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: Scientific Reports

Article Title: All-trans retinoic acid enhances cytotoxicity of CIK cells against human lung adenocarcinoma by upregulating MICA and IL-2 secretion

doi: 10.1038/s41598-017-16745-z

Figure Lengend Snippet: Effects of CIK and ATRA, alone or in combination, on the expression of MICA and MICB. Representative flow cytometric profiles of cell surface MICA ( A ) and MICB ( B ). The black profile indicates a control profile of A549 or NCI-H520 cells incubated with mouse IgG 2b . ( C ) Effects of CIK and ATRA, alone or in combination, on the secretion of soluble MICA. Cells were treated for 48 h and the concentrations of soluble MICA in the medium were determined. The data presented are mean ± SD from at least three independent experiments.

Article Snippet: After treatment for 48 h, the cells in each group were harvested and incubated with either a phycoerythrin (PE)-labeled mouse anti-human MICA mAb (clone number 159227, R&D systems, USA) and a allophycocyanin (APC)-labeled mouse anti-human MICB mAb (clone number 236511, R&D systems, USA) on ice for 30 min. As isotype controls, cells were incubated with PE- or APC- labeled mouse IgG 2b antibodies.

Techniques: Expressing, Incubation

Journal: OncoTargets and Therapy

Article Title: Potential therapeutic strategy for gastric cancer peritoneal metastasis by NKG2D ligands-specific T cells

doi: 10.2147/ott.s91122

Figure Lengend Snippet: Figure 2 ChNKG2D receptor-modified T cells released Th1 cytokines and CCL3 chemokine in response to NKG2D ligand-expressing human gastric cancer cell lines. Notes: (A) Expression of NKG2D ligands in gastric cancer cell lines. Two human gastric cancer cell lines were stained with specific antibodies recognizing MICA, MICB, ULBP1, ULBP2, or ULBP3, or matched isotype controls (filled histogram), and analyzed by flow cytometry. A murine cell line TC-1 was used as negative control. (B) Th1/ Th2 cytokine panel determination using cytometric bead array assay. ChNKG2D receptor-modified T cells were cultured with gastric cancer cell lines or TC-1 control cells for 20 hours. Supernatants were harvested and assayed to determine the concentration of six different cytokines. The beads were conjugated with antibodies against corresponding cytokines. Secondary antibody conjugated with the fluorescent dye PE was used as a detector. The dots from top to bottom represent IL-2, IL-4, CCL3, IL-10, TNF-α, and IFN-γ, respectively. Abbreviations: L, CD8α Leader; 2A, cleavable 2A-like peptide sequence; CBA, cytometric bead array; ChNKG2D, chimeric NKG2D; PE, phycoerythrin; IL, interleukin; CCL3, chemokine (C-C motif) ligand 3; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon-gamma.

Article Snippet: NKG2DL expression was analyzed with allophycocyanin (APC)-conjugated anti-human MICA (clone 159227), APC anti-human MICB (clone 236511), phycoerythrin (PE)conjugated anti-human ULBP1 (clone 170818), PE-antihuman ULBP2 (clone 165903), and APC-anti-human ULBP3 (clone 166510) (all R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Modification, Expressing, Staining, Flow Cytometry, Negative Control, Cell Culture, Control, Concentration Assay, Sequencing