Journal: Oncotarget

Article Title: Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children

doi: 10.18632/oncotarget.19821

Figure Lengend Snippet: Surface expression of human NKG2D ligands (ULBP-2/5/6, MIC A/B) and human ErbB2 (HER2/neu) on tumor cell lines

Article Snippet: Blocking of NKG2D was achieved by adding 30 μg of human NKG2D (CD314) antibody (R&D Systems, Minneapolis, MN, USA) per 4 x 10 6 CIK cells.

Techniques: Expressing

Journal: Oncotarget

Article Title: Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children

doi: 10.18632/oncotarget.19821

Figure Lengend Snippet: As a proof of concept, the NKG2D- and ErbB2-mediated cytotoxic capacity of WT and genetically modified (ErbB2-CAR and mock-vector) CIK cells was analyzed by europium release assay against (A) ErbB2-negative, NKG2D ligand-positive acute myeloid leukemia target cell line THP-1, (B) ErbB2-overexpressing, NKG2D ligand-positive breast carcinoma cell line MDA-MB-453, and (C) ErbB2-negative, NKG2D ligand-negative allogeneic PBMCs. The NKG2D-restricted cytotoxicity of CIK cells was not impaired by the genetic modification, shown by the effective cytolysis of ErbB2-negative, NKG2D ligand-positive THP-1 cells by WT, mock- vector, and ErbB2-CAR CIK cells. As expected, the cytotoxic capacity of ErbB2-CAR CIK cells against ErbB2-overexpressing target cells was significantly increased compared with mock-vector and WT CIK cells, and was low against non-malignant ErbB2-negtive, NKG2D ligand-negative target cells. (D) Stably transfected mouse renal carcinoma (Renca) cells Renca-lacZ and Renca-lacZ/erbB-2 were used for evaluation of specific ErbB2-targeting by the killer cells. WT CIK cells did not lyse Renca-lacZ and Renca-lacZ/erbB-2 cells. ErbB2-CAR-CIK cells also showed no cytotoxic capacity against Renca-lacZ cells, but displayed highly significant increased cytotoxicity against Renca-lacZ/erbB-2. (E) NKG2D mediated cytotoxicity on the cytotoxic capacity of ErbB2-CAR CIK cells against ErbB2 expressing cell line TE671 was evaluated by europium release assay after blocking of the NKG2D receptor on ErbB2-CAR CIK cells. Blocking significantly reduced the efficacy of CAR-modified CIK cells against the tumor target, suggesting that the combination of NKG2D-mediated and ErbB2-specific killing in CAR-CIK cells may reduce the impact of tumor escape mechanisms and increase killing of tumors.

Article Snippet: Blocking of NKG2D was achieved by adding 30 μg of human NKG2D (CD314) antibody (R&D Systems, Minneapolis, MN, USA) per 4 x 10 6 CIK cells.

Techniques: Genetically Modified, Plasmid Preparation, Release Assay, Modification, Stable Transfection, Transfection, Expressing, Blocking Assay

Journal: OncoImmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402x.2015.1123367

Figure Lengend Snippet: Figure 2. ADAM10 silencing leads to decreased shedding of NKG2D-L. KMH2 and L428 cells

Article Snippet: In some experiments, TGFβ (recombinant human TGFβ1, R&D) was added to either NK or γδ T cells at 10ng/ml for 12 h, in the presence or absence of saturating amounts of the anti TGFβ mAb (1μg/ml, clone 1D11, R&D), before staining with the anti-NKG2D mAb (MAB139, R&D System).

Techniques:

Journal: OncoImmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402x.2015.1123367

Figure Lengend Snippet: Figure 3. ADAM10 inhibitors reduce the shedding of NKG2D-L by HL cell lines and maintain the

Article Snippet: In some experiments, TGFβ (recombinant human TGFβ1, R&D) was added to either NK or γδ T cells at 10ng/ml for 12 h, in the presence or absence of saturating amounts of the anti TGFβ mAb (1μg/ml, clone 1D11, R&D), before staining with the anti-NKG2D mAb (MAB139, R&D System).

Techniques:

Journal: OncoImmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402x.2015.1123367

Figure Lengend Snippet: Figure 4. Exposure to ADAM10 inhibitors increases the sensitivity of HL cell lines to NKG2D-

Article Snippet: In some experiments, TGFβ (recombinant human TGFβ1, R&D) was added to either NK or γδ T cells at 10ng/ml for 12 h, in the presence or absence of saturating amounts of the anti TGFβ mAb (1μg/ml, clone 1D11, R&D), before staining with the anti-NKG2D mAb (MAB139, R&D System).

Techniques:

Journal: Nature Communications

Article Title: Aiolos restricts the generation of antigen-inexperienced, virtual memory CD8 + T cells in mice

doi: 10.1038/s41467-025-67540-8

Figure Lengend Snippet: a Heatmap showing differences in different cytokine receptors between WT and Aiolos-deficient T VM samples. b Schematic of the in vitro treatment of WT and Aiolos-deficient splenic CD8 + T cells, created in Microsoft PowerPoint. Bulk CD8 + T cells were isolated from the spleens of WT and Aiolos-deficient mice and treated with rmIL-12, rmIL-18, and IL-15C (IL-15/IL-15Rα complex) for 30 min (pY-STAT5 and pY-STAT4) or 16 h (IFN-γ, granzyme B and NKG2D) with protein transport inhibitor (PTI) added 4 h prior to harvesting the cells for flow cytometry analysis. Analysis of frequency (%) and median fluorescence intensity (MFI) fold change for ( c ) pY-STAT5 and ( d ) pY-STAT4 between WT and Aiolos-deficient T VM cells, relative to WT. Data representative of 5 independent experiments, n = 5/group, mean ± SEM; two-sided, unpaired Student’s t-test, *p ≤ 0.05 and **p ≤ 0.01. Frequency (%) and MFI fold change for e IFN-γ, f granzyme B, and g NKG2D between WT and Aiolos-deficient T VM cells, relative to WT. Data represents 6 independent experiments, n = 6/group for e , f ; and 5 independent experiments with n = 5/group for NKG2D. Data presented as mean ± SEM; two-sided, unpaired Student’s t-test, *p ≤ 0.05, ***p ≤ 0.001 and ****p ≤ 0.0001. h Percent (%) dead YAC-1 (target) cells when co-cultured with CD8 + T cells in the presence of cytokines (IL-12, Il-18, IL-15C), with or without anti-NKG2D antibody, data from 5 independent experiments with n = 5/condition. Data presented as mean ± SEM; two-way ANOVA with Tukey’s multiple comparisons test *p ≤ 0.05 and ****p ≤ 0.0001. Source data are provided as a file.

Article Snippet: For NKG2D blocking, cells were treated with anti-NKG2D antibody (10 μg/mL, clone: HMG2D; catalog # BE0111, BioXCell) or isotype control (catalog # BE0091, BioXCell) for 30 min prior to YAC-1 cells addition.

Techniques: In Vitro, Isolation, Flow Cytometry, Fluorescence, Cell Culture

Journal: Aging Cell

Article Title: Targeting TGF ‐β signaling, oxidative stress, and cellular senescence rescues osteoporosis in gerodermia osteodysplastica

doi: 10.1111/acel.14322

Figure Lengend Snippet: Gorab ‐deficient long bones are more sensitive to TGF‐β inhibition than the Gorab ‐expressing axial skeleton. (a) Treatment scheme for the Gorab Prx1 mice. 4 weeks old Gorab Prx1 mice were injected with 10 mg/kg body weight of either control IgG or 1D11 thrice a week by intraperitoneal injection for 8 weeks. (b) 3D rendering and (c) Representative microCT images showing the proximal tibia of control and Gorab Prx1 mice treated with control IgG or 1D11. Scale bar = 500 μm. Quantitation of (d) percentage of trabecular bone volume (BV/TV); (e) comparison of rescue effects in vertebrae, tibia, and femur; (f) trabecular number (Tb.N); (g) trabecular thickness (Tb.Th); and (h) trabecular separation (Tb.Sp) in proximal tibia of control IgG and 1D11 treated control and Gorab Prx1 mice ( N = 7). Scanned and analyzed with Scanco μCT40 at 10 μm. Histomorphometric analysis at the proximal tibia secondary spongiosa in control and 1D11 treated Gorab Prx1 mutants for (i) number of osteoblasts per bone perimeter (N.Ob/B.Pm); and (j) number of osteoclasts per bone perimeter (N.Oc/B.Pm). ( N = 5–6). * p < 0.05, ** p < 0.01, ***p < 0.001, ns, no statistically significant difference (unpaired Welch's t ‐test).

