mouse ifn γ secretion assay  (Miltenyi Biotec)

Journal: Hepatology (Baltimore, Md.)

Article Title: Human T cells engineered with an HLA-A2-restricted murine T-cell receptor targeting glypican 3 effectively control human hepatocellular carcinoma in mice

doi: 10.1097/HEP.0000000000001175

Figure Lengend Snippet: Identification of TCR specific for human GPC3 in HHD-DR1 mice. (A) Schematic procedure to identify immunodominant GPC3 T-cell epitopes. HHD-DR1 mice were immunized with ADV-GPC3. Eight days later, splenocytes were isolated for epitope screening. Initially, mixtures of synthetic GPC3 peptides covering the entire GPC3 protein were tested, followed by individual peptides from the reactive mixture in the first screening. Splenocytes from a naive mouse served as a negative control. The response was evaluated by intracellular staining for IFNγ and TNFα in CD8 + and CD4 + splenocytes. (B) Dot plots showing TNFα and IFNγ production by CD8 T lymphocytes upon stimulation with Mix 7, peptide 511, or medium (no Ag). Data from a representative mouse in the ADV-GPC3 group and a naive mouse are shown. Cells are gated on CD8 + . (C) Graphs displaying compiled data from the first and second screenings. The percentage of IFNγ + cells in CD8 and CD4 T lymphocytes stimulated with peptide mixes and individual peptides from Mix 7 is shown. The dotted line indicates the threshold for a positive response (3 times the % of IFNγ + cells observed in the naive mouse). Data represent one experiment out of 3. (D) Experimental design for identifying HLA-A2-restricted murine TCRs specific for pGPC3(522-530). Splenocytes from ADV-GPC3-immunized mice were stimulated with pGPC3(522-530), and specific CD8 T cells were single-cell sorted (as IFNγ high cells) and expanded. The 24 expanded clones were processed for TCR library preparation. (E) Distribution of CDR3α and CDR3β region lengths (residue numbers) and usage of TRAV, TRBV, TRAJ, TRBD (when identified), and TRBJ segments in the GPC3-TCR repertoire. Abbreviations: HHD-DR1, double transgenic mouse strain carrying both chimeric HLA-A*0201 (α1-α2) and H-2Db (α3, transmembrane, and intracytoplasmic domains), as well as HLA-DR1 molecules; NGS, NOD scid gamma; NI, nonidentified; TCR, T-cell receptor; TRAV, TCR alpha variable gene; TRAJ, TCR alpha joining gene; TRBV, TCR beta variable gene; TRBD, TCR beta diversity gene; TRBJ, TCR beta joining gene.

Article Snippet: Enriched CD8 + splenocytes were then stimulated with the FLAELAYDL peptide, labeled with Mouse IFNγ Secretion Assay kit (Miltenyi), and IFNγ-producing cells were isolated (single cells) and expanded.

Techniques: Isolation, Negative Control, Staining, Clone Assay, Residue, Transgenic Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Human T cells engineered with an HLA-A2-restricted murine T-cell receptor targeting glypican 3 effectively control human hepatocellular carcinoma in mice

doi: 10.1097/HEP.0000000000001175

Figure Lengend Snippet: Evaluation of CD8 dependency, avidity, and functional avidity of the GPC3-specific TCRs. (A) Transduction efficiency was assessed by measuring the surface expression of murine TCRβ (mTCRβ). (B) Genetically modified human T cells were stained with a saturating concentration of pGPC3(522-530)/HLA-A2 tetramer. (A and B) Dot plots are gated on CD4 (top) or CD8 (bottom) T cells. Numbers indicate the percentage of mTCRβ + cells (A) or tetramer + cells (B) within CD4 or CD8 T cells. (C) Genetically modified T cells were co-cultured with T2 cells pulsed with a saturating concentration of pGPC3(522-530) (1 µg/mL) or medium. The graphs display the response as % of CD137 + cells within transduced (mTCRβ + ) CD4 or CD8 T cells, or total CD4 or CD8 T cells (for UTD). Data are shown as mean ± SD (2 replicates per condition). (A–C) UTD: untransduced T cells. Data are representative of 1 experiment out of 3. (D) TCR avidity was assessed by staining CD8 TCR-T cells with decreasing concentrations of the pGPC3(522-530)/HLA-A2 tetramer and analyzing the % of tetramer + cells within the mTCRβ + CD8 + population. (E) Functional avidity was assessed by culturing CD8 TCR-T cells with T2 cells pulsed with serial dilutions of pGPC3(522-530) and measuring surface CD137 expression, as well as the production of IFNγ, IL-2, and TNFα (by ELISA). (D and E) Nonlinear fit curves are shown, with numbers indicating the EC50 values. Data represent the mean of 3 independent experiments with different donors. compare the different TCRs, the % of TCR + CD8 T cells in each TCR-T cell line was normalized by adding UTD CD8 T cells. Abbreviations: IFN, interferon; TCR, T-cell receptor; UTD, untransduced.

