Publications by authors named "Hsiaoyin Mao"

31 Publications

Adipocytes: A Novel Target for IL-15/IL-15Rα Cancer Gene Therapy.

Mol Ther 2019 05 16;27(5):922-932. Epub 2019 Feb 16.

The Ohio State University Comprehensive Cancer Center, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA; Department of Cancer Biology and Genetics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

IL-15 is a proinflammatory cytokine that plays an essential role in the development and activation of natural killer (NK) cells. Adipose tissue acts as an endocrine organ that secretes cytokines and is an important reservoir for lymphocytes. We hypothesized that activation of the IL-15 signaling in adipose tissue will activate and expand the NK cell population and control tumor growth. We recently developed an adipocyte-targeting recombinant adeno-associated viral (rAAV) vector with minimal off-target transgene expression in the liver. Here, we used this rAAV system to deliver an IL-15/IL-15Rα complex to the abdominal fat by intraperitoneal (i.p.) injection. Adipose IL-15/IL-15Rα complex gene transfer led to the expansion of NK cells in the adipose tissue and spleen in normal mice without notable side effects. The i.p. injection of rAAV-IL-15/IL-15Rα complex significantly suppressed the growth of Lewis lung carcinoma implanted subcutaneously and exerted a significant survival advantage in a B16-F10 melanoma metastasis model. The antitumor effects were associated with the expansion of the NK cells in the blood, spleen, abdominal fat, and tumor, as well as the enhancement of NK cell maturity. Our proof-of-concept preclinical studies demonstrate the safety and efficacy of the adipocyte-specific IL-15/IL-15Rα complex vector as a novel cancer immune gene therapy.
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http://dx.doi.org/10.1016/j.ymthe.2019.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520519PMC
May 2019

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway.

Immunity 2018 09 4;49(3):464-476.e4. Epub 2018 Sep 4.

Department of Pathology, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 USA; Comprehensive Cancer Center, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 USA. Electronic address:

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34CD117 ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34CD117 ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.
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http://dx.doi.org/10.1016/j.immuni.2018.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148384PMC
September 2018

The Fc Domain of Immunoglobulin Is Sufficient to Bridge NK Cells with Virally Infected Cells.

Immunity 2017 07;47(1):159-170.e10

The Ohio State University Comprehensive Cancer Center, The James Cancer Hospital and Solove Research Institute, Columbus, OH 43210, USA; Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43205, USA. Electronic address:

Clearance of pathogens or tumor cells by antibodies traditionally requires both Fab and Fc domains of IgG. Here, we show the Fc domain of IgG alone mediates recognition and clearance of herpes simplex virus (HSV1)-infected cells. The human natural killer (NK) cell surface is naturally coated with IgG bound by its Fc domain to the Fcγ receptor CD16a. NK cells utilize the Fc domain of bound IgG to recognize gE, an HSV1-encoded glycoprotein that also binds the Fc domain of IgG but at a site distinct from CD16a. The bridge formed by the Fc domain between the HSV1-infected cell and the NK cell results in NK cell activation and lysis of the HSV1-infected cell in the absence of HSV1-specific antibody in vitro and prevents fatal HSV1 infection in vivo. This mechanism also explains how bacterial IgG-binding proteins regulate NK cell function and may be broadly applicable to Fcγ-receptor-bearing cells.
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http://dx.doi.org/10.1016/j.immuni.2017.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568648PMC
July 2017

MicroRNA-29b mediates altered innate immune development in acute leukemia.

J Clin Invest 2016 12 24;126(12):4404-4416. Epub 2016 Oct 24.

