Multilayered Cell Sheets of Cardiac Reprogrammed Cells for the Evaluation of Drug Cytotoxicity

권성필,Song Seuk Young,유진,Kim Han Young,이주로,강미경,Sohn Hee Su,Go Seokhyoung,정문교,Hong Jihye,임송현,Kim Cheesue,문상준,차국헌,김병수 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.5

Background: Various cell-culture systems have been used to evaluate drug toxicity in vitro. However, factors that affect cytotoxicity outcomes in drug toxicity evaluation systems remain elusive. In this study, we used multilayered sheets of cardiac-mimetic cells, which were reprogrammed from human fibroblasts, to investigate the effects of the layer number on drug cytotoxicity outcomes. Methods: Cell sheets of cardiac-mimetic cells were fabricated by reprogramming of human fibroblasts into cardiac-mimetic cells via coculture with cardiac cells and electric stimulation, as previously described. Double-layered cell sheets were prepared by stacking the cell sheets. The mono- and double-layered cell sheets were treated with 5-fluorouracil (5-FU), an anticancer drug, in vitro. Subsequently, apoptosis and lipid peroxidation were analyzed. Furthermore, effects of cardiac-mimetic cell density on cytotoxicity outcomes were evaluated by culturing cells in monolayer at various cell densities. Results: The double-layered cell sheets exhibited lower cytotoxicity in terms of apoptosis and lipid peroxidation than the mono-layered sheets at the same 5-FU dose. In addition, the double-layered cell sheets showed better preservation of mitochondrial function and plasma membrane integrity than the monolayer sheets. The lower cytotoxicity outcomes in the double-layered cell sheets may be due to the higher intercellular interactions, as the cytotoxicity of 5-FU decreased with cell density in monolayer cultures of cardiac-mimetic cells. Conclusion: The layer number of cardiac-mimetic cell sheets affects drug cytotoxicity outcomes in drug toxicity tests. The in vitro cellular configuration that more closely mimics the in vivo configuration in the evaluation systems seems to exhibit lower cytotoxicity in response to drug.

MiR-30c facilitates natural killer cell cytotoxicity to lung cancer through targeting GALNT7

Gao Fei,Han Jianjun,Jia Li,He Jun,Wang Yun,Chen Mi,Liu Xiaojun,He Xia 한국유전학회 2023 Genes & Genomics Vol.45 No.2

Background MicroRNAs (miRNAs) have been reported to play important roles in regulating natural killer (NK) cell cytotoxicity to cancer cells. Objective This study aimed to investigate the effects and potential mechanism of miR-30c in regulating NK cell cytotoxicity to lung cancer cells. Methods Primary NK cells were derived from the peripheral blood of lung cancer and normal participants. Exosomes were isolated and validated via transmission electron microscopy and nanoparticle tracking analysis. The levels of miR-30c, polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) and proteins in PI3K/AKT pathway were determined using quantitative real-time polymerase chain reaction or western blot. Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) levels and the cytotoxicity of effector NK cells to target lung cancer cells were measured via enzyme linked immunosorbent assay, cell apoptosis or xenograft experiments. The relationship between miR-30c and GALNT7 was analyzed by luciferase activity, RNA pull-down and RNA immunoprecipitation assays. And a xenograft mice model was established to verify the effect of miR-30c in regulating NK cell cytotoxicity to lung cancer cells in vivo. Results NK cell-derived exosomes carrying miR-30c, and miR-30c level was significantly downregulated in primary NK cells of lung cancer patients. MiR-30c overexpression promoted TNF-α and IFN-γ secretion and enhanced the cytotoxicity of interleukin 2 (IL-2)-treated NK cells to lung cancer cells, while knockdown of miR-30c played an opposite effect in regulating the cytotoxicity of NK cells to lung cancer cells. GALNT7 was a target of miR-30c and was negatively regulated by miR-30c. Besides, miR-30c targeted GALNT7 to exert its function in regulating NK cell cytotoxicity. Furthermore, GALNT7 prompted the activation of PI3K/AKT pathway in NK cells. Additionally, miR-30c overexpression enhanced NK cell cytotoxicity to lung cancer cells and inhibited tumor growth in vivo. Conclusion miR-30c enhanced NK cell cytotoxicity to lung cancer cells via decreasing GALNT7 and inactivating the PI3K/AKT pathway, suggesting that regulating miR-30c expression maybe a promising approach for enhancing NK cell-based antitumor therapies.

