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: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 1. KSHV and EBV MiRNA-Mediated Translational Repression of MICB (A) FACS analysis of ULBP3 and of MICB expres- sion by RKO cells transduced with lentivirus encoding either the KSHV miRNA cluster or the EBV pri-miR-BART2. The gray empty histogram represents the staining of ULBP3 or of MICB in the viral miRNA-transduced cells. The black empty histogram represents the staining of cells transduced with a control miRNA (miR-K12-12). The filled gray histogram represents the staining of the secondary mAb only. (B) Western blot analysis of MICB protein levels expressed by the cells stained in (A). Quantifica- tion of relative intensity was performed by ImageJ software. (C) Quantitative real-time PCR analysis of the MICB mRNA levels in the transduced RKO cells. Relative mRNA abundance is shown as a percentage of the level of HPRT mRNA (encoding hypoxanthine guanine phosphoribosyltransfer- ase), and error bars (SD) are derived from tripli- cates. MiR-K12-12 served as negative control.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Transduction, Staining, Control, Western Blot, Software, Real-time Polymerase Chain Reaction, Derivative Assay, Negative Control

Journal: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 2. MiR-K12-7 and MiR-BART2-5p Are the Viral MiRNAs that Mediate MICB Downregulation (A) FACS analysis of MICA expression by 293T cells transduced with lentivirus encoding the above indicated KSHV miRNA. The gray empty histogram represents the staining of MICA expression by the transduced cell. The black empty histogram represents the staining of miR-K12-1, which was used as a control. The filled gray histogram represents the staining of the secondary mAb only. (B) FACS analysis of MICB expression by RKO cells transduced with lentivirus encoding the indicated KSHV miRNA. The gray empty histogram represents the stain- ing of MICB expression by the transduced cell. The black empty histogram represents the staining of miR-K12-1 or miR-K12-2 miRNAs that were used as controls. The filled gray histogram represents the staining of the secondary mAb only. The red histogram shows the MICB downregulation mediated by miR-K12-7.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Expressing, Transduction, Staining, Control

Journal: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 4. The MICB Downregulation by MiR-K12-7 and MiR-BART2-5p Results in Reduced NKG2D-Mediated Killing (A and B) Cells expressing miR-K12-7 or miR- BART2-5p exhibit reduced NK killing. Bulk NK cells were preincubated either with anti-NKG2D mAb (white) or with isotype control mAb (gray). Labeled RKO cells expressing miR-K12-7 (A), miR-BART2-5p (B), or control miRNA (miR-K12- 12) (A and B) were then added and incubated for 5 hr. Shown are mean values ± SD. Statistically significant differences are indicated (*p % 0.03, by one-tailed t test). Error bars (SD) are derived from triplicates.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Expressing, Control, Labeling, Incubation, One-tailed Test, Derivative Assay

Journal: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 5. MiR-K12-7 and MiR-BART2-5p Are Expressed and Are Functional during Authentic Viral Infection (A) Quantitative real-time PCR analysis of viral miRNA expression using custom-designed Taqman RT primer and real-time PCR probe. The KSHV-infected BCBL1 cell line was assayed for the expression of miR-K12-7. The EBV-trans- formed 721.221 cell line was assayed for the expression of miR-BART2-5p. The cell lines served as negative controls for each other. RKO cells transduced with the relevant lentivirus were used as positive controls. Error bars (SD) are derived from triplicates. (B) FACS analysis of the expression of MICA and MICB by KSHV-infected and EBV-infected cell lines. Left side shows staining of BCBL1 cells transduced with an anti-miR-K12-7-sponge (gray empty histogram) or with anti-miR-BART2-5p sponge as control (black empty histogram). The filled gray histogram is of secondary mAb staining only. Right side shows staining of 721.221 cells transduced with an anti-miR-BART2-5p sponge (gray empty histogram) or with anti-miR-K12-7 sponge as control (black empty histogram). The filled dark gray histogram is the staining of MICB expression by parental 721.221. The filled gray histogram is of secondary mAb staining only. (C) The specific effect of the antiviral miRNA sponges was tested in a dual luciferase reporter assay. 293T cells were transduced with miR- K12-7 (left) or with miR-BART2-5p (right). In addi- tion, each 293T cell was transduced with either a relevant sponge (white) or an irrelevant sponge (gray). Firefly luciferase activity was normalized to Renilla luciferase activity and then normalized to the average activity of the control reporter. Shown are mean values ± SD. Statistically signifi- cant differences are indicated (*p < 0.006, by one-tailed t test). Error bars (SD) are derived from triplicates.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Functional Assay, Infection, Real-time Polymerase Chain Reaction, Expressing, Transduction, Derivative Assay, Staining, Control, Luciferase, Reporter Assay, Activity Assay, One-tailed Test

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: VPA upregulates the expression of MICA and MICB in pancreatic cancer cells. Pancreatic cancer cells were incubated with or without 1 mM VPA for 24 h. (A) Quantitative real-time RT-PCR analysis of MICA and MICB mRNA expression. (B) Flow cytometry analysis and quantification of MICA and MICB protein expression on the surface of pancreatic cancer cells. MFI, mean fluorescence intensity. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01.

