Fig. 4: DMGOs model CAR T cell functional heterogeneity.
a, Representative multispectral 3D imaging of GD2 CAR T cells (CD3; cyan), DMG tumor cells (GFP; green) and cleaved caspase 3 (cCasp3; red) in DMGOs (n = 2 DMGOs). Scale bar, 5 µm. b, GD2 CAR T cell treatment outcome measured as a relative change in tumor GFP intensity quantified by imaging compared to the start of treatment (100%). DMGOs were left untreated (gray line; n = 1 DMGO) or treated with mock-transduced T cells (black line; n = 2) or GD2 CAR T cells (orange line; n = 4 DMGOs); for each treatment condition, a smoothed trend line of the averaged values at different time points was plotted using the locally estimated scatter plot smoothing algorithm. Arrows indicate the time points of T cell administration. c, Representative images of the tumor GFP signal at the indicated time points for a DMGO subjected to prolonged GD2 CAR T cell treatment administrated on day 0, day 8 and day 15 (n = 1 DMGO). d, Sankey plot illustrating the shift in the relative proportions of unbiasedly identified GD2 CAR T cell clusters before and after DMGO exposure. e, UMAP visualization of annotated GD2 CAR T cell clusters. f, Cytotoxic effector molecule and cytokine gene expression across the GD2 CAR T cell clusters. g, Gene expression of selected exhaustion-associated receptors, ligands and transcription factors across the GD2 CAR T cell clusters. f,g, Dot plot representing the percentage of cells expressing selected genes. The color intensity represents the average scaled gene expression. h,i, Heat map depicting the relative expression of exhaustion markers (h) and exhaustion-associated transcription factors and functional regulators (i) in nonexposed (left) and DMGO-exposed (right) GD2 CAR T cells within the TEX cluster. TF, transcription factor. j, Super-engager signature score on a blue-to-red color scale, showing the enrichment of a previously identified T cell serial killer gene set60 atop UMAP cell embeddings of the GD2 CAR T cell dataset. The dashed outline annotates the embedding of the TCYT GD2 CAR T cell cluster. For d–j, GD2 CAR T cells retrieved from n = 4 treated DMGOs and n = 2 independent batches of unexposed GD2 CAR T cells. k, Dot chart depicting the fold enrichment in tumor killing by NCAM1+ GD2 CAR T cells over NCAM1− GD2 CAR T cells quantified as the change in tumor area detected by GFP compared to the start of treatment (n = 2 DMGOs per treatment condition). l, Percentage of cells per TCYT, TEX and THS cluster for NCAM1− GD2 CAR T cells (left; retrieved from n = 2 DMGOs) and NCAM1+ GD2 CAR T cells (right; retrieved from n = 2 DMGOs).
