The study aims to explore the active molecules of traditional Chinese medicine that specifically bind to interleukin-15 receptor α (IL-15Rα) using molecular docking and surface plasmon resonance (SPR) technology. AutoDock molecular docking software was used to perform simulated docking of more than 3 000 compounds from 48 traditional Chinese medicines at IL-15Rα and screen the specific binding compounds. Then Biocore T200 biomolecular interaction analysis system of SPR was used to confirm the binding specificity of the selected target compounds. Finally, the biological effects of the target compounds on IL-15Rα were verified by cell biological experiments. The results showed that neoprzewaquinone A (Neo) possessed the highest specific binding affinity among the active molecules from traditional Chinese medicine, and the dissociation constant (K_D) value was (0.62 ± 0.20) µmol/L. The results of cell experiment showed that Neo significantly inhibited the proliferation of Mo7e cells induced by IL-15, and the IC₅₀ was 1.075 µmol/L, approximately 1/120 of the IC₅₀ of Cefazolin (IL-15 specific antagonist). These results suggest that Neo is a specific inhibitor of IL-15Rα and may be a potential active drug for the treatment of diseases related to the dysfunction of the IL-15Rα signaling.
Molecular Docking Simulation ; Interleukin-15/pharmacology* ; Surface Plasmon Resonance ; Interleukin-15 Receptor alpha Subunit/metabolism* ; Protein Binding

BACKGROUND: Asbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8 METHODS: For MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8 RESULTS: IL-15 addition partially reversed the decrease in CD3 CONCLUSION These findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.
Asbestos/adverse effects* ; CD8-Positive T-Lymphocytes/metabolism* ; Humans ; Interleukin-15/pharmacology* ; Lymphocyte Activation/immunology* ; T-Lymphocytes, Cytotoxic/metabolism*

This study was aimed to investigate the effects of different stimulatory factors on proliferation and function of cytokine induced killer (CIK) cells. Peripheral blood mononuclear cells (PBMNC) were separated by Ficoll-Hypacue gradient. According to supplement of different stimulatory factors (CD28 mAb, IL-15 and IL-21), the experiment was divided into five groups:control group (CIK), CB28+IL-15+IL-21 group, IL-15+IL-21 group, CD28+IL-15 group and CD28+IL-21 group. Effects of different stimulatory factors on the proliferation of CIK cells were assayed by an automated hematology analyzer. Changes of granzyme B,perforin and CD107a were detected by flow cytometry. IL-10, IL-12, INF-γ and TNF-α were quantified by ELISA. Cytotoxicities on lung cancer cell line A549, breast adenocarcinoma cell line MFC-7 and human melanoma cell line HME1 were examined by lactate dehydrogenase release method. The results showed that there were significant differences among different groups. The highest proliferation index on days 10 was observed in group CD28mAb, IL-15 and IL-21(255.3 ± 6.3), which was higher than control group, IL-21+IL-15 group and CD28 mAb+IL-21 group (166.6 ± 13.5, 199.4 ± 15.0 and 228.8 ± 16.6) (P < 0.05). The expression of perforin in CD28 mAb+IL-15 group was higher than the other groups. The expression of perforin,GranB and CD107a of costimulatory groups was higher than control group. The cytotoxicities of CD28 mAb+IL-15 group on A549, MFC-7 and HME1 cells (82.2%, 59.3% and 70.6%) were much higher than that of control group (60.9%, 49.6% and 48.4%) (P < 0.05). The highest IFN-γsecretion was found in CD28 mAb, IL-15 and IL-21 groups. It is concluded that there are significant difference of proliferative capacity, cytokine secretion and cytotoxicity after being activated by different stimulatory factors. Adding corresponding stimulatory factors into the culture system displays a great value for target cells culture.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cytokine-Induced Killer Cells ; cytology ; drug effects ; Humans ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-15 ; pharmacology ; Interleukins ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