Natural killer (NK) cells can have potent antileukemic activity following haplo-mismatched, T cell-depleted stem cell transplantations for the treatment of acute myeloid leukemia (AML), but they are not successful in eradicating de novo AML. Here, we have used a mouse model of de novo AML to elucidate the mechanisms by which AML evades NK cell surveillance. NK cells in leukemic mice displayed a marked reduction in the cytolytic granules perforin and granzyme B. Further, as AML progressed, we noted the selective loss of an immature subset of NK cells in leukemic mice and in AML patients. This absence was not due to elimination by cell death or selective reduction in proliferation, but rather to the result of a block in NK cell differentiation. Indeed, NK cells from leukemic mice and humans with AML showed lower levels of TBET and EOMES, transcription factors that are critical for terminal NK cell differentiation. Further, the microRNA miR-29b, a regulator of T-bet and EOMES, was elevated in leukemic NK cells. Finally, deletion of miR-29b in NK cells reversed the depletion of this NK cell subset in leukemic mice. These results indicate that leukemic evasion of NK cell surveillance occurs through miR-mediated dysregulation of lymphocyte development, representing an additional mechanism of immune escape in cancer.
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http://dx.doi.org/10.1172/JCI85413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127669PMC
December 2016

A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

Immunity 2016 05 10;44(5):1140-50. Epub 2016 May 10.

Department of Pathology, The Ohio State University, Columbus, OH 43210, USA; Comprehensive Cancer Center, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.
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http://dx.doi.org/10.1016/j.immuni.2016.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893782PMC
May 2016

Transcription factor Foxo1 is a negative regulator of natural killer cell maturation and function.

Immunity 2015 Mar 10;42(3):457-70. Epub 2015 Mar 10.

Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA; The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA; The James Cancer Hospital, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes by upregulating CD62L expression and inhibited late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21(+/-) mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions.
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http://dx.doi.org/10.1016/j.immuni.2015.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400836PMC
March 2015

Role of cysteine-rich 61 protein (CCN1) in macrophage-mediated oncolytic herpes simplex virus clearance.

Mol Ther 2014 Sep 4;22(9):1678-87. Epub 2014 Jun 4.

Department of Neurological Surgery, Dardinger Laboratory for Neuro-oncology and Neurosciences, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA.

Glioblastoma is a devastating disease, and there is an urgent need to develop novel therapies, such as oncolytic HSV1 (OV) to effectively target tumor cells. OV therapy depends on tumor-specific replication leading to destruction of neoplastic tissues. Host responses that curtail virus replication limit its efficacy in vivo. We have previously shown that cysteine-rich 61 protein (CCN1) activates a type 1 IFN antiviral defense response in glioblastoma cells. Incorporating TCGA data, we found CCN1 expression to be a negative prognostic factor for glioblastoma patients. Based on this, we used neutralizing antibodies against CCN1 to investigate its effect on OV therapy. Use of an anti-CCN1 antibody in mice bearing glioblastomas treated with OV led to enhanced virus expression along with reduced immune cell infiltration. OV-induced CCN1 increases macrophage migration toward infected glioblastoma cells by directly binding macrophages and also by enhancing the proinflammatory activation of macrophages inducing MCP-1 expression in glioblastoma cells. Activation of macrophages by CCN1 also increases viral clearance. Neutralization of integrin αMβ2 reversed CCN1-induced macrophage activation and migration, and reduced MCP-1 expression by glioblastoma cells. Our findings reveal that CCN1 plays a novel role in pathogen clearance; increasing macrophage infiltration and activation resulting in increased virus clearance in tumors.
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http://dx.doi.org/10.1038/mt.2014.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435480PMC
September 2014

DDB2 suppresses tumorigenicity by limiting the cancer stem cell population in ovarian cancer.

Mol Cancer Res 2014 May 26;12(5):784-94. Epub 2014 Feb 26.

Authors' Affiliations: Departments of Radiology and 2Pathology; 3Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, Ohio; and 4Weifang Medical University, Shandong, China.

