Journal: bioRxiv

Article Title: γδ T cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects

doi: 10.1101/2021.10.14.464229

Figure Lengend Snippet: a. Flow cytometry gating strategy on PDTO cells for analysis of surface staining. Selected cells were gated on single, live cells before quantification of staining signal. b. Histogram representation and count for surface staining of MHC-I, PD-L1, and β2m expression on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. Staining with isotype antibodies for each fluorochrome (PE, APC and FITC) were included as negative control. c. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining to test antitumor reactivity upon PDTO stimulation. Lymphocyte population was further gated on single cells, live and CD3+ cells, γδ TCR+ cells and CD8+ as well as CD8–CD4– cells. Reactivity of the sample was based on IFNγ+ cells of the selected population. d. Histogram representation and count for surface staining of NKG2D ligands MICA/B, ULBP1, ULBP2/5/6, ULBP3, and ULBP4 on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. e. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining after stimulation with PDTOs in the presence of NKG2D ligand blocking. Lymphocyte population was further gated on single cells, live and CD3+ cells, followed by γδ TCR+ and CD8+ as well as CD8– cells. Reactivity of final population was based on IFNγ+ or CD107a+ cells.

Article Snippet: Briefly, cells were incubated with human Fc receptor block (BioLegend) and stained with the different cell surface antibodies (1:10 anti-CD112-PE [clone R2.525, BD Biosciences], 1:10 anti-CD155-PE [clone 300907, R&D Systems], 1:50 anti-CD277/BTN3A1-PE [clone BT3.1, Miltenyi], 1:100 anti-HLA-A,B,C-FITC [clone W6/32, eBioscience], 1:20 anti-HLA-E­BV421 [clone 3D12, BioLegend], 1:20 anti-HLA-G-APC [clone 87G, BioLegend], 1:300 anti-MICA/B-PE [clone 6D4, BioLegend], 1:10 anti-ULBP1-PE [clone 170818, R&D Systems], 1:20 anti-ULBP2/5/6-PE [clone 165903, R&D Systems], 1:20 anti-ULBP3-PE [clone 166510, R&D Systems], or 1:20 anti-ULBP4-PE [clone 709116, R&D Systems] for 45 min at 4°C.

Techniques: Flow Cytometry, Staining, Expressing, Negative Control, Blocking Assay

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) AML cell lines (KG1a and NB4 cells) were treated with DMSO (Ctrl), DAC (1 and 5 μM) or AZA (1 and 5 μM) for 48 hours. Soluble levels of MICA, MICB, ULBP1, ULBP2 and ULBP3 ligands were quantified by sandwich ELISA. Each bar represents the mean ± SEM of at least four independent experiments. * p < 0.05 and * p < 0.001. ( B ) KG1a and NB4 cells were treated with 5 μM of DAC for 48 hours. Before and after treatment, cell surface expression of NKG2DL was analyzed by flow cytometry using monoclonal antibodies specific to each NKG2DL (MICA, MICB, ULBP1, ULBP2 and ULBP3), followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and shaded grey histograms show the NKG2DL expression. Dotted vertical lines indicate mean fluorescence intensity value in untreated samples.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Bioprocessing, Incubation, Control, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) NKL cells were co-cultured with cell-free supernatants (sn) obtained from KG1a and NB4 cells untreated (sn-DMSO) or treated with 1 μM DAC for 48 hours (sn-DAC). NKL cells grow in culture medium were considered as a control (Ctrl). NKG2D expression was analyzed by flow cytometry and represented as mean fluorescence intensity (MFI). Each bar represents the mean ± SEM of three independent experiments. * versus control and p < 0.01; # versus sn-DMSO and p < 0.05. ( B ) NKL cells were co-cultured with K562 cells at the indicated E:T ratio in a cell lysis assay, in the absence (Ctrl) or presence of cellular supernatant derived from KG1a (left panel) and NB4 (middle panel) cells previously treated with DMSO (sn-DMSO) or 1 μM DAC (sn-DAC) for 48 hours. Specificity of the NKG2D-NKG2DL interaction was corroborated using an anti-NKG2D blocking mAb and the effect of DAC was assayed to analyze the non-specific effects on the lytic capacity of NKL cells (right panel). Measurements were made in duplicate and the mean ± SEM of the two independent experiments are shown. * versus control and p < 0.05; * versus control and p < 0.01; # versus sn-DMSO and p < 0.05.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Cell Culture, Control, Expressing, Flow Cytometry, Fluorescence, Lysis, Derivative Assay, Blocking Assay