Effects of sulfated fucan from the sea cucumber <i>Stichopus japonicus</i> on natural killer cell activation and cytotoxicity

Surayot, Utoomporn,Lee, SangMin,You, SangGuan Elsevier 2018 International journal of biological macromolecules Vol.108 No.-

<P><B>Abstract</B></P> <P>The aqueous crude sulfated fucan (SF) from <I>Stichopus japonicus</I> was extracted and fractionated using anion-exchange chromatography to obtain four fractions (F<SUB>1</SUB>, F<SUB>2</SUB>, F<SUB>3</SUB> and F<SUB>4</SUB>) and to investigate their NK cell activation and cytotoxicity. The most potent NK cell cytotoxicity (45% at 250μg/mL) against HeLa cells was observed by F<SUB>1</SUB> treatment, on the other hand, F<SUB>3</SUB> and F<SUB>4</SUB> treatment exhibited strong NK cell cytotoxicity (31–34% at 250μg/mL) against HepG2 and HT-29 cells. The SF treatment enhanced the activation of NK cells through the mRNA expression of IFN-γ, an activating receptor (NKp30), lysing proteins (perforin and granzyme-B) as well as a death ligand (FasL). However, the treatment of the SF derivatives, deproteinated-F<SUB>1</SUB> and desulfated-F<SUB>3</SUB> (DP-F<SUB>1</SUB> and DS-F<SUB>3</SUB>), markedly lowered the levels of NK cell cytotoxicity and mRNA expression of the activating factors, suggesting that the protein and sulfate were pivotal for the interaction between the SF and NK cells. The antibody neutralization test revealed that complement receptor-3 (CR3) may be a critical receptor involved in NK cell activation by the SF.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Sulfated fucan (SF) from <I>Stichopus japonicus</I> increases NK cell proliferation and the cytotoxicity. </LI> <LI> Fractionated fucan effected to NK cell cytotoxicity against HeLa, HepG2 and HT-29 cells. </LI> <LI> Sulfates and proteins of SFs were required to improve NK cells cytotoxicity. </LI> <LI> NK cells are activated through the expression of NKp30, FasL, IFN-γ and granzyme-B. </LI> <LI> CR3 might be a critical receptor for the interaction between NK cells and SFs. </LI> </UL> </P>

세포장해능력 측정에서 수학적 매개변수의 이용

김재만 ( J M Kim ) 대한임상검사과학회 1991 대한임상검사과학회지(KJCLS) Vol.23 No.1

Recently, the 51Cr-release assay have been used in the measurement of cell-mediated cytotoxicity. The cell-mediated cytotoxicity is dependent on the frequency and activity of effector cells. The number of effector cells relate to the number of target cells. Generally, increase or decrease of the effector cells to the fixed number of target cells induce the increased or decreased cell-mediated cytotoxicity. But, the correlation between the number of effector cells and the number of target cells not always fixed and although the effector cells are numerous to the target cells, the effector cells can not lyse all target cells. However, in the majority of cases, cell-mediated cytotoxicity have been performed in a simple E: T ratios. This method of interpretation can not represents results that have the object because results are simple comparison at one point E : T ratio. Results of cytotoxicity should be considered and represented the two factors of frequency and activity of the effector cells. Thus a trial to interpret by scientific method results of cell-mediated cytotoxicity have been. Cell-mediated cytotoxicity is represented by Exponential fit equation Y =A (1-e-kx). The estimation unit is LU (lytic unit) and one LU defin as that 1 x 106 effector cells lyse a 30% of 5 x 10 3 target cells. In this study, LAK Oymphokine activated killer) cells and NK (natural killer) cells were used as the effector cells and the K562, Hela, MCF-7 and Raji cell-line were used as the target cells. Resuts were represented that interpretation of results utilized LU has the object better than percent(%) lysis according to E : T ratios.

Distribution of Lymphocyte Subpopulation and Natural Cytotoxicity of Peripheral Blood from Colon Cancer Patient

Park, Il Young,Park, Jang Sang,Chang, Suk Kyun,Lee, Jae Hak CATHOLIC MEDICAL CENTER 1990 Bulletin of the Clinical Research Institute Vol.18 No.1

