Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: NK cells are activated by T cell-derived soluble mediators to conduct a TRAIL-dependent cytotoxicity against HPMC (A) TRAIL expression on CD335+ ascites-derived NK cells was measured after treatment with CM from unstimulated and α-CD3 Ab stimulated T cells by flow cytometry (n = 13 different patients). The geometric MFI was calculated after subtracting the isotype control. (B) Corresponding to the TRAIL expression on NK cells induced by the CM of T cells (+/− α-CD3 Ab stimulation), HPMC apoptosis induced by these activated NK cells was also analyzed by annexin V/PI staining (n = 13 different patients). (C) CD3 activation of ascites-derived T cells induce the secretion of cytokines fitting the terms “NK cell activation” and “TRAIL” in the Genecards database. Protein signals were determined by PEA-based affinity proteomics. Results are expressed as the fold change of T cells activated with α-CD3 Ab relative to untreated T cells (n = 5 matched pairs of different patients). Boxplots show the median (line), upper and lower quartiles (box), range (whiskers) and outliers (circles). (D) Upregulation of TRAIL expression on NK cells upon stimulation with rh-TNFα and rh-IL-21 together with rh-IL-2 (n = 5 experiments) and combinations of all 3 cytokines (n = 3 experiments) was analyzed by flow cytometry. (E) Co-culture of HPMC with NK cells previously activated with rh-TNFα (n = 6 patients) and rh-IL-21 (n = 4 patients) combined with rh-IL-2 were conducted and HPMC apoptosis induction was measured by flow cytometry. (F) A TRAIL-dependent apoptosis induction in HPMC by NK cells activated with rh-IL-2/IL-21/TNFα was confirmed by adding α-TRAIL blocking Ab (n = 6 patients). (G) To evaluate the contribution of TNFα and IL-21 on NK cell activation resulting in HPMC apoptosis, CM of T cells (α-CD3 Ab stimulated) were incubated with blocking Ab against IL-21 and TNFα (Infliximab) prior to NK cell treatment. The effect on HPMC apoptosis induction was analyzed in co-culture experiments (n = 5 experiments). Horizontal bars or boxes indicate the mean and vertical error bars represent the standard deviation in panels A, B, and D–G. p values were determined by two-sided, paired t test and Benjamini-Hochberg adjustment: ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; ∗∗∗∗ FDR <0.0001.

Article Snippet: In individual experiments, NK cells were treated with the following cytokines – alone or in combination – for two days: 10 ng/mL rh-TNFα (PeproTech, Hamburg, Germany), 10 ng/mL rh-IFNγ (Biomol), 20 ng/mL rh-IL-2 (ImmunoTools, Friesoythe, Germany), and 10 ng/mL rh-IL-21 (PeproTech).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Staining, Activation Assay, Co-Culture Assay, Blocking Assay, Incubation, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4

doi: 10.1186/s13287-023-03516-z

Figure Lengend Snippet: Alteration in effector function and receptor expression of NK cells by the ADSC secretome. NK-92 cells stimulated with IL-2 were cultured for 48 h with 25 (blue) or 150 μg/ml ADSC secretome (red), or without (gray). Unstimulated NK cells were also cultured for negative control (white). A The production of effector cytokines (IFN-γ and IL-10) and cytolytic granules (granzyme B and perforin) in the supernatant was measured by ELISA. B Cytotoxicity of NK-92 cells against K562 target cells at the indicated NK:K562 (E:T, effector:target) ratios of 1:1, 2.5:1, 5:1, or 10:1 (upper). Stimulated NK cells were treated with 150 μg/ml ADSC secretome for 48 h, followed by co-culture with K562 target cells at a 10:1 ratio for 4 h (bottom). C Degranulation marker from activated NK-92 cells was assessed using flow cytometry (CD56 + CD107a + ) as shown dot blot (at a 5:1 ratio, upper), frequency of CD107a + NK cells (at the indicated ratios, bottom), and the expression level of CD107a on NK cells (at the indicated ratios, right side). D to F Flow cytometry analysis of activating receptors, such as NKG2D, NKp30, and NKp46 ( D ; histogram, MFI, and frequencies), inhibitory receptors, such as CD96, TIGIT, CD94, and Tim-3 ( E ; histogram, MFI, and frequencies), and IL-2 receptors, such as CD25, CD122, and CD132 ( F ; MFI only). Error bars represent mean ± SEM of five to seven independent experiments. Statistical tests were determined by Student’s paired two-tailed t -test; not significant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Complete growth medium was made with α minimum essential medium (α-MEM; Gibco, Grand Island, NY, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM 2-mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma-Aldrich), 1% penicillin/streptomycin (Gibco), 12.5% heat-inactivated fetal bovine serum (FBS; Corning Inc., Corning, NY, USA), 12.5% heat-inactivated horse serum (Gibco), and recombinant human (rh) IL-2 (5 ng/ml; NKMAX Co Ltd., Seongnam, Republic of Korea), essential for cell survival and activation.