Unlabelled: Ovarian cancer is an extremely aggressive disease associated with a high percentage of tumor recurrence and chemotherapy resistance. Understanding the underlying mechanism of tumor relapse is crucial for effective therapy of ovarian cancer. DNA damage-binding protein 2 (DDB2) is a DNA repair factor mainly involved in nucleotide excision repair. Here, a novel role was identified for DDB2 in the tumorigenesis of ovarian cancer cells and the prognosis of patients with ovarian cancer. Overexpressing DDB2 in human ovarian cancer cells suppressed its capability to recapitulate tumors in athymic nude mice. Mechanistic investigation demonstrated that DDB2 is able to reduce the cancer stem cell (CSC) population characterized with high aldehyde dehydrogenase activity in ovarian cancer cells, probably through disrupting the self-renewal capacity of CSCs. Low DDB2 expression correlates with poor outcomes among patients with ovarian cancer, as revealed from the analysis of publicly available gene expression array datasets. Given the finding that DDB2 protein expression is low in ovarian tumor cells, enhancement of DDB2 expression is a promising strategy to eradicate CSCs and would help to halt ovarian cancer relapse.

Implications: DDB2 status has prognostic potential, and elevating its expression eradicates CSCs and could reduce ovarian cancer relapse.
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http://dx.doi.org/10.1158/1541-7786.MCR-13-0638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096129PMC
May 2014

PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells.

J Clin Invest 2013 Oct 3;123(10):4144-57. Epub 2013 Sep 3.

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.
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http://dx.doi.org/10.1172/JCI68951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784537PMC
October 2013

MicroRNAs activate natural killer cells through Toll-like receptor signaling.

Blood 2013 Jun 11;121(23):4663-71. Epub 2013 Apr 11.

Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.

MicroRNAs (miRNAs) bind to complementary sequences of target mRNAs, resulting in translational repression or target degradation and thus gene silencing. miRNAs are abundant in circulating blood, yet it is not known whether, as a class of regulatory molecules, they interact with human natural killer (NK) cells. Here we found that the treatment of human NK cells with several mature miRNAs in the presence of a low concentration of interleukin-12 induced CD69 expression, interferon-γ production, and degranulation marker CD107a expression. In vivo, infusion of several miRNAs alone in murine peripheral blood also resulted in comparable NK-cell activation, but not T-cell activation. Furthermore, miRNA administration significantly protected mice from tumor development in an NK cell-dependent manner. Mechanistically, we found that miRNA stimulation led to downstream activation of nuclear factor κB (NF-κB), an effect that was blunted by a block in Toll-like receptor 1(TLR1) signaling and attenuated in lymphoma patients. Knockdown of TLR1 resulted in less activation by miRNAs. Collectively, we show that miRNAs have a capacity to selectively activate innate immune effector cells that is, at least in part, via the TLR1-NF-κB signaling pathway. This may be important in the normal host defense against infection and/or malignant transformation.
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http://dx.doi.org/10.1182/blood-2012-07-441360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674667PMC
June 2013

NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors.

Nat Med 2012 Dec 25;18(12):1827-34. Epub 2012 Nov 25.

Medical Scientist Training Program, Ohio State University Medical Center, Columbus, Ohio, USA.

The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1(-/-) mice and a Ncr1(-/-) NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy.
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http://dx.doi.org/10.1038/nm.3013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668784PMC
December 2012

Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver.

J Clin Invest 2012 Aug 23;122(8):2871-83. Epub 2012 Jul 23.

Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH, USA.

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.
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http://dx.doi.org/10.1172/JCI63539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408748PMC
August 2012

NKp46 identifies an NKT cell subset susceptible to leukemic transformation in mouse and human.

J Clin Invest 2011 Apr;121(4):1456-70

Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, Ohio, USA.

