Anti cd3ζ

The protocol of Western blot analysis was described previously (45 (link), 46 (link)). The antibodies used were as follows: anti-c-Met (Cell Signaling Technology, 8198S), anti-PD-L1 (Cell Signaling Technology, 13684S), anti-CD3ζ (Santa Cruz Biotechnology, sc-166275), anti-GAPDH (Proteintech, 60004-1- Ig), goat anti-mouse IgG-HRP (FDbio science, FDM007), and goat anti-rabbit IgG-HRP (FDbio science, FDR007). Final detection was performed with an enhanced chemiluminescence system (Tanon, China).

Conjugates between Jurkat cells and SEE-pulsed Raji B cells were carried out as previously described (Finetti et al., 2009 (link)). Raji cells were pulsed for 2 h with 10 μg/ml SEE (Toxin Technology, Sarasota, FL, United States) and labeled with 10 μM Cell Tracker Blue (Molecular Probes) for the last 20 min. Conjugates between T cells and unpulsed B cells were used as negative controls. SEE-pulsed or unpulsed Raji B cells were mixed with Jurkat T cells (1:1) and conjugates analyzed 15 min after their formation. Samples were allowed to adhere for 15 min on poly-L-lysine (Sigma-Aldrich)-coated wells of diagnostic microscope slides (ThermoFisher Scientific), then fixed by immersion in methanol for 10 min at -20°C. Following fixation, samples were washed in PBS and incubated with anti-pTyr (Cell Signaling, #8954) at 10 μg/mL and anti-CD3ζ at 15 μg/mL (SantaCruz, #sc-1239) in PBS 1X overnight at 4°C. After washing in PBS, samples were incubated for 45 min at room temperature with anti-rabbit Alexa-Fluor-488- and anti-mouse Alexa-Fluor-555-labeled secondary antibodies (ThermoFisher Scientific, #A11008 and #A211422, respectively).

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).