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with inhibitors specific to ADAM17 (10 μM GW280264X) and ADAM10 (50 μM of GI254023X) for 48 hours. Levels of soluble NKG2DL (sMICA/B and sULBPs1-3) were quantified by sandwich ELISA. Values are the mean ± SEM of at least three independent experiments. * p < 0.05 and * p < 0.01. ( B ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (1 μM) for 48 hours. After treatment, cell surface expression of ADAM17 was analyzed by flow cytometry using anti-human ADAM17 monoclonal antibody, followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and the shaded grey histograms show the ADAM17 expression. ( C ) ADAM17 activity in KG1a and NB4 cells was measured in whole-cell lysates after treatment with DMSO (Ctrl) or DAC (5 μM) for 48 hours. Data are expressed as relative fluorescence units (RLU) at Ex/Em=490/520 nm absorbance normalized with respect to micrograms of total protein (RLU/μg). Values are the mean ± SEM of three independent experiments. * p < 0.05 and * p < 0.01.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Incubation, Control, Activity Assay, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (0.25, 0.5 and 1 μM) for 48 hours, and TIMP3 expression was analyzed by qRT-PCR. Each bar represents the relative expression of TIMP3 normalized with respect to the reference gene (GAPDH), using the 2 −ΔCt method. MICA transcription levels in the KG1a cell line untreated (DMSO, Ctrl) or treated with DAC at different concentrations were used as a positive control. Results are summarized as the mean ± SEM of five independent experiments. * p < 0.05 and * p < 0.01. ( B ) TIMP3 protein levels were evaluated by western blot in KG1a and NB4 cells after treatment with DMSO (Ctrl) or DAC (1 μM or 5 μM) for 48 hours. * p < 0.05. ( C ) The TIMP3 methylation pattern was quantified by pyrosequencing in AML cell lines (KG1a and NB4 cells) before and after treatment with 1 μM or 5 μM DAC. Pie charts show the average percentage of methylation for the CpGs analyzed in the TIMP3 gene. ( D ) TIMP3 expression was inhibited by transfection of KG1a cells previously treated with DAC (1 μM) with a TIMP3-specific siRNA or nonspecific scramble siRNA (200 nM). * p < 0.05 ( E ) Soluble NKG2DL were quantified by sandwich ELISA after TIMP3 inhibition. Values shown are the mean ± SEM of three independent experiments. * versus control and p < 0.05; * versus control and p < 0.01 # versus nonspecific scramble siRNA and p < 0.05.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Methylation, Transfection, Sandwich ELISA, Inhibition, Control

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) Soluble NKG2DL were quantified by sandwich ELISA in sera from twelve AML patients before and after Vidaza ® treatment. Lines represent the levels of each sNKG2DL (ng/mL) before and after treatment of each individual AML patient. ( B ) Expression of NKG2DL on the cell surface of blasts from five AML patients before and after Vidaza ® treatment (left panel). The right panel shows dot plots of NKG2DL expression on the cell surface of blats from a representative patient. Numbers included in the figure quadrants indicate the percentage of positive cells for each NKG2DL.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing

Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: List of primers used in qRT-PCR.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques:

Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: Gemcitabine up-regulates NKG2D ligands in lung cancer cells. (A) qRT-PCR of H60, Raet-1 , and Ulbp1 mRNA in LLC cells. (B) qRT-PCR of major histocompatibility complex class I polypeptide-related sequence A ( MICA ), MICB , and UL16 binding protein ( ULBP ) 1-6 mRNA in A549 cells. Data are the fold-change of mRNA expression in gemcitabine- and cisplatin-treated cells relative to untreated cells. (C) Surface expression of MICA/B, ULBP1, ULBP2/5/6, and ULBP3 was measured by flow cytometry on A549 cells treated with vehicle control (DMSO), gemcitabine or cisplatin. Data are representative of three independent experiments. One-way analysis of variance (ANOVA) was used. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Immunopeptidomics, Sequencing, Binding Assay, Expressing, Flow Cytometry, Control

Journal: International journal of cancer

Article Title: Expression of the ULBP ligands for NKG2D by B-NHL cells plays an important role in determining their susceptibility to rituximab-induced ADCC.

doi: 10.1002/ijc.24351

Figure Lengend Snippet: FIGURE 2 – Correlations between the expression of MHC class I or total NKG2D ligand and rituximab-induced ADCC in B-NHL cell lines. ADCC (upper panels) or CDC (lower panels) in the presence of 10 lg/ml rituximab against 30 different CD201 B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of MHC class I (left panels) and total NKG2D ligand (right panels). Each dot plot in each panel represents 1 cell line in the same manner as in Figure 1. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel.

Article Snippet: Mouse mAbs to human UL16-binding protein (ULBP)1 (clone: 170818, IgG2a), ULBP2 (165903, IgG2a), ULBP3 (166510, IgG2a), Allophycocyanin (APC)-conjugated antihuman major histocompatibility complex (MHC) class I chain-related genes A (MICA) (159227, IgG2b), MHC class I chain-related genes B (MICB) (236511, IgG2b), Phycoerythrin (PE)-conjugated antihuman NKG2D (149810, IgG1) and appropriate isotype-matched control mAbs were purchased from R&D Systems, (Minneapolis, MN).

Techniques: Expressing, Lysis

Journal: International journal of cancer

Article Title: Expression of the ULBP ligands for NKG2D by B-NHL cells plays an important role in determining their susceptibility to rituximab-induced ADCC.

doi: 10.1002/ijc.24351

Figure Lengend Snippet: FIGURE 3 – Correlations between the expression of the NKG2D ligand, ULBP1–3, or MICA/B and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10 lg/ml rituximab against 30 different CD201 B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of ULBP1 (upper left panel), ULBP2 (upper middle panel), ULBP3 (upper right panel), MICA (lower left panel), and MICB (lower right panel). Each dot plot in each panel represents 1 cell line in the same manner as in Figure 1. The coefficients and p values assessed by Spearman rank correlation coefficient testing are indicated in each panel.

Article Snippet: Mouse mAbs to human UL16-binding protein (ULBP)1 (clone: 170818, IgG2a), ULBP2 (165903, IgG2a), ULBP3 (166510, IgG2a), Allophycocyanin (APC)-conjugated antihuman major histocompatibility complex (MHC) class I chain-related genes A (MICA) (159227, IgG2b), MHC class I chain-related genes B (MICB) (236511, IgG2b), Phycoerythrin (PE)-conjugated antihuman NKG2D (149810, IgG1) and appropriate isotype-matched control mAbs were purchased from R&D Systems, (Minneapolis, MN).

Techniques: Expressing, Lysis