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Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.
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Neoplasias Hematológicas , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Ratones , Animales , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores de Antígenos de Linfocitos T , Inmunoterapia Adoptiva , Neoplasias Hematológicas/terapia , Inmunoterapia , Antígenos CD19RESUMEN
BACKGROUND AIMS: Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. METHODS: The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. RESULTS: Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40-50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality. CONCLUSIONS: The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.
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Quinasa I-kappa B , Estomatitis Vesicular , Animales , Vectores Genéticos/genética , Humanos , Quinasa I-kappa B/genética , Células Asesinas Naturales , Lentivirus/genética , Leucocitos Mononucleares , Proteínas Serina-Treonina Quinasas/genética , Transducción GenéticaRESUMEN
Microenvironments that tumor cells encounter are different during the stages of cancer progression-primary tumor, metastasis, and at the metastatic site. This suggests potential differences in immune surveillance of primary tumor and metastasis. Epithelial-mesenchymal transition (EMT) is a key reversible process in which cancer cells transition into highly motile and invasive cells for dissemination. Only a tiny proportion successfully metastasize, supporting the notion of metastasis-specific immune surveillance. EMT involves extensive molecular reprogramming of cells conferring many clinically relevant features to cancer cells and affects tumor cell interactions within the tumor microenvironment. We review the impact of tumor immune infiltrates on tumor cell EMT and the consequences of EMT in shaping the immune microenvironment of tumors. The usefulness of EMT as a model to investigate metastasis-specific immune surveillance mechanisms are also explored. Finally, we discuss potential implications of EMT for tumor immunogenicity, as well as current immunotherapies and future strategies.
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Transición Epitelial-Mesenquimal/inmunología , Neoplasias/patología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Progresión de la Enfermedad , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunologíaRESUMEN
For 3D reconstructions of whole immune cells from zebrafish, isolated from adult animals by FAC-sorting we employed array tomography on hundreds of serial sections deposited on silicon wafers. Image stacks were either recorded manually or automatically with the newly released ZEISS Atlas 5 Array Tomography platform on a Zeiss FEGSEM. To characterize different populations of immune cells, organelle inventories were created by segmenting individual cells. In addition, arrays were used for quantification of cell populations with respect to the various cell types they contained. The detection of immunological synapses in cocultures of cell populations from thymus or WKM with cancer cells helped to identify the cytotoxic nature of these cells. Our results demonstrate the practicality and benefit of AT for high-throughput ultrastructural imaging of substantial volumes.
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Imagenología Tridimensional/métodos , Sistema Inmunológico/citología , Sistema Inmunológico/ultraestructura , Linfocitos/ultraestructura , Tomografía/métodos , Adulto , Animales , Línea Celular Tumoral , Movimiento Celular , Separación Celular , Células Cultivadas , Citometría de Flujo/métodos , Humanos , Sinapsis Inmunológicas/ultraestructura , Timo/citología , Timo/ultraestructura , Pez CebraRESUMEN
PURPOSE: Clinical efficacy of CAR T cells against pediatric osteosarcoma (OS) has been limited. One strategy to improve efficacy may be to drive chemokine-mediated homing of CAR T cells to tumors. We investigated the primary chemokines secreted by OS and evaluated efficacy of B7-H3.CAR T cells expressing the cognate receptors. EXPERIMENTAL DESIGN: We developed a pipeline to identify chemokines secreted by OS by correlating RNA-seq data with chemokines detected in media from fresh surgical specimens. We identified CXCR2 and CXCR6 as promising receptors for enhancing CAR T cell homing against OS. We evaluated the homing kinetics and efficiency of CXCR2- and CXCR6.T cells and homing, cytokine production, and antitumor activity of CXCR2- and CXCR6.B7-H3.CAR T cells in vitro and in vivo. RESULTS: T cells transgenically expressing CXCR2 or CXCR6 exhibited ligand-specific enhanced migration over T cells modified with nonfunctional receptors. Differential homing kinetics were observed, with CXCR2.T cells homing quickly and plateauing early, while CXCR6.T cells homed more slowly but achieved a similar plateau. When expressed in B7-H3.CAR T cells, CXCR2- and CXCR6 modification conferred enhanced homing towards OS in vitro and in vivo. CXCR2- and CXCR6-B7-H3.CAR treated mice experienced prolonged survival in a metastatic model compared to B7-H3.CAR T cell treated mice. CONCLUSIONS: Our patient-based pipeline identified targets for chemokine receptor modification of CAR T cells targeting OS. CXCR2 and CXCR6 expression enhanced homing and anti-OS activity of B7-H3.CAR T cells. These findings support clinical evaluation of CXCR-modified CAR T cells to improve adoptive cell therapy for OS patients.
