Journal: Molecular Medicine Reports

Article Title: MG132 selectively upregulates MICB through the DNA damage response pathway in A549 cells

doi: 10.3892/mmr.2018.9676

Figure Lengend Snippet: Mean fluorescence intensity of NK group 2, member D ligands on non-small cell lung cancer cells.

Article Snippet: Mouse anti-human MICA (catalog no. sc-23870), MICB (catalog no. sc-80527), ULBP1 (catalog no. sc-53131), ULBP2 (catalog no. sc-53135), ULBP3 (catalog no. sc-53132) and ULBP4 (catalog no. sc-53133) monoclonal antibodies (mAbs) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Fluorescence

Journal: Journal of Immunotherapy

Article Title: Modulation of NKG2D-ligand Cell Surface Expression Enhances Immune Cell Therapy of Cancer

doi: 10.1097/cji.0b013e31820e1b0d

Figure Lengend Snippet: FIGURE 1. Histone deactylase inhibitors enhance sensitivity of some cancer cells to CIK-mediated killing through NKG2D ligand expression. A, Different human ovarian and cervical cancer cell lines were treated with different histone deactylase inhibitors for 24 hours as indicated. At the end of this time, cells were stained with a pan anti-MICAS/MICB antibody and percentage positive cells determined by flow cytometry (a typical flow cytometry plot is shown for UCI-101 cells with (gray) or without (black) TSA treatment. Isotype is thin black plot). B, Enhanced MICA/MICB expression correlates with enhanced sensitivity to CIK-mediated killing. Cells treated as before (and expressing luciferase) were mixed with human CIK cells at an effector to target ratio of 5 : 1 for 4 hours before luciferase signal was determined after luciferin addition. Luciferase signal [relative to target cells only (0%) or ethanol treated target cells (100%)] was used to determine percentage killing. C, This enhanced killing is mostly mediated through MICA/MICB expression levels. Cells (UCI- 101) were treated with TSA or anti-MICA/MICB siRNA, or both, and levels of MICA/B surface expression by antibody staining and flow cytometry (nontargeting siRNA was used as a control) (left panel); CIK cell killing was determined as before for cells treated with TSA with or without MICA/MICB siRNA (right panel). CIK indicates cytokine induced killer; TSA, trichostatin A.

Article Snippet: For flow cytometry, cells were stained with phycoerythrin (PE)-conjugated antihumanMICA/MICB (eBioscience, San Diego, CA) for 30 minutes on ice or stained by PEconjugated mouse anti-human MICA and allophycocyaninconjugated mouse anti-human MICB separately (R and D systems, Minneapolis, MN).

Techniques: Expressing, Staining, Flow Cytometry, Luciferase, Control

Journal: Journal of Immunotherapy

Article Title: Modulation of NKG2D-ligand Cell Surface Expression Enhances Immune Cell Therapy of Cancer

doi: 10.1097/cji.0b013e31820e1b0d

Figure Lengend Snippet: FIGURE 2. Histone deactylase inhibitor treatment increases MICA/B level and CIK antitumor effects in vivo in mouse tumor models. A, Anti-MICA/B staining shows upregulation of surface expression of these ligands. UCI-101 tumors implanted subcutaneously into athymic nu-/nu- mice were treated with TSA (IP injection) and mice were killed after 24 hours. Sections were stained with anti-MICA/ MICB antibody (green), anti-CD31 antibody (red) to show vasculature, and with Hoescht 3323 (blue) (magnification 200), whereas flow cytometry of dissociated UCI-101 cells from the same tumors stained with antibodies specific for MICA or MICB shows that both ligands are upregulated (right panels; red is no TSA; blue is with TSA). B, TSA treatment also increases the numbers of CIK cells within tumors. Athymic nu-/nu- mice implanted with UCI-101 cells as before and treated with TSA (IP, day 0) and CIK cells IV, day 1), were sacrificed on day 4 and tumors dissociated. Numbers of CIK cells in the tumor were determined after staining with an antihuman CD-56 antibody. C, Antitumor effects are enhanced when CIK cells and TSA are combined. Athymic nu-/nu- mice implanted with UCI-101 tumors as before were treated with PBS, TSA (IP, day 0), CIK cells (day 1, IV) or both. The combined effect is significantly greater than any therapy used as a single agent (P = 0.046) (n = 8 per group). D, Combining TSA with CIK cells preinfected with the oncolytic vaccinia virus vvB18R (in the same tumor model) also leads to significantly enhanced antitumor effects relative to the same therapy without TSA (P = 0.014). In this case, 8 of 10 mice had complete responses (n = 10 per group). CIK indicates cytokine induced killer; IP, intraperitoneal; TSA, trichostatin A.

Article Snippet: For flow cytometry, cells were stained with phycoerythrin (PE)-conjugated antihumanMICA/MICB (eBioscience, San Diego, CA) for 30 minutes on ice or stained by PEconjugated mouse anti-human MICA and allophycocyaninconjugated mouse anti-human MICB separately (R and D systems, Minneapolis, MN).

