Fig. 1: Base-editing screens of the ABD of PI3Kδ in 1928z and 19BBz CAR T cells identify beneficial point mutations.

a, Schematic of CD28 and 4-1BB signaling in T cells. The drawing was adapted from a previous study³¹. b, Flow-cytometry-based analysis of pAKT S473 in 1928z and 19BBz CAR T cells without stimulation and 1 h after coculture with Nalm6 cells; stim., stimulated. Data are representative of n = 2 biological independent samples. c, Representative flow-cytometry-based analysis of CD3 surface expression in primary 19BBz CAR T cells after AncBE4max-mediated KO of TRBC1 and TRBC2 versus mock-electroporated CAR T cells. A modal y-axis representation is shown. Data are representative of n = 3 biologically independent samples. d, Schematic of the ABE and CBE screens performed in primary human 1928z and 19BBz CAR T cells. e, Schematic representation of the PIK3CD domains with indicated amino acid positions adapted from a previous study³¹.The screened ABD is highlighted in red. f, Fold-change enrichment of the respective point mutation in 1928z or 19BBz CAR T cells at the end of the screen relative to day 0 for biologically independent donor A and donor B. g, Differential enrichment of individual point mutations in 1928z versus 19BBz CAR T cells, calculated as the enrichment score in 1928z minus the corresponding score in 19BBz. Positive values indicate mutations enriched in 1928z over 19BBz CAR T cells, while negative values indicate enrichment in 19BBz over 1928z CAR T cells. Donors A and B are biologically independent samples. h, Structural model of the interaction of p110δ (gray) with p85 (light blue) (PDB 7JIS). The protein tertiary structure is shown for ABD and PIK3R1. The side chains for P32 (green) and K81 (red) substitutions are shown with their closest amino acid compared to the respective WT amino acid (light red). i, Flow-cytometry-based analysis of pAKT T308 in 19BBz and 1928z CAR T cells without (mock) and with the E81K and L32P substitutions 30 min after Nalm6 tumor cell stimulation at an E:T ratio of 1:1. Data are shown in technical triplicates from one representative donor of three biologically independent donors. j, Schematic representation of PI3K signaling activity in BBz-based and 28z-based CAR T cells, illustrating the impact of E81K and L32P substitutions on phosphorylation of AKT at T308. The drawing was adapted from a previous study⁷⁷.