Extended Data Fig. 5: The modified KG2032 CAR T cells react with AML cells, but not with normal epithelial cells even when stimulated with IFN-γ.
a. Constructs of VHVL-KG2032 CAR. b. Flow cytometric analysis of CAR transduction efficiencies in CAR T cells. c. Secretion of IFN-γ by the indicated CAR T cells after co-culture with KG1a AML cells. Mock-transduced T cells were used as controls (n = 2 technical replicates). d. Constructs of the VHVL86-KG2032 CAR e,f. The same analysis as b and c, comparing the VHVL-KG2032 CAR and VHVL86-KG2032 CAR. g. Constructs of the VHVL86-1XXKG2032 CAR h,i. The same analysis as b and c, comparing the VHVL86-KG2032 CAR and VHVL86-1XX-KG2032 CAR. j. Flow cytometric analysis of KG2032 reactivity to the indicated cells. k. 51Cr release assay using HLA-DRB1*04:05-expressing U937 cells as target (n = 3 technical replicates from a single experiment, repeated two times with similar results). E/T, effector/target. l. 51Cr release assay using primary AML patients’ BM cells as target (n = 5 technical replicates from a single experiment). m. Cytotoxicity assay performed via flow cytometry using either the CD4+ or CD8+ fraction of the modified KG2032 CAR or control T cells. Results of an experiment are shown. n. KG2032 or L243 reactivity to KO52 cells treated with IFN-γ for 48 h. Data are representative of two independent experiments. o-q. Secretion of IFN-γ and IL-2 by modified KG2032 CAR T cells after co-culture with (o) normal intestinal epithelial cells purified from the unaffected region of small bowel in patients with colon cancer (n = 3 technical replicates from a single experiment), (p) HT29 colon epithelial cell line or H1975 lung epithelial cell line stimulated with IFN-γ for 48 h (n = 3 technical replicates from a single experiment, repeated two times with similar results), or (q) intestinal organoid–derived epithelial cells stimulated with IFN-γ for 48 h (n = 1 for donor 1; n = 3 technical replicates for donor 2 from a single experiment for each). In each case, co-culture with KG1a cells was performed as a positive control.
