Attenuated anti‑tumor activity of NK‑92 cells by invasive human breast carcinoma MDA‑MB‑231 cells

Hwan Hee Lee,Hyosun Cho 대한독성 유전단백체 학회 2020 Molecular & cellular toxicology Vol.16 No.2

Background Natural killer (NK) cells are typical innate lymphocytes that directly kill cancer cells. However, the anti-tumor function of NK cells can be modulated by various cancers in a tumor microenvironment. Objective We investigated whether the functional characteristics of NK cells are affected by coculture with human breast carcinoma MCF-7 or MDA-MB231 cells and further examined the underlying molecular mechanisms that are associated with attenuated anti-tumor activity of NK cells. NK-92 cells were cocultured with MCF-7 or MDA-MB-231 cells at a ratio of 1:2 (target:effector) for 6 h. The cytotoxic effect of NK-92 cells was measured using CytoTox96 assay. The frequency of CD56, NKp30+, NKp44+, NKG2D+, and NKG2A+ in NK-92 cells was investigated using individual fluorescent antibodies and flow cytometry. In addition, the production of cytokines was measured using Human Cytokine Array C1 kit. Proteins in cancer cell lysates were quantified and the expressions of PI3K, pAkt, Stat3, and pStat3 were examined by western blot analysis. Results The cytotoxicity of NK-92 cells is greater against MCF-7 than against MDA-MB-231 cells and cocultures with MCF-7 or MDA-MB-231 cells increased the frequency of CD56dim population in NK-92 cells as well as IFN-γ production. The frequency of the NKp30+ or NKG2D+ population in NK-92 cells was significantly decreased in coculture with MCF-7 or MDA-MB-231 cells. However, the frequency of NKG2A+ expression in NK-92 cells was significantly increased in coculture with MCF-7 or MDA-MB-231 cells. The secretions of IL-6, IL-8, monocyte chemoattractant protein-1, and granulocyte– macrophage colony-stimulating factor were significantly higher in supernatant from coculture of NK-92 and MDA-MB-231 cells than in supernatants from NK-92 cell single culture or coculture of NK-92 and MCF-7 cells, which positively correlates with the enhanced expression of pStat3 in MDA-MB-231 cells cocultured with NK-92 cells. Conclusion These findings demonstrate that the anti-tumor function of NK-92 cells is significantly suppressed by an invasive human breast carcinoma, MDA-MB-231, and the impairment of NK-92 cell function is associated with the upregulation of the IL-6/Stat3 signaling pathway.

NK Cell-Based Immunotherapies in Cancer

신민화,Junghee Kim,Siyoung A. Lim,Jungwon Kim,김성진,이경미 대한면역학회 2020 Immune Network Vol.20 No.2

With the development of technologies that can transform immune cells into therapeutic modalities, immunotherapy has remarkably changed the current paradigm of cancer treatment in recent years. NK cells are components of the innate immune system that act as key regulators and exhibit a potent tumor cytolytic function. Unlike T cells, NK cells exhibit tumor cytotoxicity by recognizing non-self, without deliberate immunization or activation. Currently, researchers have developed various approaches to improve the number and anti-tumor function of NK cells. These approaches include the use of cytokines and Abs to stimulate the efficacy of NK cell function, adoptive transfer of autologous or allogeneic ex vivo expanded NK cells, establishment of homogeneous NK cell lines using the NK cells of patients with cancer or healthy donors, derivation of NK cells from induced pluripotent stem cells (iPSCs), and modification of NK cells with cutting-edge genetic engineering technologies to generate chimeric Ag receptor (CAR)-NK cells. Such NK cell-based immunotherapies are currently reported as being promising anti-tumor strategies that have shown enhanced functional specificity in several clinical trials investigating malignant tumors. Here, we summarize the recent advances in NK cell-based cancer immunotherapies that have focused on providing improved function through the use of the latest genetic engineering technologies. We also discuss the different types of NK cells developed for cancer immunotherapy and present the clinical trials being conducted to test their safety and efficacy.

