Fig 1: Flow cytometry data validate the transcriptional characterization of CD8+ CAR T cells. A, Box plots comparing the percentage of IFN?-producing CD8+ CAR T cells (top) and TOX-producing CD8+ CAR T cells (bottom) between effector precursor preinfusion cells (Tigit+, CD62Llo, CD27-; red) and preinfusion cells with the opposing surface phenotype (Tigit-, CD62Lhi, CD27+; blue) cocultured either with CD19 knockout (KO) tumor (left) or CD19+ tumor (right). Dashed lines link subsets from the same patient and sample. B, Representative flow cytometry data visualizing GzmB-BV421 and GzmK-FITC staining of preinfusion CD8+ CAR T cells (left) stimulated with either CD19+ tumor (red) or CD19 KO tumor (blue) and unstimulated postinfusion CD8+ CAR T cells (right).
Fig 2: A subset of GMP CAR T cells is uniquely poised to give rise to cytotoxic effectors. A, Dot plot comparing relative expression of 14 genes differentially expressed between GMP effector precursors (labeled as “precursors”), as defined by aßTCR lineage tracing, and all other CD8+ GMP CAR T cells (labeled as “nonprecursors”). B, Box plot comparing the proportion of GMP to postinfusion lineages that ended up in effector clusters 3 and 8 between responders and nonresponders. C, UMAP based on SCENIC transcriptional factor regulatory network analysis conducted on effector precursors (labeled as “precursors”; red) and a random subset of other CD8+ GMP CAR T cells (labeled as “nonprecursors”; blue). D, Violin plots comparing activity levels of regulons based on SCENIC analysis. Asterisks indicate the degree of significance. ****, Padj < 1E-15; ***, Padj < 1E–10 (>1E-15); **, Padj < 1E-5 (>1E-10); *, Padj < 0.05 (>1E-5). E, AUC of the best performing iteration of an SVM classifier trained on the top 100 differentially expressed genes between GMP effector precursors and all other CD8+ GMP T cells. The single-cell data set was randomly downsampled for 1,000 iterations, with LOOCV in each iteration. F, Flow cytometry data from an aliquot of patient 11's GMP preinfusion product, visualizing CD8+ CAR T cells with the effector precursor surface phenotype (TIGIT+, CD62Llo, and CD27-) and the opposite noneffector associated surface phenotype (TIGIT-, CD62hi, and CD27hi). G, Bar plot comparing the proportion of bulk ß TCRs sequenced that matched those from postinfusion CD8 transcriptional clusters. Differences are represented as log2 fold change for the proportions of TCRs matching each cluster. Colors correspond to the precursor effector signature (red) or the opposing, noneffector signature (blue). Asterisks indicate the degree of significance. ****, Padj < 1E-15; ***, Padj < 1E-10 (>1E-15); **, Padj < 1E-5 (>1E-10); *, Padj < 0.05 (>1E-5); Padj < 0.1 (>0.05). H, Cumulative ß clone sizes of postinfusion CAR T cells that share ß TCRs with GMP product CAR T cells sorted by the precursor effector phenotype (red) or the opposing, noneffector surface phenotype (blue). The graph represents the proportion of cells (y-axis) cumulatively encompassed by increasing clone sizes.
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