Journal: Molecules

Article Title: Effects of a Peripherally Restricted Hybrid Inhibitor of CB1 Receptors and iNOS on Alcohol Drinking Behavior and Alcohol-Induced Endotoxemia

doi: 10.3390/molecules26165089

Figure Lengend Snippet: Intracerebroventricular microinfusion of JD5037 increases anxiety-related behavior in the elevated plus maze test. Heat maps ( a ) and summary of mouse activities in individual compartments ( b – g ) of the elevated plus maze. Animals received i.c.v. infusion of JD5037 (1 µg; JD) or its vehicle (3% DMSO, 8% Tween 80, 30% PEG-400, 59% saline; V). They were tested in the elevated plus maze 1 h later. The computerized EthoVision video tracking system was used for data collection and analysis. Bars are mean ± s.e.m. * p < 0.05, ** p < 0.01, compared with vehicle.

Article Snippet: The behavior of each mouse was monitored by a computer-assisted video tracking system EthoVision XT (Noldus, Leesburg, VA, USA).

Techniques:

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: CD2, CD3, and CD7 are expressed on all T-cell subsets and CD2 as well as CD7 show an upregulation on specifically activated T cells. (A) Analysis of CD2, CD3, and CD7 expression on the surface of different T-cell subsets from freshly isolated peripheral blood mononuclear cells (PBMC). Mean fluorescence intensity (MFI) of PE-labeled anti-CD2, anti-CD3, and anti-CD7 antibodies is shown normalized to PE-isotype control for cells pre-gated on CD4 or CD8. T-cell subpopulations include T CM (central memory T cells; CD45RA - CD62L + ), T EFF (effector T cells; CD45RA + CD62L - ), T EM (effector memory T cells; CD45RA - CD62L - ), T N (naive T cells; CD45RA + CD62L + ), and T Reg (regulatory T cells; CD4 + CD25 + CD127 low ). Analysis of CD2 and CD7 on T-cell subpopulations of two other donors revealed the same pattern for each subset at other MFI values. Flow cytometry data for LSR II was evaluated using FlowJoSoftware7.6.5 and is shown for cells pre-gated on single and 7-AAD - cells. (B) CD2, CD3, and CD7 molecules on the surface of T cells and tumor cells (control). TCR2.5D6iRFP or iRFP T CM (both GFP - ) were co-cultivated for 24 h with ML2-B7 or ML2-B15 (both GFP + ) tumor cells as indicated and subsequently analyzed for surface expression with respective PE-labeled antibodies. For calculation of the number of surface molecules, detected geometric mean (GM) was related to GM of PE quantification beads and the labeling efficiency of antibodies was determined by nanophotometer.

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Isolation, Fluorescence, Labeling, Control, Flow Cytometry

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: OKT11 (anti-CD2) and T3-3A1 (anti-CD7) IgG and F(ab´) 2 show high specificity and affinity to selected antigens on T cells, with highest internalization effects observed for CD7 binding. (A) Affinity binding curves of OKT11 (anti-CD2) and T3-3A1 (anti-CD7) IgG and F(ab´) 2 to PBMC-derived T cells at different concentrations. OKT3 (anti-CD3)-IgG was used as a positive control. Flow cytometric data (performed in triplicates) are depicted as semi-logarithmic plots in comparison to the isotype. Each value is shown as mean ± standard deviation (SD). Histograms within the graphs show specific binding of tested PacBlue-labeled IgG and F(ab´) 2 (colored filled histograms) to PBMC-derived T cells at a concentration of 100 nM in comparison to respective PacBlue-isotype controls (gray histogram). Constant of dissociation (K d ) values are depicted in the table. (B) Internalization of IgG and F(ab´) 2 after incubation with PBMC-derived T cells. MFI values of CD2, CD7 and CD3 staining with respective PE-labeled antibodies at various time points after application of indicated IgG or F(ab´) 2 were normalized to MFI of control sample without addition of proteins beforehand. The experiment was performed in duplicates and results are shown as mean ± SD (n = 3).

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Derivative Assay, Positive Control, Comparison, Standard Deviation, Labeling, Concentration Assay, Incubation, Staining, Control

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: OKT11 (anti-CD2) and T3-3A1 (anti-CD7) IgG and F(ab´) 2 show no functional T-cell impairment during short-term co-incubation. (A-B) PBMC-derived T cells were incubated for 18 h at 37 °C with various concentrations of IgG and F(ab´) 2 in triplicates. Respective isotypes served as negative control, whereas OKT3 was used as positive control. Mean ± SD is depicted (n = 3). (A) Percentage of apoptotic PBMC-derived T cells after co-incubation at indicated protein concentrations. Values are based on flow cytometry analysis identifying annexin V-APC or 7-AAD positive cells of all cells detected. (B) Corresponding IFNγ release of PBMC-derived T cells in supernatants after incubation with indicated concentrations of IgG or F(ab´) 2 determined by ELISA. (C) CTV-labeled PBMC-derived T cells were incubated with 100 nM of the indicated antibodies or F(ab´) 2 at 37 °C over a period of 3 days. Incubation with isotypes served as negative control and OKT3 as positive control. Decrease of MFI of CTV indicates T-cell proliferation in response to indicated IgG or F(ab´) 2 depicted as mean ± SD from triplicates (n = 3).

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: Functional Assay, Incubation, Derivative Assay, Negative Control, Positive Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: T3-3A1 (anti-CD7) IgG and F(ab´) 2 have no impact on the cytotoxic efficacy of specifically activated TCR2.5D6iRFP T CM in contrast to co-incubation with OKT11 (anti-CD2) resulting in minor changes in tumor cells killing and IFNγ secretion. TCR2.5D6iRFP T CM were co-cultured with tumor cells bearing either the respective restriction element HLA-B7 or the irrelevant HLA-B15 for 24 h at 37 °C. As control, iRFP T CM were co-incubated with ML2-B7 tumor cells. Each condition is indicated below the graph. After incubation of the co-culture with respective IgG or F(ab´) 2 , cells were harvested for flow cytometric analysis and IFNɣ secretion in supernatants was analyzed by ELISA. The experiment was performed in triplicates and results are shown as mean ± SD (n = 3). One representative experiment out of three is depicted. Statistically significant differences were observed in single but not all experiments for full IgG and F(ab´) 2 . (A) GFP + tumor cells per microliter calculated from flow cytometric analysis. (B) Corresponding IFNγ cytokine secretion in co-culture supernatant.

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Cell Culture, Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: In vivo imaging of T cells at the specific tumor site by immuno-PET using 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 and 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 . (A) 3D-maximum intensity projection (MIP) pictures of PET/CT imaging of mice. Mice bearing ML2-B7 or ML2-B15 tumors that received TCR2.5D6iRFP T CM or PBS and 89 Zr-labeled F(ab´) 2 as indicated. Red arrows point at the ML2-B7 tumor site recognized by TCR2.5D6iRFP T CM and white arrows mark the ML2-B15 control tumor site. Animals injected with PBS served as negative controls. The scale bar shows 0-20% of percentage injected dose per gram (%ID/g). (B) Biodistribution analysis based on activity accumulation measurement by a gamma counter of the depicted organs for animals that received 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 (left panel) or 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 (right panel). %ID/g is depicted as mean ± SD for mice injected with TCR2.5D6iRFP T CM (n = 3 each) or PBS (n = 1 each) as control for various organs as indicated. Unpaired t -test: ** p < 0.01 and **** p < 0.0001. (C) Respective organ-to-blood ratio from %ID/g values shown as mean ± SD for 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 (left panel) and 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 (right panel). Unpaired t -test: *** p < 0.001. (D) Percentage of T cells detected by flow cytometry in tumors and spleen for animals injected with TCR2.5D6iRFP T CM and either 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 or 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 . Unpaired t -test: ** p < 0.01.

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: In Vivo Imaging, Positron Emission Tomography-Computed Tomography, Imaging, Labeling, Control, Injection, Activity Assay, Flow Cytometry

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: Immunohistochemistry confirmed the presence of CD2 + and CD7 + T cells in the ML2-B7 tumor specifically recognized by TCR2.5D6 transduced T CM . Tumor tissue from 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 (left panel) or 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 (right panel) injected mice was harvested after imaging. The bars in the upper right corner represent 100 µm. (A) ML2-B7 tumor slides stained for CD2, CD7, and CD3, as well as ML2-B15 tumor slides stained for CD3 as control. (B) ML2-B7 and ML2-B15 tumor slides stained for CD3 (brown)/ cleaved caspase 3 (red).

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: Immunohistochemistry, Injection, Imaging, Staining, Control

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: Injection of OKT11 (anti-CD2)-F(ab´) 2 , but not T3-3A1 (anti-CD7)-F(ab´) 2 , impaired T-cell function in vivo . After injection of ML2-B7 tumor cells into the right flank and ML2-B15 tumor cells into the left flank, as well as total body irradiation, mice received 2×10 7 TCR2.5D6iRFP T CM . Comparable to the imaging experiments, three days after T CM injection, mice received 20 µg of OKT11 (anti-CD2)-F(ab´) 2 , T3-3A1 (anti-CD7)-F(ab´) 2 , or isotype-F(ab´) 2 (n = 6 for each group). Animals were sacrificed on day 11 after T-cell administration (Figure B). Unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (A) Tumor growth kinetics of ML2-B7 (left side) and ML2-B15 control tumors (right side) for mice injected as indicated. The arrow indicates the time point of respective F(ab´) 2 injection. Tumor size is shown in mm 2 as mean ± SD over different days post intravenous T CM injection. (B) Weight of tumors and spleen 11 days after T CM injection for mice receiving different F(ab´) 2 constructs as indicated. The weight of the organs is shown in milligram as mean ± SD. (C) Percentage of human T cells in indicated organs analyzed by flow cytometry on day 11 post T CM injection. The percentage of CD45 + GFP - CD3 + cells of all 7-AAD - cells is depicted as mean ± SD for the indicated groups. (D) The percentage of TCR2.5D6iRFP + T CM of all 7-AAD - cells in indicated organs is shown as mean ± SD.

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: Injection, Cell Function Assay, In Vivo, Irradiation, Imaging, Control, Construct, Flow Cytometry

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: T-cell tracking by immuno-PET using 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 reveals prominent signals corresponding to accumulation of specific tumor infiltrating lymphocytes at the tumor site. Mice were treated following the imaging protocol depicted in Figure A. (A) Representative 3D-MIP PET images of ML2-B7 and ML2-B15 tumor-bearing mice injected with either TCR2.5D6iRFP T CM (n = 7) or iRFP T CM (n = 6). Animals received 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 intravenously and PET imaging was performed 48 h post injection. Red arrows point at ML2-B7 tumor site, and white arrows mark ML2-B15 control tumor site. The scale bar represents 0-20% ID/g. (B) Ex vivo biodistribution analysis of different organs analyzed on the day of imaging. Mean ± SD of %ID/g is shown for different organs from mice receiving either TCR2.5D6iRFP or iRFP T CM . Unpaired t -test: *** p < 0.001. (C) Respective organ-to-blood ratio for TCR2.5D6iRFP or iRFP T CM treated mice. Unpaired t -test: *** p < 0.001. (D) Flow cytometric analysis of T cells detected in the ML2-B7, ML2-B15 control tumor, or the spleen. Mean ± SD of percentage of CD45 + CD5 + GFP - T cells of 7-AAD - cells is shown for the two groups. Unpaired t -test: ** p < 0.01 and *** p < 0.001. (E) Percentages of TCR2.5D6iRFP transduced T cells of all viable cells analyzed by flow cytometric analysis of respective organs. Mean ± SD is depicted. Unpaired t -test: * p < 0.05 and ** p < 0.01 (F) Percentages of GFP + tumor cells of all viable cells in ML2-B7 tumors and ML2-B15 control tumors evaluated by flow cytometry. Mean ± SD is shown. Unpaired t -test: *** p < 0.001.

Article Snippet: Cells were stained with T3-3A1 (anti-CD7) antibody with and without pre-incubation of a polyclonal sheep anti-human CD7 antibody (R&D Systems, Minneapolis, MN).

Techniques: Cell Tracking Assay, Imaging, Injection, Control, Ex Vivo, Flow Cytometry

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: CD2, CD3, and CD7 are expressed on all T-cell subsets and CD2 as well as CD7 show an upregulation on specifically activated T cells. (A) Analysis of CD2, CD3, and CD7 expression on the surface of different T-cell subsets from freshly isolated peripheral blood mononuclear cells (PBMC). Mean fluorescence intensity (MFI) of PE-labeled anti-CD2, anti-CD3, and anti-CD7 antibodies is shown normalized to PE-isotype control for cells pre-gated on CD4 or CD8. T-cell subpopulations include T CM (central memory T cells; CD45RA - CD62L + ), T EFF (effector T cells; CD45RA + CD62L - ), T EM (effector memory T cells; CD45RA - CD62L - ), T N (naive T cells; CD45RA + CD62L + ), and T Reg (regulatory T cells; CD4 + CD25 + CD127 low ). Analysis of CD2 and CD7 on T-cell subpopulations of two other donors revealed the same pattern for each subset at other MFI values. Flow cytometry data for LSR II was evaluated using FlowJoSoftware7.6.5 and is shown for cells pre-gated on single and 7-AAD - cells. (B) CD2, CD3, and CD7 molecules on the surface of T cells and tumor cells (control). TCR2.5D6iRFP or iRFP T CM (both GFP - ) were co-cultivated for 24 h with ML2-B7 or ML2-B15 (both GFP + ) tumor cells as indicated and subsequently analyzed for surface expression with respective PE-labeled antibodies. For calculation of the number of surface molecules, detected geometric mean (GM) was related to GM of PE quantification beads and the labeling efficiency of antibodies was determined by nanophotometer.

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: Expressing, Isolation, Fluorescence, Labeling, Control, Flow Cytometry

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: OKT11 (anti-CD2) and T3-3A1 (anti-CD7) IgG and F(ab´) 2 show high specificity and affinity to selected antigens on T cells, with highest internalization effects observed for CD7 binding. (A) Affinity binding curves of OKT11 (anti-CD2) and T3-3A1 (anti-CD7) IgG and F(ab´) 2 to PBMC-derived T cells at different concentrations. OKT3 (anti-CD3)-IgG was used as a positive control. Flow cytometric data (performed in triplicates) are depicted as semi-logarithmic plots in comparison to the isotype. Each value is shown as mean ± standard deviation (SD). Histograms within the graphs show specific binding of tested PacBlue-labeled IgG and F(ab´) 2 (colored filled histograms) to PBMC-derived T cells at a concentration of 100 nM in comparison to respective PacBlue-isotype controls (gray histogram). Constant of dissociation (K d ) values are depicted in the table. (B) Internalization of IgG and F(ab´) 2 after incubation with PBMC-derived T cells. MFI values of CD2, CD7 and CD3 staining with respective PE-labeled antibodies at various time points after application of indicated IgG or F(ab´) 2 were normalized to MFI of control sample without addition of proteins beforehand. The experiment was performed in duplicates and results are shown as mean ± SD (n = 3).

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: Binding Assay, Derivative Assay, Positive Control, Comparison, Standard Deviation, Labeling, Concentration Assay, Incubation, Staining, Control

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: OKT11 (anti-CD2) and T3-3A1 (anti-CD7) IgG and F(ab´) 2 show no functional T-cell impairment during short-term co-incubation. (A-B) PBMC-derived T cells were incubated for 18 h at 37 °C with various concentrations of IgG and F(ab´) 2 in triplicates. Respective isotypes served as negative control, whereas OKT3 was used as positive control. Mean ± SD is depicted (n = 3). (A) Percentage of apoptotic PBMC-derived T cells after co-incubation at indicated protein concentrations. Values are based on flow cytometry analysis identifying annexin V-APC or 7-AAD positive cells of all cells detected. (B) Corresponding IFNγ release of PBMC-derived T cells in supernatants after incubation with indicated concentrations of IgG or F(ab´) 2 determined by ELISA. (C) CTV-labeled PBMC-derived T cells were incubated with 100 nM of the indicated antibodies or F(ab´) 2 at 37 °C over a period of 3 days. Incubation with isotypes served as negative control and OKT3 as positive control. Decrease of MFI of CTV indicates T-cell proliferation in response to indicated IgG or F(ab´) 2 depicted as mean ± SD from triplicates (n = 3).

