Journal: Blood cancer discovery

Article Title: An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities

doi: 10.1158/2643-3230.BCD-19-0059

Figure Lengend Snippet: A) Self-reactive antibodies of the IgM isotype were visualized by a Kallestad HEp-2 assay adapted to the murine system and mean fluorescence intensities (MFI) were quantified (n = 6 per genotype, two exemplary cases per genotype are shown). B) Quantification of the observed staining patterns. C) Autoreactive IgG immunoglobulins were visualized by an adapted Kallestad HEp-2 assay and MFI values were quantified (wt, n = 7; MC, n = 8; BC, n = 8; MBC, n = 9. Two exemplary cases per genotype are shown). D) Quantification of the observed staining patterns. E) and F) wt, MC, BC and MBC animals (n = 3) were immunized intraperitoneally with either NP-Ficoll (50 μg) or NP-CGG (50 μg) at day 0 and the NP-specific IgM and IgG levels in the sera of animals were measured at days 0, 4, 7, 10, 21 and 40 after immunization by ELISA. Envelopes represent SEM.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; Welch’s unpaired two-tailed t-test.

Article Snippet: HEp2 assay Serum was diluted 1:40 in PBS and 30 μl per sample were added on a Kallestad HEp-2 slide (Biorad, Cat. No. 26101), incubated for 20 min in a wet chamber and washed with PBS for 10 min.

Techniques: Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell Reports

Article Title: Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice

doi: 10.1016/j.celrep.2022.110399

Figure Lengend Snippet:

Article Snippet: Anti-nuclear antigen/HEp-2 cell-lysate ELISAs were performed using the ANA-HEp2 Screen ELISA kit (Tecan, RE70151), as described above.

Techniques: Activation Assay, Recombinant, Reverse Transcription, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: Science translational medicine

Article Title: Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

doi: 10.1126/scitranslmed.3006034

Figure Lengend Snippet: (A) Biophysical parameters describing the interaction between 12 MAGE A3 affinity-enhanced TCRs and the HLA-A*01–restricted MAGE A3 peptide antigen. Affinity-enhanced TCRs were produced by phage display, and affinity and half-life were calculated from SPR measurements. (B) Sensitivity of TCR-engineered T cells to antigen. The TCRs from the panel were used to produce TCR-engineered T cells via lentivirus-mediated transduction. Sensitivity was assessed by IFN-γ release, using HEP2 cells HLA-A*01+ pulsed with a titration of MAGE A3 peptide, as targets. Control measurements were carried out in the absence of peptide. For clarity, data are shown for 8 of the 12 TCRs. TCRs with α chain mutations are shown in the top panel and those with β chain mutations in the lower panel. Experiments were carried out in triplicate. Error bars indicate SEM. wt, wild type; ntc, absence of T cells.

Article Snippet: HEP2 cells were obtained from ScienCell and maintained in hepatocyte medium (ScienCell).

Techniques: Produced, Transduction, Titration, Control

Journal: Science translational medicine

Article Title: Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

doi: 10.1126/scitranslmed.3006034

Figure Lengend Snippet: (A) Activity and specificity of engineered T cells expressing affinity-enhanced TCRs against various tumor cell lines and normal human hepatocytes (HEP2). The cell line is indicated above each graph along with HLA-A*01 status and an indication of MAGE A3 expression (shown in brackets as +/++/+++). IFN-γ release (left column) was determined by ELISpot, and cytotoxicity (right column) was determined using the IncuCyte platform (no. of apoptotic cells). Controls were carried out using nontransduced cells (ntd) and in the absence of T cells (ntc). Experiments were carried out in triplicate. Error bars indicate SEM. (B) Degranulation. T cell lines carrying the a3a or b2a affinity-enhanced TCRs were assessed for surface accumulation of CD107a in the presence of MAGE A3+ OV79 cells and MAGE A3− M108. Nontransduced T cells (ntd) and T cell carrying the wt TCR were included for comparison. Data were obtained using flow cytometry. Samples were initially gated on lymphocytes followed by gating on CD8+ and CD4+ events and finally a gate on Vβ5.1. Control measurements were carried out using PMA and ionomycin (PMA+I) and unstimulated T cells (No stim).

Article Snippet: HEP2 cells were obtained from ScienCell and maintained in hepatocyte medium (ScienCell).

Techniques: Activity Assay, Expressing, Enzyme-linked Immunospot, Comparison, Flow Cytometry, Control

Journal: Science translational medicine

Article Title: Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

doi: 10.1126/scitranslmed.3006034

Figure Lengend Snippet: (A) Each residue in the MAGE A3 peptide sequence was sequentially replaced with alanine. Alanine-substituted peptides were pulsed onto HLA-A*01 HEP2 target cells, and IFN-γ release from a3a-engineered T cells was assessed. The residue replaced by alanine is indicated under each bar. (B) The same procedure was followed for glycine-substituted peptides, except substitution of the native glycine residue at position 6 was omitted. (C) Activation of a3a-engineered T cells by motif-containing peptides. Peptides were produced synthetically and pulsed onto HLA-A*01 HEP2 target cells. Activation of a3a-engineered T cell patient product and a3a-engineered T cells prepared from a healthy donor was determined by IFN-γ release. Nontransduced T cells (ntd) (from a healthy donor) were used as a control. (D) Activation of a3a-engineered T cells by mouse Titin. IFN-γ release was used to compare activation of a3a-engineered T cells against HEP2 cells pulsed with mouse and human Titin peptide. MAGE A3–pulsed cells are also included for comparison. All data shown were obtained from three independent measurements and represent means ± SEM.

Article Snippet: HEP2 cells were obtained from ScienCell and maintained in hepatocyte medium (ScienCell).

Techniques: Residue, Sequencing, Activation Assay, Produced, Control, Comparison