Fig. 3

C215Fab-SEA promoted CD8 T-cell cytotoxic function and reduced the Treg cell number in the TME. A Heatmap displaying the mean log2-fold-change values of the expression of selected genes associated with T cells in tumors following different treatments (left). n = 3–4/group/cycle. The color gradient indicates the fold increase relative to the designated control. The right heatmap shows the p values of the significant differences in the left heatmap. White represents a nonsignificant expression change compared to the matched control. Differential expression analysis was performed with Welch’s t test. B Representative immunohistochemical (IHC) double staining analysis (n = 2–3/group/cycle) of CD8 (red) and granzyme B (green). Frozen sections of MC38-hEpCAM tumors from the control and treatment groups were taken 24 h after the completion of cycle 3 for IHC analysis. Scale bar, 100 µm. C Violin plots show the percentage of CD25⁺Foxp3⁺ cells among the total CD4⁺ T cells in the tumors as determined by FC. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. n = 3–4/group/cycle. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control; for C4 Combo versus C215Fab-SEA. D Pseudocolor plots showing Treg gating strategy and representative FC scatter plots (n = 3–4/group/cycle) demonstrating the reduction in Tregs in the TME following C215Fab-SEA. E Graph displaying CD8/Treg ratios in the TME according to NanoString gene expression analysis (left) and FC (right) of MC38-hEpCAM tumors after the completion of each treatment cycle. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. n = 3–4/group/cycle. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control, for C4 Combo versus C215Fab-SEA. In all figures, timepoints are referred to as C1-C4 (cycles 1-4, respectively).