Extended Data Fig. 9: Design and optimization of photoswitchable optoCAR constructs.

a, Design of split CAR candidate constructs to enable light-inducible assembly of intact chimeric antigen receptors (optoCARs). The optical heterodimerization domains ssrA-cpLOV2/LOV2-ssrA and sspB are included in Part A and Part B, respectively. The A1 and B2 construct highlighted in red showed the best performance. B5 without the T cell activation ___domain CD3ζ was constructed as negative control. b, Light-inducible recruitment of Part B (red) to the PM-resident Part A (green) visualized in Jurkat T cells. Scale bar, 5 μm. c, NFAT-dependent luciferase expression in Jurkat T cells transduced with the indicated combinations of optoCAR components. Jurkat T cells were transduced with viruses encoding WT CAR or optoCARs (A1 + B combinations shown in panel a) and co-cultured with CD19⁺ Raji cells. Cell were either shielded or exposed to pulsed blue light illumination overnight. T cell activation was measured by the activity of the Ca²⁺/NFAT-dependent luciferase reporter gene (n = 3 independent biological replicates; mean ± s.e.m.). d, Immunoblot analysis of cell lysates from Jurkat T cells to confirm the expression of each CAR component. Antibodies against GFP and mCherry were used to probe the expression of Part A and Part B, respectively. e, Flow cytometry analysis of cell surface CD69 expression in engineered human CD4⁺ T cells. Under dark (gray) or lit (blue) states, human primary CD4⁺ T cells were transduced with retroviruses expressing WT CAR, optoCAR, or defective CAR constructs, and co-cultured with CD19-negative K562 cells or CD19-positive Raji cells.