Fig. 4: Unique targeting features of TEG subpopulations and serial killer potential.

a, Images of CD4⁺ (blue) and CD8⁺ (red) TEGs and their full tracks (up to 10 h) cocultured with 13T organoids (gray surface rendering at t = 0). Scale bars, 50 µm (main image), 30 µm (zoomed-in images). b, Relative behavioral cluster distribution of TEGs cocultured with various organoids. c, Behavioral cluster size difference (%) between CD4⁺ and CD8⁺ TEGs cocultured with the indicated organoid cultures from b (n = 33 wells pooled from the five organoid cultures shown in b; see Supplementary Table 8 for replicate specifics; mean ± s.e.m.). Linear regression model fitting with each well as a random effect: CL9, P = 7.52 × 10^–6 (***); CL8, P = 0.0034 (**); CL7, P = 0.00018 (***); CL6, P = 0.000023 (***); CL5, P = 0.0062 (**); CL4, P = 0.01 (*); CL3, P = 0.001 (**); CL1, P = 3.01 × 10^–6 (***). d, A CD4⁺ TEG killing a 13T tumor cell in a first organoid and a second tumor cell in a neighboring organoid (upper), and a CD8⁺ TEG killing a complete 13T organoid over 11 h (lower). Scale bars, 30 µm; time, h. e, Processed images from d showing 3D-rendered organoids (gray) at t = 0 and the CD4⁺ TEG or CD8⁺ TEG with their full track. Scale bars, 10 µm. f, UMAP embedding showing expression levels of NCAM1. Color gradient represents log₂-transformed normalized counts of genes. g, Quantification of the percentage of dying 13T organoids (of total) after 10 h of coculture with either sorted NCAM1^–CD8⁺ TEGs or NCAM1⁺CD8⁺ TEGs (n = 5 independent experiments, mean ± s.e.m.). Two-tailed unpaired t-test, P = 0.0001036. h, Schematic representation of fluorescent labelling strategy for CD8⁺ TEGs. i, Behavioral cluster difference (%) between NCAM1^–CD8⁺ TEGs and NCAM1⁺CD8⁺TEGs cocultured with 13T organoids (n = 6 independent experiments, mean ± s.e.m.). Linear regression model fitting with each experimental replicate as a random effect: CL9, P = 0.0002 (***); CL8, P = 0.07 (·) ; CL2, P = 0.005 (**); CL1, P = 0.02 (*). j, Images of 13T organoids (gray) with NCAM⁺ super engager CD8⁺TEGs (top) and NCAM^– lazy and dying CD8⁺TEGs (bottom). Scale bars, 10 µm. a,d,j, Representative of n = 5, 3 and 5 independent experiments, respectively).