Cd137 apc

To assess whether EGFRvIII/CD3 TandAb-induced activation of T-cells is dependent on the presence of target cells, primary human PBMC (4 × 10⁵/well) were cultured in the presence of increasing concentrations EGFRvIII/CD3 TandAbs in the absence of EGFRvIII-positive target cells. After 5 days incubation, proliferation was assessed in a BrdU incorporation assay as previously described (28 (link)).
T-cell activation marker induction by EGFRvIII/CD3 TandAb was analyzed by FACS: primary human T-cells were cultured for 24 h with or without 10 µg/mL of TandAb in the presence or absence of F98^EGFRvIII target cells at an E:T ratio of 1:1 before T-cells were analyzed by flow cytometry for expression of the activation markers CD25, CD137, and CD69 after staining of the cell cultures with CD4-FITC/CD8-FITC (Beckman-Coulter) in combination with CD25-PE (Beckman-Coulter), CD137-APC (BD Biosciences), or CD69-APC-Cy7 (BD Biosciences). As a control, cells were cultured in the absence of antibodies and in the absence of target cells.

The lytic granule loading assay was performed as previously described1. Briefly, 10⁶ PBMCs were seeded into a 96-well plate. For antigen-specific responses, cells were stimulated 120 hours with HPV16/18 E6 and E7 antigens, while R10 media alone was used as a negative control, OVA was used as an irrelevant peptide control and concanavalin A was used as a positive control (Sigma-Aldrich). At the end of the 5-day incubation period, all samples were washed with phosphate buffered saline and subjected to staining for CD3-APCH7, CD4-PerCPCy5.5, CD8-FITC, CD137-APC (BD Biosciences), Granzyme B-PETR and Perforin-PE using the same protocol as listed above. Staining was performed once per time point per sample listed.

The following antibodies were used for flow cytometry: CD3-PerCP-Cy5.5 (clone SK7; eBioscience; used 1:20); CD4-FITC (clone RPA-T4; BD Biosciences; used 1:20), CD4-APC (clone RPA-T4; BD Biosciences; used 1:30), CD4-BV421 (clone SK3, Biolegend; used 1:100), CD8-BV421 (clone RPA-T8; BD Biosciences; used 1:50), CD14-APC-H7 (clone MoP9, BD Biosciences; used 1:100), CD16-APC-H7 (clone 3G8, BD Biosciences; used 1:100), CD19-FITC (clone 4G7, BD Biosciences; used 1:30), CD137-BV421 (clone 4B4-1; Biolegend; used 1:200), CD137-APC (clone 4B4-1; BD Biosciences; used 1:30), OX40-PE-Cy7 (clone Ber-ACT35, Biolegend), CD107-PE (clone H4A3, BD Biosciences; used 1:150) and PE-conjugated anti-mouse TCRβ constant domain (clone H57-597; BD Biosciences; used 1:150). The viability stain IR-Dye (Thermo Fisher, used 1:2,000) was used to identify live cells.