Techniques: Expressing, Cell Culture, Negative Control, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Marker, Flow Cytometry, Dot Blot, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4

doi: 10.1186/s13287-023-03516-z

Figure Lengend Snippet: The ADSC secretome suppresses JAK-STAT and AKT signaling pathway by upregulation of regulator CIS. IL-2-stimulated NK cells were treated with or without 150 μg/ml ADSC secretome for 36 h. Unstimulated NK cells were also cultured for negative control. Immunoblot analysis of A STAT5, AKT, ERK, B JAK1, JAK3, C SHP1, SHP2, SHIP1, and D CIS expressions in NK-92 cells. Full-length blots were presented in Additional file : Figure S3 to S6. In the groups A , B , and C , the p- prefix denotes a phosphorylated protein. Error bars represent mean ± SEM of three to four independent experiments. Statistical tests were determined by Student’s paired two-tailed t -test; not significant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Complete growth medium was made with α minimum essential medium (α-MEM; Gibco, Grand Island, NY, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM 2-mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma-Aldrich), 1% penicillin/streptomycin (Gibco), 12.5% heat-inactivated fetal bovine serum (FBS; Corning Inc., Corning, NY, USA), 12.5% heat-inactivated horse serum (Gibco), and recombinant human (rh) IL-2 (5 ng/ml; NKMAX Co Ltd., Seongnam, Republic of Korea), essential for cell survival and activation.

Techniques: Cell Culture, Negative Control, Western Blot, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4

doi: 10.1186/s13287-023-03516-z

Figure Lengend Snippet: Efficacy of the ADSC secretome to effector function of NK-92 cells when each immunomodulatory candidate within the ADSC secretome was blocked by neutralizing antibody. A to C NK-92 cells stimulated with IL-2 (5 ng/ml) were incubated for 48 h with the ADSC secretome (150 μg/ml) and neutralizing antibodies (1 μg/ml) to each candidate to examine recovery of cellular activity. Unstimulated NK cells were cultured for negative control. A The concentrations of IFN-γ and IL-10 in the culture supernatants was measured by ELISA. Error bars represent mean ± SEM of five independent experiments. Statistical tests were determined by Student’s paired two-tailed t-test; * P < 0.05. Anti-ANXA1, anti-annexin A1 antibody; anti-LGALS3BP, anti-galectin-3-binding protein antibody. B After incubation, NK-92 cells were co-cultured with K562 target cells for 4 h at the indicated E:T ratios, followed by measurement of cytotoxicity of NK-92 cells against K562 cells. C NK-92 cells were stained with PE-conjugated CD107a antibodies and co-cultured with K562 cells for 4 h at the indicated E:T ratios to measure the level of exocytosis of NK cells toward target cells. Degranulation marker CD107a was assessed using flow cytometry as shown dot blot (left) and frequency of CD107a + NK cells (right). Representative data were shown

Article Snippet: Complete growth medium was made with α minimum essential medium (α-MEM; Gibco, Grand Island, NY, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM 2-mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma-Aldrich), 1% penicillin/streptomycin (Gibco), 12.5% heat-inactivated fetal bovine serum (FBS; Corning Inc., Corning, NY, USA), 12.5% heat-inactivated horse serum (Gibco), and recombinant human (rh) IL-2 (5 ng/ml; NKMAX Co Ltd., Seongnam, Republic of Korea), essential for cell survival and activation.

