Extended Data Fig. 1: Generating a non-immunogenic lung tumor model targeted by CAR T cells.
a, Flow cytometry of hCD19-expressing KP lung adenocarcinoma clone expressing GFP-Luciferase. b, Bioluminescence kinetics of KP-hCD19+ GFP-Luciferase+ clonal cell line after tail-vein injection into C57BL/6 mice, quantified in right panel, n = 4 mice. c, Survival of mice after tail-vein injection (TVI) of KP lung adenocarcinoma cells expressing the CAR target hCD19 compared to founder KP clones that are negative for hCD19 expression (Target−). Two dose levels were used as indicated. Expression of hCD19 did not alter growth kinetics of KP tumor cells suggesting that this human gene did not induce an endogenous adaptive immune response. n = 5 mice per group. d, Irradiation protocol. Middle figure showed bioluminescence imaging of KP-hCD19+ GFP-Luciferase+ clonal cell line three weeks after tail-vein injection into C57BL/6 mice, illustrating that the majority of lung tumor bioluminescence is captured within the “radiation portal” shown in the right panel. Anesthetized mice were placed supine on an aluminum shelf plate under a cone shaped irradiation field (left panel) in a RS2000 Small Animal Irradiator (Rad Source Technologies Inc.) to deliver 160 kV photons at a 25 mA, with a dose rate of 2.1 Gy/min. Mice were treated with a half-beam block technique (right panel) eliminating divergence into the neck superiorly. Radiation was delivered to the thorax through an aperture formed between two 2 mm lead sheets placed above the mice, that shielded the head and neck superior to the apex of the thorax, and tissues inferior to the xiphoid process. e-g, Quantification of numbers of CD8+ and CD4+ CAR T cells in the lungs (e) or thoracic LNs (f,g) of KP19-tumor bearing mice harvested 3 days after adoptive transfer of CAR T cells. n = 5 mice per group, two-sided t-test. h-i, numbers of CD8+ and CD4+ CAR T cells (h) or endogenous T cells (i) in thoracic LNs of KP19-tumor bearing mice harvested 9 days after adoptive transfer of CAR T cells. n = 5 mice per group, two-sided t-test. j-k, Quantification of numbers of A20 (j) or hCD19+ KP cells (k) 2 days after plating. Cells were exposed to indicated doses of irradiation, or not, 24 hours prior to harvesting (KP cells trypsinized) and quantification. n = 6 technical replicates, mean ± SEM. Results representative of three independent experiments. l, Survival of mice after TVI 5×105 of hCD19+ KP lung adenocarcinoma cells. Mice were randomized to treatment with 8 Gy of thoracic irradiation or not, then followed for survival. TVI, tail vein injection; RT, radiotherapy. n = 5 mice per group. Logrank Mantel-Cox test ** p value 0.0027.