IL-15 may have a role in the development of T cell large granular lymphocyte (T-LGL) or NKT leukemias. However, the mechanisms of action and the identity of the cell subset that undergoes leukemic transformation remain elusive. Here we show that in both mice and humans, NKp46 expression marks a minute population of WT NKT cells with higher activity and potency to become leukemic. Virtually 100% of T-LGL leukemias in IL-15 transgenic mice expressed NKp46, as did a majority of human T-LGL leukemias. The minute NKp46+ NKT population, but not the NKp46⁻ NKT population, was selectively expanded by overexpression of endogenous IL-15. Importantly, IL-15 transgenic NKp46⁻ NKT cells did not become NKp46+ in vivo, suggesting that NKp46+ T-LGL leukemia cells were the malignant counterpart of the minute WT NKp46+ NKT population. Mechanistically, NKp46+ NKT cells possessed higher responsiveness to IL-15 in vitro and in vivo compared with that of their NKp46⁻ NKT counterparts. Furthermore, interruption of IL-15 signaling using a neutralizing antibody could prevent LGL leukemia in IL-15 transgenic mice. Collectively, our data demonstrate that NKp46 identifies a functionally distinct NKT subset in mice and humans that appears to be directly susceptible to leukemic transformation when IL-15 is overexpressed. Thus, IL-15 signaling and NKp46 may be useful targets in the treatment of patients with T-LGL or NKT leukemia.
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http://dx.doi.org/10.1172/JCI43242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069763PMC
April 2011

The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells.

Blood 2011 Feb 14;117(8):2378-84. Epub 2010 Dec 14.

Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Comprehensive Cancer Center, 460 West 12th Ave., Columbus, OH 43210, USA.

The ability of natural killer (NK) cells to kill malignant or infected cells depends on the integration of signals from different families of cell surface receptors, including cytokine receptors. How such signals then regulate NK-cell cytotoxicity is incompletely understood. Here we analyzed an endogenous inhibitor of protein phosphatase 2A (PP2A) activity called SET, and its role in regulating human NK-cell cytotoxicity and its mechanism of action in human NK cells. RNAi-mediated suppression of SET down-modulates NK-cell cytotoxicity, whereas ectopic overexpression of SET enhances cytotoxicity. SET knockdown inhibits both mRNA and protein granzyme B expression, as well as perforin expression, whereas SET overexpression enhances granzyme B expression. Treatment of NK cells with the PP2A activator 1,9-dideoxy-forskolin also inhibits both granzyme B expression and cytotoxicity. In addition, pretreatment with the PP2A inhibitor okadaic acid rescues declining granzyme B mRNA levels in SET knockdown cells. Down-modulation of SET expression or activation of PP2A also decreases human NK-cell antibody-dependent cellular cytotoxicity. Finally, the induction of granzyme B gene expression by interleukin-2 and interleukin-15 is inhibited by SET knockdown. These data provide evidence that granzyme B gene expression and therefore human NK-cell cytotoxicity can be regulated by the PP2A-SET interplay.
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http://dx.doi.org/10.1182/blood-2010-05-285130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062407PMC
February 2011

In vivo role of Flt3 ligand and dendritic cells in NK cell homeostasis.

J Immunol 2010 Mar 8;184(6):2769-75. Epub 2010 Feb 8.

Department of Molecular Virology, The Ohio State University, Columbus, OH43210, USA.

IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Ralpha-chain to cells expressing the IL-15Rbetagamma complex, we postulated that certain IL-15-bearing cells must be required for NK cell homeostasis. Using IL-15(WT/WT) and IL-15(-/-) mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11c(hi) dendritic cells (DCs), we demonstrate that ablation of the resting CD11c(hi) DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11c(hi) DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11c(hi) DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.
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http://dx.doi.org/10.4049/jimmunol.0900685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924750PMC
March 2010

CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets.

Blood 2010 Jan 6;115(2):274-81. Epub 2009 Nov 6.

Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH 43210, USA.

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.
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http://dx.doi.org/10.1182/blood-2009-04-215491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808153PMC
January 2010

CD94 defines phenotypically and functionally distinct mouse NK cell subsets.

J Immunol 2009 Oct;183(8):4968-74

Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.