Techniques: Incubation, Activity Assay, Cell Culture, Negative Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Binding Assay, Staining, Marker, Flow Cytometry, Dot Blot

Journal: Stem Cell Research & Therapy

Article Title: ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4

doi: 10.1186/s13287-023-03516-z

Figure Lengend Snippet: Human NK cells showed transcriptome changes that include the expression of cell activity– regulated genes and upregulation of DUSP4 expression caused by the ADSC secretome. Human primary NK cells isolated from PBMCs of a healthy donor were cultured for 48 h with 150 μg/ml of the ADSC secretome or without in the presence of 10 ng/ml of rhIL-2 ( n = 6). A A total of 352 differentially expressed genes (DEGs) were indicated into 99 down-regulated genes (blue) and 253 up-regulated genes (yellow) in secretome-treatment group compared to IL-2 alone control group based on fold change and P value ( P < 0.05, and |fc|≥ 2). B 352 DEGs indicated in A were grouped by the similarity of expression level and visualized in the heat map of the one-way Hierarchical Clustering using Z-score for normalized value (log 2 based), ranging from low expression (blue) to high expression (yellow). C Volcano plot of gene expression of human primary NK cells using mRNA-seq ( n = 6), indicating selected genes (red) associated with regulation of cell activity. Down-regulated (blue) and up-regulated (mustard) differentially expressed genes in secretome-treatment group were indicated. D mRNA levels of up-regulated differentially expressed genes that are phosphatase or ubiquitin ligase among the genes displayed in C . E Immunoblot analysis of protein levels of DUSP4 (left) and DTX1 (right) in NK cells from human blood. Full-length blots were presented in Additional file : Figure S7. RNA-seq data satisfying the conditions of adjusted P < 0.05, and |fc|≥ 2 were shown. Error bars represent mean ± SEM of four healthy donors. Statistical tests were determined by Student’s paired two-tailed t -test; not significant (ns), ** P < 0.01

Article Snippet: Complete growth medium was made with α minimum essential medium (α-MEM; Gibco, Grand Island, NY, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM 2-mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma-Aldrich), 1% penicillin/streptomycin (Gibco), 12.5% heat-inactivated fetal bovine serum (FBS; Corning Inc., Corning, NY, USA), 12.5% heat-inactivated horse serum (Gibco), and recombinant human (rh) IL-2 (5 ng/ml; NKMAX Co Ltd., Seongnam, Republic of Korea), essential for cell survival and activation.

Techniques: Expressing, Activity Assay, Isolation, Cell Culture, Control, Western Blot, RNA Sequencing Assay, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4

doi: 10.1186/s13287-023-03516-z

Figure Lengend Snippet: Possible mechanism of the regulation of IL-2 signaling pathway via CIS and DUSP4 increased by the ADSC secretome. The ADSC secretome induces upregulation of CIS and DUSP4 in NK cells, leading to attenuated effector function of NK cells while maintaining cell proliferation. CIS binds to pJAK1 and pJAK3 and targets them to proteasomal degradation to suppress their enzymatic activity. DUSP4 dephosphorylates pSTAT5 and pAKT to weaken the amplitude of IL-2 signaling cascade

Article Snippet: Complete growth medium was made with α minimum essential medium (α-MEM; Gibco, Grand Island, NY, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM 2-mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma-Aldrich), 1% penicillin/streptomycin (Gibco), 12.5% heat-inactivated fetal bovine serum (FBS; Corning Inc., Corning, NY, USA), 12.5% heat-inactivated horse serum (Gibco), and recombinant human (rh) IL-2 (5 ng/ml; NKMAX Co Ltd., Seongnam, Republic of Korea), essential for cell survival and activation.

Techniques: Activity Assay