polyclonal anti rae 1γ antibodies  (R&D Systems)

Journal: Experimental Hematology & Oncology

Article Title: Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation

doi: 10.1186/s40164-022-00289-8

Figure Lengend Snippet: CML-RAE-1γ-Dex were isolated from DCs loaded with lysates of RAE-1γ-expressing CML cells. a Flow cytometric analysis of the expression of RAE-1γ in parental and transfected CML cells. b Flow cytometric analysis of the percentages of cells with positive expression of CD11c, CD80, CD86 and MHC class I/II before and after cytokine induction. c The morphology of Dex was visualized by TEM. Scale bar, 100 nm. d The particle size of Dex was measured by NTA. e The expression of HRS, Alix, TSG101, Cytochrome C, and RAE-1 in DCs and various types of Dex was measured by western blot analysis. f NK cells were incubated with Dex labeled with PKH26 (red). After fixation, the nuclei were stained with DAPI (blue). Finally, representative images were obtained by confocal microscopy. Red fluorescent particles represent PKH26-labeled Dex. Scale bar, 5 μm. Values are presented as the mean ± SD. *p < 0.05, ***p < 0.001

Article Snippet: Cells were stained with a PE-conjugated anti-RAE-1γ antibody (eBioscience, CA, USA) or PE-conjugated rat IgG2b kappa antibody (eBioscience, USA) and then subjected to flow cytometric detection.

Techniques: Isolation, Expressing, Transfection, Western Blot, Incubation, Labeling, Staining, Confocal Microscopy

Journal: Experimental Hematology & Oncology

Article Title: Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation

doi: 10.1186/s40164-022-00289-8

Figure Lengend Snippet: CML-RAE-1γ-Dex promoted NK-cell activation and proliferation. a-e Flow cytometric analysis of CD69 ( a ), CD137 ( b ), CD107a ( c ), perforin ( d ) and GzmB ( e ) expression in NK cells after exposure to exosomes for 6 h. f NK-cell proliferation after 72 h of culture with Dex was evaluated by flow cytometric analysis. *p < 0.05 vs. the PBS group. g , h NK cells were treated with exosomes for 6 h and then seeded in ELISPOT plates overnight. ELISPOT assays were employed to compare TNF-α and IFN-γ production by activated NK cells among the groups. i A cytotoxicity assay was used to measure the killing ability of effector NK cells prestimulated with exosomes for 6 h against BP210 (left) or BP210-T315I (right) target cells. Cytotoxic activity was detected at the effector-to-target (E:T) ratios of 12.5:1, 25:1, 50:1, and 100:1, respectively. Values are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the mock group

Article Snippet: Cells were stained with a PE-conjugated anti-RAE-1γ antibody (eBioscience, CA, USA) or PE-conjugated rat IgG2b kappa antibody (eBioscience, USA) and then subjected to flow cytometric detection.

Techniques: Activation Assay, Expressing, Enzyme-linked Immunospot, Cytotoxicity Assay, Activity Assay

Journal: Experimental Hematology & Oncology

Article Title: Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation

doi: 10.1186/s40164-022-00289-8

Figure Lengend Snippet: Anti-tumor immune responses induced by BP210-T315I-RAE-1γ-Dex in the BCR-ABL T315I -induced CML mouse model. a Peripheral WBC counts were analyzed. b , c The weights of the liver ( b ) and spleen ( c ) were recorded. d Wright’s staining showed abnormal leukemic cells (arrows) in bone marrow, spleen and liver tissues. Scale bar, 10 μm. e Histological sections stained with H&E showed the infiltration of leukemic cells (arrows). Scale bar, 10 μm. f The BCR-ABL oncoprotein was identified with a fluorophore-labeled antibody in the liver and spleen and observed under a microscope. Scale bar, 25 μm. g The survival rates of tumor-bearing mice were measured by Kaplan–Meier methods. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cells were stained with a PE-conjugated anti-RAE-1γ antibody (eBioscience, CA, USA) or PE-conjugated rat IgG2b kappa antibody (eBioscience, USA) and then subjected to flow cytometric detection.

Techniques: Staining, Labeling, Microscopy

Journal: Experimental Hematology & Oncology

Article Title: Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation

doi: 10.1186/s40164-022-00289-8

Figure Lengend Snippet: Therapeutic effects of BP210-RAE-1γ-Dex in a murine BP210 tumor model. a The average of the maximum WBC count of every group was calculated. b , c The liver ( b ) and spleen ( c ) were harvested and weighed. d Representative images of tissue smears stained with Wright’s staining are shown. The black arrows point to abnormal cells invading tissues. Scale bar, 10 μm. e Hematoxylin and eosin-stained liver and spleen tissues were used to confirm pathological characteristics. The arrows indicate leukemic cells. Scale bar, 10 μm. f Immunofluorescence assays were conducted to determine the level of BCR-ABL expression. Scale bar, 25 μm. g Kaplan–Meier survival analysis was performed to analyze the survival times of treated mice. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cells were stained with a PE-conjugated anti-RAE-1γ antibody (eBioscience, CA, USA) or PE-conjugated rat IgG2b kappa antibody (eBioscience, USA) and then subjected to flow cytometric detection.

Techniques: Staining, Immunofluorescence, Expressing

Journal: Experimental Hematology & Oncology

Article Title: Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation

doi: 10.1186/s40164-022-00289-8

Figure Lengend Snippet: Long-term immunological effect of CML-RAE-1γ-Dex in vivo. Blank A and Blank B were inoculated with BP210 and BP210-T315I cells, respectively. a The maximum WBC count was recorded. b , c The weights of the liver ( b ) and spleen ( c ) in the four groups were measured. d Wright’s staining was carried out to examine leukemic cell infiltration in tissues. Microscopy images show the typical morphology of leukemic cells (arrows). Scale bar, 10 μm. e Tissue sections from the liver and spleen were stained H&E and showed infiltrated leukemic cells. Scale bar, 10 μm. f Cross-sectional microscopy images showing immunofluorescence staining were used to evaluate the BCR-ABL protein in bone marrow, spleen and liver tissues. Scale bar, 25 μm. g The Kaplan–Meier survival method was conducted to compare differences in survival among groups. **p < 0.01, ***p < 0.001

Article Snippet: Cells were stained with a PE-conjugated anti-RAE-1γ antibody (eBioscience, CA, USA) or PE-conjugated rat IgG2b kappa antibody (eBioscience, USA) and then subjected to flow cytometric detection.

Techniques: In Vivo, Staining, Microscopy, Immunofluorescence

Journal: Experimental Hematology & Oncology

Article Title: Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation

doi: 10.1186/s40164-022-00289-8

Figure Lengend Snippet: Schematic illustration of CML-RAE-1γ-Dex-based cancer immunotherapy. CML-RAE-1γ-Dex augment NK-cell and CD4 + and CD8 + T-cell activation in an NKG2D-dependent manner and amplify the immune response by promoting NK/T-cell proliferation. Created with BioRender.com

Article Snippet: Cells were stained with a PE-conjugated anti-RAE-1γ antibody (eBioscience, CA, USA) or PE-conjugated rat IgG2b kappa antibody (eBioscience, USA) and then subjected to flow cytometric detection.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: The Tumorigenicity of Mouse Embryonic Stem Cells and In Vitro Differentiated Neuronal Cells Is Controlled by the Recipients' Immune Response

doi: 10.1371/journal.pone.0002622

Figure Lengend Snippet: (A) Mean of specific lysis and standard deviation (SD) of triplicates of ES or in vitro differentiated cells at three effector∶target (E∶T) ratios. Effector cells were lymphocytes obtained from spleens of 10 individual LOU/c rats by density gradient centrifugation on Biocoll. Results obtained with lymphocytes from female rats are indicated by open symbols and from male rats by filled symbols. (B) The mean specific lysis and SD of triplicates of ES, YAC-1, and RMA target cells at three effector∶target (E∶T) ratios by freshly isolated NK cells or NK cell-depleted splenocytes from naïve LOU/c rats are shown. The NK cell enriched fraction contained 86% and the NK cell depleted fraction 3% NKR-P1A-positive cells as determined by flow cytometry. The results are representative for 3 independent experiments. (C) The mean specific lysis and SD of triplicates of ES and YAC-1 cells at three effector∶target (E∶T) ratios by freshly isolated (open symbols) or 3 days in vitro with 1000 U/ml IL-2 stimulated (closed symbols) mouse and rat NK cells are shown. The NK cell-enriched fractions contained more than 80% NK cells as determined by flow cytometry. The results shown are representative for 3 independent experiments. (D) The expression of MHC class I molecules on ES and differentiated cells was analyzed by flow cytometry using anti-H2K b and anti-H2D b Abs (full lines). The stainings with the isotype control are shown by the dotted lines. RMA cells (H2 b ) served as positive control for these antibodies. NKG2D ligands were stained with a recombinant mouse NKG2D-Fc chimeric protein and a FITC-conjugated goat anti-human IgG antibody as secondary reagent (full lines). Stainings with the secondary reagent only are shown by the dotted lines. YAC-1 cells (H2 a ) served as positive controls for these stainings. The results shown are representative for more than 3 independent experiments. (E) ES and as positive control YAC-1 cells were stained with the NKG2D-Fc chimeric protein and with mAbs specific for the NKG2D ligands H60, MULT-1, and RAE-1. The mean+SD of the proportion of positive cells and the mean fluorescence intensity+SD determined in 6 independent experiments is shown. (F) The mean inhibition+SD of specific lysis of ES and YAC-1 cells by soluble mouse NKG2D is shown as determined in 3 experiments. The mean of specific lysis of the target cells by rat NK cells at an effector to target ratio of 20∶1 was determined as described above and adjusted to 100%. For inhibition of lysis a soluble mouse NKG2D protein was added to the test at a concentration of 3 µg/ml. The relative lysis of the target cells exposed to NKG2D was calculated. (G) The mean inhibition+SD of specific lysis of ES cells by soluble mouse NKG2D and various inhibitory antibodies against NKG2D ligands is shown. The mean of specific lysis of the target cells by IL-2 activated mouse NK cells at an effector to target ratio of 10∶1 was determined as described above and adjusted to 100%. For inhibition of lysis a soluble mouse NKG2D protein or the indicated mAbs were added to the test at a concentration of 3 µg/ml. The relative lysis of the target cells exposed to NKG2D or the mAbs was calculated.

Article Snippet: Similarly, antibodies which block the binding of NKG2D to RAE-1β (MAB17581, clone 199205, rat IgG 2a , R&D Systems), RAE-1α and RAE-1γ (MAB 1758, clone 199215, ratIgG 2a , R&D Systems), and H60 (clone 205326, rat IgG 2a , R&D Systems) were used for blocking experiments.

Techniques: Lysis, Standard Deviation, In Vitro, Gradient Centrifugation, Isolation, Flow Cytometry, Expressing, Control, Positive Control, Staining, Recombinant, Fluorescence, Inhibition, Concentration Assay