Depression of general immune reactivity has been well documented in patients with colorectal cancer as well as in patient with other types of solid tumors. The main effector of immune surveillance against cancer have been considered to be sensitized T lymphocyte and activated macrophages. Recently there has been increasing recognition that natural cell-mediated cytotoxicity is potentially an important antitumor mechanism especially in recurrence, metastasis and prognosis. Authors investigated to study the relation between subpopulation, stimulated or non-stimulated natural cytotoxicity of lymphocyte and stage of disease, serum CEA levels, or differentiation of cancer. The results were as follows; 1. The peripheral blood lymphocyte count was significantly decreased only in group 4 as compared with control. 2. The natural cytotoxicity against K_562 cells were significantly decreased in group 2 and group 4 than that of control. But except for group 3, there were no significant differences in natural cytotoxicity against SBA cells. 3. The natural cytotoxieity against K_562 cells of interleukin-2 stimulated lymphocyte was significantly increased than that of non-stimulated lymphocytes in both of control and stage Ⅳ patients group. 4. The distributions of lymphocyte subpopulation (CD_4, CD_8, CD_16) were slightly decreased in group 3 and decrease of CD_4, CD_16 with increase of CD_8 in group 4 without statistical significancy. But in group 3, CD_4 cells were significantly increased than that of control and CD_8 cells were significantly increased as compared with control. 5. The ratio of CD_4 to CD_8 was significantly decreased in group 3 than that of control, but no significant differences in group 2 and 4. 6. The serum CEA was positive (>5 ng/ml) in 43% of patients and the level was increased with advance of diseases. But there were no correlation between serum CEA level, tumor cell differentiation and natural cytotoxicity, CD_4 to CD_8 ratio or K_562 cytotoxicity to CD_15 cells. With above results, authors insist that the natural cytotoxicity by natural killer (NK) cells is decreased but not by cytotoxic T-cell in colon cancer patients and these cytotoxic activities can be enhenced by interleukin-2 stimulation. The NK cells was slightly increased in all stage of disease, but the decrease of helper T cell and increase of suppressor T-cell were significant in patients with lymph node metastasis.

MiR-506 Promotes Natural Killer Cell Cytotoxicity against Human Hepatocellular Carcinoma Cells by Targeting STAT3

Zhixiong Su,Xinping Ye,Liming Shang 연세대학교의과대학 2019 Yonsei medical journal Vol.60 No.1

Purpose: It is well documented that natural killer (NK) cytotoxicity against hepatocellular carcinoma (HCC) cells is impaired inHCC, which might account for the failure of anti-tumor immune response. miRNAs are considered as important regulators forthe development and functions of NK cells. However, the entire role of miR-506 in NK cells remains far from being addressed. Materials and Methods: The expressions of miR-506 and signal transducer and activator of transcription 3 (STAT3) mRNA in primaryNK cells from HCC patients and healthy controls were detected by quantitative real-time PCR. NK cell cytotoxicity was assessedby CFSE/7AAD cytotoxicity assay and lactate dehydrogenase assay. Luciferase reporter assay, RNA immunoprecipitationassay, and western blot were conducted to confirm the interaction between miR-506 and STAT3. Results: miR-506 expression was downregulated and STAT3 mRNA was upregulated in primary NK cells from HCC patients. PrimaryNK cells from HCC patients showed remarkably reduced cytotoxicity against SMMC7721 or HepG2 cells. NK cell cytotoxicitywas positively correlated with miR-506 expression and negatively correlated with STAT3 mRNA expression. Additionally, miR-506 overexpression enhanced NK cell cytotoxicity against HCC cells, while miR-506 inhibitor showed the reverse effect. Moreover,miR-506 could suppress STAT3 expression by directly targeting 3'-untranslated regions of STAT3. A negative correlation betweenmiR-506 and STAT3 mRNA expression in HCC patients was observed. Mechanistically, overexpressing STAT3 greatly reversedmiR-506-mediated promotion of NK cell cytotoxicity against HCC cells. Conclusion: miR-506 enhanced NK cell cytotoxicity against HCC cells by targeting STAT3, suggesting that modulating miR-506expression maybe a promising approach for enhancing NK cell-based antitumor therapies.

