Figure 3

Characterization of immunomodulating effects of S. aureus molecules on tumor immune microenvironment. (A) Six days after 4T1 tumors were injected with 20 μg of PSM⍺3 (in green, n = 5), 4 μg of Protein A (in blue, n = 5), or vehicle control (Con, in black, n = 5), flow cytometric analysis was performed to determine total T cells and CD8⁺ T cells in tumors. (B–H), Six days after 4T1 tumors were injected with 1 μg of HLA (in orange, n = 5) or vehicle control (in black, n = 5), flow cytometric analysis was performed to enumerate the immune cells in tumors, which includes the percentage of total T cells and CD8⁺ T cells among viable cells (B), the percentage of CD8⁺ and CD4⁺ T cells among total T cells as well as CD8/CD4 and CD8/regulatory T cells (Tregs) ratios (C), the percentage of GzmB⁺ , TNFα⁺ , and IFNγ⁺ CD8 T cells among viable cells (D), the percentage of M1-like (M1, CD206^- MHC-II^high), M2-like (M2, CD206⁺ MHC-II^low), CD40⁺ macrophages (MACs) among macrophages, M1/M2 ratio, and CD86 median fluorescence intensity (MFI) of macrophages (E), the percentage of total dendritic cells (DCs) and CD40⁺ DCs among viable cells (F), the percentage of gMDSCs and PMNs among viable cells (G), and the percentage of PD-L1⁺ immune and nonimmune cells among viable cells (H). (I–K), Six days after the EO771 tumors were injected with 2 μg of HLA (in red, n = 5) or vehicle control (in gray, n = 5), flow cytometric analysis was performed to determine the immune cells in tumors, which includes the percentage of CD8⁺ T cells among viable cells (I), the percentage of CD8⁺ and CD4⁺ T cells among total T cells (J), and the percentage of PD-L1⁺ macrophages among viable cells and PD-L1⁺ non-immune cells among non-immune cells (K). Data are presented as median with quartiles (truncated violin plots). Unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ns, not significant.