Publications by authors named "Ilwoong Hwang"

7 Publications

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Expansion of cytotoxic natural killer cells in multiple myeloma patients using K562 cells expressing OX40 ligand and membrane-bound IL-18 and IL-21.

Cancer Immunol Immunother 2021 Jul 20. Epub 2021 Jul 20.

Research Center for Cancer Immunotherapy, Gwangju, South Korea.

Background: Natural killer (NK) cell-based immunotherapy is a promising treatment approach for multiple myeloma (MM), but obtaining a sufficient number of activated NK cells remains challenging. Here, we report an improved method to generate ex vivo expanded NK (eNK) cells from MM patients based on genetic engineering of K562 cells to express OX40 ligand and membrane-bound (mb) IL-18 and IL-21.

Methods: K562-OX40L-mbIL-18/-21 cells were generated by transducing K562-OX40L cells with a lentiviral vector encoding mbIL-18 and mbIL-21, and these were used as feeder cells to expand NK cells from peripheral blood mononuclear cells of healthy donors (HDs) and MM patients in the presence of IL-2/IL-15. Purity, expansion rate, receptor expression, and functions of eNK cells were determined over four weeks of culture.

Results: NK cell expansion was enhanced by short exposure of soluble IL-18 and IL-21 with K562-OX40L cells. Co-culture of NK cells with K562-OX40L-mbIL-18/-21 cells resulted in remarkable expansion of NK cells from HDs (9,860-fold) and MM patients (4,929-fold) over the 28-day culture period. Moreover, eNK cells showed increased expression of major activation markers and enhanced cytotoxicity towards target K562, U266, and RPMI8226 cells.

Conclusions: Our data suggest that genetically engineered K562 cells expressing OX40L, mbIL-18, and mbIL-21 improve the expansion of NK cells, increase activation signals, and enhance their cytolytic activity towards MM cells.
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http://dx.doi.org/10.1007/s00262-021-02982-9DOI Listing
July 2021

A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation.

Front Immunol 2020 14;11:1851. Epub 2020 Aug 14.

Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, South Korea.

Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay requires large amounts of blood and a rapid PBMC isolation step. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases. Peripheral blood of healthy volunteers ( = 28) and patients with liver diseases, including hepatocellular carcinoma ( = 19) and liver cirrhosis ( = 7), was analyzed for complete blood count, absolute NK cell count, and NK cell activity (NKA). NKA was evaluated in three assay types: an FC-based overnight WB NK cytotoxicity assay using carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells in the presence of various cytokine combinations [including interleukin (IL)-2, IL-18, and IL-21], an FC-based 4-h PBMC NK cytotoxicity assay, and an FC-based CD107a degranulation assay using WB and PBMCs. Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). A good correlation was observed between WB and PBMC NK cytotoxicity assays; absolute NK cell counts were better correlated with the WB NK cytotoxicity assay than with the PBMC NK cytotoxicity assay. This WB NK cytotoxicity assay showed that patients with liver diseases had significantly lower NK cytotoxicity than healthy volunteers, under stimulation with various cytokines ( < 0.001). The proposed FC-based overnight WB NK cytotoxicity assay correlates well with the conventional 4-h PBMC NK cytotoxicity assay, demonstrating future potential as a supportive assay for clinical laboratory research and observational studies.
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http://dx.doi.org/10.3389/fimmu.2020.01851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457041PMC
April 2021

Phenotypic and Functional Analysis of Human NK Cell Subpopulations According to the Expression of FcεRIγ and NKG2C.

Front Immunol 2019 6;10:2865. Epub 2019 Dec 6.

Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, South Korea.

Human memory-like NK cells are commonly defined by either a lack of FcεRIγ or gain of NKG2C expression. Here, we investigated the heterogeneity of human CD56 NK cell subpopulations according to the expression of FcεRIγ and NKG2C in a large cohort ( = 127). Although the frequency of FcεRIγ and NKG2C NK cells positively correlated, the FcεRIγ and NKG2C NK cell populations did not exactly overlap. The FcεRIγNKG2C, FcεRIγNKG2C, and FcεRIγNKG2C NK cell populations were only evident after HCMV infection, but each had distinct characteristics. Among the subpopulations, FcεRIγNKG2C NK cells exhibited the most restricted killer immunoglobulin-like receptor repertoire, suggesting clonal expansion. Moreover, FcεRIγNKG2C NK cells exhibited the lowest Ki-67 and highest Bcl-2 expression, indicating the long-lived quiescent memory-like property. Functionally, FcεRIγNKG2C NK cells had weak natural effector function against K562 but strong effector functions by CD16 engagement, whereas FcεRIγNKG2C NK cells had strong effector functions in both settings. Anatomically, the FcεRIγNKG2C, FcεRIγNKG2C, and FcεRIγNKG2C NK cell populations were present in multiple human peripheral organs. In conclusion, we demonstrate the heterogeneity of memory-like NK cells stratified by FcεRIγ and NKG2C and suggest both markers be utilized to better define these cells.
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http://dx.doi.org/10.3389/fimmu.2019.02865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908468PMC
November 2020

Epigenetic modification and antibody-dependent expansion of memory-like NK cells in human cytomegalovirus-infected individuals.

Immunity 2015 Mar;42(3):431-42

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. Electronic address:

Long-lived "memory-like" NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion.
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http://dx.doi.org/10.1016/j.immuni.2015.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537797PMC
March 2015

Cutting edge: antibody-dependent memory-like NK cells distinguished by FcRγ deficiency.

J Immunol 2013 Feb 23;190(4):1402-6. Epub 2013 Jan 23.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

Because NK cells lack gene-recombination machinery and are thought to be relatively short-lived, it is unclear whether NK cells can mount long-term effective recall responses to reinfections by diverse pathogens. In this article, we report that FcRγ-deficient NK cells, which we recently identified and termed g(-)NK cells, possess distinct memory features directed by FcR-mediated Ab-dependent target recognition. The presence of g(-)NK cells was associated with prior human CMV (HMCV) infection, yet g(-)NK cell responses were not restricted to HCMV-infected target cells. In the presence of virus-specific Abs, g(-)NK cells had greatly enhanced functional capabilities, superior to conventional NK cells, and were highly responsive to cells infected with either HCMV or HSV-1. Remarkably, the g(-)NK cell subset persisted long-term at nearly constant levels in healthy individuals. Therefore, FcRγ deficiency distinguishes an Ab-dependent memory-like NK cell subset with enhanced potential for broad antiviral responses.
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http://dx.doi.org/10.4049/jimmunol.1203034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623944PMC
February 2013

Activation mechanisms of natural killer cells during influenza virus infection.

PLoS One 2012 31;7(12):e51858. Epub 2012 Dec 31.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA.

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051858PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534084PMC
July 2013

Identification of human NK cells that are deficient for signaling adaptor FcRγ and specialized for antibody-dependent immune functions.

Int Immunol 2012 Dec 7;24(12):793-802. Epub 2012 Sep 7.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

NK cells respond to tumor and virus-infected cells directly through several activation receptors, including natural cytotoxicity receptors, or indirectly through the activating Fc receptor CD16 for antibody-coated cells. Triggering of NK-cell effector functions through these receptors depends on physically associated transmembrane signaling adaptors, such as FcRγ (also known as FcεRIγ) and CD3ζ, both of which have been traditionally believed to be expressed by all mature NK cells. However, we have identified a distinct subset of human NK cells that are deficient for FcRγ expression but express normal levels of CD3ζ. FcRγ-deficient NK cells were readily detectable in about one-third of the healthy individuals examined. The deficiency was confined to the CD56(dim) population and was due to low FcRγ mRNA. FcRγ-deficient NK cells displayed dramatically reduced expression of the natural cytotoxicity receptors NKp46 and NKp30 but still expressed substantial levels of CD16. Compared to FcRγ-expressing NK cells, FcRγ-deficient NK cells showed poor direct reactivity toward tumor targets as measured by cytokine production and degranulation. Unexpectedly, however, FcRγ-deficient NK cells exhibited significantly more robust responsiveness upon stimulation through CD16, particularly for cytokine production, compared to FcRγ-expressing NK cells. Thus, our study reveals FcRγ-deficient NK cells as a novel subset of human NK cells that have remarkably potent responses toward antibody-coated targets. These findings also illustrate a differential contribution of FcRγ and CD3ζ for the expression and functional activity of their associated receptors.
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http://dx.doi.org/10.1093/intimm/dxs080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3621379PMC
December 2012
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