Inflammatory Mediators Modulate NK Cell-stimulating Activity of Dendritic Cells by Inducing Development of Polarized Effector Function

Kim, Kwang-Dong,Choi, Seung-Chul,Lee, Eun-Sil,Kim, Ae-Yung,Lim, Jong-Seok The Korean Association of Immunobiologists 2007 Immune Network Vol.7 No.3

Background: It is well established that cross talk between natural killer (NK) cells and myeloid dendritic cells (DC) leads to NK cell activation and DC maturation. In the present study, we investigated whether type 1-polarized DC (DC1) matured in the presence of IFN-${\gamma}$ and type 2-polarized DC (DC2) matured in the presence of PGE2 can differentially activate NK cells. Methods: In order to generate DC, plastic adherent monocytes were cultured in RPMI 1640 containing GM-CSF and IL-4. At day 6, maturation was induced by culturing the cells for 2 days with cytokines or PGE2 in the presence or absence of LPS. Each population of DC was cocultured with NK cells for 24 h. The antigen expression on DC was analyzed by flow cytometry and cytokine production in culture supernatant was measured by ELISA or a bioassay for TNF-${\alpha}$ determination. NK cell-mediated lysis was determined using a standard 4h chromium release assay. Results: DC2, unlike DC1, had weak, if any, ability to induce NK cell activation as measured by IFN-${\gamma}$ production and cytolytic activity. DC2 were weakly stimulated by activated NK cells compared to DC1. In addition, IFN-${\gamma}$-primed mature DC appeared to be most resistant to active NK cell-mediated lysis even at a high NK cell/DC ratio. On the other hand, PGE2-primed DC were less resistant to feedback regulation by NK cells than IFN-${\gamma}$-primed mature DC. Finally, we showed that the differential effect of two types of DC population on NK cell activity is not due to differences in their ability to form conjugates with NK cells. Conclusion: These results suggest that different combinations of inflammatory mediators differentially affect the effector function of DC and, as a result, the function of NK cells, eventually leading to distinct levels of activation in adaptive immunity.

The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells

Park, J.Y.,Shin, D.J.,Lee, S.H.,Lee, J.J.,Suh, G.H.,Cho, D.,Kim, S.K. Elsevier Scientific Pub. Co 2015 Veterinary microbiology Vol.176 No.3

Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3<SUP>-</SUP>CD5<SUP>-</SUP>CD21<SUP>-</SUP> NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions.

전신성 홍반성 낭창 환자에서 Natural Killer 세포와 Natural Killer T 세포의 분포와 기능에 관한 연구

김현리 ( Hyun Lee Kim ),김성균 ( Seong Gyun Kim ),김연수 ( Yon Su Kim ),이동섭 ( Dong Sup Lee ),정경천 ( Kyeong Cheon Jung ),정종훈 ( Jong Hoon Chung ),안규리 ( Curie Ahn ),한진석 ( Jin Suk Han ),김성권 ( Suhng Gwon Kim ),이정상 ( 대한신장학회 2003 Kidney Research and Clinical Practice Vol.22 No.2