Strategies for ex vivo Expansion and Cytotoxicity Enhancement of NK Cells Using Mechanical Stimuli

Myeongkwan SONG,Soonjo KWON 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Adoptive cell therapy, also known as cellular immunotherapy, has recently attracted attention as a new cancer treatment method. Natural killer (NK) cells with the advantage of allogenic transfer are used as a cell therapy strategy along with Tumor-Infiltrating Lymphocyte (TIL), Engineered T Cell Receptor (TCR), and Chimeric Antigen Receptor (CAR) T Cell. However, for clinical applications, NK cells have problems with low ex vivo expansion and NK cell mediated cytotoxicity. To enhance NK cell expansion and cell-mediated cytotoxicity, we exposed NK cells to two types of mechanical stimuli: vibration and hyper-gravity. Vibration stimulus was calculated by sinusoidal acceleration, and hyper-gravity stimulus was calculated as g-force. CCK-8 assay was performed to determine whether vibration and hyper-gravity stimulus affect cell viability. There was no statistically significant decrease in cell viability at all intensities. We analyzed changes in the expression of cell-mediated cytotoxicity-related genes (granzyme B, perforin, TNF-α, and IFN-γ) and apoptosis related genes (BCL-2, BAX) following exposure of NK cells to two types of mechanical stimuli. In both mechanical stimuli, cytotoxicity enhancement and apoptosis inhibitory effects were observed. In addition, we are observing the changes in cell-mediated cytotoxicity compared to the control group by LDH assay in cancer cells in co-cultures. These findings provide useful insight for improving ex vivo expansion of NK cells and enhancing cell-mediated cytotoxicity.

1, 2-Hexanediol과 1, 2-Hexanediol Galactoside의 HaCaT Cell에 대한 세포독성

김준섭 ( Jun-sub Kim ),정경환 ( Kyung-hwan Jung ) 대한화장품학회 2018 대한화장품학회지 Vol.44 No.3

화장품에 방부제(살균/보존제)로 사용되는 1, 2-hexanediol (HD)로 인한 부작용을 극복하기 위하여, Escherichia coli (E. coli)의 β-galactosidase (β-gal)를 이용하여 transgalactosylation 반응으로 1, 2-hexanediol galactoside (HD-Gal)를 합성하였다. 본 연구에서는 합성된 HD-Gal의 인간 피부세포에 대한 독성이 어느 정도인지를 HD와 비교하여 관찰하였다. HD-Gal과 HD의 세포독성은 인간 피부각질형성세포(HaCaT cell line)에 HD와 HD-Gal을 처리한 후, cell proliferation assay를 이용하여 비교 분석하였다. 또한 이때, 위상차 현미경으로 HD-Gal과 HD로 처리한 세포의 상태를 비교 관찰하였다. 그 결과, HD-Gal은 42.2 mM에서 211 mM의 농도 범위에서 세포독성이 관찰되지 않았으며, 현미경 관찰에서도 큰 변화를 관찰할 수 없었다. 그러나, HD의 경우에는 저농도에서(42.2 mM and 84.4 mM)는 세포독성이 관찰되지 않았으나, 고농도(168.8 and 211 mM)에서 매우 높은 세포독성을 나타내었고, 현미경 관찰에서는 고농도에서는 물론이고, 세포독성이 관찰되지 않은 HD의 저농도에서도 세포모양과 세포 수에서의 변화가 관찰되었다. 앞으로 세포독성이 감소된 HD-Gal이 HD의 대체제로서 안전, 건강 및 웰빙 개념의 새로운 용도로 개발될 수 있을 것으로 생각된다. We synthesized 1, 2-hexanediol galactoside (HD-Gal) from HD using Escherichia coli (E. coli) β-galactosidase (β-gal), in which the reaction is generally called as transgalactosylation (reverse hydrolysis). In this study, we investigated how much HD-Gal and HD had a cytotoxic effect on HaCaT cell, in order to compare HD-Gal with HD in terms of the cytotoxicity of human skin cell. Cell proliferation assay and phase-contrast microscope observation were used for investigating the cytotoxicity. As a result, HD-Gal had not cytotoxic effect on HaCaT cell in the concentration range from 42.2 to 211 mM. In addition, when we observed the cells using microscopy, there was no change in the cell morphology. Meanwhile, when 42.2 mM and 84.4 mM HD were treated on HaCaT cell, we did not observe the cytotoxicity; however, when 168.8 mM and 211 mM HD were on HaCaT cell, HD had a higher cytotoxic effect on HaCaT cell. In addition, when HD was treated on the cells regardless of the concentration of HD, there were obvious changes in cell morphology and cell number. It was expected hopefully that HD-Gal would be applicable as a substitute for HD as a less toxic preservative in views of safety, health, and well-being.

