Publications by authors named "Minh-Trang Thi Phan"

11 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

Expanded natural killer cells augment the antimyeloma effect of daratumumab, bortezomib, and dexamethasone in a mouse model.

Cell Mol Immunol 2021 Jul 12;18(7):1652-1661. Epub 2021 May 12.

Research Center for Cancer Immunotherapy, Chonnam National University Hwasun Hospital and Chonnam National University Medical School, Gwangju, Korea.

The use of natural killer (NK) cells is a promising and safe immunotherapeutic approach in the field of cancer immunotherapy. However, combination treatments are required to enhance the effector functions and therapeutic efficacy of NK cells. In this study, we investigated the potential of daratumumab (Dara), bortezomib, and dexamethasone (Dvd) to augment the antitumor effects of NK cells in a multiple myeloma (MM) xenograft mouse model. NK cells were expanded and activated using the K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells from MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγ (NSG) mice. Tumor-bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNKs), Dara, Dara + eNKs, Dvd, and Dvd + eNKs. Dvd treatment strongly enhanced the cytotoxicity of eNKs by upregulating expression of NK cell activation ligands, downregulating expression of NK cell inhibitory ligands, and promoting antibody-dependent cellular cytotoxicity. The combination of eNKs with Dvd significantly prolonged mouse survival and reduced the tumor burden and serum M-protein level. Furthermore, Dvd pretreatment significantly increased eNK persistence and homing to MM sites. Our findings suggest that Dvd treatment potentiates the antimyeloma effects of NK cells expanded and activated ex vivo by modulating immune responses in MM-bearing mice.
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http://dx.doi.org/10.1038/s41423-021-00686-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245645PMC
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

Double-filtered leukoreduction as a method for risk reduction of transfusion-associated graft-versus-host disease.

PLoS One 2020 26;15(3):e0229724. Epub 2020 Mar 26.

Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunwan University School of Medicine, Seoul, Korea.

Background: Transfusion-associated graft-versus-host disease (TA-GvHD) is caused by leukocytes, specifically T cells within a transfused blood product. Currently, the prevention of transfusion-associated graft-versus-host disease is performed by irradiation of blood products. With a sufficient reduction of leukocytes, the risk for TA-GvHD can be decreased. With consistent advances in current state-of-the-art blood filters, we herein propose that double filtration can sufficiently reduce leukocytes to reduce the risk for TA-GvHD.

Materials: Thirty RBC concentrates were filtered with leukocyte filters, followed by storage at 1-6 oC for 72 hours, and then a second filtration was performed. Residual leukocytes in the double-filtered RBC units (n = 30) were assessed with flow cytometric methods, and an additional assay with isolated peripheral blood mononuclear cells (PBMCs) (n = 6) was done by both flow cytometric methods and an automated hematology analyzer. Quality of the RBCs after filtration was evaluated by hematological and biochemical tests. In vitro T cell expansion was performed using anti-CD3/CD28-coated Dynabeads or anti-CD3 (OKT3). In vivo experiment for GvHD was performed by using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice.

Results: Double-filtered blood products showed residual leukocyte levels below detection limits, which calculated to be below 1200-2500 cells per blood unit. In vitro expansion rate of T cells showed that 6x103 and 1x103 cell-seeded specimens showed 60.8±10.6 fold and 10.2±9.7-fold expansion, respectively. Cell expansion was not sufficiently observed in wells planted with 1x102 or 10 cells. In vivo experiments showed that mice injected with 1x105 or more cells cause fatal GvHD. GvHD induced inflammation was observed in mice injected with 1x104 or more cells. No evidence of GvHD was found in mice injected with 103 cells.

Conclusions: Our study suggests that additional removal of contaminating lymphocytes by a second leukodepletion step may further reduce the risk for TA-GvHD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229724PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098637PMC
June 2020

Expansion of Human NK Cells Using K562 Cells Expressing OX40 Ligand and Short Exposure to IL-21.

Front Immunol 2019 24;10:879. Epub 2019 Apr 24.

Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea.

Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction. K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4-5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells. The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation. Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.
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http://dx.doi.org/10.3389/fimmu.2019.00879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491902PMC
October 2020

X-ray as Irradiation Alternative for K562 Feeder Cell Inactivation in Human Natural Killer Cell Expansion.

Anticancer Res 2018 Oct;38(10):5767-5772

Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, Republic of Korea

Background/aim: γ-Irradiation has been proven to be the most effective method to inactivate K562 cells, but γ-irradiators are not available in some institutes. This study was designed to compare the effects of X-ray and γ-irradiation on K562 cells in natural killer (NK) cell expansion.

Materials And Methods: To expand NK cells, isolated peripheral blood mononuclear cells (PBMCs) were co-cultured with γ-irradiated or X-ray-treated K562 cells plus IL-2 and IL-15. Characteristics of expanded NK cells were identified by flow cytometry.

Results: NK cell expansion rate tended be to lower in the X-ray-treated group (68.9±32.6) than the γ-irradiated group (78±28.7), but the difference was not significant (p=0.39). Furthermore, NK cell functions or receptor expression were similar in the two groups.

Conclusion: Our results suggest that X-ray treatment can be used as an alternative to γ-irradiation for K562 cells inactivation in human NK cell expansion.
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http://dx.doi.org/10.21873/anticanres.12915DOI Listing
October 2018

The first case of congenital blood chimerism in two of the triplets in Korea.

J Clin Lab Anal 2018 Oct 24;32(8):e22580. Epub 2018 May 24.

Department of Laboratory Medicine, International St. Mary's Hospital, Catholic Kwandong University College of Medicine, Incheon, Korea.

Background: Chimeras are composed of two or more different populations that originated from different zygotes. Blood chimerism arising from twins have been reported in the literature. Herein, we report the first blood group chimerism in triplets.