Understanding of heterogeneous NK subsets is important for the study of NK cell biology and development, and for the application of NK cell-based therapies in the treatment of disease. Here we demonstrate that the surface expression of CD94 can distinctively divide mouse NK cells into two approximately even CD94(low) and CD94(high) subsets in all tested organs and tissues. The CD94(high) NK subset has significantly greater capacity to proliferate, produce IFN-gamma, and lyse target cells than does the CD94(low) subset. The CD94(high) subset has exclusive expression of NKG2A/C/E, higher expression of CD117 and CD69, and lower expression of Ly49D (activating) and Ly49G2 (inhibitory). In vivo, purified mouse CD94(low) NK cells become CD94(high) NK cells, but not vice versa. Collectively, our data suggest that CD94 is an Ag that can be used to identify functionally distinct NK cell subsets in mice and could also be relevant to late-stage mouse NK cell development.
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http://dx.doi.org/10.4049/jimmunol.0900907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924742PMC
October 2009

A novel mouse model for the aggressive variant of NK cell and T cell large granular lymphocyte leukemia.

Leuk Res 2010 Feb 5;34(2):203-9. Epub 2009 Aug 5.

Department of Molecular Virology, Immunology, and Medical Genetics, Department of Internal Medicine, Division of Hematology/Oncology, The Ohio State University College of Medicine, Columbus, OH, United States.

Murine models of disease are vital to the understanding of pathogenesis and the development of novel therapeutics. We have previously established interleukin (IL)-15 transgenic (tg) mice that demonstrate rapid proliferation of natural killer (NK) and T cells, followed by spontaneous transformation to lethal leukemia. Herein, we have characterized this model, which has many features in common with the aggressive variants of NK and T large granular lymphocyte leukemia (LGLL) in humans. The LGLL blasts are cytolytic and produce IFN-gammaex vivo. Cytogenetic analysis revealed trisomy of chromosome 17 and/or 15. This model should provide opportunities to develop effective standard therapies for this fatal disease.
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http://dx.doi.org/10.1016/j.leukres.2009.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2814907PMC
February 2010

TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia.

Blood 2009 May 27;113(22):5558-67. Epub 2009 Mar 27.

Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.

Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22 is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22 promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22 expression. The TSC-22 deregulation was reversed in vivo by a 5-aza-2'-deoxycytidine therapy of T or NK LGL leukemia, which significantly increased survival of the mice bearing this disease. Ectopic expression of TSC-22 in mouse leukemia or lymphoma cell lines resulted in delayed in vivo tumor formation. Targeted disruption of TSC-22 in wild-type mice enhanced proliferation and in vivo repopulation efficiency of hematopoietic precursor cells (HPCs). Collectively, our data suggest that TSC-22 normally contributes to the regulation of HPC function and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia.
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http://dx.doi.org/10.1182/blood-2009-02-205732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689053PMC
May 2009

Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH 17 cytokine interleukin-22.

Blood 2009 Apr 24;113(17):4008-10. Epub 2009 Feb 24.

Integrated Biomedical Graduate Program, The Ohio State University College of Medicine, Columbus, Ohio, USA.

Considerable functional heterogeneity within human natural killer (NK) cells has been revealed through the characterization of distinct NK-cell subsets. Accordingly, a small subset of CD56(+)NKp44(+)NK cells, termed NK-22 cells, was recently described within secondary lymphoid tissue (SLT) as IL-22(-) when resting, with a minor fraction of this population becoming IL-22(+) when activated. Here we discover that the vast majority of stage 3 immature NK (iNK) cells in SLT constitutively and selectively express IL-22, a T(H)17 cytokine important for mucosal immunity, whereas earlier and later stages of NK developmental intermediates do not express IL-22. These iNK cells have a surface phenotype of CD34(-)CD117(+)CD161(+)CD94(-), largely lack expression of NKp44 and CD56, and do not produce IFN-gamma or possess cytolytic activity. In summary, stage 3 iNK cells are highly enriched for IL-22 and IL-26 messenger RNA, and IL-22 protein production, but do not express IL-17A or IL-17F.
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http://dx.doi.org/10.1182/blood-2008-12-192443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673127PMC
April 2009

TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells.

