Journal: Frontiers in Oncology

Article Title: Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells

doi: 10.3389/fonc.2023.1124272

Figure Lengend Snippet: Dual peptide CAR-T cell design. (A) Diagram of the CAR construct designs. The E-28t28z and E-8t28z constructs use CD28 and CD8 as the hinge/transmembrane domains respectively, while both constructs employed CD28 for the co-stimulatory domain. The E-28t28z-tCD34 and E-8t28z-tCD34 constructs are identical to the E-28t28z and E-8t28z constructs but feature co-expression of truncated CD34 as an extracellular marker. (B) Diagram of a CAR-T cell with the different dual peptide-based constructs as the antigen-recognition domain. (C) CD34+ expression analysis by flow cytometry allows for a built-in quantification of transduction efficiency. (D) ELISA results displaying IFN-gamma secretion from CAR- or mock-transduced (GFP) T cells co-cultured overnight against T387 GSCs or cell-free media, **** = p < 0.0001. Values shown in the bar chart represent the average of two independent experiments. (E) CD3ζ western blotting with GAPDH as a housekeeping gene, used for additional support to confirm the presence or lack of the CAR following transduction. LTR, Long terminal repeat; S, signal peptide; P2A, Porcine teschovirus-1 2A self-cleaving peptide; tCD34, Truncated CD34.

Article Snippet: Membranes were incubated with 0.2 μg/mL mouse anti-human CD3ζ (Santa Cruz Biotechnology, SC-166275, Dallas, TX) and 0.04 μg/mL rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, SC-25778, Dallas, TX).

Techniques: Construct, Expressing, Marker, Flow Cytometry, Transduction, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot