Fig 2: Mice injected with single KO of SOX4 or ID3 eventually experience tumor recurrence(A) Experimental design for recurrent tumor model.(B) KO efficiency in sorted T cells from relapsing tumors. Each dot represents a technical replicate (independent sequencing for KO assessment from one mouse per group); mean ± SEM.(C) CD3+CD45+ T cell infiltration in ND561 WT and SOX4KO relapsing tumors (left) and percent of CD3+CD45+ surface huCART-meso in relapsing tumors and infused products (right). Each dot represents a technical replicate (n = 1 mouse per group); Mann-Whitney U test (left) and two-Way ANOVA and Sidak post hoc test (right); mean ± SEM.(D) Total number of MSLN+Epcam+ cells (#) per gram of tumor (left) and representative flow cytometry plots (right) of MSLN expression in ND561 WT and SOX4KO-treated relapsing tumors (n = 1 mouse per group). AsPC-1 cells were used as positive control and MSLN minus as negative control for MSLN staining; Mann-Whitney U test. Error bars represent variation between technical replicates; mean ± SEM.(E) UMAP projection of scRNA-seq data from recurrent TILs harvested from mice injected with WT CAR T cells (ND561, n = 1 donor) and SOX4-KO CAR T cells (ND561 and ND539, n = 2 donors).(F) UMAP projection with broad T cell subsets labeled. NK-like T cells clustered on the left (cluster 2) and all other clusters were defined as GZMK+ exhausted T cells (Tex), right.(G) huCART-meso expression in recurrent tumor TILs.(H) Cluster distribution depicting the frequency that WT and SOX4KO-injected recurrent tumor TILs were found within the broad clusters defined in (F). y axis represents the percent of cells found within each cell type cluster for each condition (WT or SOX4KO). The percent was calculated by taking the number of cells per condition within each cell type cluster and dividing by the total number of cells in that condition. SOX4KO condition was averaged for the two mice and visualized via bar plot, and circles depict the individual values for each mouse; moderated t test using the propeller method.(I) Gene expression levels of TNFRSF9, TIGIT, LAG3, and HAVCR2 in recurrent tumor CARpos TILs extracted from mice injected with WT and SOX4KO CAR T cells.∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ns: not significant. See also Figure S4.