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The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.
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Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Humanos , Linfocitos T , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Chaperón BiP del Retículo Endoplásmico , Receptores Quiméricos de Antígenos/metabolismo , Leucemia Mieloide Aguda/patologíaRESUMEN
Host hematopoietically derived APCs play a vital role in the initiation of GVH responses. However, the APC autonomous molecular mechanisms that are critical for the induction of GVHD are not known. We report here that the Ikaros-Notch axis in host hematopoietically derived APCs regulates the severity of acute GVHD across multiple clinically relevant murine models of experimental bone marrow transplantation. In the present study, Ikaros deficiency (Ik(-/-)) limited to host hematopoietically derived APCs enhanced donor T-cell expansion and intensified acute GVHD, as determined by survival and other GVHD-specific parameters. The Ik(-/-) conventional CD8(+) and CD8(-)CD11c(+) dendritic cells (DCs), the most potent APCs, showed no increase in the expression of activation markers or in response to TLR stimulation compared with wild-type controls. However, Ik(-/-) DCs demonstrated an enhanced stimulation of allogeneic T cells. Deficiency of Ikaros in the conventional CD8(+) and CD8(-)CD11c(+) DCs was associated with an increase in Notch signaling, the blockade of which mitigated the enhanced in vitro and in vivo allostimulatory capacity. Therefore, the Ikaros-Notch axis is a novel pathway that modulates DC biology in general, and targeting this pathway in host hematopoietically derived APCs may reduce GVHD.
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Células Dendríticas/inmunología , Enfermedad Injerto contra Huésped/inmunología , Factor de Transcripción Ikaros/inmunología , Receptor Notch1/inmunología , Transducción de Señal/inmunología , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Células Dendríticas/citología , Modelos Animales de Enfermedad , Femenino , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/fisiopatología , Hematopoyesis/inmunología , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Inmunofenotipificación , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMEN
While miRNAs are increasingly linked to various immune responses, whether they can be targeted for regulating in vivo inflammatory processes such as endotoxin-induced Gram-negative sepsis is not known. Production of cytokines by the dendritic cells (DCs) plays a critical role in response to endotoxin, lipopolysaccharide (LPS). We profiled the miRNA and mRNA of CD11c⺠DCs in an unbiased manner and found that at baseline, miR-142-3p was among the most highly expressed endogenous miRs while IL-6 was among the most highly expressed mRNA after LPS stimulation. Multiple computational algorithms predicted the IL-6 3' untranslated region (UTR) to be a target of miR-142-3p. Studies using luciferase reporters carrying wild-type (WT) and mutant IL-6 3'UTR confirmed IL-6 as a target for miR-142-3p. In vitro knockdown and overexpression studies demonstrated a critical and specific role for miR142-3p in regulating IL-6 production by the DCs after LPS stimulation. Importantly, treatment of only WT but not the IL-6-deficient (IL-6(â»/â»)) mice with locked nucleic acid (LNA)-modified phosphorothioate oligonucleotide complementary to miR 142-3p reduced endotoxin-induced mortality. These results demonstrate a critical role for miR-142-3p in regulating DC responses to LPS and provide proof of concept for targeting miRs as a novel strategy for treatment of endotoxin-induced mortality.
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Células Dendríticas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/biosíntesis , Lipopolisacáridos/toxicidad , MicroARNs/biosíntesis , Sepsis/metabolismo , Regiones no Traducidas 3'/genética , Animales , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Femenino , Regulación de la Expresión Génica/genética , Interleucina-6/genética , Ratones , Ratones Noqueados , MicroARNs/genética , Sepsis/inducido químicamenteRESUMEN
Chimeric antigen receptor (CAR) technologies have been clinically implemented for the treatment of hematological malignancies; however, solid tumors remain resilient to CAR therapeutics. Natural killer (NK) cells may provide an optimal class of immune cells for CAR-based approaches due to their inherent anti-tumor functionality. In this study, we sought to tune CAR immune synapses by adding an intracellular scaffolding protein binding site to the CAR. We employ a PDZ binding motif (PDZbm) that enables additional scaffolding crosslinks that enhance synapse formation and NK CAR cell polarization. Combined effects of this CAR design result in increased effector cell functionality in vitro and in vivo. Additionally, we used T cells and observed similar global enhancements in effector function. Synapse-tuned CAR immune cells exhibit amplified synaptic strength, number and abundance of secreted cytokines, enhanced killing of tumor cells and prolonged survival in numerous different tumor models, including solid tumors.
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BACKGROUND: CD47 is an attractive immunotherapeutic target because it is highly expressed on multiple solid tumors. However, CD47 is also expressed on T cells. Limited studies have evaluated CD47-chimeric antigen receptor (CAR) T cells, and the role of CD47 in CAR T-cell function remains largely unknown. METHODS: Here, we describe the development of CD47-CAR T cells derived from a high affinity signal regulatory protein α variant CV1, which binds CD47. CV1-CAR T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo. The role of CD47 in CAR T-cell function was examined by knocking out CD47 in T cells followed by downstream functional analyses. RESULTS: While CV1-CAR T cells are specific and exhibit potent activity in vitro they lacked antitumor activity in xenograft models. Mechanistic studies revealed CV1-CAR T cells downregulate CD47 to overcome fratricide, but CD47 loss resulted in their failure to expand and persist in vivo. This effect was not limited to CV1-CAR T cells, since CD47 knockout CAR T cells targeting another solid tumor antigen exhibited the same in vivo fate. Further, CD47 knockout T cells were sensitive to macrophage-mediated phagocytosis. CONCLUSIONS: These findings highlight that CD47 expression is critical for CAR T-cell survival in vivo and is a 'sine qua non' for successful adoptive T-cell therapy.
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Receptores Quiméricos de Antígenos , Linfocitos T , Humanos , Inmunoterapia Adoptiva/métodos , Antígeno CD47/genética , Antígeno CD47/metabolismo , Leucocitos Mononucleares/metabolismo , Supervivencia Celular , Línea Celular TumoralRESUMEN
Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.
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Quimiocina CCL22 , Células Asesinas Naturales , Trastornos Linfoproliferativos , Animales , Quimiocina CCL22/genética , Células Asesinas Naturales/patología , Activación de Linfocitos , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Ratones , MutaciónRESUMEN
During epithelial-mesenchymal transition (EMT) epithelial cancer cells transdifferentiate into highly motile, invasive, mesenchymal-like cells, giving rise to disseminating tumor cells. Few of these disseminated cells successfully metastasize. Immune cells and inflammation in the tumor microenvironment were shown to drive EMT, but few studies investigated the consequences of EMT for tumor immunosurveillance. In addition to initiating metastasis, we demonstrate that EMT confers increased susceptibility to natural killer (NK) cells and contributes, in part, to the inefficiency of the metastatic process. Depletion of NK cells allowed spontaneous metastasis without affecting primary tumor growth. EMT-induced modulation of E-cadherin and cell adhesion molecule 1 (CADM1) mediated increased susceptibility to NK cytotoxicity. Higher CADM1 expression correlates with improved patient survival in 2 lung and 1 breast adenocarcinoma patient cohorts and decreased metastasis. Our observations reveal a novel NK-mediated, metastasis-specific immunosurveillance in lung cancer and present a window of opportunity for preventing metastasis by boosting NK cell activity.
