Fig 2: The alterations of immune cell populations during induced expression of dcIL12IL2GMCSF. The inducible B16 cells were subcutaneously inoculated into the flanks of C57BL/6 mice. At different times after dox administration, splenocytes and tumor-infiltrating lymphocytes were isolated and subjected to flow cytometry analysis. The gated CD45+ cells were analyzed with the groups of NK1.1, CD3 (A) or CD11C, MHCII (B). The experiments were performed twice. *p < 0.05; **p < 0.01; ***p < 0.001. n = 4

Fig 3: Overview of Flow Cytometry Gating Strategy and Data Analysis(A) Example of the gating strategy used to assess CD8+ T cells by flow cytometry. Total lymphocytes are gated by forward scatter-area (FSC-A) and side scatter-area (SSC-A) characteristics. Doublets are then excluded by FSC-A and FSC-width (FSC-W). Viable cells are defined as those excluding a viability dye. CD3+CD8+ T cells are gated and then analyzed for expression of IFN? and TNF-a. (B) An example of the data analysis workflow following gating and export from flow cytometry analysis software. Total percentage and number of IFN?+ and IFN?+ TNF-a+ T cells from the non-co-cultured controls are subtracted from the co-culture data to remove background.

Fig 4: LCMV Infection of White Adipose TissueB6 mice were given LCMV-Armstrong (2 3 105 PFUs, intraperitoneal [i.p.]), and levels of virus in adipose and spleen and leukocyte abundance in adipose were determined.(A) The line graphs represent virus levels mean (±SEM) in the spleen or perigonadal white adipose tissue (WAT) at the indicated days after infection. The dotted horizontal line indicates the limit of detection. Data are pooled from two experiments with three to six mice per group.(B) Perigonadal fat pads were isolated from LCMV-infected mice at day 5 and assessed for viral infection. Gentle centrifugation was used to separate low density mature adipocytes from denser stromal vascular fraction (SVF) cell populations. The graph shows quantification of infectious virus (n = 3, one experiment), with an example of plaques developing in Vero cells.(C) Perigonadal adipose tissue was harvested at the indicated days of infection and analyzed by microscopy. Top: representative sections stained with H&E; scale bar, 120 mm. Bottom: whole-mount staining reveals the lipid droplet in adipocytes (BODIPYC12; red), cell nuclei (Hoechst; blue), and infiltrating T cells (anti-CD3; green); scale bar, 100 mm. Data are representative of two experiments with two or three mice per group.(D) Total number of leukocytes, CD8+ T cells, and CD4+ T cells recovered from fat pads before infection (n = 8) and at day 8 post-infection (n = 6). Data were analyzed using unpaired Student’s t test with **p < 0.01 and ****p < 0.0001.

Fig 5: Immunomodulatory effect in vivo.(A) Statistical analysis of flow cytometry of the uptake of NS-mediated nanozymes in different immune cells of the spleen in the subcutaneous tumor model (n = 3). (B) Statistical analysis of flow cytometry of the uptake of NS-mediated nanozymes in different immune cells of the spleen in the metastatic tumor model (n = 3). (C) Flow cytometry and corresponding statistical analysis of CD11c+MHC II+CD80+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (D) Flow cytometry and corresponding statistical analysis of CD11c+MHC II+CD86+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (E) Statistical analysis of flow cytometry of CD11b+Gr-1+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (F) Statistical analysis of flow cytometry of CD3+CD8+T-bet+ and CD3+CD8+Eomes+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (G) Statistical analysis of flow cytometry of CD3+CD8+CD107a+, CD3+CD8+IFN-?+, and CD3+CD8+TNF-a+ splenocytes of mice in six groups with different stimulations for the early-phase subcutaneous tumor treatment model (n = 3).