자살유전자 도입이 사람의 glioma cell의 성장에 미치는 영향

홍승희 대한구강악안면병리학회 2015 대한구강악안면병리학회지 Vol.39 No.3

Malignant gliomas and glioblastomas are the most common type of primary brain tumors. The treatment of malignant glioma involves surgery, radiation, and chemotherapy. These therapies have not been successful in curing malignant glioma and typically associated with dismal prognosis. Therefore, we can investigate thymidine kinase activity and cytotoxic effect after transfer suicide gene in U-251 glioma cells. We assessed expression patterns of green fluorescence protein(GFP) after infected with adenovirus in U-251 cells. After infection of HSV-tk in U-251 cells, we observed thymidine kinase activity with [3H]-penciclovir and cytotoxic effect by treated with ganciclovir. We could observe that expression level of GFP was increased according to infected concentrations in U-251 cells. GFP was not expressed in 1moi and 10moi, and slightly expressed in 30moi. Expression level of GFP was largely increased in 50moi and almost cells expressed GFP in 100moi. GFP expression has shown clear image in 100moi compared with other concentrations. We also investigated thymidine kinase activity using [3H]-penciclovir after infection of suicide gene HSV-tk into U-251 cells. Thymidine kinase activity increased in 10moi concentration compared with empty adenovirus infection. We could find that thymidine kinase activity was elevated proportional to HSV-tk infection amount in 30moi and 50moi. For evaluation of cellular cytotoxic effect of HSV-tk, we treated ganciclovir to U-251 cells and assessed cytotoxicity by using MTT assay. We could identifiy that cytotoicity appeared in very low concentration of HSV-tk compared with cancer cells originated with other organs. Cytotoxic effect was shown about 15% of U-251 cells of total cells in 5moi. By infection 10moi of HSV-tk, cytotoxic effect was intensively increased and about 60% of U-251 cells became extinct. About 70% cells exhibited cytotoxic effect in 30moi and more than 80% cells also appeared cytotoxic effect by infection of HSV-tk in 50moi, 100moi, and 200moi. Therefore, we could confirm to gene expression in U-251 cells was increased proportional to infected gene concentrations. Also we could find that thymidine kinase activity elevated with according to infected concentration and cellular cytotoxic effect was shown in very low concentration and higher cytotoxic effect also appeared by infection of suicide gene HSV-tk into U-251 glioma cells. These results suggest that gene therapy with suicide gene will be successful in curing brain tumors containing malignant glioma and glioblastoma.

