Fig. 3: Intratumoral CD8 T cell response in mice challenged with the original and CRISPR edited Meth A cell lines.

Tumors from mice challenged with MUT1, REV, and MUT2 Meth A cell lines were harvested ten days after the tumor challenge. Tumor infiltrating CD45 + cells were sorted (Supplementary Fig. 9b) and analyzed by scRNA sequencing, as described in Methods. Combined scRNA sequencing data from the three resulting libraries were analyzed. a Top panel shows a simple hierarchical clustering of MUT1, REV and MUT2 libraries, based on the normalized average expression vector of top ~1,500 informative genes (selected by the highest average TF-IDF score) where, the Y value reflects the distance between clusters. The bottom panel represents the violin heat map plots of the top average TF-IDF scoring gene expression for the three libraries (expression of 26 genes is shown). Significant difference in distance of the REV versus MUT1 and MUT2 in the hierarchy is indicated by asterisk (please refer to Sup Fig. 6 for more details). b Top panel depicts the expression percentage of the genes involved in CD8 T cell activation of CD8 T cells that are derived from MUT1, MUT2, and REV libraries. Bottom panel shows the violin plots for the expression of genes involved in cytotoxicity, early response and other effector functions of CD8 T cells that are derived from the three libraries. c Top panel illustrates clone networks resulting from applying GLIPH-algorithm to the mixed pool of T cells TCR sequencing data, using igraph R package. Each node, represented by a circle, is a TCR clone. The diameter of a node is representative of the number of cells with the same TCR. Existence of a link between two nodes indicates global or local similarity between the two nodes as defined by the GLIPH algorithm. Further, a dense cluster in the network, characterized by high number of connections within a cluster and a low number of connections to neighboring clusters, suggest higher similarity and hence higher specificity within the cluster. A large number of dense clusters might suggest higher diversity in the network. Bottom panel represents the violin plots for the expression of genes involved in cytotoxicity, early response and other effector functions of the top 10 clonally expanded CD8 T cells that are derived from each library.