Article Snippet: 1D11 (Cat#BP0057, Bioxcell, New Hampshire, USA) treatment was done by intraperitoneal injection of 10 mg antibody per kg body weight thrice a week from 4 to 12 weeks of age.

Techniques: Inhibition, Expressing, Injection, Control, Quantitation Assay, Comparison

Journal: Aging Cell

Article Title: Targeting TGF ‐β signaling, oxidative stress, and cellular senescence rescues osteoporosis in gerodermia osteodysplastica

doi: 10.1111/acel.14322

Figure Lengend Snippet: Elevated TGF‐β signaling caused increased Nox4 expression and elevated oxidative stress in GO. (a) 1D11 treatment inhibited the Nox4 upregulation in Gorab Prx1 mice ( N = 5) (b) Representative images and (c) quantitation of Mitosox intensity in GO patient fibroblasts ( N = 3–6) treated with TGF‐β inhibitor SB431542. (d) SB431542 reduced Nox4 expression in GO patient fibroblasts ( N = 3–4). (e) Coculturing of control fibroblasts with GO patient fibroblasts caused upregulation of mitosox signal in the control fibroblasts, which can be prevented by SB431542 ( N = 4–5). (f) Increased in mitosox signal in the bone marrow of tibiae of 4 weeks old Gorab Prx1 (N = 4) in vivo. (g) Knockdown of gorab in zebrafish by TALEN resulted in upregulation of serpine1 and nox4 expression. (h) Representative picture and (i) quantitation of Mitosox positive cells by flow cytometry in gorab TALEN zebrafish embryos. * p < 0.05, ** p < 0.01, ns, no statistically significant difference (unpaired Welch's t ‐test). Scale bar = 100 μm.

Article Snippet: 1D11 (Cat#BP0057, Bioxcell, New Hampshire, USA) treatment was done by intraperitoneal injection of 10 mg antibody per kg body weight thrice a week from 4 to 12 weeks of age.

Techniques: Expressing, Quantitation Assay, Control, In Vivo, Knockdown, Flow Cytometry

Journal: Aging Cell

Article Title: Targeting TGF ‐β signaling, oxidative stress, and cellular senescence rescues osteoporosis in gerodermia osteodysplastica

doi: 10.1111/acel.14322

Figure Lengend Snippet: Evidence of DNA damage and cellular senescence in GORAB‐deficient cells and bone tissue. (a) Percentage of cells with 53BP1 nuclear foci in GO patient fibroblasts ( N = 4). (b) Representative images of nuclear 53 bp1 staining in the secondary spongiosa of proximal tibia in 4 weeks old Gorab Prx1 mice. Scale bar = 50 μm. (c) Increase in γH2AX expression in femur diaphysis of 4 weeks old Gorab Prx1 mice ( N = 3). (d) Relative expression of Cdkn2a in control and Gorab Prx1 mice treated with control IgG or 1D11 ( N = 5). (e) Representative images and quantitation of senescence associated beta‐galactosidase (SA‐βGal) positive cells in GO patient fibroblasts ( N = 4). Scale bar =20 μm. (f) BrdU positive cells in Control and GO patient fibroblasts after 24 h BrdU labeling ( N = 4). * p < 0.05, ** p < 0.01, ns, no statistically significant difference (unpaired Welch's t ‐test).

Article Snippet: 1D11 (Cat#BP0057, Bioxcell, New Hampshire, USA) treatment was done by intraperitoneal injection of 10 mg antibody per kg body weight thrice a week from 4 to 12 weeks of age.

Techniques: Staining, Expressing, Control, Quantitation Assay, Labeling