J Immunol 2008 Sep;181(6):3784-92

Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH 43210, USA.

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924753PMC
http://dx.doi.org/10.4049/jimmunol.181.6.3784DOI Listing
September 2008

The PP2A inhibitor SET regulates natural killer cell IFN-gamma production.

J Exp Med 2007 Oct 17;204(10):2397-405. Epub 2007 Sep 17.

Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.

Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.
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http://dx.doi.org/10.1084/jem.20070419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2118465PMC
October 2007

Transcriptional control of human T-BET expression: the role of Sp1.

Eur J Immunol 2007 Sep;37(9):2549-61

Division of Hematology/Oncology, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA.

Murine T-bet (T-box expressed in T cells) is a master regulator of IFN-gamma gene expression in NK and T cells. T-bet also plays a critical role in autoimmunity, asthma and other diseases. However, cis elements or trans factors responsible for regulating T-bet expression remain largely unknown. Here, we report on our discovery of six Sp1-binding sites within the proximal human T-BET promoter that are highly conserved among mammalian species. Electrophoretic mobility shift assays demonstrate a physical association between Sp1 and the proximal T-BET promoter with a direct dose response between Sp1 expression and T-BET promoter activity. Ectopic overexpression of Sp1 also enhanced T-BET expression and cytokine-induced IFN-gamma secretion in NK cells and T cells. Mithramycin A, which blocks the binding of Sp1 to the T-BET promoter, diminished both T-BET expression and IFN-gamma protein production in monokine-stimulated primary human NK cells. Collectively, our results suggest that Sp1 is a positive transcriptional regulator of T-BET. As T-BET and IFN-gamma are critically important in inflammation, infection, and cancer, targeting Sp1, possibly with mithramycin A, may be useful for preventing and/or treating diseases associated with aberrant T-BET or IFN-gamma expression.
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http://dx.doi.org/10.1002/eji.200737088DOI Listing
September 2007

Hlx homeobox transcription factor negatively regulates interferon-gamma production in monokine-activated natural killer cells.

Blood 2007 Mar 16;109(6):2481-7. Epub 2006 Nov 16.

Medical Scientist Program, Department of Internal Medicine, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA.

Natural killer (NK) cells contribute to host immunity, including tumor surveillance, through the production of interferon gamma (IFN-gamma). Although there is some knowledge about molecular mechanisms that induce IFN-gamma in NK cells, considerably less is known about the mechanisms that reduce its expression. Here, we investigate the role of the Hlx transcription factor in IFN-gamma production by NK cells. Hlx expression is induced in monokine-activated NK cells, but with delayed kinetics compared to IFN-gamma. Ectopic Hlx expression decreases IFN-gamma synthesis in primary human NK cells and IFN-gamma promoter activity in an NK-like cell line. Hlx protein levels inversely correlate with those of STAT4, a requisite factor for optimal IFN-gamma transcription. Mechanistically, we provide evidence indicating that Hlx overexpression accelerates dephosphorylation and proteasome-dependent degradation of the active Y693-phosphorylated form of STAT4. Thus, Hlx expression in activated NK cells temporally controls and limits the monokine-induced production of IFN-gamma, in part through the targeted depletion of STAT4.
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http://dx.doi.org/10.1182/blood-2006-10-050096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852195PMC
March 2007

Pro- and antiinflammatory cytokine signaling: reciprocal antagonism regulates interferon-gamma production by human natural killer cells.

Immunity 2006 May;24(5):575-90

Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine and School of Public Health, Columbus, Ohio 43210, USA.