동종항원 자극에 대한 제대혈 및 성인 말초혈액 단핵세포에서의 Th/Tc 및 세포독성 반응

김연우,박현진,한윤수 대한조혈모세포이식학회 2001 대한조혈모세포이식학회지 Vol.6 No.2

연구배경: 제대혈이식이 골수이식으로 치료될 수 있는 여러 질병에서 골수이식을 대체하여 시술되고 있으며, 골수이식에 비해 이식편대 숙주반응 (graft versus host disease, GVHD)이 상대적으로 적다고 보고되고 있다. 이에 대한 이유로 제대혈 T 림프구가 interferon-γ(IFN-γ)를 생성하는 능력이 저하되어 있기 때문이라는 보고들이 있었으나, 최근에 골수이식시 이식편대 숙주반응의 발생이 IFN-γ에 의해 감소한다는 사실이 보고되었다. 따라서 제대혈 T 림프구의 IFN-γ 생성 능력과 제대혈이식시 나타나는 이식편대 숙주반응의 감소와의 관계를 관찰하는 새로운 연구가 필요하다. 재료 및 방법: 성인 및 제대혈 T 림프구를 동종항원으로 자극한 후 IFN-γ 생성 정도와 세포살해능을 관찰하여 성인 및 제대혈 T 림프구의 IFN-γ 생성 정도와 그에 따른 세포살해능의 변화를 비교하고자 하였다. 또한 각각의 T 림프구를 동종항원으로 자극할 때 IFN-γ 생성 억제인자로 알려진 interleukin-4 (IL-4)와 anti- IFN-γ monoclonal antibody (MoAb)를 첨가하여 그에 따른 세포살해능의 변화를 관찰하고자 하였다. 결과: 제대혈 단핵세포를 4일 동안 동종항원으로 자극하였을 때 상층액에서 IFN-γ는 측정되지 않았으며 Th1/Th2 및 Tc1/Tc2 비율이 낮아 Th2/Tc2 세포의 우월성이 관찰되었다. 그러나 6일 동안 자극하였을 때에는 상층액에서 IFN-γ의 농도, Th1/Th2 및 Tc1/Tc2 비율이 현저히 증가하여 Th1/Tc1 세포들의 우월성이 관찰되었다. 성인의 말초혈액 단핵세포를 동종항원으로 4일 동안 자극하였을 때 제대혈 단핵세포에 비하여 상층액에서의 IFN-γ 농도와 Th1/Th2 및 Tc1/Tc2 비율이 현저히 높았으며, 6일 동안 자극하였을 때에는 상층액에서의 IFN-γ 농도는 제대혈 단핵세포에 비해 높았으나 Th1/Th2 비율은 더 낮았으며 Tc1/Tc2 비율은 거의 대등하였다. 제대혈 및 성인의 말초혈액 단핵세포 모두에서 동종항원으로 4일 동안 자극했을 때에 비해 6일 동안 자극하였을 때 세포살해능이 더 증가하였으며, 모든 경우에서 성인의 말초혈액 단핵세포에 비해 제대혈 단핵세포에서의 세포살해능이 낮았다. 동종항원으로 6일 동안 자극하였을 때 제대혈 및 성인의 말초혈액 단핵세포 모두에서 IL-4와 anti-IFN-γ MoAb를 첨가한 경우 첨가하지 않았을 때에 비해 세포살해능이 증가하였으며 그 정도는 제대혈에서 더 현저하였다. 결론: 제대혈 T 림프구는 자극되지 않았을 때에는 IL-4를 생성하는 Th2 및 Tc2 세포의 우월성을 나타내지만 동종항원으로 충분히 자극되었을 때는 Th2 및 Tc2 세포의 우월성이 소실되고 IFN-γ를 생성하는 Th1 및 Tc1 림프구의 우월성을 나타낸다는 것을 알 수 있었다. 이와 같은 동종항원 자극에 의한 제대혈에서의 우월한 Th1 및 Tc1 림프구의 생성은 성인의 말초혈액 단핵세포에 비교할 때 대등하거나 현저하였으나 이들 세포에 의한 세포살해능은 성인에 비해 낮았다. 또한 동종항원 자극시 IFN-γ 생성 억제가 세포살해능의 감소보다는 증가와 관련된 것으로 관찰되어 이식편대 숙주반응에 있어서 IFN-γ의 역할에 대해서 향후 더 많은 연구가 필요할 것으로 생각된다. Background: The reduced incidence of graft versus host disease (GVHD) following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Although it has been reported that the reduced incidence of GVHD following cord blood transplantation is due to decreased ability of cord blood T lymphocytes to produce interferon-γ(IFN-γ), others have also reported that GVHD associated with bone marrow transplantation is suppressed by IFN-γ. So it is necessary to investigate the relationship between the ability of cord blood T lymphocytes to produce IFN-γ and lower risk of GVHD following cord blood transplantation. Methods: We examined Th/Tc and cytotoxic responses in cord and adult blood mononuclear cells after stimulation with alloantigen. We also observed the changes of cytotoxicity after stimulation of cord and adult blood mononuclear cells with alloantigen in the presence or absence of interleukin-4 (IL-4) and anti-IFN-γ monoclonal antibody (MoAb). Results: Cord blood mononuclear cells (CBMC) did not produce IFN-γ and showed very low Th1/Th2 and Tc1/Tc2 ratios after 4 days of allogeneic stimulation. However, CBMC, following 6 day stimulation, acquired the ability to secrete IFN-γ and showed marked increases in Th1/Th2 and Tc1/Tc2 ratios. In contrast, adult peripheral blood mononuclear cells (PBMC) which were stimulated with alloantigen produced substantial amounts of IFN-γ and showed markedly higher Th1/Th2 and Tc1/Tc2 ratios than CBMC. Although PBMC were found to produce more IFN-γ following 6 day stimulation, they showed lower Th1/Th2 ratio and equivalent Tc1/Tc2 ratio compared with CBMC. Following stimulation with alloantigen, CBMC and PBMC produced cytotoxicity which was more remarkable in 6 days than in 4 days. In addition, cytotoxic activities of adult blood mononuclear cells were always higher than those of cord blood mononuclear cells. Addition of IL-4 and anti-IFN-γ MoAb enhanced cytotoxic activity by 8.9% in cord blood mononuclear cells and by 5.4% in adult blood mononuclear cells when stimulated with alloantigen for 6 days. Conclusion: These results demonstrate that (a) cord blood mononuclear cells willingly differentiate to Th1 and Tc1 cells and show lower cytotoxic activity than adult blood mononuclear cells in response to allogeneic stimulation, and (b) induction of IFN-γ-producing cells is associated with lower risk of GVHD rather than higher risk of GVHD.