Fig. 4: Characterization of immunosuppressive ESCC tumor microenvironment.

a tSNE plots of 69,278 T cells, colored by cell type (left) and tissue type (top right). The proportions of cell types in normal and tumor tissues are shown on the bottom right (colors as in the left panel). See Fig. 1b legend for the line indications. b Boxplots showing exhaustion score (left) and Treg score (right) of T cells are differentially distributed in patients at stage I (N = 16) and stage II/III (N = 44). P values of two-sided Wilcoxon test are shown. Boxplots show the median (central line), the 25–75% interquartile range (IQR) (box limits), the ±1.5 times IQR (Tukey whiskers), and all data points, among which the lowest and the highest points indicate minimal and maximal values, respectively. c tSNE plot of T cells colored by clone size. d Bar plot showing fractions of proliferating cells in each T cell subtype. The cell numbers of T cell subtypes are labeled on the right. e tSNE plots of 19,273 myeloid cells, colored by cell type (left) and tissue type (top right). The proportions of cell types in normal and tumor tissues are shown on the bottom right (colors as in the left panel). See Fig. 1b legend for the line indications. f Violin plots showing the expression distribution of selected genes involved in immune suppression and angiogenesis in the monocyte and TAM clusters. g Violin plots showing the expression distribution of selected genes involved in DC maturation and immunosuppression in the three DC clusters. h Boxplots showing the expression levels of PD-L1 and PD-L2 in tDC are higher in tumor tissues (N = 60) compare with normal tissues (N = 4; two-sided Student’s t-test). The data presented as mean ± SEM. i, j 100,000 autologous CD8+ T cells isolated from human patients’ peripheral blood and tDCs were co-cultured in a 96-well plate at a ratio of 10:1 with Human T-Activator CD3/CD28. Anti-PD-L1 was administered at 100 μg/ml. T cell number was counted by hemocytometer at day 1 and day 2 (i). IL-2 and IFN-γ levels of the coculture supernatant at day 2 were measured by BD Cytometric Bead Array (CBA) kit (j). In (i, j), P values are derived from two-sided Wilcoxon test; *P < 0.05, **P < 0.01, ***P < 0.001. The data presented as mean ± SD and mean ± SEM in (i) and (j), respectively. Data in (i, j) are representative of three independent experiments for each group. k Clustered heatmap showing two-sided Spearman correlation coefficients between proportions of immune cell subtypes in tumor samples. P < 0.05 is used as the cutoff value.