Activated monocytes produce proinflammatory cytokines (monokines) such as interleukin (IL)-12, IL-15, and IL-18 for induction of interferon-gamma (IFN-gamma) by natural killer (NK) cells. NK cells provide the antiinflammatory cytokine transforming growth factor (TGF)-beta, an autocrine/negative regulator of IFN-gamma. The ability of one signaling pathway to prevail over the other is likely important in controlling IFN-gamma for the purposes of infection and autoimmunity, but the molecular mechanism(s) of how this counterregulation occurs is unknown. Here we show that in isolated human NK cells, proinflammatory monokines antagonize antiinflammatory TGF-beta signaling by downregulating the expression of the TGF-beta type II receptor, and its signaling intermediates SMAD2 and SMAD3. In contrast, TGF-beta utilizes SMAD2, SMAD3, and SMAD4 to suppress IFN-gamma and T-BET, a positive regulator of IFN-gamma. Indeed, activated NK cells from Smad3(-/-) mice produce more IFN-gamma in vivo than NK cells from wild-type mice. Collectively, our data suggest that pro- and antiinflammatory cytokine signaling reciprocally antagonize each other in an effort to prevail in the regulation of NK cell IFN-gamma production.
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http://dx.doi.org/10.1016/j.immuni.2006.03.016DOI Listing
May 2006

Evidence for discrete stages of human natural killer cell differentiation in vivo.

J Exp Med 2006 Apr 10;203(4):1033-43. Epub 2006 Apr 10.

Medical Scientist Program, The Ohio State University, Columbus, OH 43210, USA.

Human natural killer (NK) cells originate from CD34(+) hematopoietic progenitor cells, but the discrete stages of NK cell differentiation in vivo have not been elucidated. We identify and functionally characterize, from human lymph nodes and tonsils, four NK cell developmental intermediates spanning the continuum of differentiation from a CD34(+) NK cell progenitor to a functionally mature NK cell. Analyses of each intermediate stage for CD34, CD117, and CD94 cell surface expression, lineage differentiation potentials, capacity for cytokine production and natural cytotoxicity, and ETS-1, GATA-3, and T-BET expression provide evidence for a new model of human NK cell differentiation in secondary lymphoid tissues.
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http://dx.doi.org/10.1084/jem.20052507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2118285PMC
April 2006

The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein.

Cancer Cell 2005 Nov;8(5):355-68

Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio 43210, USA.

The oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.
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http://dx.doi.org/10.1016/j.ccr.2005.10.015DOI Listing
November 2005

A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells.

Immunity 2005 Mar;22(3):295-304

Department of Molecular Virology, Immunology, and Medical Genetics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43210, USA.

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.
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http://dx.doi.org/10.1016/j.immuni.2005.01.013DOI Listing
March 2005

Efficient infection of human natural killer cells with an EBV/retroviral hybrid vector.

J Immunol Methods 2005 Jan 8;296(1-2):115-23. Epub 2004 Dec 8.

Medical Scientist Program, Integrated Biomedical Graduate Program, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA.

Molecular characterization of human natural killer (NK) cells will require targeted gene delivery to inhibit and activate specific signaling pathways, yet to our knowledge, an effective means to deliver such products for long-term gene expression without disrupting normal cellular processes has not been described. In this study, we have developed a retroviral strategy to effectively express gene products in the NK cell, whereby its effector functions of cytotoxicity and cytokine production remain intact. Using an EBV/retroviral hybrid vector, we demonstrate infection of human peripheral blood NK cells with simultaneous expression of a marker for infection--the enhanced green fluorescent protein (EGFP)--along with various genes of interest. This technique results in successful infection of the CD56dim NK population that predominates among human peripheral blood NK and is the effector of antibody-dependent cellular cytotoxicity and natural killing. In addition, we demonstrate infection of the CD56bright NK subset as well as the NK-92 cell line. In summary, we have devised an efficient and reproducible methodology for the targeted delivery of gene products to human NK cells that should now provide opportunities to dissect the molecular processes critical to normal NK cell physiology.
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http://dx.doi.org/10.1016/j.jim.2004.11.012DOI Listing
January 2005