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: Frontiers in Immunology

Article Title: Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping

doi: 10.3389/fimmu.2019.00655

Figure Lengend Snippet: HCMV-seropositive and -seronegative healthy human donors' reactivity to 20 HCMV antigens represented by peptide pools.

Article Snippet: This HCMV pp65 495−503 peptide, along with additional HCMV, EBV, and influenza virus peptides specified in Figure 3 , , , respectively, were purchased from Panatecs (Heilbronn, Germany) at >95% purity according to the manufacturer's specification.

Techniques:

Journal: Frontiers in Immunology

Article Title: Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping

doi: 10.3389/fimmu.2019.00655

Figure Lengend Snippet: CD8+ T cell epitopes of HCMV pp65 protein recognized in A * 02:01-positive healthy donors as established by brute force epitope mapping.

Article Snippet: This HCMV pp65 495−503 peptide, along with additional HCMV, EBV, and influenza virus peptides specified in Figure 3 , , , respectively, were purchased from Panatecs (Heilbronn, Germany) at >95% purity according to the manufacturer's specification.

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping

doi: 10.3389/fimmu.2019.00655

Figure Lengend Snippet: The T cell recall response to single peptide pp65 495−503 vs. the pp65 peptide pool in HLA-A * 02:01-positive test subjects. Both antigens were tested in an IFN-γ ELISPOT assay on 16 HCMV-seronegative subjects (A) and 32 HCMV-seropositive donors (B) . The results are expressed as SFU/ 1 × 10 6 PBMC. Colored lines link each test subject's response to the two antigens. Panel (C) shows the normalized data for the seropositive cohort, with the SFU counts elicited by the pp65 peptide pool set as 100% for each donor, to which the magnitude of the pp65 495−503 triggered SFU counts in the same donor was compared.

Article Snippet: This HCMV pp65 495−503 peptide, along with additional HCMV, EBV, and influenza virus peptides specified in Figure 3 , , , respectively, were purchased from Panatecs (Heilbronn, Germany) at >95% purity according to the manufacturer's specification.

Techniques: Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping

doi: 10.3389/fimmu.2019.00655

Figure Lengend Snippet: Representative raw data for pp65 495−503 single peptide-reactive CD8+ cells vs. pp65 peptide pool-specific T cells in HLA A * 02:01-positive donors. In row (A) , IFN-γ ELISPOT well images are shown for a donor with matching SFU counts: pp65 495−503 appears to be immune dominant in this donor. In the donor shown in row (B) , pp65 495−503 single-peptide reactive CD8+ T cells constitute a fraction of the pp65 peptide pool-reactive T cells. The donor shown in row (C) did not display pp65 495−503 single-peptide reactive CD8+ T cells, although a response to the pp65 peptide pool was observed: in this donor apparently, other epitopes are targeted as well by CD8+ T cells.

Article Snippet: This HCMV pp65 495−503 peptide, along with additional HCMV, EBV, and influenza virus peptides specified in Figure 3 , , , respectively, were purchased from Panatecs (Heilbronn, Germany) at >95% purity according to the manufacturer's specification.

Techniques: Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping

doi: 10.3389/fimmu.2019.00655

Figure Lengend Snippet: Previously reported HCMV epitopes frequently do not recall a CD8+ T cell response in HCMV-seropositive individuals expressing the matching HLA class I allele. The specified peptides were tested in an IFN-γ ELISPOT assay on PBMC of HCMV-seropositive donors expressing HLA-B35 (A) , HLA-B44 (B) or HLA-B7 (C) .

Article Snippet: This HCMV pp65 495−503 peptide, along with additional HCMV, EBV, and influenza virus peptides specified in Figure 3 , , , respectively, were purchased from Panatecs (Heilbronn, Germany) at >95% purity according to the manufacturer's specification.

Techniques: Expressing, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections

doi: 10.1038/srep04166

Figure Lengend Snippet: Cloned Virus-specific T cell receptors

Article Snippet: HBV peptides: Env 183–191 (FLLTRILTI), Env 171–180 (FLGPLLVLQA), Env 370–379 (SIVSPFIPLL), Core 18–27V (FLPSDFFPSV); HCMV peptides pp65 495–505 (NLVPMVATV), pp65 501–509 (ATVQGQNLK), ie1 42–50 (KEVNSQLSL); EBV peptide EBNA-4NP 399–408 (AVFDRKSDAK); SARS peptide NP 216–225 (GETALALLLL) and Influenza peptide M1 58–66 (GILGFVFTL) were purchased from Genscript.

Techniques: Clone Assay, Virus, Sequencing