ABSTRACT
Natural killer (NK) cells with adaptive immunological properties expand and persist in response to human cytomegalovirus. Here, we explored the metabolic processes unique to these cells. Adaptive CD3-CD56dimCD57+NKG2C+ NK cells exhibited metabolic hallmarks of lymphocyte memory, including increased oxidative mitochondrial respiration, mitochondrial membrane potential, and spare respiratory capacity. Mechanistically, we found that a short isoform of the chromatin-modifying transcriptional regulator, AT-rich interaction domain 5B (ARID5B), was selectively induced through DNA hypomethylation in adaptive NK cells. Knockdown and overexpression studies demonstrated that ARID5B played a direct role in promoting mitochondrial membrane potential, expression of genes encoding electron transport chain components, oxidative metabolism, survival, and IFN-γ production. Collectively, our data demonstrate that ARID5B is a key regulator of metabolism in human adaptive NK cells, which, if targeted, may be of therapeutic value.
Subject(s)
Cytomegalovirus Infections , DNA-Binding Proteins , Killer Cells, Natural , Lymphocyte Activation , Transcription Factors , Cell Survival/genetics , Cell Survival/immunology , Cytomegalovirus/immunology , Cytomegalovirus/metabolism , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/metabolism , Cytomegalovirus Infections/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Female , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interferon-gamma/immunology , K562 Cells , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Killer Cells, Natural/pathology , Male , Membrane Potential, Mitochondrial/genetics , Membrane Potential, Mitochondrial/immunology , Oxidation-Reduction , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolismABSTRACT
Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664-75. ©2017 AACR.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Immunotherapy, Adoptive/methods , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , A549 Cells , Animals , Antibody-Dependent Cell Cytotoxicity/drug effects , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Female , Glycogen Synthase Kinase 3/immunology , Humans , Interleukin-15/pharmacology , K562 Cells , Killer Cells, Natural/enzymology , Mice , Mice, Inbred NOD , Ovarian Neoplasms/immunology , Ovarian Neoplasms/therapy , Pyridines/pharmacology , Pyrimidines/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The discovery of chemoresistant cancer stem cells (CSCs) in carcinomas has created the need for therapies that specifically target these subpopulations of cells. Here, we characterized a bispecific targeted toxin that is composed of two antibody fragments and a catalytic protein toxin allowing it to bind two CSC markers on the same cell killing this resistant subpopulation. CD133 is a well-known CSC marker and has been successfully targeted and caused regression of head and neck squamous cell carcinoma (HNSCC) in vivo. To enable it to bind a broader range of CSCs, an anti-epithelial cell adhesion molecule (EpCAM) scFv was added to create dEpCAMCD133KDEL, a deimmunized bispecific targeted toxin on a single amino acid chain. This bispecific potently inhibited protein translation and proliferation in vitro in three different types of carcinoma. Furthermore, in a CSC spheroid model dEpCAMCD133KDEL eliminated Mary-X spheroids, an inflammatory breast carcinoma. Finally, this bispecific also caused tumor regression in an in vivo model of HNSCC. This represents the first bispecific CSC-targeted toxin and warrants further development as a possible therapy for carcinoma.
Subject(s)
Antibodies, Bispecific/pharmacology , Antigens, CD/immunology , Antigens, Neoplasm/immunology , Carcinoma, Squamous Cell/therapy , Cell Adhesion Molecules/immunology , Glycoproteins/immunology , Head and Neck Neoplasms/therapy , Immunotoxins/pharmacology , Peptides/immunology , AC133 Antigen , Animals , Antibodies, Bispecific/immunology , Caco-2 Cells , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/pathology , Cell Proliferation , Epithelial Cell Adhesion Molecule , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/pathology , Humans , Immunotoxins/immunology , Mice , Mice, Nude , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/pathology , Squamous Cell Carcinoma of Head and Neck , TransfectionABSTRACT
⢠Availability of growth limiting resources may alter root dynamics in forest ecosystems, possibly affecting the land-atmosphere exchange of carbon. This was evaluated for a commercially important southern timber species by installing a factorial experiment of fertilization and irrigation treatments in an 8-yr-old loblolly pine (Pinus taeda) plantation. ⢠After 3 yr of growth, production and turnover of fine, coarse and mycorrhizal root length was observed using minirhizotrons, and compared with stem growth and foliage development. ⢠Fertilization increased net production of fine roots and mycorrhizal roots, but did not affect coarse roots. Fine roots had average lifespans of 166 d, coarse roots 294 d and mycorrhizal roots 507 d. Foliage growth rate peaked in late spring and declined over the remainder of the growing season, whereas fine roots experienced multiple growth flushes in the spring, summer and fall. ⢠We conclude that increased nutrient availability might increase carbon input to soils through enhanced fine root turnover. However, this will depend on the extent to which mycorrhizal root formation is affected, as these mycorrhizal roots have much longer average lifespans than fine and coarse roots.