Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 2. Expression of CD70 on solid tumors and confirmation of target expression and CAR T cell transduction efficiency. (A) Gene expression of CD70 in various solid tumors compared to normal matched tissues was analyzed using data from The University of Alabama at Birmingham Cancer data analysis (UALCAN) and The Cancer Genome Atlas program (TCGA). B-C) Top: confirmation of CD70 (target) expression on cancer cells post transduction – (B) glioblastoma (GBM) and (C) osteosarcoma (OS) models - and Bottom: transduction of CAR construct in (B) C57BL/6 mice - derived T cells (CAR T Kr158B ) and (C) in Balb/c mice - derived T cells (CAR T K7M2 ) as indicated by GFP reporter. On average, (D) the mean CD70 expression was found to be 73% for GBM and 99% for OS models. (E) The mean transduction efficiency was 66% for CAR T Kr158B and 60% for CAR T K7M2 . The names of the cell lines and tumor models, GBM for Kr158B and OS for K7M2, will be used interchangeably in the study. Number of biological replicates (N) is indicated on the graphs.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Expressing, Transduction, Gene Expression, Construct, Derivative Assay

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 3. CAR T cell exhibit directed motion towards the target tumor. A) Representative confocal snapshots showed CD70-specific CAR T cells (green) navigating through supported COL1-LLS RhB microgels (red) and infiltrating the target GBM tumor (white), with blue arrows indicating the paths of CAR T cell migration. (B, D) Mean velocity of CAR T cells co-cultured with CD70-positive tumors and the wild-type (WT) control were quantified for GBM (B) and OS (D) tumor models. The number of tracks (n) and biological replicates (N) were indicated on the plots, and statistical significance was determined using an unpaired two-tailed Student’s t-test with p values indicated on the plots. (C, E) Tumor-infiltrating CAR T cells were quantified as a percentage of total CAR T cells on average from 0 to 72 h for GBM (C) and OS (E) tumors, displayed as box plots showing 25th and 75th percentiles, median, and mean, with whiskers representing the minimum to maximum observations. An unpaired two-tailed Student’s t-test was performed for statistical analysis, and p values were indicated on the plots. (F) Top panel: maximum intensity z projection showed snapshots of CAR T – GBM tumor interaction at 0, 24, and 72 h. Middle panel: the segmentation of CAR T with colors indicating individual CAR T cells at each frame. Bottom panel: maximum intensity projection of segmented CAR T cell velocity tracks over time with color-coded velocity gradient, revealing accumulation of CAR T cells inside the tumor. The segmentation employed a deep learning-based method, as discussed in the methods section, and the cells were tracked using a Linear Assignment Problem (LAP) tracker with maximum frame-to-frame linking and allowable track segment gap closing of 150 μm. (G) Evidence of chemotaxis and upregulation in migratory pathways for CAR T cells co-cultured with their target tumors was demonstrated for GBM (top panel) and OS (bottom panel) tumor models. Notably, evidence of immune-mediated cytotoxic function was shown through IFN γ detection. Error bars represent standard deviation.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Migration, Cell Culture, Control, Two Tailed Test, Chemotaxis Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 4. CAR T expansion, activation, and killing. (A) Representative confocal time-lapse images of the FITC channel (green) illustrating immune activation, expansion, and killing of the target tumors. CAR T cell clustering and rapid expansion were observed in almost all conditions with efficient anti-tumor activity. White arrows indicate CAR T cell clusters. (B, C) The number of CAR T cell clusters rapidly increased within the first 24 h and remained steady for over 72 h in (B) GBM and (C) OS models. (D, E) Cluster size in all co-cultures with target tumors steadily increased over time, but not in WT controls. (F, G) Expansion of CAR T cell clusters revealed an inverse correlation with tumor size. n = 3 for all CD70 pos samples and n = 2 for all WT samples. (H, I) Endpoint flow cytometry data measuring CAR T cell expansion after 96 h of co-culture with cancer cells in the 2D assay for both tumor models. (J, K) A comparison in IFN γ secretion (pg/mL) between 2D and 3D cultures for (J) GBM and (K) OS. Error bars indicate SD and the number of biological replicates is n = 2. (L) Top row: confocal 3D snapshots of GBM tumors after co-cultured with CAR T cells (E:T = 1:4) for 24 h, showing highly tortuous tumor margins in CD70 pos tumors compared to WT counterparts. Bottom row: cross-section view (at z depth: 70 μm) of the sample on the top row revealing infiltrating CAR T cells within the tumor mass. (M) Measurement of tumor tortuosity factors revealed more than 3-fold change for the CD70 pos tumors within the first 48 h of co-culture. The tumor tortuosity factor was calculated as the ratio between the perimeter of the tumor outline and the perimeter of a circle with the same pixel area. Data was obtained from GBM samples ( n = 3) at an initial E:T ratio of 1:2 and from osteosarcoma (K7M2) samples ( n = 3) at an initial E:T ratio of 1:1. Error bars represent standard deviation and biological samples ( n = 3) for each group were performed unless indicated otherwise.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activation Assay, Activity Assay, Cytometry, Co-Culture Assay, Two-Dimensional Assay, Comparison, Cell Culture, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 5. Quantification of CAR T–tumor interactions . Top rows: confocal timelapse images at 0, 24, 48, and 72 h showing dynamic immune-cancer interactions and antitumor activities of CAR T cells in (A) GBM and (B) OS tumor models. The bottom row of each group shows a digital reconstruction of the confocal data with distinct segmentation of tumor and immune cell populations. The initial CAR T to tumor cell ratio or effector-to-tar get (E:T) ratio is indicated on the graph. (C, D) Quantification of CAR T–tumor interactions revealing an inverse correlation between tumor size and infiltrating CAR T cells for (C) GBM and D) OS tumors. The percentage of tumor size (left axis) and CAR T cells infiltrating the tumor (right axis) were normalized to the original tumor size at time 0. (E, F) Killing rates over time were calculated as derivatives from C and D, respectively. (G, H) The measured CAR T to cancer cell area ratios (E:T) as a function of time. The E:T ratios dynamically changed over time, while for WT tumors, the ratios remained constant. (I) RNA sequencing data from single CAR T cells isolated from a 3D co-culture with target CD70 pos versus WT tumors. All genes shown in the results have a statistical significance of p ≤0.05. Error bars represent standard deviation and biological samples n = 3 for all CD70 pos samples and n = 2 for all WT samples.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: RNA Sequencing, Isolation, Co-Culture Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 7. Sensitivity of anti-tumor activity to various CAR T: cancer cell (E:T) ratios. (A,B) confocal time-lapse images of CAR T – tumor co-culture in iVITA at different E:T ratios. (C,D) show the digital image reconstruction of confocal data quantifying bulk tumor mass, migrating single cancer cells, immune cells, and immune cell clusters, and killing activity over time. (A,C) GBM CD70 pos tumors and (B,D) OS CD70 pos tumors were co-cultured with their respective CAR T cells at different concentrations corresponding to E:T of 1:4, 1:2, and 4:1 for the GBM model and E:T of 1:4, 1:1, and 4:1 for the OS model. (E–H) CAR T expansion as a function of initial E:T seeding ratios. (E,F) CAR T expansion on average in both models from 0 to 72 h. For each seeding E:T, CAR T expansion at each time point was normalized to the CAR T at time 0, and the average was calculated for all frames. G,H) the number of CAR T clusters counted every 1.5 h for 72 h for each group. The box plots display 25th and 75th percentiles, a line at the median a plus sign at the mean, from the minimum to the maximum observation. (I,J) Quantification of tumor size over time at the initial E:T = 1:4, 1:2, and 4:1 for I) GBM model and at E:T = 1:4, 1:1, and 4:1 for (J) OS model. (K,L) The E:T ratio dynamically changed over time. The CAR T expansion and tumor-killing were presented by the exponential increase of E:T ratios. (M,N) tumor killing rates were calculated as derivatives from (I) and (J), respectively. Error bars represent standard deviation. Statistical analysis was performed using Ordinary One-Way ANOVA. ( n = 3 unless indicated otherwise, ∗∗= p < 0.01, and ∗∗∗∗= p < 0.0 0 01).

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activity Assay, Co-Culture Assay, Cell Culture, Standard Deviation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: The CD70 SNP (single nucleotide polymorphism) associates with vascular disease in PheWAS (Phenome-Wide Association Study) and loss of CD70 impairs endothelial wound recovery. Using an unbiased phenome-wide analysis of SNPs of CD70, rs11458827 showed a significant association with vascular disease phenotypes in humans (* P <9.4×10 −5 corrected for multiple comparisons) ( A ). Gene map showing rs1145887 is an intronic SNP that occurs in a region with acetylated histone H3 marks (H3K27Ac) and within a Dnase I hypersensitivity domain, typical of a regulatory element ( B ). Human pulmonary artery endothelial cells treated with CD70 siRNA demonstrated reduced wound recovery after scratch compared with control cells ( C and D ). *** P <0.001.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques: Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown reduced cellular NO levels and eNOS (endothelial nitric oxide synthase) expression. Real-time intracellular levels of NO in human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs) were measured following stimulation with 30 μM ATP. A and B , Show representative averaged time curves for baseline fluorescence, ATP treatment, and washout, with greater fluorescence change (%ΔF intensity ) correlating with greater NO levels. NO levels were reduced following siRNA-mediated CD70 (siCD70) knockdown compared with control-siRNA-treated cells (siCtrl) for HAECs and HPAECs ( C and G ). For HAECs, data represent the median of 89 cells for siCtrl and 129 cells for siCD70, collected over 3 independent experiments. For HPAECs, data represent the median of 105 cells for siCtrl and 106 cells for siCD70 collected over 4 independent experiments. Protein levels ( D and H ) mRNA ( E and I ) for eNOS were decreased after treatment with siCD70 compared with siCtrl. Protein expression of the eNOS chaperone Hsp90 (heat shock protein 90) was reduced with siCD70 treatment in HAECs and HPAECs ( F and J ). For eNOS and Hsp90, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For eNOS mRNA, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques: Knockdown, Expressing, Fluorescence, Control, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: 3-Nitrotyrosine and cellular superoxide are increased and cGMP levels are decreased after CD70 knockdown. Human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs) were treated with control (siCtrl) vs CD70-directed siRNA (siCD70). Immunoblotting for 3-nitrotyrosine adducts revealed two major species in endothelial cells: a higher molecular weight band corresponding to ≈ 45 kD and a lower molecular weight band corresponding to ≈ 20 kD ( A ). Both bands were increased in siCD70 cells as compared with siCtrl in HAECs ( B and C ) and HPAECs ( D and E ). Total intracellular cGMP levels were likewise decreased in siCD70 cells compared with siCtrl ( F and G ). CD70-knockdown HPAECs loaded with dihydroethidium (DHE) demonstrated increased basal fluorescence in the expected spectrum (excitation 488 nm, emission 588 nm) for 2-hydroethidium, the product of superoxide’s reaction with DHE ( H ). Cells loaded with DHE and then subsequently stimulated with menadione showed increased fluorescence in the setting of CD70 knockdown; furthermore, the reduction in fluorescence with PEG-SOD (pegylated superoxide dismutase) was greater in these cells compared with control cells, supporting the specificity of this increased fluorescence to enhanced superoxide levels. Nitrite/nitrate levels are unchanged in control vs CD70-knockdown cells ( J ). For 3-nitrotyrosine, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques: Knockdown, Control, Western Blot, Molecular Weight, Fluorescence

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: Reduced CD70 expression leads to increased cytosolic and caveolar hydrogen peroxide levels in endothelial cells. Human pulmonary artery endothelial cells (HPAECs) were treated with control (siCtrl) vs CD70-directed siRNA (siCD70) and subsequently transfected with the genetically encoded biosensor Hyper7.2 for real-time intracellular monitoring of hydrogen peroxide (H 2 O 2 ) in the cytosol ( A , B , and E ) and plasmalemmal caveolae ( C , D , and F ). Hydrogen peroxide was assessed by the ratio of fluorescence emission following excitation at 490 nm and 420 nm normalized to baseline fluorescence (normalized R/R 0 ). Following treatment with 1 μM auranofin, cells were treated with 25 μM H 2 O 2 as a positive control. The rate of rise of intracellular H 2 O 2 , calculated as the slope of the linear portion of the auranofin-treatment curve over the final 300 seconds of drug treatment (ΔF intensity /s), was greater in siCD70-treated cells compared with control cells for both cytosolic ( A and B ) and caveolar H 2 O 2 ( C and D ). Treatment with histamine similarly led to greater cytosolic ( E ) and caveolar H 2 O 2 ( F ) levels in CD70-knockdown cells compared with control. All panels were analyzed by unpaired Mann-Whitney U test. *** P <0.001, **** P <0.0001.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques: Expressing, Control, Transfection, Fluorescence, Positive Control, Knockdown, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown is associated with enhanced NOX (NADPH oxidase) expression. Following CD70 knockdown (siCD70), human pulmonary artery endothelial cells (HPAECs) demonstrated increased NOX (NADPH oxidase) 1 transcript ( A ) and protein ( B ) expression, and NOXA1 mRNA ( C ). Transcript levels of gp91phox, the catalytic subunit of NOX2, were enhanced ( D ), along with the corresponding protein levels of gp91phox ( E ). For NOX1 and gp91phox protein levels, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For NOX1 and NOXA1, mRNA levels are normalized to β-actin mRNA, and for gp91phox, mRNA levels are normalized to RNA polymerase II subunit A mRNA; data are expressed as fold change compared with siCtrl. Data presented as mean±SE.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques: Knockdown, Expressing, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: Reduction in CD70 alters endothelial anti-oxidant enzyme expression. In comparison to control-treated cells (siCtrl), CD70-knockdown cells (siCD70) showed enhanced SOD1 (superoxide dismutase 1) mRNA ( A and H ) and protein ( B and I ) levels in both human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs). Catalase protein expression was reduced in siCD70 samples ( C and J ); conversely, GPx-1 (glutathione peroxidase 1) mRNA ( D and K ) and protein levels ( E and L ) were elevated. Total intracellular GPx activity was augmented after treatment with siCD70 ( F and M ). Nrf2 (nuclear factor-erythroid factor 2-related factor 2) transcript levels were elevated in siCD70-treated cells ( G and N ). For SOD1, catalase and GPx-1 protein levels, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For SOD1, GPx-1, and Nrf2 mRNA levels, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques: Expressing, Comparison, Control, Knockdown, Activity Assay, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown leads to increased mitochondrial hydrogen peroxide levels. Following treatment with CD70 siRNA, human pulmonary artery endothelial cells (HPAECs) showed faster accumulation of hydrogen peroxide in the mitochondrial matrix in response to treatment with 1 mmol/L auranofin ( A and B ). SOD2 transcript ( C and E ) and protein ( D and F ) expression were correspondingly increased after CD70 knockdown compared with control cells in human aortic endothelial cells (HAECs) and HPAECs. Representative Western blots are shown for SOD2 along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For SOD2 mRNA, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques: Knockdown, Expressing, Control, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: Working model for changes in endothelial cell signaling induced by CD70 ablation. Loss of CD70 leads to reduced eNOS (endothelial nitric oxide synthase) protein and decreased NO levels with concomitant increase in intracellular reactive oxygen species from several sources.

Article Snippet: OnTARGETplus short-interfering RNA (siRNA) smart pool was purchased from Dharmacon (Lafayette, CO) to target human CD70 (L009952-00-0005; siCD70).

Techniques:

Journal: JCI Insight

Article Title: CD34 hi subset of synovial fibroblasts contributes to fibrotic phenotype of human knee osteoarthritis

doi: 10.1172/jci.insight.183690

Figure Lengend Snippet: ( A ) Dot plots of representative DEGs from fibrotic group synovium in 11 synovial cell subsets . ( B ) Expression levels of CD70 in 2 OA synovium groups, determined by knee OA synovium RNA-Seq. All data are expressed as dot plots and mean ± SEM. Mann-Whitney U test. * P < 0.05. ( C ) Expression levels of CD70 in 4 OA synovium subgroups, determined by knee OA synovium RNA-Seq. All data are expressed as dot plots and mean ± SEM. Kruskal-Wallis test. * P < 0.05. ( D ) UMAP of 2 CD34 hi fibroblast clusters identified by scRNA-Seq. ( E ) Percentage of fibroblast subsets in the 4 synovium samples. Infla, inflammatory group; Fibro, fibrotic group. ( F ) Dot plots of stable state marker genes and CD70 in CD34 hi CD70 lo fibroblasts and CD34 hi CD70 hi fibroblasts. ( G ) Feature plots of stable state marker genes and CD70 in CD34 hi CD70 lo fibroblasts and CD34 hi CD70 hi fibroblasts. ( H ) Immunofluorescence images of Podoplanin (PDPN), CD34, and CD70 in human knee OA synovium tissue around vessels. Arrowheads indicate PDPN + CD34 + CD70 + cells. Scale bar: 100 μm.

Article Snippet: In the coculture experiments, after 2 days of culture, synovial and cartilage tissues were transferred to coculture plates, and each well was treated with anti–human CD70 antibody (NOVUS Bio, NBP3-11587) or isotype control (R&D Systems, MAB002) at a concentration of 5 μg/mL.