Article Snippet: PANC-1, MIA PaCa-2, and BxPC-3 cells were cultured to 80-90% confluence, trypsinized, washed twice with phosphate buffer solution (PBS), re-suspended in PBS at 1 × 10 6 cells/100 μl, incubated with PE-anti-human MICA and MICB antibody or an isotype control antibody for 30 min, and then analyzed on a Becton Dickson LSR II flow cytometer (BD, Franklin Lakes, NJ, USA).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Flow Cytometry, Fluorescence

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: PI3K/Akt signaling is required for VPA-induced upregulation of MICA and MICB in pancreatic cancer cells. (A, B) Quantitative real-time RT-PCR analysis. The VPA-induced upregulation of MICA and MICB mRNA expression were inhibited by the HER2/HER3 inhibitor lapatinib and the PI3K inhibitor LY294002, but not by the ATM/ATR inhibitor caffeine. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. * P < 0.05; ** P < 0.01; ns P > 0.05. (C) Flow cytometry analysis. The VPA-induced upregulation of MICA and MICB protein expression on the cell surface were inhibited by the PI3K inhibitor LY294002. MFI, mean fluorescence intensity; * P < 0.05. (D) Western blotting analysis. Transfection of the PI3KCA siRNA inhibited PI3KCA protein expression at 48 h post-transfection. NC, negative control; siR1-3, PI3KCA_siR sequence 1-3; ** P < 0.01. (E) Quantitative real-time RT-PCR analysis. VPA-induced upregulation of MICA and MICB mRNA expression were attenuated in PI3KCA-knockdown cells; Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01. (F) Flow cytometric analysis. VPA-induced upregulation of MICA and MICB protein expression on the cell surface were attenuated in PI3KCA-knockdown cells. MFI, mean fluorescence intensity; * P < 0.05; ** P < 0.01. (G) LDH release assay. The VPA-induced susceptibility of cancer cells to NK cell-mediated cell lysis was reduced in PI3KCA-knockdown cells. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01 and * P < 0.05 for NC vs. siR2; ▲▲ P < 0.01 and ▲ P < 0.05 for NC vs. siR3. siR2, PI3KCA siR sequence 2; siR3, PI3KCA siR sequence 3.

Article Snippet: PANC-1, MIA PaCa-2, and BxPC-3 cells were cultured to 80-90% confluence, trypsinized, washed twice with phosphate buffer solution (PBS), re-suspended in PBS at 1 × 10 6 cells/100 μl, incubated with PE-anti-human MICA and MICB antibody or an isotype control antibody for 30 min, and then analyzed on a Becton Dickson LSR II flow cytometer (BD, Franklin Lakes, NJ, USA).

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Western Blot, Transfection, Negative Control, Sequencing, Knockdown, Lactate Dehydrogenase Assay, Lysis

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: VPA sensitizes pancreatic cancer cells to NK cell-mediated lysis in vivo by upregulating the expression of MICA and MICB. (A, B) Excised xenograft tumors and growth curves for the xenografts; VPA enhanced the growth inhibitory effect of NK cells in vivo ; this effect was attenuated by the PI3K inhibitor LY294002. Data are mean ± SD of five mice per group; * P < 0.05 for NK-92 vs. NK-92 + VPA; Δ P < 0.05 for NK-92 + VPA + LY294002 vs. NK-92 + VPA. (C) Immunohistochemical staining and quantification of MICA and MICB expression in the xenograft tumors; VPA significantly upregulated the expression of MICA and MICB in the pancreatic cancer xenograft tumors in vivo , this effect was attenuated by the PI3K inhibitor LY294002.

Article Snippet: PANC-1, MIA PaCa-2, and BxPC-3 cells were cultured to 80-90% confluence, trypsinized, washed twice with phosphate buffer solution (PBS), re-suspended in PBS at 1 × 10 6 cells/100 μl, incubated with PE-anti-human MICA and MICB antibody or an isotype control antibody for 30 min, and then analyzed on a Becton Dickson LSR II flow cytometer (BD, Franklin Lakes, NJ, USA).

Techniques: Lysis, In Vivo, Expressing, Immunohistochemical staining, Staining