Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CMVpp65 vaccine enhances the antitumor efficacy of adoptively transferred CD19-redirected CMV-specific T cells

doi: 10.1158/1078-0432.CCR-14-2920

Figure Lengend Snippet: (A) 7 days after the second CD19 Ag stimulation, T cells were stained with HLA A2 restricted pp65 tetramer, cetuximab-biotin, anti-CD8 and antibodies specific to central memory T cell surface markers. Percent positive cells are indicated after dead cell exclusion with DAPI, gating based on pp65 tetramer and cetuximab double-positivity, and isotype-matched stained samples. (B) Four-hour 51Cr release assays were performed using the bi-specific T cells and indicated 51Cr-labeled target cells at different effector : target (E:T) ratios. OKT3-expressing LCLs were used as positive controls, KG1A and U251T as negative controls. CD19+ LCL and engineered pp65U251T cells were used as target for CD19 and CMV-specific T cells, respectively. Data from a representative donor is presented. (C) Bi-specific T cells (105) were activated overnight with 105 LCL-OKT3, LCL, or KG1a in 96-well tissue culture plates and 105 U251T and engineered pp65 expressing U251T cells (pp65U251T) in 24-well tissue culture plates. Supernatants were collected after overnight co-incubation of bi-specific T cells and stimulators. Cytokine levels with indicated stimulators (means ±SEM of triplicate wells) were determined using cytometric bead array.

Article Snippet: MP1 GIGFVFTL peptide (HLA-A 0201 influenza) was synthesized at the City of Hope DNA/RNA Peptide Synthesis Facility, (Duarte, CA). pepMix HCMVA (pp65) (pp65pepmix) was purchased from JPT peptide Technologies (GmbH, Berlin Germany).

Techniques: Staining, Labeling, Expressing, Incubation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CMVpp65 vaccine enhances the antitumor efficacy of adoptively transferred CD19-redirected CMV-specific T cells

doi: 10.1158/1078-0432.CCR-14-2920

Figure Lengend Snippet: (A) pp65 tetramer analysis of expanded bi-specific T cells was performed before and after each CD19 Ag stimulation by flow cytometry. Percentages of pp65 tetramer and CD8 double-positive cells are indicated based on negative tetramer and isotype gating. (B) Bi-specific T cells (105) were activated overnight with 105 of LCL-OKT3, LCL, KG1a in 96-well tissue culture plates and 105 U251T and engineered pp65 expressing U251T cells (pp65U251T) in 24-well tissue culture plates. Co-cultures were fixed and permeabilized using the BD Cytofix/Cytoperm kit according to manufacturer’s instructions. After fixation and permeabilization, the T cells were stained with anti-IFNγ. Before fixation, anti-cetuximab-biotin and anti-CD3 staining was used to analyze surface expression of CAR and T cells. Percentages of positive cells on gated CD3 T cells are presented based on that stained with isotype antibodies.

Article Snippet: MP1 GIGFVFTL peptide (HLA-A 0201 influenza) was synthesized at the City of Hope DNA/RNA Peptide Synthesis Facility, (Duarte, CA). pepMix HCMVA (pp65) (pp65pepmix) was purchased from JPT peptide Technologies (GmbH, Berlin Germany).

Techniques: Flow Cytometry, Expressing, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CMVpp65 vaccine enhances the antitumor efficacy of adoptively transferred CD19-redirected CMV-specific T cells

doi: 10.1158/1078-0432.CCR-14-2920

Figure Lengend Snippet: (A) NSG mice were injected i.v. on day 0 with 2.5×106 GFPffluc+ LCL cells. Three days after tumor inoculation, recipient mice were injected i.v. with 2×106 bi-specific cells that underwent 2 rounds of CD19 stimulation. Vaccine was given by i.v. injection of peptide pulsed autologous T cells. Fourteen to seventeen days post T cell infusion, 5×106 pp65pepmix (B) or pp65 peptide (C) (or MP1) loaded autologous T cells were irradiated and injected (iv) into T-cell-engrafted mice as vaccine. pp65 vaccine was also supplemented to the mice that were treated with 10×10^6 CMV-specific T cells from the same donor and untreated mice were used as another type of control. Tumor growth was evaluated by Xenogen® imaging. N=5 for each group in the experiments. The Mann Whitney test was used for statistical analysis.

