Extended Data Fig. 8: Additional confocal images and Grb2 mechanistic data.
From: The BTLA–HVEM axis restricts CAR T cell efficacy in cancer
a, Graphical abstract of lipid bilayer platform and confocal imaging to colocalize BTLA and SHP-1/SHP-2 within BOX and FOXY CART19. b, Representative confocal images of SHP-1 and BTLA. c. Representative raw images from confocal studies. d,e, Quantification of phosphorylated-Akt (p-Akt) following CD19 and HVEM stimulation. Briefly, human CD19-transduced HVEM+ DM-6 melanoma cells were seeded into culture dishes and allowed to form a stable monolayer for 48 h. 5 × 105 BOX or FOXY CART19 cells were settled onto CD19+ HVEM+ DM-6 monolayers for 0 m, 15 m, and 30 m, and subsequently rinsed off using cold 1× PBS. Cells were then centrifuged, washed twice with cold 1× PBS, and then lysed in buffer (RIPA + protease inhibitor + phosphatase inhibitor). d, Standard western blot was conducted, first to measure p-Akt. Membranes were then stripped, and re-stained for total Akt. Blots were read and quantification was performed using the LI-COR imaging system (Image StudioTM). e, Quantification of p-Akt/Akt for n = 2 independent experiments, using two independent biological donors. f-h, Jurkat-NFAT-GFP reporter cells were transduced with either the BOX CAR19 or FOXY CAR19 construct. f, BOX-Jurkat-CART19 or FOXY-Jurkat-CART19 were cocultured with Nalm6-HVEM in a 1:1 effector:target ratio and monitored each in real-time for the first 15 minutes of coculture via flow cytometry (gated on CD3+ > GFP+). This is a single, representative example. g, BOX-Jurkat-CART19 or FOXY-Jurkat-CART19 were cocultured with either Nalm6 or Nalm6-HVEM (effector: target = 0.25, n = 5 technical replicates per condition), and GFP+ MFI was measured at 6 h using flow cytometry. h, Same experiment and data as in g, but now including the 20 h time point. Statistical significance was determined by two-way ANOVA (g,h).