This study was purposed to investigate the amplification of CD3(-)CD56(+)NK cells in umbilical cord blood and their change of immunophenotype and cytotoxicity after stimulation with IL-2 and IL-15. Mononuclear cells were isolated from umbilical cord blood and cultured in serum-free medium supplemented with IL-2 or (and) IL-15 for 14 d. The subset level of CD3(-)CD56(+)NK cells and expression of CD16, CD62L, NKG2A, NKG2D, NCR44, NCR46, granzyme B and perforin were analyzed by flow cytometry. The cytotoxicity of NK cells to K562 was detected by WST-1 method. The results showed that NK cells stimulated with IL-2, IL-15 and IL-2/IL-15 were amplified by 10.78 ± 2.51, 10.42 ± 3.72, and 10.54 ± 6.24 times respectively after 14 d, there was no statistically significant difference between these three groups. The expression of CD16 decreased obviously in NK cells after amplification; there was significant difference between IL-2 and IL-15 groups. The expression of CD62L was not changed statistically after stimulation with cytokines, the IL-2 down-regulated the expressions of NKG2A and NCR46, while IL-15 showed the opposite effect. IL-2 or IL-15 displayed upregulation effect on the expression of NKG2D, perforin and NCR44, but there was statistically significant difference between effects of these two cytokines. IL-15 up-regulated the expression of granzyme B on NK cells. The cytotoxicity of NK cells stimulated and amplified by cytokines significantly increased, but there was no statistically significant difference between IL-2 and IL-15. It is concluded that IL-2 or IL-15 can effectively amplify umbilical cord blood NK cells under serum-free conditions. Although the immunophenotype associated with NK cells function showed different characteristics between them, however, cytotoxicity of NK cells increased obviously after amplification and there is no statistically significant difference between effect of these two cytokines, their synergistic effect is not obvious. The cytotoxicity of NK cells is the result from combined effect of all active molecules.
Cells, Cultured ; Cytotoxicity, Immunologic ; drug effects ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Immunophenotyping ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; K562 Cells ; Killer Cells, Natural ; drug effects

The aim of this study was to explore the cytotoxicity of fresh cord blood(CB) NK cells and the influence of IL-12 and IL-15 on activity of the NK cells killing K562 and Jurkat cells lines. The NK cells were isolated from cord blood by depleting CD3(+) cells and then enriching CD56(+) cells using sorting with immunomagnetic beads. The experiment was divided into 3 groups: group A (fresh CB-NK cells without cytokines), group B (CB-NK cells cultured by IL-2) and group C (CB-NK cells cultured by IL-2 and IL-15). The purity of NK cells was determined by flow cytometry; the cytotoxity of fresh and different cytokine-treated CB-NK cells on K562 and Jurkat cell lines was detected by LDH release test. The results showed that the purity of NK cells before and after sorting was 14.88 ± 9.2% and 92.39 ± 0.8% respectively. After culture for 3 days, NK-forming colony amounts in group B and group C were 148.60 ± 13.0 and 831.80 ± 23.0 respectively, the comparison between group B and group C showed the significant difference (p < 0.05). The cytotoxicities of NK cells in group A, B and C on K562 and Jurkat cell lines were 27.76 ± 8.8%, 61.90 ± 9.1% and 87.62 ± 3.7%; 29.32 ± 2.5%, 69.43 ± 4.4% and 92.95 ± 3.2% respectively, the difference was significant (p < 0.05). It is concluded that the fresh isolated CB-NK cells show low cytotoxic activity. After stimulated with IL-2 or IL-2 plus IL15, cytotoxicity of CB-NK cells increases obviously, the effect of IL-2 plus IL-15 is much better than IL-2 alone for promoting the growth and enhancing the cytotoxicity of CB-NK cells.
Cytotoxicity, Immunologic ; drug effects ; immunology ; Fetal Blood ; drug effects ; immunology ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Jurkat Cells ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology

This study was aimed to investigate the expression level of perforin in cord blood NK cells and the relation of perforin expression after IL-2, IL-15 stimulation to cytotoxicity of NK cells. NK cells were isolated from cord blood MNC by depleting CD3(+) cells and then enriching CD56(+) cells using immunomagnetic separation (CD3 and CD56 cell isolation kit, autoMACS, miltenyi). The purity was analysed by flow cytometry. According to the different combination of cytokines, there were two groups: group A (IL-2) and group B (IL-2 + IL-15). The cytotoxicity and perforin expression rate of fresh and different cultured CB-NK cells against K562/Jurkat cell lines were estimated by LDH release test (cytotoxic 96 non-radioactive cytotoxicity assay). The results showed that the purity of NK cells after separation was more than 90%. The cytotoxicity towards both tumor lines in group B was higher than that in group A (p < 0.05), and cytotoxicity in group A was higher than that of fresh NK cells (p < 0.05). Perforin expression rate of group A (84.55%) was higher than that of fresh NK cells (67.21%) (p < 0.05), and there was no significant difference between group A and B (84.55% versus 87.22%) Cytotoxic activity of CB-NK cells was positively correlated with perforin expression rate (r = 0.886, p < 0.05). It is concluded that IL-2 can enhance cytotoxicity of CB/BM-NK cells by increasing the perforin expression.
CD56 Antigen ; metabolism ; Cells, Cultured ; Cytotoxicity, Immunologic ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; K562 Cells ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; Perforin ; metabolism

Mounting evidence has demonstrated that CD4(+) T cells play an important role in anti-tumor immune responses. Thus, adoptive transfer of these cells may have great potential for anti-cancer therapy. However, due to the difficulty to generate sufficient tumor-specific CD4(+) T cells, the use of CD4(+) T cells in tumor therapy is limited. It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8(+) T cells, but the effect of IL-15 transfection on CD4(+) T cells remains unknown. Here, the effects of retrovirus-mediated IL-15 expression in Ova-specific CD4(+) T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4(+) T cells expressed both soluble and membrane-bound IL-15. Retrovirus-mediated IL-15 expression led to a selective expansion of antigen-specific CD4(+) T cells by inhibiting their apoptosis. In vivo IL-15 transfected CD4(+) T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones. To ensure the safety of the method, the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4(+) T cells following ganciclovir treatment. Together, we show that IL-15 transfection induced a selective expansion of antigen-specific CD4(+) T cells ex vivo and enhanced their tumor-suppression effects in vivo. This has an important significance for improving the efficacy of adoptive T cell therapy.
Animals ; Antineoplastic Agents ; pharmacology ; CD4-Positive T-Lymphocytes ; cytology ; drug effects ; immunology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Humans ; Interleukin-15 ; metabolism ; Mice ; Mice, Transgenic ; Neoplasms ; drug therapy ; Recombinant Fusion Proteins ; genetics ; Retroviridae ; genetics

The aim of this study was to compare cell proliferation and function of the T cells acquired under various culture conditions for establishing a simple, safe and efficient cell expansion protocol in vitro. The peripheral blood mononuclear cells (PBMNC) were isolated and stimulated with autologous dendritic cells (DC) and EBV-transformed B lymphoblastoid cell line (BLCL) weekly. The cell proliferation test, flow cytometry with PI and Annexin V double staining, Cr release test and ELISPOT test were used to detect the cell expansion level, frequency of IFN-γ producing T cells, killing activity of antigen-specific T cells, cell apoptotic status and cell differentiation potential, respectively. The results indicated that use of IL-2 combined with IL-7 and IL-15 resulted in the highest cell expansion comparing to the use of IL-2 alone and the use of CD3/28 Microbeads. Also the cells obtained under cultivating with IL-2, IL-7 and IL-15 together showed high frequency of IFN-γ producing cells, strong killing activity, high viability and high differentiation potential with large portion of CD3(+)CD8(+) population among the T cells. It is concluded that a protocol is established in which the use of IL-2 combined with IL-7 and IL-15 induces the biggest cell expansion, expanded cells show high viability, strong differentiation potential, high frequency of IFN-γ producing cells and strong killing activity.
Cell Line, Transformed ; Cell Proliferation ; Cell Separation ; Dendritic Cells ; cytology ; metabolism ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; T-Lymphocytes ; cytology ; drug effects ; metabolism

