Characterization of intratumoral T cell subtypes and their heterogeneity by CyTOF. CyTOF proteomic analysis using a T cell antibody panel for TILs from mice challenged with B16-F10 cells and given indicated treatments as described in the Materials and methods. (A) t-SNE plot of an equal number of tumor-infiltrating CD3 T cells from each group and overlaid with color-coded clusters. (B–D) Expression of (B) T cell signature genes, (C) T cell cosignaling receptors, and (D) transcription factors projected onto t-SNE plot in A. (E) t-SNE plots with clusters of T cells in each indicated treatment group. (F) Bar plot of the frequency of each T cell cluster. Cluster names are indicated on the y axis, and frequencies of each cluster are on the x axis. Data represent three independent experiments (n = 4–7 mice per group; t test, *P < 0.05), and significance is shown between αCTLA-4 and αCTLA-4 + IVAX treatment groups. Error bars represent means ± SEM. Irradiated parental B16 tumor cells (Vac) were used as a control for the IVAX group.