Fig. 7

Single-cell RNA-sequencing analysis showed that anti-PD-L1 treatment increased different populations of T cells in TCh3 tumors. A Transcriptional data of CD45⁺ tumor-infiltrating immune cells from 4 samples (TAb2, TAb2-anti-PD-L1, TCh3, and TCh3-anti-PD-L1, n = 1 per sample) were integrated using Seurat’s integration algorithm and clustered using UMAP. Cluster phenotyping identified 20 functional clusters colored based on gene expression. B The same UMAP clusters of CD45⁺ tumor-infiltrating immune cells are shown for individual TAb2, TAb2-anti-PD-L1, TCh3 and TCh3-anti-PD-L1 sample colored based on sample type. C Each cluster represented as a proportion of total cells from TAb2 (red), TAb2-anti-PD-L1 (orange), TCh3 (blue) and TCh3-anti-PD-L1 (green) group. D Violin plots showing Cd8a, Ifng, and Csf1r expression in TAb2, TAb2-anti-PD-L1, TCh3 and TCh3-anti-PD-L1 group across all UMAP clusters. Gene expression level shown on the y-axis, and the binned cell count as the width shown on the x-axis. Black dots indicate the mean in each group. Groups were compared using one-way ANOVA. E Volcano plot of DEGs in CD45⁺ tumor-infiltrating immune cells between different groups. Left panel: TAb2 control (blue) vs. TAb2-anti-PD-L1 (red). Right panel: TCh3 control (blue, meaning upregulated in TCh3 control) vs. TCh3-anti-PD-L1 (red, meaning upregulated in TCh3-anti-PD-L1). Difference between DEGs in the two groups was plotted against a threshold of log2(fold change) = 1 and BH adjusted p-value = 0.05. F The category network plot shows the relationship between significant genes (threshold is defined as log2(fold change) = 1 and BH adjusted p-value = 0.05) and top 5 most significant GO terms in the comparison of TCh3-anti-PD-L1 vs TCh3 control group. Within each connected network, node (pathway) size is proportional to the number of neighbors (interacting genes of each node) that are upregulated in TCh3-anti-PD-L1 compared to TCh3 control. Color scheme indicates fold change of upregulation in TCh3-anti-PD-L1 (red) vs. TCh3 control (green)