This study was purposed to explore the changes in biological functions of human peripheral blood derived NK Cells after ex vivo expansion with different combinations of interleukin IL2 and/or IL12, IL15. According to different combination of cytokines, cultured NK cells were divided into 4 groups: group IL2, group IL2 + IL12, group IL2 + IL15 and group IL2 + IL15 + IL12. The group in which NK cells were cultured without cytokines was used as control. The cytotoxicity of cultured NK cells to target K562 cells was determined by using cell counting kit-8; the level of IFN-gamma in supernatants of NK cell culture was detected by ELISA; the perforin and granzyme B mRNA expressions were assayed by competitive quantitative RT-PCR. The results showed that the cytotoxicity of expanded NK cells in groups cultured with cytokines at different E:T ratio was significantly higher than that in group without cytokines (p < 0.01), although the cytotoxicity of NK cells in IL2 + IL15 + IL12 group seem to be slightly higher than that in IL2 + IL15 group, but there was no statistic difference (p > 0.05). The IFN-gamma levels in the supernatants of NK cell culture in the presence of cytokines significantly increased, and the IFN-gamma levels in IL2 + IL15 + IL12 group and IL2 + IL12 group were significantly higher than that in others (p < 0.01). The expressions of perforin and granzyme B mRNA of expanded NK cells in groups cultured with cytokines was significantly higher than that in control group (p < 0.01), and was consistent with cytotoxicity of NK cells. It is concluded that there are differences in the functions of NK cells cultured with different cytokines. IL2 and IL15 have synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion. However, the main function of IL12 promotes NK cells to secrete IFN-gamma, which plays a role in immunoregulation.
Humans ; Interferon-gamma ; secretion ; Interleukin-12 ; administration & dosage ; pharmacology ; Interleukin-15 ; administration & dosage ; pharmacology ; Interleukin-2 ; administration & dosage ; pharmacology ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology

This study was purposed to investigate the phenotype, in vitro expansion and cytokine secretion profile of Valpha24(+) NKT cells from cord blood (CB), peripheral blood (PB), and granulocyte colony stimulating factor-mobilized peripheral blood mononuclear cells (G-PBMNCs). Fresh mononuclear cells (MNCs) were isolated by the method of gradient centrifugation and then cultured with alpha-GalCer (100 ng/ml), IL-2 (50 U/ml), IL-15 (50 ng/ml) for 12 days. Valpha24(+) NKT cells were purified by anti-Vbeta11 TCR McAb and goat anti-mouse IgG magnetic beads. The phenotype and purity of Valpha24(+) NKT cells were determined by flow cytometry. Cytokine production was analyzed by ELISA. The results showed that Valpha24(+) NKT cells in CB, PB and G-PBMNCs were expanded by 221.5 (95 - 501), 456.5 (101 - 2207), and 756.38 (82 - 20373)-fold respectively. After stimulation by phorbol-12-myristate-13-acetate (PMA) for 24 hours, IL-4 and IFN-gamma produced by Valpha24(+) NKT cells from CB and PB were 180.33 (144.67 - 2253.48) vs 190.67 (110.07 - 6060.16) ng/ml, 864.33 (401.33 - 3386.67) vs 508.49 (253.82 - 8840.00) ng/ml respectively, with IL-4/IFN-gamma ratio of 0.503 +/- 0.642 vs 0.455 +/- 0.562 respectively. After expansion of Valpha24(+) NKT cells from G-PBMNCs, IL-4 and IFN-gamma produced by Valpha24(+) NKT cells at day 9 and day 12 were 139.08 (7.62 - 606) vs 89.3 (0 - 729.2) ng/ml, 14264.8 (1168 - 18059) vs 14488 (1041 - 18261) ng/ml respectively, with IL-4/IFN-gamma ratio of 0.0531 +/- 0.1081 vs 0.0376 +/- 0.1148 respectively. It is concluded that in presence of IL-2 and IL-15, alpha-GalCer can facilitate the rapid short-term expansion of Valpha24(+) NKT cells from CB, PB, and G-PBMNCs. Valpha24(+) NKT cells from G-PBMNCs show much high potential of expansion in comparison to the counterparts from CB or PB (p < 0.05). The activated Valpha24(+) NKT cells can secrete IFN-gamma and IL-4 in large amounts, with IFN-gamma in particular.
Cell Culture Techniques ; Fetal Blood ; cytology ; Galactosylceramides ; pharmacology ; Humans ; Interferon-gamma ; secretion ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-4 ; secretion ; Leukocytes, Mononuclear ; cytology ; Lymphocyte Activation ; Natural Killer T-Cells ; metabolism