Techniques: In Vivo, Staining, Expressing, Injection, Flow Cytometry, Virus

Journal: Journal of Immunotherapy

Article Title: Modulation of NKG2D-ligand Cell Surface Expression Enhances Immune Cell Therapy of Cancer

doi: 10.1097/cji.0b013e31820e1b0d

Figure Lengend Snippet: FIGURE 3. Shedding of soluble MICA or MICB can be blocked by PAO or MMPi treatment, leading to enhanced antitumor effects. A, HeLa and UCI-101 cells were grown for 24 hours in culture, before the media was sampled and assayed for sMICA and sMICB by ELISA. HeLa cells showed greatest shedding of soluble MICA/MICB domains, with sMICA being the major shed domain. B, Use of either PAO or MMPi can significantly reduce accumulation of sMICA in the media. HeLa cells were cultured as before, only in some wells PAO or MMPi III were added. ELISA was used to assay sMICA levels in the media after 24 hours. C, The same experiment was repeated for UCI-101 cells with or without TSA treatment. TSA leads to upregulation of sMICA shedding that can be reduced with combination with MMPi. D, CIK cells were mixed with media from HeLa cells (cultured for 24 h) or media from HeLa cells treated with MMPi before being added to UCI- 101-luciferase target cells (at effector:target of 5:1). After 4 hours, UCI-101 cell survival was determined by imaging of luciferase signal. CIK indicates cytokine induced killer; ELISA, enzyme-linked immunosorbent assay; MMPi, metalloproteinase inhibitors; PAO, phenylarsine oxide; TSA, trichostatin A.

Article Snippet: For flow cytometry, cells were stained with phycoerythrin (PE)-conjugated antihumanMICA/MICB (eBioscience, San Diego, CA) for 30 minutes on ice or stained by PEconjugated mouse anti-human MICA and allophycocyaninconjugated mouse anti-human MICB separately (R and D systems, Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Luciferase, Imaging

Journal: Journal of Immunotherapy

Article Title: Modulation of NKG2D-ligand Cell Surface Expression Enhances Immune Cell Therapy of Cancer

doi: 10.1097/cji.0b013e31820e1b0d

Figure Lengend Snippet: FIGURE 4. MMPi treatment in vivo leads to increased MICA expression in tumors, reduced serum levels of sMICA and enhanced antitumor effects of CIK cells. A, serum was taken from athymic nu-/nu- mice at different times after subcutaneous implantation with HeLa cells and sMICA levels determined by ELISA. At the same time tumor measurement was determined by caliper measurement, and the correlation between these 2 plotted. B, In HeLa tumor models as before, serum levels of sMICA (determined by ELISA) were adjusted for tumor volume (caliper measurement) for mice 24 hours after IP MMPi or PBS treatment (n = 5 per group) (C) Cell surface expression of MICA and MICB from cells dissociated from HeLa tumors in athymic nu-/nu- mice formed by subcutaneous implantation. Single cell suspensions were stained with anti-MICA or anti-MICB antibodies and levels determined by flow cytometry. D, Athymic nu/nu mice were implanted subcutaneously with UCI-101 cells and treated with TSA, MMPi, or TSA and MMPi once palpable tumors had formed. Serum was drawn after 24 hours and sMICA levels determined by ELISA. Serum levels were normalized for tumor volume as before. E, Anti-tumor effect of athymic nu-nu- mice bearing subcutaneous HeLa tumors and treated with CIK cells alone (day 1), or in combination with MMPi (day 0, and CIK cells at day 1). Combination therapy led to significantly enhanced therapeutic benefit relative to CIK therapy alone (P = 0.015 at day 21) (n = 8 per group). CIK indicates cytokine induced killer; ELISA, enzyme-linked immunosorbent assay; MMPi, metalloproteinase inhibitors; TSA, trichostatin A.

Article Snippet: For flow cytometry, cells were stained with phycoerythrin (PE)-conjugated antihumanMICA/MICB (eBioscience, San Diego, CA) for 30 minutes on ice or stained by PEconjugated mouse anti-human MICA and allophycocyaninconjugated mouse anti-human MICB separately (R and D systems, Minneapolis, MN).

Techniques: In Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: JCI insight

Article Title: Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion.

