Clone 6b10

Viral binding assays were performed as described under “Flow cytometry analysis of GP binding” above with the additional step being performed prior to harvesting cell lysates. Following incubation on ice, cells were either immediately trypsinized or shifted to 37°C for 1 h to promote internalization of virions prior to trypsin digestion; nontrypsinized samples were used as a control. Cells were pelleted, washed in PBS, lysed as described under “BSL-4 work” above, and analyzed by Western blotting with antibodies specific to EBOV NP (IBT Bioservices; catalog no. 301-012), CD3ε (Santa Cruz Biotech; clone M-20), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling; clone 14C10). To further confirm reduction in TCR levels, EBOV stimulation was followed by Western blot analysis with antibodies specific for CD3ζ (Santa Cruz Biotech; clone 6B10.2).

To demonstrate that CD133 CAR-T and PD-1 s cells can secrete PD-1 blocking scFv, CAR-T cells were centrifuged at 2000 × g for 20 min at 4 °C. Then, the supernatant of Mock T, CD133 CAR-T, and CD133 CAR-T and PD-1 s cells was concentrated using a Millipore system (10 kd), with centrifugation at 4000 g for 15–20 min until the remaining liquid was approximately 200 µl. Then, RIPA (Sigma) containing protease inhibitor was added on ice for 15 min, and PD-1 blocking scFv was detected using a rabbit anti-c-Myc-tag antibody (CST, Cat 14,038, 1:1000). Detection of antibody was achieved with Pierce ECL western blot substrate (Tanon). To detect whether the CAR genes integrated into T cells, endogenous CD3 zeta and exo-CD3zeta were detected using anti-CD3 ζ (Santa Cruz Biotechnology, Clone 6B10.2, 1:200).