Methods: ABO blood grouping was carried out by manual tile methods (Merck Millipore, UK) and micro-column agglutination method (Bio-Rad, Cressier sur Morat, Switzerland). Flow cytometric analysis was performed with Anti-A-PE conjugated monoclonal antibodies (BD Biosciences, San Jose, CA, USA) and FACS Canto II (BD Biosciences). Molecular analysis was performed with allele-specific polymerase chain reaction (AS-PCR) and direct sequencing of the exons 6 and 7.

Results: Mixed-field agglutination and weak agglutination against anti-A were revealed by ABO blood grouping. Flow cytometric analysis revealed the presence of both A cells and O cells. AS-PCR and sequencing showed two neonates with chimerism, with each neonate`s genotype being A102/O01/O02.

Conclusion: This is the first recorded case of blood chimera from a triplet in Korea. We recommend full investigation of blood group chimerism in neonates with ABO discrepancy, as blood chimerism is subject to certain caution in the clinical environment.
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http://dx.doi.org/10.1002/jcla.22580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816996PMC
October 2018

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor.

Cytotherapy 2016 12 6;18(12):1532-1542. Epub 2016 Oct 6.

Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; Stem Cell and Regenerative Medicine Institute, Samsung Medical Center, Seoul, South Korea. Electronic address:

Background Aims: Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells.

Methods: The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays.

Results: The activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R = 0.91; P <0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines.

Conclusions: Our data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer.
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http://dx.doi.org/10.1016/j.jcyt.2016.08.006DOI Listing
December 2016

Expansion of NK Cells Using Genetically Engineered K562 Feeder Cells.

Methods Mol Biol 2016 ;1441:167-74

Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul, 135-710, South Korea.

Natural killer (NK) cells can be expanded upon activation by proliferative cytokines (such as IL-2 and IL-15). The NK cell expansion can be greatly enhanced by proteins from feeder cells such as tumor cell lines or PBMCs. Therefore, coculture systems of irradiated feeder cells and NK cells in media containing IL-2 and IL-15 have been developed to generate large numbers of NK cells, although NK cell expansion protocol using anti-CD3 antibody (OKT-3) without feeder cells has also been developed. Commonly used feeder cell lines are RPMI8866, Epstein-Barr lymphoblastoid cell line (EBV-LCL), and K562. Stimulation with NK-sensitive K562 cells is known to augment NK cell proliferation to IL-2, IL-15, and IL-21 in combination.Recently, remarkable NK cell-expansion rates are achieved when genetically engineered (GE) feeder cells are used. Dr. Dario Campana's group found that membrane-bound IL-15 and 4-1BBL, coexpressed by K562 cells, acted synergistically to augment K562-specific NK stimulatory capacity, resulting in vigorous expansion of peripheral blood CD56(+) CD3(-) NK cells without concomitant growth of T lymphocytes. Here, we describe an in vitro expansion method of human NK cells among PBMCs by coculturing with GE_K562 cells.
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http://dx.doi.org/10.1007/978-1-4939-3684-7_14DOI Listing
December 2017

Comparison of FcRγ-Deficient and CD57+ Natural Killer Cells Between Cord Blood and Adult Blood in the Cytomegalovirus-Endemic Korean Population.

Ann Lab Med 2015 Jul 21;35(4):423-8. Epub 2015 May 21.

Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea. ; Center for Creative Biomedical Scientists, Chonnam National University, Gwangju, Korea. ; Research Center for Cancer Immunotherapy, Chonnam National University Hwasun Hospital, Hwasun, Korea.

Background: FcRγ-deficient natural killer (NK) cells (g(-)NK cells) have been associated with cytomegalovirus (CMV) infection. However, the frequency of g(-)NK cells in a CMV-endemic area (i.e., Korea) has not yet been studied. We examined the frequency of g(-)NK cells and expression of CD57 on NK cells in cord blood (CB) and adult blood (AB).

Methods: Of the 24 AB samples collected, 95.8% (23/24) were CMV IgG(+)/IgM(-), while 100% of the 13 healthy CB samples were CMV IgG(+)/IgM(-). We performed whole-blood flow cytometry assays to analyze intracellular FcRγ and CD3ζ expression of CD3(-)/CD56(dim) NK cells from 13 CB and 24 AB samples, and surface CD57 expression on CD3(-)/CD56(dim)/CD16(+) NK cells from 13 CB and 19 AB samples.

Results: All CMV seropositive AB samples contained g(-)NK cells (23/23), and the median proportion of g(-)NK cells in the CD3(-)/CD56(dim) NK cell pool was 35.0% (range: 11-77%). CD57(+) NK cells in the CD3(-)/CD56(dim)/CD16(+) NK cell population were detected in all 19 AB samples tested, but not in any CB samples.

Conclusions: Our data suggest that g(-)NK cells and CD57(+) NK cells are present at a very high frequency in CMV-seropositive AB, but rare in CMV-naïve CB.
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http://dx.doi.org/10.3343/alm.2015.35.4.423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446581PMC
July 2015

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture.

Cytotherapy 2014 Oct 18;16(10):1419-30. Epub 2014 Jun 18.

Research Center for Cancer Immunotherapy, Chonnam National University, Hwasun Hospital, Jeollanam-do, Korea; Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea; Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, Korea. Electronic address:

Background Aims: Interleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture.

Methods: IL-2/IL-15-activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry.

Results: Compared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the "first week" group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the "first week" group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of "first week" NK cells that received IL-21 treatment for an additional 2 days compared with the "first week" NK cells (P < 0.05).

Conclusions: These data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.
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http://dx.doi.org/10.1016/j.jcyt.2014.04.008DOI Listing
October 2014