Anti human cd137

Antibodies were obtained as follows: anti-human CD3 (BD Biosciences, 555335), anti-human CD8 (BD Biosciences, 555366), anti-human CD107a (BD Biosciences, 555801), and anti-human CD137 (BD Biosciences, 555956). The antibodies were incubated with T cells at 4°C for 25 min and washed twice (PBS with 2% FBS). Mesothelin CAR, ErbB2 CAR, CD19 CAR, and OKT3-28BB expression were detected by biotin-labeled polyclonal anti-mouse F(ab)2 antibody (Jackson Immunoresearch). Samples were then stained with phycoerythrin-labeled streptavidin (eBioscience, 17-4317-82). Flow cytometry acquisition was performed on either a BD FACSCalibur or Accuri C6 Cytometer (BD Biosciences). Analysis was performed using FlowJo software (Treestar).

Cells, 1.0 × 10⁵, were seeded and grown as a monolayer on 8‐well chambered slides (LAB‐TEK) for 3 weeks. Cells were washed with PBS and fixed with one of the following steps: (1) preextracted with 0.2% TritonX/PBS on ice followed by 1% paraformaldehyde/PBS for 15 min at RT; (2) fixed directly with 1% paraformaldehyde/PBS for 15 min at RT; (3) fixed directly with 100% methanol for 5 min on ice; and (4) fixed directly with 100% Acetone for 5 min on ice. Cells were permeabilized with 0.5% Tween/PBS and blocked in 0.1% Tween/casein/PBS before primary antibody staining. Tissue postfixation was done with 4% paraformaldehyde/PBS and mounted with Prolong Gold antifade reagent with DAPI (Life Technologies). Images were obtained using a spinning disk BD CARVII Confocal Imager (BD Biosystems, San Jose, CA) on a Zeiss Axio Observer inverted microscope (Carl Zeiss Microscopy, Thornwood, NY) at 63X objective lens controlled by MetaMorph imaging software (Molecular Devices, Sunnyvale, CA). Image Z resolution was further optimized with Volocity software (PerkinElmer, Waltham, MA). For non‐Z‐stacked higher resolution images, a Leica SP5 inverted laser confocal micorscope was used. Antibodies used: Anti‐claudin 3(#ab15102, AbCam, Cambridge, MA), anti‐claudin4 (#329400, Life Technologies), Anti‐human CD137 (#552533, BDBioscience), phalloidin‐Alexa 647(A22287, Life Technologies).