Article Snippet: Enriched CD8 + splenocytes were then stimulated with the FLAELAYDL peptide, labeled with Mouse IFNγ Secretion Assay kit (Miltenyi), and IFNγ-producing cells were isolated (single cells) and expanded.

Techniques: Functional Assay, Transduction, Expressing, Genetically Modified, Staining, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Human T cells engineered with an HLA-A2-restricted murine T-cell receptor targeting glypican 3 effectively control human hepatocellular carcinoma in mice

doi: 10.1097/HEP.0000000000001175

Figure Lengend Snippet: TCR-engineered human CD8 T cells recognize GPC3 + HLA-A2 + tumor cells. (A) Relative expression of GPC3 versus HLA-A2 molecules in different tumor cell lines was used in this study. GPC3 expression is shown as reverse transcription-quantitative PCR CT values and HLA-A2 expression is shown as median fluorescence intensity (MFI). (B) Heat map showing the average % of CD137 + cells within mTCRβ + CD8 T cells stimulated with GPC3 + HLA-A2 + and GPC3 + HLA-A2 - tumor cells. For comparison, the average % of CD137 + cells in UTD CD8 T cells cultured under similar conditions is also shown. Data are compiled from 3 experiments for TCR-A and 7 experiments for TCR-B, TCR-C, and UTD. (C) CD8 TCR-T cells were co-cultured (6 h) with GPC3-silenced tumor cell lines and their respective shREN-treated control cells. (D) GPC3 + HLA-A2 + and GPC3 + HLA-A2 − tumor cells were incubated with anti-pan-HLA-I, anti-HLA-A2, or control IgG (mouse IgG1) mAbs before co-culture with CD8 TCR-T cells. (C and D) The response was evaluated by measuring CD137 expression in mTCRβ + cells (upper panels) and IFNγ release (lower panels). PLCPRF5 cells were used as a negative control for TCR-A and TCR-B, and HEP3B-A2 cells for TCR-C. Data are shown as mean ± SD (2 replicates per condition) and are representative of two experiments (C) or 3 experiments (D). To compare different TCRs, % of TCR + CD8 T cells in each TCR-T cell line were normalized by adding UTD CD8 T cells. Abbreviations: HLA, human leukocyte antigen; TCR, T-cell receptors; UTD, untransduced.

Article Snippet: Enriched CD8 + splenocytes were then stimulated with the FLAELAYDL peptide, labeled with Mouse IFNγ Secretion Assay kit (Miltenyi), and IFNγ-producing cells were isolated (single cells) and expanded.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Fluorescence, Comparison, Cell Culture, Control, Incubation, Co-Culture Assay, Negative Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Human T cells engineered with an HLA-A2-restricted murine T-cell receptor targeting glypican 3 effectively control human hepatocellular carcinoma in mice

doi: 10.1097/HEP.0000000000001175

Figure Lengend Snippet: Effector and proliferative capacity of GPC3 TCR-T cells in response to GPC3 + HLA-A2 + tumor cell recognition. (A and B) CD8 TCR-T (5 × 10 4 mTCRb + /well) cells from 6 donors were co-cultured with serially diluted tumor cells for 24 hours. CD137 expression was detected by FACS (A), and IFNγ in the supernatant was measured by ELISA (B). (A) Percentages of CD137 + cells within TCRβ + CD8 T cells are shown. (C) TCR-T cells (5×10 4 mTCRβ + T cells/well) from 6 donors were co-cultured with luciferase-expressing target cells at varying E:T ratios for 10 hours, and % cytotoxicity was calculated as described in Experimental Procedures. (A–C) Data shown are from donor 1, with data for donors 2–6 available in Supplemental Figure S7, http://links.lww.com/HEP/J637 . (D) CTV-labeled CD8 TCR-T (5 × 10 4 mTCRb + /well) cells were cultured alone (medium) or with irradiated tumor cells (1.25 × 10 4 cells/well) with or without anti(α)-pan-HLA-I mAbs. IL-2 (10 U/mL) was added 48 hours poststimulation. After 96 hours, cells were collected, stained with anti-mTCRβ mAb, and analyzed by FACS. The graph shows the % of proliferating T cells (CTV low ) within mTCRβ + CD8 T cells for donor 1. (E) Effector functions and proliferation of GPC3 TCR-T cells across donors in response to GPC3 + HLA-A2 + tumor cells. For each donor, percentages of TCR+ CD8 T cells were normalized using autologous UTD CD8 T cells. (A-E) Data are shown as mean ± SD. (E) Statistical significance was calculated using Two-way ANOVA. **** p <0.0001. Abbreviations: CTV, CellTrace Violet; IFN, interferon; TCR, T-cell receptor.

Article Snippet: Enriched CD8 + splenocytes were then stimulated with the FLAELAYDL peptide, labeled with Mouse IFNγ Secretion Assay kit (Miltenyi), and IFNγ-producing cells were isolated (single cells) and expanded.

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Luciferase, Labeling, Irradiation, Staining