Techniques: Expressing, RNA Sequencing, MANN-WHITNEY, Marker, Immunofluorescence

Journal: JCI Insight

Article Title: CD34 hi subset of synovial fibroblasts contributes to fibrotic phenotype of human knee osteoarthritis

doi: 10.1172/jci.insight.183690

Figure Lengend Snippet: ( A ) T cell proliferation assay cocultured with CD70 transcript variant 1 or 2 or with GFP-transfected fibroblasts cell line (MRC-5). ( B ) The percentage of CD45 + cells and CD4 + T cells in live cells cocultured with CD70 transcript variant 1 or 2 or with GFP-transfected MRC-5 cells. ( C ) Graphical schema of OA synovial and cartilage tissues coculture experiments. ( D ) Time-course mRNA levels of IL1B , IL6 , and TNF in cocultured synovial tissues and COL2A1 , ACAN , and SOX9 mRNA levels in cocultured cartilage tissues with anti-CD70 antibody or isotype control. n = 6 biologically independent experiments. ( E ) The percentage of CD45 + cells and T cell subsets in cocultured synovial tissues with anti-CD70 antibody or isotype control. n = 10 biologically independent experiments. ( F ) The percentage of IL-1B + DCs, macrophages (Mφ), and T cells in cocultured synovial tissues with anti-CD70 antibody or isotype control. n = 6 biologically independent experiments. Data are expressed as dot plots and mean ± SEM. Mann-Whitney U test. * P < 0.05, ** P < 0.01.

Article Snippet: In the coculture experiments, after 2 days of culture, synovial and cartilage tissues were transferred to coculture plates, and each well was treated with anti–human CD70 antibody (NOVUS Bio, NBP3-11587) or isotype control (R&D Systems, MAB002) at a concentration of 5 μg/mL.

Techniques: Proliferation Assay, Variant Assay, Transfection, Control, MANN-WHITNEY

Journal: Oral oncology

Article Title: CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma

doi: 10.1016/j.oraloncology.2018.01.024

Figure Lengend Snippet: Analysis of candidate CAR-T targets, especially CD70, in head and neck cancers. (A) Analysis of The Cancer Genome Atlas database identified nine cell surface protein candidate CAR-T targets with significantly elevated expression in 303 tumors (Ca) compared to 37 adjacent tissue controls (Ctrl). (B) The CD70 mRNA expression data were further analyzed by tumor types and CD70 was significantly elevated in several tumor subtypes (T) compared to controls (N). Note that the normal controls for base of tongue and floor of mouth tumors only included two samples and hence not showing statistical differences to corresponding tumors. (C) CD70 mRNA was heterogeneously overexpressed in 22 individual comparisons of head and neck tumors (black bars, Ca) vs. adjacent normal tissues (white bars, Ctrl). All results are presented as mean ± standard error. Statistical significance was determined by the Mann-Whitney U test (**P<0.05, ***P<0.0005).

Article Snippet: CD70 mRNA expression was measured using TaqMan human CD70 gene expression assay (Assay ID Hs00174297_m1, Thermo Fisher Scientific).

Techniques: Expressing, MANN-WHITNEY

Journal: Oral oncology

Article Title: CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma

doi: 10.1016/j.oraloncology.2018.01.024

Figure Lengend Snippet: Heterogeneity of CD70 mRNA and protein overexpression in several representative head and neck cancer cell lines. (A) qRT-PCR results are expressed as mean ±SEM from total six experiments. Statistical significance was determined by one-way ANOVA multiple comparison test comparing to K562 (***, P<0.0005, **, P<0.005, *, P<0.05; ns, not significant). (B) Cell surface CD70 protein expression in seven head and neck cancer cell lines was determined by flow cytometry. Gray-filled histograms represent signal without antibody, while red-line histograms show staining with PE-conjugated anti-CD70 mAb. Data were collected from at least two independent experiments. RS4 and HOS cells were used as high CD70 expression controls and K562 as a negative control.

Article Snippet: CD70 mRNA expression was measured using TaqMan human CD70 gene expression assay (Assay ID Hs00174297_m1, Thermo Fisher Scientific).

Techniques: Over Expression, Quantitative RT-PCR, Comparison, Expressing, Flow Cytometry, Staining, Negative Control

Journal: Oral oncology

Article Title: CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma

doi: 10.1016/j.oraloncology.2018.01.024

Figure Lengend Snippet: CD70 expression in a representative patient tumor tissue positive for CD70. A HNSCC tumor section was stained with H&E (upper left panel) and adjacent serial section co-stained with anti-CD70 (red, upper middle panel; shown with DAPI counterstain, upper right panel), and an unrelated anti-giantin (green, lower left panel; arrows show Golgi staining). Arrows in enlarged inset show staining consistent with cell surface expression. Other sections were also stained without anti-CD70 (negative control, lower middle panel) or with anti-EGFR (red, positive control, lower right panel). All panels are shown at 200× original magnification. Scale bar: 20 μm.

Article Snippet: CD70 mRNA expression was measured using TaqMan human CD70 gene expression assay (Assay ID Hs00174297_m1, Thermo Fisher Scientific).

Techniques: Expressing, Staining, Negative Control, Positive Control

Journal: Oral oncology

Article Title: CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma

doi: 10.1016/j.oraloncology.2018.01.024

Figure Lengend Snippet: Demonstration of in vitro killing of two head and neck cancer cell lines by CD70 CAR-T cells. (A) CD70-positive luciferase-expressing OQ01 (OQ01.Luc) and CAL27 (CAL27.Luc) cells were co-cultured with either CD70 CAR-T cells or non-transduced T cells (NT) at indicated effector-to-target (E:T) ratios for 24 hours. Specific killing activity of CD70 CAR-T cells was measured by bioluminescence assay. Cultures were set up in duplicate and mean ± error are shown. Similar results were obtained in independent experiments from three healthy T cell donors. (B) IFN-γ secretion of CD70 CAR-T cells in co-culture supernatants after 1 and 4 days was measured by ELISA. Cultures were set up in triplicate and mean ±SEM are shown. Statistical significance was determined by Student’s unpaired t test (*P<0.05; ***P<0.005).

Article Snippet: CD70 mRNA expression was measured using TaqMan human CD70 gene expression assay (Assay ID Hs00174297_m1, Thermo Fisher Scientific).

Techniques: In Vitro, Luciferase, Expressing, Cell Culture, Activity Assay, ATP Bioluminescent Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Tumor necrosis factor receptor superfamily costimulation couples T cell receptor signal strength to thymic regulatory T cell differentiation

doi: 10.1038/ni.2849

Figure Lengend Snippet: Neonatal Foxp3-GFP thymic lobes from one day-old pups were separated and plated in organ cultures with isotype controls or neutralizing antibodies to GITR-L, OX40-L, and CD70, or the combination of anti-GITR-L, OX40-L, CD70, and TNFR2. After 14 days, lobes were dissociated and T reg development was analyzed by flow cytometry. ( a ) A representative comparison of CD4SP thymocytes from isotype- vs. antibody-treated thymic lobes is shown. ( b ) Shown are the percentages of CD25 + Foxp3 + T reg cells amongst total CD4SP thymocytes (mean ± SEM, n=21 over four experiments for isotype controls, n=16 over three experiments for anti-GITR-L, OX40-L, and TNF; n=5 over two experiments for anti-GITR-L, OX40-L, CD70, and TNFR2; * p < 0.05 calculated by ANOVA with Bonferroni’s multiple comparison test). ( c,d ) The expression of CD25, Foxp3, CD73, and FR4 expression in CD4 + Foxp3 + cells from TOCs treated with isotype control antibodies- (solid gray) or TNFSF neutralizing Abs treated (open black) is displayed in the histograms and corresponding bar graphs below (n=16 from three independent experiments, mean ± SEM). P-values were generated using paired, two-tailed T tests ( * P < 0.0001).

Article Snippet: Purified T reg progenitors were incubated in complete RPMI and supplemented with human IL-2 (1 Unit/mL, from the NIH repository at Frederick National Laboratory) with or without 100 nM recombinant GITR-L–Fc, OX40-L–Fc (both from Imgenex, San Diego, CA), recombinant murine TNF (Peprotech, Rocky Hill, NJ), recombinant CD70-Fc (Sino Biologicals, Beijing, China), soluble CD70 (Enzo Life Sciences, Farmingdale, NY), agonist CD27 mAb (RM27-3E5) , or recombinant mouse CD30-L and 4-1BBL (the latter two of which were crosslinked using 5 ug/ml anti-poly His antibody, both from R&D Systems, Minneapolis, MN).

Techniques: Flow Cytometry, Expressing, Generated, Two Tailed Test