Fig 2: SGT-53 enhances immune responses in 4T1 tumor in vivo. Tumor cells were dissociated and immune cell infiltration was assessed via FACS. (A) Infiltrating immune cells were identified by gating CD45+ live cells including DCs (CD11c+I-A/I-E+), macrophages (CD11b+F4/80+), activated CD4 TILs (CD3+CD4+CD107a+), and activated CD8 TILs (CD3+CD8+CD107a+). Infiltrating cells are shown in terms of absolute number of cells per 1 × 104 live cells collected (n = 6–8). (B) Representative FACS plots (left panels) and graphs (right panels) of CTLs (CD3+CD8+GzmB+ or CD3+CD8+IFNγ+) are shown. Numbers in the plots indicate the percentage of cells (n = 6–8). (C) Immunosuppressive MDSCs (CD11b+Gr1+) and Tregs (CD3+CD4+FoxP3+) were identified (n = 6–8). (D) Altered expression of genes associated with immunosuppression in the tumor tissue were assessed via NanoString. (E) Expression of genes associated with immunosuppressive enzymes was assessed by RT-PCR in the tumors (n = 6–8). The fold-change relative to untreated tumors is shown. Data are shown as mean ± SEM. *p < 0.05, 1-way ANOVA with Bonferroni t-test.

Fig 3: mregDCs are enriched upon sepsis induction with dual immunoregulatory and immunogenic functions on CD4+ T cell responses. A. Representative contour plots elucidating flow cytometry-based gating strategy for spleen-derived cDCs in wild-type mice. cDCs were defined as CD45+ Lin (CD3e, CD19, CD49b, Ly6C)- CD11c+ MHC-II+ cells, in which cDC1 and cDC2 were defined CD45+ Lin- CD11c+ MHC-II+ CD8a+ CD11b- and CD45+ Lin- CD11c+ MHC-II+ CD8a- CD11b+ cells, respectively. B, C. Frequency of splenic mregDCs as a proportion of total cDCs at disparate time points after CLP operation. mregDCs were defined as CD45+ Lin- CD11c+ MHC-II+ CD274hi CD80hi cells. Contour plots B. and quantitative bar charts C. showing alterations in mregDCs proportion upon sepsis induction measured by flow cytometry analysis. D. Scatter plot showing mregDCs proportion in cDC1 and cDC2 subsets independently between sham and sepsis groups. E. Quantitative bar plots displaying and comparing mregDCs proportion between wild-type, Batf3-/- and Irf4-/- mice. F. Histograms with quantitative bar charts showing proportion of CCR7, CD80, and CD274 positive cells measured by flow cytometry, across distinct time points after onset of sepsis, as a percentage of total cDCs. G. Multiplex immunofluorescence images demonstrating the in situ existence of mregDCs in spleen after septic challenge, using antibodies, including CD11c, MHC-II, CD80, and CD274. Scale bar, 100 μm. H. Quantitative bar charts showing the level of multiple cytokines in supernatants between mregDCs and non-mregDCs groups, including interleukin (IL)-2, IL-4, IL-10, IL-12, and IFN-γ. I. Quantitative bar chart displaying the results from cell counting kit-8 (CCK-8) assay. J. Histogram with quantitative bar plot illustrating and comparing the proliferative activity of naïve CD4+ T cells co-cultured with either mregDCs or non-mregDCs based on carboxyl fluorescein succinyl ester staining (CFSE) assay. K. Contour plots with quantitative bar chart showing the proportion of CD4+CD25+Foxp3+ Tregs between mregDCs and non-mregDCs groups. One-way ANOVA with Tukey HSD test C, F, J, K; Unpaired two-sided Student's t test D, H, I. Data were expressed as means ± SEM. *P<0.05; **P <0.01; ***P<0.001; ****P<0.0001.

Fig 4: IL1R2 is preferentially expressed on regulatory T cells. (A) IL1R2 expression on Tregs in the TME. Analysis of integrated published scRNA-seq dataset showed different populations of infiltrating immune cells from human CRC tissues (GSE108989), HCC tissues (GSE98638), and NSCLC tissues (GSE99254), respectively. (B) UMAP analysis showing specific expression of Il1r2 on tumor-infiltrating Foxp3+ Tregs. (C and D) MC38 tumor model was established (2×106 cells inoculatedintradermally (i.d.) into the right flank) in wide type C57BL/6 mice (n=8) for 2 weeks, then the spleens and tumors were collected to examine the IL1R2 expression on Tregs. *P<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by Student’s t-test. CRC, colorectal cancer; HCC, hepatocellular cell carcinoma; IL1R2, IL1 receptor 2; NSCLC, non-small cell lung cancer; scRNA-seq, single-cell RNA-seq; TME, tumor microenvironment; Tregs, regulatory T cells;

Fig 5: Hyperoside suppresses T cell proliferation while increasing Th2 or Foxp3+ Treg generation in the co-culture with M2 macrophages. T cells cocultured with M2 macrophages at a ratio of 1:10 (T cell/M2 macrophages) were treated with high-dose HPS (HHPS) (50uM) or low-dose HPS (LHPS) (12.5uM) for 72hrs, and then analyzed via flow cytometry. (A, D) For proliferation assays, T cells were first labeled with CFSE before the co-culture, and the CFSE dilution of CFSE-labeled CD4+T cells was detected by flow cytometry. Flow cytometric analysis showed T-cell proliferation was inhibited by addition of M2 macrophages to the coculture system, and the inhibition was further enhanced by HPS. (B, E) The percentage of IL4-expressing CD4+ T cells was increased by HPS treatment compared with a control group (MT). (C, F) The percentage of CD4+Foxp3+ T cell subsets was also increased by HPS treatment compared with a control group (MT). Differences between experimental groups were evaluated using ANOVA (# P < 0.05 and ## P < 0.01 vs T group; *P < 0.05 and **P < 0.01 vs MT group). T, T cells alone; MT, T cells cocultured with M2 macrophages; MT+LHPS, T cells cocultured with M2 macrophages treated with low-dose HPS (12.5 μM); MT+HHPS, T cells cocultured with M2 macrophages treated with high-dose HPS (50 μM).