Fig 2: Functional activation of ALDH1-specific CD8+ cells in AGI-101H-treated melanoma patients upon vaccine administration. A. Representative dot plots presenting unstimulated, ALDH1A1 peptide stimulated, MAA peptides stimulated, and anti-CD3/anti-CD28 stimulated CD8+ T cells (from AGI-101H treated melanoma patient on the day of vaccine administration) stained with anti-CD107a and anti-IFN-?. B. Representative dot plots presenting unstimulated, ALDH1A1 peptide stimulated, MAA peptides stimulated, and anti-CD3/anti-CD28 stimulated CD8+ T cells (from AGI-101H treated melanoma patient 6 days after vaccine administration) stained with anti-CD107a and anti-IFN-?. C. Percentage of CD107a+IFN-?+ cells in unstimulated, ALDH1A1 peptide stimulated, MAA peptides stimulated, and anti-CD3/anti-CD28 stimulated CD8+ T cells (from AGI-101H treated melanoma patient on the day of vaccine administration and 6 days later).

Fig 3: Mice expressing RNase1 favor an antitumor TME. (A) A heatmap of different marker intensities in CD45+ tumor-infiltrating immune cell clusters identified by CyTOF data analysis using t-SNE and FlowSOM. (B and C) Annotation of t-SNE plots of immune cells overlaid with color-coded clusters based on marker expression as shown in (A). Mf, macrophage; MDSC, myeloid-derived suppressor cell; NK, natural killer cell; DC, dendritic cell; CD8 T, CD8+ T cell; Mono, monocytes; CD4 T, CD4+ T cell. (D) Box plots of frequency of CD45+ tumor-infiltrating immune cell clusters as indicated. n = 6 mice per group. Box plots indicate minima (lower end of whisker), maxima (upper end of whisker), median (center), 25th percentile (bottom of box), and 75th percentile (top of box). *p < 0.05, **p < 0.01, ***p < 0.001, individually compared with the percentage at the first bar of each cluster (4T1-vector), one-tailed unpaired t test.

Fig 4: BCAT2 varies expression patterns of CD8+T-cell-related chemokines and inhibits cytotoxic capacity of CTLs. A,B) Heatmaps of ProcartaPlex multiple immunoassays display secretion variation of common cytokines and chemokines in BCAT2 OE, BCAT2 KD and negative control groups (n = 3 per group). C,D) Histograms show normalized mRNA expression levels and protein secretion concentrations of CCL3, CCL4, CCL5, CXCL9, and CXCL10 in BCAT2 OE (left), BCAT2 KD (right) and negative control groups (n = 3 per group). *p < 0.05; **p < 0.01; ***p < 0.001. E) Chemotaxis assay indicates different chemotaxis ability of CTLs in BCAT2 OE, BCAT2 KD, and negative control groups (n = 3 per group). *p < 0.05; **p < 0.01. F) T-cell-mediated cancer cell killing assay indicates different killing abilities of T cells cocultured with BCAT2 OE, BCAT2 KD, and negative control cell lines (n = 3 per group). G) Flow cytometry analysis indicates different activities of CD8+T cells in different coculture groups (n = 3 per group).

Fig 5: BCAT2 negatively correlates with anticancer immunity in TME of BLCA. A) Expression pattern of chemokines, chemokine receptors, MHC molecules, and immunostimulators in high and low BCAT2 groups. B) Activity of cancer immunity cycle in high and low BCAT2 groups. Seven colors represent seven steps in the cycle. *p < 0.05; **p < 0.01; ***p < 0.001. C) Correlations between BCAT2 and multiple types of immune cell (CD8+T cell, CD4+T cell, DC cell, and NK cell) in six independent algorithms. D) Expression patterns of multiple subtypes of immune cell (CD8+T cell, NK cell, Macrophage, Th1 cell, and DC cell) related effector genes in high and low BCAT2 groups. E) Correlations between BCAT2 and ICB related effector genes. Number in the circle means correlation coefficient.