doi: 10.1172/jci.insight.150079

Figure Lengend Snippet: Figure 1. Prolonged stimulation through activating receptors induces NK cell exhaustion. (A) Schematic representing the in vitro model of exhaus- tion. Agonists of NKp46 (anti-NKp46) and NKG2D (MICA and MICB) were adsorbed onto tissue culture plates and used to stimulate NK cells for 7 days. Plate-bound isotype IgG served as a control. Both groups received 1 ng/mL IL-15. (B) NK cells harvested from isotype-coated and exhaustion plates (day 7) were incubated with K-562 targets for 4 hours (E/T: 2:1). Cytokine production (IFN-γ and TNF-α) and degranulation (CD107a) were measured via flow cytometry. Isotype NK cells (top row) in blue, exhausted NK cells (bottom row) in red. E/T, effector/target. (C–E) Quantification of cytokine production and degranulation as percentage of parent population (live, CD3–CD56+ cells) and mean fluorescence intensity (MFI) (n = 4). Paired t tests were used for comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: A total of 300 μL of the following PBS solutions were added to 24-well plates and incubated at 4°C overnight: anti-NKp46 (5 μg/mL; [R&D Systems; catalog MAB1850]), recombinant MICA Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1300-MA]), recombinant MICB 1 1 R E S E A R C H A R T I C L E JCI Insight 2022;7(15):e150079 https://doi.org/10.1172/jci.insight.150079 Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1599-MB]), and isotype (20 μg/mL IgG2b; [R&D Systems catalog MAB004]); anti-NKp46 (5 μg/mL; [R&D Systems catalog MAB1850]), recombinant MICA Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1300-MA]), recombinant MICB Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1599-MB]), and anti-NKG2A (20 μg/mL; [Beckman Coulter catalog IM2750]); or isotype only (25 μg/mL IgG2b [R&D Systems catalog MAB004]).

Techniques: In Vitro, Control, Incubation, Flow Cytometry, Fluorescence

Journal: Chest

Article Title: Soluble Major Histocompatibility Complex Class I-Related Chain B Molecules Are Increased and Correlate With Clinical Outcomes During Rhinovirus Infection in Healthy Subjects

doi: 10.1378/chest.13-2247

Figure Lengend Snippet: – RV infection increases in vitro levels of soluble MICA and MICB in bronchial respiratory epithelial cells. BEAS-2B respiratory epithelial cells were infected with RV1B and RV16 at different concentrations, or cultured with medium alone or filtered virus, and soluble MIC levels measured in culture supernatants by enzyme-linked immunosorbent assay at different time points. A-B, RV1B and RV16 increased soluble MICA (A) and soluble MICB (B) in BEAS-2B culture supernatants. C-D, RV1B increased levels of soluble MICA and MICB in a dose-responsive manner. Data are means ± SEMs of three to eight experiments. An asterisk (*) over columns represent comparisons with medium alone. * P < .05; ** P < .01; *** P < .001. MIC = major histocompatibility complex class I-related chain; MOI = multiplicity of infection; RV = rhinovirus .

Article Snippet: Surface MICA and MICB were detected on cells by direct staining using mouse anti-human antibodies: MICA (phycoerythrin) and MICB (allophycocyanin) (all from R&D Systems, Inc).

Techniques: Infection, In Vitro, Cell Culture, Virus, Enzyme-linked Immunosorbent Assay, Immunopeptidomics

Journal: Chest

Article Title: Soluble Major Histocompatibility Complex Class I-Related Chain B Molecules Are Increased and Correlate With Clinical Outcomes During Rhinovirus Infection in Healthy Subjects

doi: 10.1378/chest.13-2247

Figure Lengend Snippet: – RV infection increases IL-15 protein levels, and IL-15 increases MIC levels in bronchial respiratory epithelial cells. A, BEAS-2B cells were treated with RV1B MOI 1 or medium alone up to 72 h and IL-15 levels determined at different time points. B, BEAS-2B cells were infected with RV1B at different concentrations, filtered virus or medium alone, supernatants were harvested at 48 h, and levels of IL-15 measured. C-D, BEAS-2B cells were treated with RV1B MOI 1, filtered virus, IL-15, and RV1B + IL-15, supernatants harvested at 48 h, and levels of MICA and MICB determined. Data are means ± SEMs of three to six experiments. An asterisk (*) over columns represent comparisons with medium alone. * P < .05; ** P < .01; *** P < .001. See legend for expansion of abbreviations .

Article Snippet: Surface MICA and MICB were detected on cells by direct staining using mouse anti-human antibodies: MICA (phycoerythrin) and MICB (allophycocyanin) (all from R&D Systems, Inc).

Techniques: Infection, Virus

Journal: Chest

Article Title: Soluble Major Histocompatibility Complex Class I-Related Chain B Molecules Are Increased and Correlate With Clinical Outcomes During Rhinovirus Infection in Healthy Subjects

doi: 10.1378/chest.13-2247

Figure Lengend Snippet: – A-F, Soluble MICA and soluble MICB levels in biologic fluids during an experimental RV16 infection in normal subjects and patients with asthma. Levels of soluble MICA and soluble MICB in normal subjects and patients with asthma at baseline, during the acute experimental RV infection, and at convalescence (6 weeks postinfection) in (A, B) induced sputum, (C, D) BAL fluid, and (E, F) serum. Data are mean and SEM. * P < .05. See legend for expansion of abbreviations .

Article Snippet: Surface MICA and MICB were detected on cells by direct staining using mouse anti-human antibodies: MICA (phycoerythrin) and MICB (allophycocyanin) (all from R&D Systems, Inc).

Techniques: Infection