TCR-KO reporter cells were activated with blinatumomab or staphylococcal enterotoxin E in the presence of Raji target cells (4:1 E:T ratio) in 96-well white flat-bottom assay plates (Corning). After a 6-hour incubation at 37°C, 5% CO2, luciferase reporter induction was determined by addition of Bio-Glo-NL detection reagent (Promega). Plates were read on a GloMax Discover luminometer (Promega). Fold induction was calculated: RLUagonist/RLUno agonist. 4-PL curves were generated by GraphPad Prism Software.
A375 or SK-MEL5 cells were plated overnight to test the TCR-mediated reporter activation by peptide presented by APCs. The following day, serial titrations of the peptides were added followed by TCR-expressing TCR-KO cells (2.5:1 E:T ratio) as indicated. After a 6-hour incubation at 37°C, 5% CO2, luciferase reporter induction was determined as previously shown. In some cases, SK-MEL5 cells were treated with 200 ng/mL of interferon-gamma (IFN-γ; Peprotech) during the overnight plating.
To test the TCR-mediated reporter activation by processed whole antigen protein, THP-1-derived dendritic cells (THP-1-DCs) were generated as described32 (link) with slight modification. Briefly, immature-like DCs were generated by treatment with 100 ng/mL granulocyte-macrophage colony-stimulating factor and 100 ng/mL IL-4 (both from Peprotech) for 5 days at 37°C. Then, cells were harvested and matured with 100 ng/mL granulocyte-macrophage colony-stimulating factor, 200 ng/mL IL-4, 15 ng/mL tumor necrosis factor-alpha (Peprotech), and 268 μM Ionomycin (Sigma) in the presence of titrations of recombinant H3 or 2020 Fluzone Quadrivalent. After 48 hours at 37°C, loaded THP-1-DCs were co-cultured with TCR-expressing reporter cells as previously shown.
To test the TCR-mediated reporter activation by translation and antigen presentation of transfected nucleic acids, A375 cells were transfected with a titration of HPV16 E6 expression plasmid complexed with ViaFect transfection reagent (Promega). After 24 hours at 37°C, transfected A375 cells were co-cultured with HPV16 E7 TCR-expressing reporter cells as previously shown.
A375 or SK-MEL5 cells were plated overnight to test the TCR-mediated reporter activation by peptide presented by APCs. The following day, serial titrations of the peptides were added followed by TCR-expressing TCR-KO cells (2.5:1 E:T ratio) as indicated. After a 6-hour incubation at 37°C, 5% CO2, luciferase reporter induction was determined as previously shown. In some cases, SK-MEL5 cells were treated with 200 ng/mL of interferon-gamma (IFN-γ; Peprotech) during the overnight plating.
To test the TCR-mediated reporter activation by processed whole antigen protein, THP-1-derived dendritic cells (THP-1-DCs) were generated as described32 (link) with slight modification. Briefly, immature-like DCs were generated by treatment with 100 ng/mL granulocyte-macrophage colony-stimulating factor and 100 ng/mL IL-4 (both from Peprotech) for 5 days at 37°C. Then, cells were harvested and matured with 100 ng/mL granulocyte-macrophage colony-stimulating factor, 200 ng/mL IL-4, 15 ng/mL tumor necrosis factor-alpha (Peprotech), and 268 μM Ionomycin (Sigma) in the presence of titrations of recombinant H3 or 2020 Fluzone Quadrivalent. After 48 hours at 37°C, loaded THP-1-DCs were co-cultured with TCR-expressing reporter cells as previously shown.
To test the TCR-mediated reporter activation by translation and antigen presentation of transfected nucleic acids, A375 cells were transfected with a titration of HPV16 E6 expression plasmid complexed with ViaFect transfection reagent (Promega). After 24 hours at 37°C, transfected A375 cells were co-cultured with HPV16 E7 TCR-expressing reporter cells as previously shown.
