Figure 4.

FKBP1A-disrupted CAR-T cells resist rapamycin and tacrolimus immunosuppression in vitro

(A) Frequency of maximum on-target nucleotide conversion by next-generation sequencing (NGS) in T cells base edited with individual FKBP1A-specific sgRNAs complexed with mRNA encoding an adenosine (ABE8.20m) or cytosine (BE4) base editor. (B) Frequency of maximum on-target A>G nucleotide conversion by NGS in T cells base edited with TSBTx1538 sgRNA and ABE8.20m mRNA (FKBP1A^KO). Symbols indicate independent donors. (C and D) FACS plots (C) and summarized data (D) indicate frequency of phosphorylated S6 protein in unmodified and FKBP1A^KO T cells that were activated and treated with rapamycin (RPM) or DMSO vehicle (VEH) control. (E and F) FACS plots (E) and summarized data (F) indicate frequency of NFAT-driven GFP expression in reporter T cells that were unmodified or FKBP1A^KO after treatment with tacrolimus (TAC) or VEH. (G) Percentage change in total CD19-specific CAR-T cells (19CAR) counts 1 week post-treatment with RPM or TAC relative to VEH. Symbols represent three independent donors in duplicate. (H and I) Intracellular cytokine expression was measured in unmodified and FKBP1A^KO 19CAR-T cells after stimulation with JeKo-1 tumor cells in the presence of RPM, TAC, or VEH. FACS plots indicate frequency of IFN-γ⁺ and TNF-α⁺ 19CAR-T cells (H) and summarized data show percentage change in cytokine expression in RPM- and TAC-treated conditions relative to VEH (I). (J) Incucyte cytotoxicity assay as described in materials and methods. Tumor burden quantified as green calibrated units (GCUs) derived from the fluorescence intensity of GFP⁺ JeKo-1 tumors that were cultured in triplicate with either untransduced (UTD) T cells, unmodified 19CAR-T cells, or FKBP1A^KO 19CAR-T cells at a 0.25:1 ratio. Solid lines represent mean GCU from images taken every 4 h, dotted lines show ±SEM, and vertical lines indicate redosing with VEH, RPM, or TAC. (D, F, and I) Symbols represent three independent donors in duplicate. For all data, lines and bars represent mean and error bars show ±SEM.