Article Snippet: MP1 GIGFVFTL peptide (HLA-A 0201 influenza) was synthesized at the City of Hope DNA/RNA Peptide Synthesis Facility, (Duarte, CA). pepMix HCMVA (pp65) (pp65pepmix) was purchased from JPT peptide Technologies (GmbH, Berlin Germany).

Techniques: Injection, Irradiation, Imaging, MANN-WHITNEY

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CMVpp65 vaccine enhances the antitumor efficacy of adoptively transferred CD19-redirected CMV-specific T cells

doi: 10.1158/1078-0432.CCR-14-2920

Figure Lengend Snippet: 2×106 CMV-specific or bi-specific T cells from the same donor were adoptively transferred into CD19 tumor-bearing NSG mice. 2 weeks post T cell infusion, mice received either pp65 vaccine or MP1 vaccine. (A) Percentages of human T cells pooled from blood, bone marrow and spleen from multiple mice (N=4) and (B) GFP+ tumor cells in the mouse spleen were determined by flow cytometry. The Mann Whitney test was used for statistical analysis. (C) CMVpp65 tetramer and CAR double positive cells in the spleen of mice were analyzed by flow cytometry after labeling with antibodies specific to human CD45, pp65 tetramer and EGFR, 28 days post bi-specific T cell infusion. The percentages of CMVpp65 tetramer+ CAR+ T cells in the human T cell population of a representative mouse are presented. (D) 1×106 bi-specific T cells were adoptively transferred into CD19 tumor-bearing NSG mice. 2 weeks post T cell engraftment, mice received cetuximab (Erbitux™) 1mg/day i.p. injection for 4 days. One day after the last injection, CD45+GFP− human T cells and CD45+CAR+ T cells in the bone marrow were analyzed by flow cytometry after staining with antibodies specific to human CD45 and cetuximab-biotin. Representative FACS data from cetuximab-treated and untreated mice are depicted on the left and percentages of CAR+ T cells in the mouse bone marrow from multiple mice are presented on the right.

Article Snippet: MP1 GIGFVFTL peptide (HLA-A 0201 influenza) was synthesized at the City of Hope DNA/RNA Peptide Synthesis Facility, (Duarte, CA). pepMix HCMVA (pp65) (pp65pepmix) was purchased from JPT peptide Technologies (GmbH, Berlin Germany).

Techniques: Flow Cytometry, MANN-WHITNEY, Labeling, Injection, Staining

Journal: The Journal of Biological Chemistry

Article Title: Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi

doi: 10.1074/jbc.RA118.004498

Figure Lengend Snippet: Calcium sensitivity of recombinant TcPDP and TbPDP. A–D, purification of TcPDP (A) and TbPDP (C) recombinant proteins by immobilized metal affinity chromatography. Total lysate (Lys), flow through (FT) and eluted/desalted fractions (rPDP and TbPDP) were analyzed by SDS-PAGE on 10% gel stained with Coomassie Brilliant Blue. Molecular weight (MW) markers are shown on the left side of panels A and C. Recombinant TcPDP (B) and TbPDP (D) enzymatic activities were assayed at different Ca2+ concentrations. E, enzyme kinetics of TcPDP. Recombinant TcPDP phosphatase activity was assayed in buffer containing 50 mm Hepes, pH 7.0, 50 μm MgCl2, 2 mm DTT, 100 nm free Ca2+, 120 μg of BSA and different concentrations of TcPDH E1α synthetic phosphopeptide as substrate. Error bars are smaller than the symbols used for some data points. Kinetic parameters of recombinant TcPDP obtained from linear regression using Michaelis-Menten equation are shown in the plot. Values are mean ± S.D. of three independent experiments (B, D, and E).

Article Snippet: TbPDP and TcPDP belong to the PP2C/PPM family, which is a group of Mg 2+ /Mn 2+ -dependent serine/threonine phosphatases essential for regulation of cell cycle and stress signaling pathways in different cell types (InterPro protein sequence analysis and classification) ( 33 ).

Techniques: Recombinant, Purification, Affinity Chromatography, SDS Page, Staining, Molecular Weight, Activity Assay, Phospho-proteomics