목적 : T 세포 중 표지자인 NK1.1 단백을 표현하는 T 세포를 NKT 세포라고 한다. 이 NKT 세포의 기능에 대하여 정확하게 밝혀지지는 않았지만 최근 들어서 생체 내에서 면역학적 반응을 조절하는 기능을 지니고 있다고 알려지고, 이에 대한 연구가 진행되고 있다. 저자들은 전신성 홍반성 낭창 (SLE)의 발병에 관여하는 NKT 세포의 면역조절기능을 알아보기 위해 말초 혈액 내 NK, NKT 세포의 분포와 외부자극에 대한 NKT 세포들의 기능을 평가하고자 본 연구를 수행하였다. 방법 : SLE 환자 22명과 정상 대조군 18명을 대상으로 NK 표지자 (CD56, CD94)와 T 세포 표지자 (CD3)를 이용한 유세포 분석을 통해 NK와 NKT 세포의 말초혈액 내 분포를 확인하고 이를 질환의 임상지표와 비교하였다. SLE 환자와 정상 대조군의 NKT 세포 기능은 PMA와 ionomycin 자극에 대한 NKT 세포 내의 cytokine (IL-4, IFN-γ)의 발현으로 비교하였다. 결과 : SLE 환자와 정상 대조군을 대상으로 말초혈액 내 NK 세포와 NKT 세포의 수적변화를 비교하였을 때 SLE 환자의 말초혈액 내 NK 세포 (CD3-/CD56+ 4.93±1.30%; CD3-/CD94+ 4.03±1.00%)는 정상 대조군 (CD3-/CD56+ 11.28±1.77%; CD3-/CD94+; 8.15±1.40%)에 비해 감소되어 있었으며 p<0.01), NKT 세포 또한 SLE군이 대조군에 비해 유의하게 감소되어 있었다 (CD3+/CD56+; 1.79±0.42 vs 5.04±0.44%: CD3+/CD94+; 1.21±0.27 vs 4.39±0.45%, p<0.01). NK 세포의 수적변화와 SLE 환자의 임상상과의 연관성을 분석하였을 때 항 ds-DNA 항체가의 증가, ESR의 증가와 C4의 감소와 유의한 연관성을 보였으나 (p<0.05), 신질환의 활동성과는 상관관계를 보이지 않았다. 말초 혈액을 분리, 자극한 후 NK 및 NKT 세포 내 IL-4, IFN-γ를 측정하였을 때, 발현정도는 SLE 환자(n=6)와 정상 대조군(n=6) 사이에 유의한 차이가 없었다. 결론 : SLE 환자에서 면역조절 세포의 일부인 NKT 세포의 수적인 감소는 NKT 세포가 SLE 병인에 밀접한 관계가 있음을 시사한다. 하지만 감소된 NKT 세포의 기능은 정상으로 유지되고 있는 점으로 보아 SLE 환자에서 NKT 세포의 기능적 결함보다는 NKT 세포의 단순한 수의 감소에 의한 면역조절능의 감소가 SLE의 병의 발병및 경과에 영향을 미칠 것으로 판단된다. Background : Systemic lupus erythematosus (SLE) is one of chronic autoimmune diseases of which the central pathophysiologic derangement has not been yet established. Recently, it has been suggested that immune-regulatory cells might affect the development of autoimmune diseases such as SLE and RA. NKT cells were reported to be strong candidate for regulatory cells to regulate immune responses in vivo. To elucidate the roles of immune regulatory cells in the pathogenesis of SLE, we investigated the fractional distribution and functional status of NK and NKT cells in peripheral blood mononuclear cells (PBMC) of SLE patients and healthy volunteers. Methods : Twenty-two SLE patients and 18 agematched healthy volunteers were included in this study. The analysis for NK and NKT cells fraction in PBMCs of patients and normal controls were performed by flow cytometric analysis. In addition, to explore the functional status of these cells in SLE patients, we stimulated PBMCs using phorbol ester and ionomycin and measured cytoplasmic IL-4 and IFN-γ by flow cytometry. Results : The number (percentage) of NK cells was lower in SLE patients (CD3- CD56+ :4.93±1.30%, CD3-CD94+:4.03±1.00%) than in controls (11.28±1.77%, 8.15±1.40%; p<0.01, respectively). Peripheral NK cell numbers negatively correlated with antidsDNA Ab levels (r=0.431, p<0.05) and ESR (r=-0.475, p,0.05). However, the percentage of these cells was not correlated with renal activity or corticosteroid doses. SLE patients showed, compared with controls, significantly decreased numbers of NKT cells (CD3+CD56+ :1.79±0.42% vs 5.04±0.44%, CD3+CD94+ :1.21±0.27% vs 4.39±0.45%; p<0.01, respectively). The cytoplasmic expression of IL-4 and IFN-γ in NK and NKT cells of SLE patients stimulated using phorbol ester and ionomycin were almost sililar to those of normal controls, suggesting the NKT cells from SLE patients are functionally intact. Conclusion : Our results suggested that the decreased numbers of immune regulatory cells were associated with the immune dysregulation of SLE patients. The cellular replacement of NKT cells may be one of useful therapeutic approaches for autoimmune diseases such as SLE.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy

민보경,최하나,허정현,정미영,김효진,정미영,이은경,조성유,황유경,신의철 대한면역학회 2018 Immune Network Vol.18 No.4

Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD3+ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy

Min, Bokyung,Choi, Hana,Her, Jung Hyun,Jung, Mi Young,Kim, Hyo-Jin,Jung, Mi-young,Lee, Eun-Kyoung,Cho, Sung Yoo,Hwang, Yu Kyeong,Shin, Eui-Cheol 한국조명·전기설비학회 2018 한국조명·전기설비학회 학술대회논문집 Vol. No.

<P>Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD3<SUP>+</SUP> T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. <I>In vivo</I> antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.</P>

중증 기관지천식 환자의 말초단핵구에서 자연살해세포의 비율과 자연살해세포에서 interferon gamma 의 생산

천은미(Eun Mi Chun),김미선(Mi Sun Kim),장윤혜(Yoon Hae Chang),박성숙(Sung Sook Park),조영주(Young Joo Cho) 대한천식알레르기학회 2000 천식 및 알레르기 Vol.20 No.3

N/A Background: and objective: The natural killer (NK) cells which play an important role in defense immune system are supposed to be involved in the pathogenesis of bronchial asthma. The goal of this study is to analyze the role of NK cells in the pathogenesis of bronchial asthma by examining the proportion of NK cells in peripheral blood mononucler cells (PBMCs) and production of interferon gamma (IFN-γ) in NK cells between normal group and asthmatic group. Methods: Ten patients with moderate to severe persistent asthma sensitive to house dust mite were enrolled as asthmatic group. PBMCs were activated by 12-0-tetracanoylphorbol-13- acetate (TPA) and calcium inonophore for 18 hours. Surface CD3 and CD56 antigens with intracytoplasmic IFN-γ staining were performed simultaneously and the results were analyzed by three color fiow cytometer. Results : The percentage of CD56+ positive NK cells in PBMCs from asthma group was lower compared to control group (15.4±3.9% vs 19.8±4.5%). However, There was no signficant difference of IFN-γ production in CD56+ NK cells between two groups (30.3±3.9% vs 25.9±5.8%. P>0.05). IFN-γ producing CD3+ T cells were significantly higher in asthma group compared with normal control group (36.3±1.8% vs 28.4±5.7%, p<0.05). The ratio of TNK cells expressing both CD56 and CD3 was not different between asthma group and control group (4.7±1.4 % vs 5.9±1.8%, p<0.05). Conclusion : The results suggest that aggravation of asthma symptoms in severe asthma may be caused partly by decrease in NK cells. The increased production of IFN-γ in asthma patients suggest that IFN-γ may function as inflammatory cytokine.

Immunohistochemical Study on the Activation of Splenic Cell Mediated Immunity in Mouse by Sarcoma 180 Cells : Based on the change of T lymphocytes,IL-2 receptors, and NK cells T 림프구, IL-2 수용기 및 NK 세포의 변화를 중심으로

Kim, Jin-Taek,Ahn, Sang-Hyun,Park, In-Sick,Choi, Nan-Hee,Kim, Dong-Hoan 동국대학교 경주대학 1997 東國論集 Vol.16 No.2

비정상적인 성장을 하고 전이를 통해서 다른 정상적인 조직까지도 파괴하는 것으로 알려진 암이 ICR계 생쥐 비장의 세포성 면역활성에 미치는 영향을 조사하기 위해서 복수암세포 sarcoma 180 cells를 1 X 10^6 cells씩 복강주사하여 비장을 적출한 후 항 CD4항체, 항 CD8항체, 항 IL-2 receptor항체 그리고 항 NK-1.1항체와 같은 monoclonal antibody를 사용한 ABC법으로 면역조직화학적 염색을 한 후 T 림프구, IL-2 수용기 그리고 자연살해세포의 변화를 관찰하였다. 그 결과 비장에서의 도움 T 림프구, 세포독성 T 림프구, IL-2 receptor 그리고 NK 세포는 복수암세포 접종 후 3일까지는 별 변화가 없었으나 7일 이후 서서히 증가하기 시작하여 21일에는 양성반응 세포수와 반응성이 최고에 이으렀다. 그리고 이들 양성반응세포의 분포 지역은 백색수질의 림프성동맥주위집, 적색수질의 붓털소동맥 주변부였다. 아울러 백색수질의 가장자리부분 크기와 적색수질에는 큰대식세포수도 증가한 것으로 관찰되었다. 이상의 결과로 볼 때 이종 암항원은 면역계에 7일 이후 노출되어 비장에서 T 림프구와 NK 세포의 활성과 증식이 일어나며, 이것은 암세포를 파괴하기 위한 세포성 면역반응의 활성에 의한 것으로 사료된다. Splenic tissues of ICR mouse intraperitoneally injected with sarcoma 180 cells were immunohistochemically observed to investigate the change of splenic T lymphocytes and NK cells by tumor cell. The spleens were obtained at day-3, 7, 14, and 21 after sarcoma 180 cell's injection and embedded with paraffin, and then stained by following ABC method that used monoclonal antibody including L3T4, Ly-2, IL-2 receptor, and NK 1,1. There were little changes of helper T lymphocytes, cytotoxic T lymphocytes, IL-2 receptors positive reacted cells, and NK cells at day-3 after the initial injection of sarcoma 180 cells, but they began to increase at day-7. The number of positive reacted cells and reactive degree were peaked at day-21. Helper T lymphocytes, cytotoxic T lymphocytes, IL-2 receptors expressed cells, and NK cells were found in PALS of white pulp and penicilla artery of red pulp. The size of marginal zone in white pulp was enlarged and the number of macrophages in red pulp was increased. These results indicates that immune system begin to detect the tumor antigen at day-7 after the initial induction with tumor cells and subsequently to activate and proliferate splenic T lymphocytes and NK cells as component of cell mediated immunity for destroying tumor cells.

Interleukin-21 induces proliferation and modulates receptor expression and effector function in canine natural killer cells

Shin, D.J.,Lee, S.H.,Park, J.Y.,Kim, J.S.,Lee, J.J.,Suh, G.H.,Lee, Y.K.,Cho, D.,Kim, S.K. Elsevier 2015 Veterinary immunology and immunopathology Vol. No.

<P>Interleukin (IL)-21 is an important modulator of natural killer (NK) cell function. However, little is known about IL-21 function in canine NK cells because the phenotype of these cells remains undefined. In this study, we selectively expanded non-B and non-T large granular NK lymphocytes (CD3(-)CD21(-)CD5(-)CD4(-)TCR alpha beta-TCR gamma delta(-)) ex vivo from the peripheral blood mononuclear cells (PBMCs) of healthy dogs using a combination of IL-2, IL-15, and IL-21 in the presence of 100 Gy-irradiated K562 cells. We investigated the effects of varying the duration and timing of IL-21 treatment on stimulation of proliferation, expression of NK-related receptors, anti-tumor activity and production of interferon (IFN)-gamma. The expanded NK cells in each treatment group became enlarged and highly granular after 21 days in culture. NK cells proliferated rapidly in response to activation by IL-21 for 3 weeks, and IL-21 was able to induce changes in the mRNA expression of NK cell-related receptors and enhance the effector function of NK cells in perforin- and granzyme-B-dependent manners. The duration, frequency and timing of IL-21 stimulation during culture affected the rate of proliferation, patterns of receptor expression, cytokine production, and anti-tumor activity. The optimal conditions for maximizing the IL-21-induced proliferation and effector function of NK cells in the presence of IL-2 and IL-15 were seen in cells treated with IL-21 for the first 7 days of culture but without any further IL-21 stimulation other than an additional 2-day treatment prior to harvesting on day 21. The results of this study suggest that synergistic interactions of IL-21 with IL-2 and IL-15 play an important role in the proliferation, receptor expression, and effector function of canine NK cells. (C) 2015 Elsevier B.V. All rights reserved.</P>