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

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

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

To explore the efficacy of the different combinations of stem cell factor (SCF), FLT3 ligand (FL) and interleukin (IL) 2, 7, 15 on the induction and expansion of cord blood (CB) derived CIK/ NK cells in vitro, according to the different combinations of cytokines, combinations of cytokines were divided into 3 groups: group A (SCF + IL2 + IL7 + IL15), group B (SCF + FL + IL2 + IL7 + IL15) and group C (IL2 + IL7 + IL15 as control group). At 21 days of culture, the proportion and the expansion rate of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells were measured. The results showed that at 21 days in culture, the percentages of CD3(+)CD56(+) CIK cells derived from CB-MNC in group A, B and C were (19.84 +/- 2.93)%, (26.20 +/- 4.05)%, (24.03 +/- 4.99%) and that of NK cells were (49.60 +/- 1.40)%, (51.16 +/- 6.45)% and (30.85 +/- 8.12)%, respectively. The proliferation of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells was the most effective in group B (SCF + FL + IL2 + IL7 + IL15). Expansion multiple of CIK cell number increased from 575.81 +/- 221.72 (control) to 796.09 +/- 278.47 with peak value of 1 313, and that of NK cells increased from 30.39 +/- 14.47 (control) to 65.85 +/- 30.83 with peak value of 121.06. In conclusion, a proper cytokines (SCF + FL + IL2 + IL7 + IL15) culture system can effectively induce and expand CB CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells simultaneously.
CD3 Complex ; analysis ; CD56 Antigen ; analysis ; Cytokines ; pharmacology ; Fetal Blood ; cytology ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; Killer Cells, Natural ; drug effects ; Membrane Proteins ; pharmacology ; Stem Cell Factor ; pharmacology

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

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*

IL-15 and IL-15 receptors (IL-15R) play a crucial role in the pathogenesis of adult T-cell leukemia (ATL), multiple myeloma and inflammatory autoimmune diseases. To develop a novel therapeutic agent capable of eliminating IL-15R-over-expressing abnormal cells, the gene coding for human IL-15 antagonist (IL-15M) was fused with a DNA fragment coding for the mutated form of Pseudomonas exotoxin, PEdelta293. The resulting gene fusion was cloned into pET16b under the control of T7 promoter, giving rise to the expression plasmid pET-IL15M-PEdelta293. Using Ni2+ -NTA affinity chromatography, IL15M-PEdelta293 was purified from E. coli BL21 (DE3) pLysS transformed with pET-IL15M-PEdelta293. The fusion toxin showed cytotoxicity to IL-15R-bearing myelogenous leukemia cell line K562 and K562-derived multidrug resistant cell line K562/AO2. However, IL-15R negative cell line Jurkat was insensitive to IL15M-PEdelta293. In addition, the toxic effect of IL15M-PEdelta293 on K562 was completely blocked by excessive amount of recombinant human IL-15. These results demonstrated that the selective cytotoxicity of IL15M-PEdelta293 correlated with the appropriate IL-15R expression on target cells. The present data suggest that the chimeric toxin constructed in this report may have therapeutic potential in the treatment of diseases associated with abnormal expression of IL-15/IL-15R, even in the treatment of chemotherapy refractory tumors.
Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; metabolism ; Humans ; Interleukin-15 ; antagonists & inhibitors ; biosynthesis ; genetics ; K562 Cells ; Pseudomonas aeruginosa ; genetics ; metabolism ; Receptors, Interleukin-15 ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo explore the expansion method of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after ex vivo expansion.

METHODSNK cells were isolated from peripheral blood mononuclear cells (PBMNCs) by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II, and cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were semi-exchanged with fresh media and cytokines every 3 days. Evaluation for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-gamma secretion before and after the culture period.

RESULTSCD3(-) CD56(+) cells concentration increased from (11.2 +/- 5.2)% to (94.2 +/- 3.5)%. In group IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group, cells were expanded 50.5 +/- 4.3 and 52.3 +/- 6.7 - fold, respectively, being significantly higher than that in other three groups [(15.4 +/- 1.1 fold in IL-2 group, 19.9 +/- 3.9 fold in IL-2 + IL-12 group, 6.1 +/- 1.0 fold in control group)] (P<0.01), but no significant difference between each other (P>0.05). The purity of CD3(-) CD56(+) NK cells was over 94% in all groups except the control. The perforin and granzyme B mRNA expressions of expanded NK cells in four experimental groups were significantly higher than those of before expansion (P<0.01) and the expressions in IL-2 + IL-15 and in IL-2 + IL-12 + IL-15 group were significant higher than in other three groups (P<0.01) while no significant difference between each other (P>0.05). IFN-gamma levels in the supernatants of four experiment groups were significantly higher than that in control group (P<0.01) and its levels order was IL-2 + IL-15 + IL-12 group > IL-2 + IL-12 group > IL-2 + IL-15 group > IL-2 group (P<0.01).

CONCLUSIONHigh purity NK cells isolated by negative selection using miniMACS can be efficiently expanded with IL-2 + IL-15, and their biological functions were enhanced.

Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; pharmacology ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukins ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; metabolism ; Perforin ; metabolism

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