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: Functional Assay, Incubation, Derivative Assay, Negative Control, Positive Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: T3-3A1 (anti-CD7) IgG and F(ab´) 2 have no impact on the cytotoxic efficacy of specifically activated TCR2.5D6iRFP T CM in contrast to co-incubation with OKT11 (anti-CD2) resulting in minor changes in tumor cells killing and IFNγ secretion. TCR2.5D6iRFP T CM were co-cultured with tumor cells bearing either the respective restriction element HLA-B7 or the irrelevant HLA-B15 for 24 h at 37 °C. As control, iRFP T CM were co-incubated with ML2-B7 tumor cells. Each condition is indicated below the graph. After incubation of the co-culture with respective IgG or F(ab´) 2 , cells were harvested for flow cytometric analysis and IFNɣ secretion in supernatants was analyzed by ELISA. The experiment was performed in triplicates and results are shown as mean ± SD (n = 3). One representative experiment out of three is depicted. Statistically significant differences were observed in single but not all experiments for full IgG and F(ab´) 2 . (A) GFP + tumor cells per microliter calculated from flow cytometric analysis. (B) Corresponding IFNγ cytokine secretion in co-culture supernatant.

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: Incubation, Cell Culture, Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: In vivo imaging of T cells at the specific tumor site by immuno-PET using 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 and 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 . (A) 3D-maximum intensity projection (MIP) pictures of PET/CT imaging of mice. Mice bearing ML2-B7 or ML2-B15 tumors that received TCR2.5D6iRFP T CM or PBS and 89 Zr-labeled F(ab´) 2 as indicated. Red arrows point at the ML2-B7 tumor site recognized by TCR2.5D6iRFP T CM and white arrows mark the ML2-B15 control tumor site. Animals injected with PBS served as negative controls. The scale bar shows 0-20% of percentage injected dose per gram (%ID/g). (B) Biodistribution analysis based on activity accumulation measurement by a gamma counter of the depicted organs for animals that received 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 (left panel) or 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 (right panel). %ID/g is depicted as mean ± SD for mice injected with TCR2.5D6iRFP T CM (n = 3 each) or PBS (n = 1 each) as control for various organs as indicated. Unpaired t -test: ** p < 0.01 and **** p < 0.0001. (C) Respective organ-to-blood ratio from %ID/g values shown as mean ± SD for 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 (left panel) and 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 (right panel). Unpaired t -test: *** p < 0.001. (D) Percentage of T cells detected by flow cytometry in tumors and spleen for animals injected with TCR2.5D6iRFP T CM and either 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 or 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 . Unpaired t -test: ** p < 0.01.

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: In Vivo Imaging, Positron Emission Tomography-Computed Tomography, Imaging, Labeling, Control, Injection, Activity Assay, Flow Cytometry

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: Immunohistochemistry confirmed the presence of CD2 + and CD7 + T cells in the ML2-B7 tumor specifically recognized by TCR2.5D6 transduced T CM . Tumor tissue from 89 Zr-OKT11 (anti-CD2)-F(ab´) 2 (left panel) or 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 (right panel) injected mice was harvested after imaging. The bars in the upper right corner represent 100 µm. (A) ML2-B7 tumor slides stained for CD2, CD7, and CD3, as well as ML2-B15 tumor slides stained for CD3 as control. (B) ML2-B7 and ML2-B15 tumor slides stained for CD3 (brown)/ cleaved caspase 3 (red).

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: Immunohistochemistry, Injection, Imaging, Staining, Control

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: Injection of OKT11 (anti-CD2)-F(ab´) 2 , but not T3-3A1 (anti-CD7)-F(ab´) 2 , impaired T-cell function in vivo . After injection of ML2-B7 tumor cells into the right flank and ML2-B15 tumor cells into the left flank, as well as total body irradiation, mice received 2×10 7 TCR2.5D6iRFP T CM . Comparable to the imaging experiments, three days after T CM injection, mice received 20 µg of OKT11 (anti-CD2)-F(ab´) 2 , T3-3A1 (anti-CD7)-F(ab´) 2 , or isotype-F(ab´) 2 (n = 6 for each group). Animals were sacrificed on day 11 after T-cell administration (Figure B). Unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (A) Tumor growth kinetics of ML2-B7 (left side) and ML2-B15 control tumors (right side) for mice injected as indicated. The arrow indicates the time point of respective F(ab´) 2 injection. Tumor size is shown in mm 2 as mean ± SD over different days post intravenous T CM injection. (B) Weight of tumors and spleen 11 days after T CM injection for mice receiving different F(ab´) 2 constructs as indicated. The weight of the organs is shown in milligram as mean ± SD. (C) Percentage of human T cells in indicated organs analyzed by flow cytometry on day 11 post T CM injection. The percentage of CD45 + GFP - CD3 + cells of all 7-AAD - cells is depicted as mean ± SD for the indicated groups. (D) The percentage of TCR2.5D6iRFP + T CM of all 7-AAD - cells in indicated organs is shown as mean ± SD.

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: Injection, Cell Function Assay, In Vivo, Irradiation, Imaging, Control, Construct, Flow Cytometry

Journal: Theranostics

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

doi: 10.7150/thno.27275

Figure Lengend Snippet: T-cell tracking by immuno-PET using 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 reveals prominent signals corresponding to accumulation of specific tumor infiltrating lymphocytes at the tumor site. Mice were treated following the imaging protocol depicted in Figure A. (A) Representative 3D-MIP PET images of ML2-B7 and ML2-B15 tumor-bearing mice injected with either TCR2.5D6iRFP T CM (n = 7) or iRFP T CM (n = 6). Animals received 89 Zr-T3-3A1 (anti-CD7)-F(ab´) 2 intravenously and PET imaging was performed 48 h post injection. Red arrows point at ML2-B7 tumor site, and white arrows mark ML2-B15 control tumor site. The scale bar represents 0-20% ID/g. (B) Ex vivo biodistribution analysis of different organs analyzed on the day of imaging. Mean ± SD of %ID/g is shown for different organs from mice receiving either TCR2.5D6iRFP or iRFP T CM . Unpaired t -test: *** p < 0.001. (C) Respective organ-to-blood ratio for TCR2.5D6iRFP or iRFP T CM treated mice. Unpaired t -test: *** p < 0.001. (D) Flow cytometric analysis of T cells detected in the ML2-B7, ML2-B15 control tumor, or the spleen. Mean ± SD of percentage of CD45 + CD5 + GFP - T cells of 7-AAD - cells is shown for the two groups. Unpaired t -test: ** p < 0.01 and *** p < 0.001. (E) Percentages of TCR2.5D6iRFP transduced T cells of all viable cells analyzed by flow cytometric analysis of respective organs. Mean ± SD is depicted. Unpaired t -test: * p < 0.05 and ** p < 0.01 (F) Percentages of GFP + tumor cells of all viable cells in ML2-B7 tumors and ML2-B15 control tumors evaluated by flow cytometry. Mean ± SD is shown. Unpaired t -test: *** p < 0.001.

Article Snippet: In addition, the first in vitro experiments were performed with anti-CD3 antibodies of the clones BC3 (BioLegend, San Diego, CA), VIT3b (kindly provided by Institute of Immunology, Medical University Vienna), and OKT3 (BioLegend, San Diego, CA), as well as another anti-CD7 antibody (clone 4H9; Caprico Biotechnology, Norcross, GA).

Techniques: Cell Tracking Assay, Imaging, Injection, Control, Ex Vivo, Flow Cytometry