-2, and NKT-17 subsets, respectively. The molecular pathways regulating iNKT-cell fate are not fully defined. Recent studies implicate Rictor, a required component of mTORC2, in the development of select iNKT-cell subsets, however these reports are conflicting. To resolve these questions, we used Rictorfl/fl CD4cre+ mice and found that Rictor is required for NKT-17 cell development and normal iNKT-cell cytolytic function. Conversely, Rictor is not absolutely required for IL-4 and IFN-γ production as peripheral iNKT-cells make copious amounts of these cytokines. Overall iNKT-cell numbers are dramatically reduced in the absence of Rictor. We provide data indicating Rictor regulates cell survival as well as proliferation of developing and mature iNKT-cells. Thus, mTORC2 regulates multiple aspects of iNKT-cell development and function.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejos Multiproteicos/metabolismo , Células T Asesinas Naturales/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Inmunidad Adaptativa , Animales , Proteínas Portadoras/genética , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Citotoxicidad Inmunológica/genética , Inmunidad Innata , Activación de Linfocitos/genética , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antígeno Nuclear de Célula en Proliferación/genética , Quimera por Radiación , Proteína Asociada al mTOR Insensible a la RapamicinaRESUMEN
Genetic aberrations responsible for soft-tissue sarcoma formation in adults are largely unknown, with targeted therapies sorely needed for this complex and heterogeneous family of diseases. Here we report that that the Hippo pathway is deregulated in many soft-tissue sarcomas, resulting in elevated expression of the effector molecule Yes-Associated Protein (YAP). Based on data gathered from human sarcoma patients, a novel autochthonous mouse model, and mechanistic analyses, we determined that YAP-dependent expression of the transcription factor forkhead box M1 (FOXM1) is necessary for cell proliferation/tumorigenesis in a subset of soft-tissue sarcomas. Notably, FOXM1 directly interacts with the YAP transcriptional complex via TEAD1, resulting in coregulation of numerous critical pro-proliferation targets that enhance sarcoma progression. Finally, pharmacologic inhibition of FOXM1 decreases tumor size in vivo, making FOXM1 an attractive therapeutic target for the treatment of some sarcoma subtypes.
Asunto(s)
Carcinogénesis , Factores de Transcripción Forkhead/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Sarcoma/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/fisiología , Vía de Señalización Hippo , Humanos , Fosfoproteínas/metabolismo , Sarcoma/patología , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1α promotes HSC adaptation to stress. Because the related factor HIF-2α is also expressed in HSCs, the combined role of HIF-1α and HIF-2α in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-α dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1α and HIF-2α and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1α and Hif-2α phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Supervivencia Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Akt1 and Akt2, isoforms of the serine threonine kinase Akt, are essential for T cell development. However, their role in peripheral T cell differentiation remains undefined. Using mice with germline deletions of either Akt1 or Akt2, we found that both isoforms are important for Th17 differentiation, although Akt2 loss had a greater impact than loss of Akt1. In contrast to defective IL-17 production, Akt2 -/- T cells exhibited enhanced IL-4 production in vitro under Th2 polarizing conditions. In vivo , Akt2 -/- mice displayed significantly diminished IL-17A and GM-CSF production following immunization with myelin oligodendrocyte glycoprotein (MOG). This dampened response was associated with further alterations in Th cell differentiation including decreased IFNγ production but preserved IL-4 production, and preferential expansion of regulatory T cells compared to non-regulatory CD4 T cells. Taken together, we identify Akt2 as an important signaling molecule in regulating peripheral CD4 T cell responses.
RESUMEN
The adapter protein Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adapter proteins that function together to activate second messengers required for TCR signal propagation. The N terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with ADAP and HPK1. Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production, and phosphorylation of protein kinase D and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Fosfoproteínas/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Dominios Homologos src/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular/genética , Humanos , Células Jurkat , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Dominios Homologos src/genéticaRESUMEN
SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) nucleates a signaling complex critical for T-cell receptor (TCR) signal propagation. Mutations in the tyrosines of SLP-76 result in graded defects in TCR-induced signals depending on the tyrosine(s) affected. Here we use 2 strains of genomic knock-in mice expressing tyrosine to phenylalanine mutations to examine the role of TCR signals in the differentiation of effector and memory CD8(+) T cells in response to infection in vivo. Our data support a model in which altered TCR signals can determine the rate of memory versus effector cell differentiation independent of initial T-cell expansion. Furthermore, we show that TCR signals sufficient to promote CD8(+) T-cell differentiation are different from those required to elicit inflammatory cytokine production.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Fosfoproteínas/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/fisiología , Animales , Western Blotting , Linfocitos T CD8-positivos/virología , Diferenciación Celular , Proliferación Celular , Citometría de Flujo , Activación de Linfocitos , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Tirosina/genéticaRESUMEN
Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-ß. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-ß receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion, >98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-ß-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Masculino , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Linfocitos T , Factor de Crecimiento Transformador beta/metabolismo , Microambiente TumoralRESUMEN
Hypoxia inducible factors (HIFs) regulate adaptive responses to changes in oxygen (O(2)) tension during embryogenesis, tissue ischemia, and tumorigenesis. Because HIF-deficient embryos exhibit a number of developmental defects, the precise role of HIF in early vascular morphogenesis has been uncertain. Using para-aortic splanchnopleural (P-Sp) explant cultures, we show that deletion of the HIF-beta subunit (ARNT) results in defective hematopoiesis and the inhibition of both vasculogenesis and angiogenesis. These defects are rescued upon the addition of wild-type Sca-1(+) hematopoietic cells or recombinant VEGF. Arnt(-/-) embryos exhibit reduced levels of VEGF protein and increased numbers of apoptotic hematopoietic cells. These results suggest that HIF coordinates early endothelial cell emergence and vessel development by promoting hematopoietic cell survival and paracrine growth factor production.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/fisiología , Vasos Sanguíneos/embriología , Factores de Crecimiento de Célula Hematopoyética/fisiología , Animales , Apoptosis , Translocador Nuclear del Receptor de Aril Hidrocarburo/deficiencia , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Secuencia de Bases , Células de la Médula Ósea/fisiología , Técnicas de Cocultivo , ADN/genética , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/fisiología , Femenino , Hematopoyesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Embarazo , Proteínas Recombinantes/farmacología , Técnicas de Cultivo de Tejidos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.