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Carcinoma Pulmonar de Lewis/inmunología , Transición Epitelial-Mesenquimal/inmunología , Inmunidad Celular , Vigilancia Inmunológica , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/inmunología , Microambiente Tumoral/inmunología , Células A549 , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Molécula 1 de Adhesión Celular/genética , Molécula 1 de Adhesión Celular/inmunología , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Células Asesinas Naturales/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunologíaRESUMEN
Malignant gliomas are resistant to natural killer (NK) cell immune surveillance. However, the mechanisms used by these cancers to suppress antitumor NK cell activity remain poorly understood. We have recently reported on a novel mechanism of innate immune evasion characterized by the overexpression of the carbohydrate-binding protein galectin-1 by both mouse and rat malignant glioma. Here, we investigate the cytokine profile of galectin-1-deficient GL26 cells and describe the process by which these tumors are targeted by the early innate immune system in RAG1(-/-) and C57BL/6J mice. Our data reveal that galectin-1 knockdown in GL26 cells heightens their inflammatory status leading to the rapid recruitment of Gr-1(+)/CD11b(+) myeloid cells and NK1.1(+) NK cells into the brain tumor microenvironment, culminating in tumor clearance. We show that immunodepletion of Gr-1(+) myeloid cells in RAG1(-/-) mice permits the growth of galectin-1-deficient glioma despite the presence of NK cells, thus demonstrating an essential role for myeloid cells in the clearance of galectin-1-deficient glioma. Further characterization of tumor-infiltrating Gr-1(+)/CD11b(+) cells reveals that these cells also express CCR2 and Ly-6C, markers consistent with inflammatory monocytes. Our results demonstrate that Gr-1(+)/CD11b(+) myeloid cells, often referred to as myeloid-derived suppressor cells (MDSCs), are required for antitumor NK cell activity against galectin-1-deficient GL26 glioma. We conclude that glioma-derived galectin-1 represents an important factor in dictating the phenotypic behavior of monocytic Gr-1(+)/CD11b(+) myeloid cells. Galectin-1 suppression may be a valuable treatment approach for clinical glioma by promoting their innate immune-mediated recognition and clearance through the concerted effort of innate myeloid and lymphoid cell lineages.
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Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the ß-galactoside-binding lectin galectin-1. Conversely, galectin-1-deficient glioma cells could be eradicated by host NK cells before the initiation of an antitumor T-cell response. In vitro experiments demonstrated that galectin-1-deficient GL26-Cit glioma cells are â¼3-fold more sensitive to NK-mediated tumor lysis than galectin-1-expressing cells. Our findings suggest that galectin-1 suppression in human glioma could improve patient survival by restoring NK immune surveillance that can eradicate glioma cells. Cancer Res; 74(18); 5079-90. ©2014 AACR.
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Neoplasias Encefálicas/inmunología , Galectina 1/deficiencia , Glioma/inmunología , Células Asesinas Naturales/inmunología , Inmunidad Adaptativa/inmunología , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Femenino , Galectina 1/inmunología , Galectina 1/metabolismo , Glioma/metabolismo , Glioma/patología , Glioma/terapia , Humanos , Vigilancia Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Linfocitos T/inmunologíaRESUMEN
PURPOSE: The graft-versus-tumor (GVT) effect is a potent form of immunotherapy against many hematologic malignancies and some solid tumors. The beneficial GVT effect after allogeneic bone marrow transplantation (BMT) is tightly linked to its most significant complication, graft-versus-host disease (GVHD). The role of interleukin-6 (IL-6) after allogeneic BMT is not well understood. This study used a series of complementary knockout and antibody blockade strategies to analyze the impact of IL-6 in multiple clinically relevant murine models of GVHD and GVT. EXPERIMENTAL DESIGN: We examined the effect of the source of IL-6 by analyzing the role IL-6 deficiency in donor T cells, donor bone marrow or in host tissues. We confirmed and extended the relevance of IL-6 deficiency on GVHD and GVT by treating BMT recipients with anti-mouse IL-6 receptor (IL-6R), MR16-1. RESULTS: Deficiency of IL-6 in donor T cells led to prolongation of survival. Total inhibition of IL-6 with MR16-1 caused an even greater reduction in GVHD-induced mortality. The reduction in GVHD was independent of the direct effects on T effector cell expansion or donor regulatory T cells. GVT responses were preserved after treatment with MR16-1. CONCLUSION: MR16-1 treatment reduced GVHD and preserved sufficient GVT. Tocilizumab, a humanized anti-IL-6R monoclonal antibody (mAb), is approved in several countries including the United States and European Union for the treatment of rheumatoid arthritis and other inflammatory diseases. Blockade of IL-6 with anti-IL-6R mAb therapy may be testable in clinical trials as an adjunct to prevent GVHD in BMT patients without a significant loss of GVT.