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: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: CD19/NKG2DL tandem CAR design. Schematic representation of single and tandem CAR constructs. scFv FMC63, single-chain variable fragment anti-CD19; NKG2D EC, extracellular domain of human NKG2D; (G 4 S) 3 , Glycine-Serine linker; CD8α H 55 , 55 amino acids hinge from human CD8α; CD8α H 12 , 12 amino acids hinge from human CD8α; IgG4 H 12 , 12 amino acids hinge from human IgG4; CD8α TM, transmembrane region from human CD8α; 4-1BB, intracellular domain from 4-1BB; CD3ζ, intracellular domain from human CD3ζ; 2A, 2A self-cleaving peptide; tCD34, truncated CD34; shRNA MICA/B-CD3ζ , shRNA duplex targeting MICA/B and CD3ζ.

Article Snippet: MICA coating was performed by incubating recombinant human MICA (rhMICA-Fc, 1300-MA-050, R&D systems) overnight at 4°C while CD19 coating was performed as described elsewhere ( ).

Techniques: Construct, shRNA

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: Cytokine secretion, cytolytic activity and proliferation of CD19/NKG2DL tandem CAR T-cells co-cultured with CD19+ and CD19- Nalm-6 cells. (A) Expression of CD19 and NKG2DL in Nalm-6 and Nalm-6 CD19 KO cells. (B) Expression of individual NKG2DL using antibodies directed against MICA, MICB, ULBP1, ULBP3 or ULBP2-5-6 (grey histograms) vs autofluorescence (white histograms). (C) Secretion of IFN-γ, TNF-α and IL-2 cytokines after a 24-hour co-culture at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells. (D) Cytolytic activity of CAR T-cells against Nalm-6 and Nalm-6 CD19 KO cells at 1:1 and 0.1:1 E:T ratio. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (E) Representative experiment showing CTV histograms of CAR T-cells after 4 days of co-culture without cancer cells or at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells (4 individual experiments were performed on 4 different donors). Adjusted P values (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data presented as means (SD) of n=5. Each symbol denotes a different PBMC donor.

Article Snippet: MICA coating was performed by incubating recombinant human MICA (rhMICA-Fc, 1300-MA-050, R&D systems) overnight at 4°C while CD19 coating was performed as described elsewhere ( ).

Techniques: Activity Assay, Cell Culture, Expressing, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: Cytokine secretion of CD19/NKG2DL tandem CAR T-cells upon stimulation with coated CD19 or coated MICA. IFN-γ secretion after a 24-hour exposure with increasing concentrations of (A) coated rhCD19-Avi or (B) coated rhMICA-Fc.

Article Snippet: MICA coating was performed by incubating recombinant human MICA (rhMICA-Fc, 1300-MA-050, R&D systems) overnight at 4°C while CD19 coating was performed as described elsewhere ( ).

Techniques: