Journal: Journal of Translational Medicine

Article Title: Endometrial regenerative cells as a novel cell therapy attenuate experimental colitis in mice

doi: 10.1186/s12967-014-0344-5

Figure Lengend Snippet: ERCs modulate the levels of immune cells in the spleen of colitis mice. The levels of immune cells were analyzed by flow cytometry in different groups. A) The level of CD3 + CD25 + cells in the spleen from normal, untreated and ERC-treated groups. B) The level of CD3 + CD8 + cells in the spleen from normal, untreated and ERC-treated groups. C) The level of CD4 + CD25 + Foxp3 + Tregs in the spleen from normal, untreated and ERC-treated groups. D) The level of CD11c + MHC-II + DCs in the spleen from normal, untreated and ERC-treated groups. (n = 8 mice per group) * p < 0.01, ERC-treated group vs. untreated group.

Article Snippet: Fluorescent antibodies, including CD3ε-FITC, CD8a-PerCP, CD4-PE, CD25-APC, Foxp3 + -FITC, CD11c-PE, MHC-II-FITC (Miltenyi Biotec, Germany), were added to the suspensions respectively.

Techniques: Flow Cytometry

Journal: PLoS ONE

Article Title: The novel multi-cytokine inhibitor TO-207 specifically inhibits pro-inflammatory cytokine secretion in monocytes without affecting the killing ability of CAR T cells

doi: 10.1371/journal.pone.0231896

Figure Lengend Snippet: A) Effects of PSL and TO-207 on the cytotoxicity of CAR T cells. K562/CD19/fLucEGFP cells (3 × 10 3 ) were co-cultured with CD4 + or CD8 + CAR T cells (1.5 × 10 4 ), and treated with different concentrations of PSL and TO-207. Viable target cells were quantified using the luciferase assay after 72 h of co-culture. Values were normalized to viable K562/CD19/fLucEGFP cells cultured alone. The error bars represent SDs from three independent experiments. B) Effects of PSL and TO-207 on CAR T cell degranulation. K562/CD19 cells (5 × 10 5 ) and CAR T cells (5 × 10 5 ) were co-cultured in 500 μl T-cell expansion medium in the absence or presence of PSL or TO-207. Following 68 h of co-culture, monensin (2 μM) and APC–anti-human CD107a antibody were added. Cells were incubated for an additional 4 h, and membrane expression of CD107a was determined by flow cytometry. Representative data of four independent experiments are shown. C) Mean fluorescence intensity (MFI) of samples in Fig 5B. The error bars represent SEs from four independent experiments. D) Sustained cytotoxicity of CAR T cells following TO-207 treatment. K562/CD19/fLucEGFP cells (1 × 10 4 ), CAR T cells (5 × 10 4 ), and CD14 + cells (5 × 10 4 ) were co-cultured in the absence or presence of PSL or TO-207 for 72 h, and viable target cells were quantified by the luciferase assay. Values were normalized to the well containing K562/CD19/fLucEGFP cells alone. The error bars represent SDs from three independent experiments. The linear dose-response relationship was assessed using log-transformed dose values (to the base 10) in a mixed model, in which the zero dose was replaced by the log (minimal dose) - 1. P < 0.05 was considered statistically significant. n.s.: not significant.

Article Snippet: Flow cytometry was performed using allophycocyanin (APC)-conjugated IgG 1 (BioLegend), APC–anti-human CD19 antibody (BioLegend), APC–anti human CD107a (BioLegend), APC–Cy7–anti-human CD3 antibody (BioLegend), V500–anti-human CD4 antibody (BD Biosciences, San Jose, CA, USA), and PerCP–Cy5.5–anti-human CD8 antibody (TONBO Biosciences, San Diego, CA, USA).

Techniques: Cell Culture, Luciferase, Co-Culture Assay, Incubation, Membrane, Expressing, Flow Cytometry, Fluorescence, Transformation Assay

Journal: Molecular Therapy Oncolytics

Article Title: Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers

doi: 10.1016/j.omto.2018.12.001

Figure Lengend Snippet: CD8 + T cm Can Be Infected with VSV and Transfer Virions to Susceptible Cells CD8 + T cm were infected with indicated MOIs of rVSV vectors expressing GFP and cultured until the indicated time points. (A) CD8 + T cm were counted. Percentage of viable cells normalized to time point 0 hr are shown. Means ± SD of three independent experiments, performed in triplicates from three different CD8 + T cm donors, are shown. Statistical analysis was performed by two-way ANOVA with Bonferroni post-test (*p < 0.05). (B) TCID 50 assays were performed from tissue culture supernatants at the indicated time points. The experiment was performed three times in triplicates from different donors, and the means ± SD are shown. Two-way ANOVA with Bonferroni post-test showed no significant difference between the different MOIs at 24 hr after infection. (C) Infected CD8 + T cm were co-cultured with BHK-21 cells. The percentage of GFP + BHK-21 cells was used to calculate the viral load on the CD8 + T cm surface that could be transferred to other cells. The experiment was performed in triplicates using CD8 + T cm derived from three individual donors. Means ± SD are shown. The best-fitting equation is given.

Article Snippet: For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences).

Techniques: Infection, Expressing, Cell Culture, Derivative Assay

Journal: Molecular Therapy Oncolytics

Article Title: Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers

doi: 10.1016/j.omto.2018.12.001

Figure Lengend Snippet: ML2B7-Fluc Leukemia Cells Are Susceptible to Treatment with VSV and CD8 + T cm ML2B7-Fluc cells were infected with indicated MOIs of rVSV-tk. (A) Cell viability was assayed by measuring the luciferase activity of viable tumor cells. The means ± SD from three independent experiments, performed in triplicates, are shown. Two-way ANOVA with Bonferroni post-test was performed to determine statistical significance (**p < 0.01; ****p < 0.0001). (B) Virus titers in culture supernatants at the indicated time points were assayed by TCID 50 . Means ± SD from three individual experiments, performed in triplicates, are shown. (C) Luciferase assays were performed to determine the number of viable ML2B7-Fluc tumor cells 24 hr after treatment with indicated treatment groups. TCR transduced and control T cells were uninfected or infected with rVSV-tk at an MOI of 0.1 before being added in a 1:20 ratio to the tumor cells. Individual replicates and means ± SD from three experiments performed in triplicates from three different T cell donors are shown. The percentage of living cells was normalized to untreated control cells. One-way ANOVA with Bonferroni post-test was performed (*p < 0.05; ***p < 0.001). (D) Viral titers from co-culture supernatants were measured by TCID 50 . Individual replicates and means ± SD are shown. Statistical analysis was performed using unpaired t test (**p < 0.01). (E) IFN-γ concentrations in coculture supernatants were measured by ELISA assay. Means ± SD are shown.

Article Snippet: For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences).

Techniques: Infection, Luciferase, Activity Assay, Virus, Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers

doi: 10.1016/j.omto.2018.12.001

Figure Lengend Snippet: Loading of VSV onto T cm Reduces Virus Toxicity in NSG Mice (A) Increasing doses of naked rVSV vectors expressing GFP or rVSV vectors expressing GFP loaded onto CD8 + T cm were injected by tail-vein injection in NSG mice (n = 3–6). Mice were followed for up to 21 days and then censored. Kaplan-Meier survival curves are shown. The virus dose that was loaded onto T cells was calculated using the equation derived from Figure 2 . (B) TCID 50 assays were performed on homogenized brain tissue. The percentage of mice that had a replication-competent virus in the brain is shown (10 4 PFU VSV-GFP: n = 5; 3.3*10 4 PFU VSV-GFP: n = 6; 10 7 CD8 + T cm, MOI 0.1 of VSV-GFP: n = 3; 10 7 CD8 + T cm, MOI 1 of VSV-GFP: n = 5). Brains were collected upon euthanasia at the time point of toxic events or at the end of the 21-day monitoring period.

Article Snippet: For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences).

Techniques: Virus, Expressing, Injection, Derivative Assay

Journal: Molecular Therapy Oncolytics

Article Title: Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers

doi: 10.1016/j.omto.2018.12.001

Figure Lengend Snippet: Testing of Different Doses of Free and Cell-Bound VSV in an MTD Study in NSG Mice

Article Snippet: For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences).

Techniques:

Journal: Molecular Therapy Oncolytics

Article Title: Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers

doi: 10.1016/j.omto.2018.12.001

Figure Lengend Snippet: TCR CD8 + T cm Deliver Virus to the Tumor and Proliferate Specifically in Target ML2B7 Cells Tumor-bearing NSG mice were treated systemically with the indicated T cells and/or rVSV-tk and euthanized after 120 hr. (A) The percentages of intratumoral CD8 + T cells was determined by FACS analysis. *The p value for the TCR T cell group compared with PBS, 10 4 PFU VSV-tk, and control T cells after 120 hr is <0.05, as determined by one-way ANOVA with Bonferroni post-test. Means ± SD are shown (n = 3–5). (B) Intratumoral viral titers at 72 hr post-treatment were measured by TCID 50 analysis of tumor homogenates. Means ± SD are shown. Statistical analysis was performed by Mann-Whitney test (*p < 0.05; n = 4–6). (C) Immunohistochemical staining for CD3 + cells in tumor tissue at 120 hr post-treatment was performed. Representative images are shown: 1, TCR T cells; 2, TCR T cells infected with rVSV-tk at MOI 0.1; 3, 10 4 PFU rVSV-tk; 4, control T cells; 5, control T cells infected with rVSV-tk at MOI 0.1; 6, PBS. Scale bars indicate 100 μm.

Article Snippet: For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences).

Techniques: Virus, Control, MANN-WHITNEY, Immunohistochemical staining, Staining, Infection

Journal: Molecular Therapy Oncolytics

Article Title: Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers

doi: 10.1016/j.omto.2018.12.001

Figure Lengend Snippet: Treatment with TCR CD8 + T cm Loaded with VSV Causes Rapid Necrosis in ML2B7 Tumors NSG mice bearing ML2B7 tumors were treated by tail-vein injection with T cells and/or rVSV-tk as indicated. (A) The percentage of GFP + tumor cells at the indicated time points was determined by flow cytometry after exclusion of dead cells by staining with Viobility 405/520 fixable dye. Mean ± SD are shown (n = 3–5). (B) Tumor sections from mice euthanized at the indicated time points were analyzed histologically for quantification of necrosis. Three individual sections of each tumor were analyzed. Mean ± SD are shown (n = 3–6). (C) Representative histological images from each treatment group are shown: 1, TCR T cells; 2, TCR T cells infected with rVSV-tk at MOI 0.1; 3, 10 4 PFU rVSV-tk; 4, control T cells; 5, control T cells infected with rVSV-tk at MOI 0.1; 6, PBS. Scale bars indicate 100 μm. (D) Tumor sizes were measured daily with a caliper until day 10 after therapy was started. Tumor area is expressed as mean ± SD (n = 4–6). Two-way ANOVA with Bonferroni post-test was performed (****p < 0.0001). PBS and control T cell groups were not significantly different from each other. They were both significantly (****) different from all other groups. The TCR T cell group was significantly (****) different from the VSV therapy group.

Article Snippet: For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences).

Techniques: Injection, Flow Cytometry, Staining, Infection, Control

Journal: Molecular Therapy Oncolytics

Article Title: Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers

doi: 10.1016/j.omto.2018.12.001

Figure Lengend Snippet: TCR CD8 + T cm Monotherapy Leads to Tumor Relapse in Target ML2B7 Tumors in NSG Mice ML2B7 tumor-bearing NSG mice were treated 10 days after tumor implantation by tail-vein injection and were euthanized at humane endpoints due to poor health or when the tumors reached a diameter of 2 cm. (A) Survival times were plotted by Kaplan-Meier curve (n = 5–6). Long-term surviving TCR T cell-treated mice with relapsing tumors were administered a second round of treatment on day 52 after initial therapy as indicated. ***p < 0.0001 by log-rank [Mantel-Cox] test. (B) Tumor sizes of individual mice treated with TCR T cells were plotted until the time of euthanasia. The dotted line indicates the time point of the second treatment with TCR T cells. (C) Relapsed tumors were analyzed by flow cytometry to determine presence of the targeted HLA, using the co-expressed GFP as a surrogate marker. Representative FACS dot plots of control tumor that expressed the HLA (left) and a relapsed tumor lacking GFP expression (right) are shown.

Article Snippet: For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences).

Techniques: Tumor Implantation, Injection, Flow Cytometry, Marker, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Reduced Immunosenescence of Peripheral Blood T Cells in Parkinson’s Disease with CMV Infection Background

doi: 10.3390/ijms222313119

Figure Lengend Snippet: The proportion of T cells (CD3 + ), including CD4 + and CD8 + subsets, and NK cells in PD patients, HD group and CMV-seropositive HD individuals.

Article Snippet: The following mouse anti-human fluorescent-labeled antibodies were used for surface cell staining: CD56-APC (clone N901, Beckman Coulter, Miami, FL, USA), CD4-FITC (clone RPA-T4, Sony Biotechnology, San Jose, CA, USA), CD57-PE (clone HCD57, Sony Biotechnology, San Jose, CA, USA), NKG2C-PE (clone 134591, R&D Systems, Minneapolis, MN, USA), CD3-PerCP (clone HIT3a, Sony Biotechnology, San Jose, CA, USA), CD3-FITC (clone FIT3a, Sony Biotechnology, San Jose, CA, USA), CD3-APC (clone F OKT3, Sony Biotechnology, San Jose, CA, USA) CD8-PerCP (clone SK1, Sony Biotechnology, San Jose, CA, USA), CD45RA-APC (clone HI100, Sony Biotechnology, San Jose, CA, USA), CD197-FITC (clone G043H7, Sony Biotechnology, San Jose, CA, USA), CD56-APC-Vio770 (clone REA196, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: