Fig 1: Cellularity of Peyer's patches from WT and IE-Cpr-null mice.Peyer's patches from 8 wk-old WT (Panels A, C) and IE-Cpr-null (Panels B, D) mice were prepared and immuno-stained as described in the Methods. Cryosections were labeled with fluorophore-conjugated antibodies as indicated below. (Panels A, B) Cryosections were stained with antibodies against CD11c (magenta), B220 (blue), and CD3 (green). (Panels C, D) Cryosections were stained with antibodies against IgA (green) and CD11c (red). (Panel E) Monodisperse cell suspensions of total Peyer's patch cells from WT (open bars) and IE-Cpr-null (solid bars)mice were subjected to flow cytometry as a means to enumerate B and T cell subsets. Cell suspensions were labeled with fluorophore-conjugated antibodies against epitopes B220, CD19, CD3, CD4, or CD8. At least four mice were used for each analysis and the experiments were repeated at least three times.
Fig 2: The TME Is Dynamically Modified Post-dual Therapy(A–F) MC38-luc tumor-bearing mice were sacrificed 1 day after the first or second injection of CpG in different groups (n = 6 in each group), and tumor nodules on both sides were collected and digested for qRT-PCR to explore the cancer-immune set point. The expression levels of all markers are relative to the housekeeping gene, HPRT1. (A–C) Results show the levels of perforin (A), granzyme B (B), and IFN-γ (C), 1 day after the first injection of CpG in the treated side. (D–F) 1 day after the second injection of CpG, the levels of CD8 (D), TGF-β (E), and CD105 (F) from the untreated side are also presented. (G–N) MC38-luc tumor-bearing mice were euthanized 1 day after finishing the whole treatment, and tumor nodules from both sides were collected and lysed for flow cytometry to analyze the TME. Data are quantified (n = 8 in each group). (G–L) Quantities of activated TNF-α+CD8+ T cells (G) and IFN-γ+CD4+ T cells (H); the ratio of more severely exhausted PD-1+CTLA-4+CD8+ T cells (I); and quantities of NKs (defined as CD45+CD3−NK1.1+) (J), macrophages (defined as CD45+CD11b+ F4/80+) (K), and Tregs (defined as CD45+CD4+FOXP3+) (L) from both sides are shown. (M and N) On the untreated side, the number of myeloid-derived suppressor cells (MDSCs) (defined as CD45+CD11b+Gr-1+) (M) and DCs (defined as CD45+CD11c+) (N) is also presented. Values are presented as mean ± SD. Two-way ANOVA was used to analyze the data from (G) to (L). One-way ANOVA was used to analyze the statistical significance in other panels (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; and ns, not significant).
Fig 3: Type I interferon (IFN) and its receptor (IFNAR) contribute to the cGAS pathway in BMDCs. (A) The expression of IFN-β was assayed after anti-mouse IFNAR monoclonal antibody (mAb; 10 ng/mL) treatment. (B) The cell surface markers of CD40, CD80, CD86, and MHC class II were analyzed by flow cytometry in BMDCs transfected with siCon or sicGAS and then infected for 24 h with M. bovis (MOI 5). The CD11c marker was used to set the gate for flow cytometric analysis. The interferon receptor was treated with neutralizing anti-mouse IFNAR mAb (10 ng/mL) to block and then infected for 24 h with M. bovis (MOI 5). (C) The positive cell rate of surface markers in each group was calculated, and histograms were generated by FlowJo software. (D) Culture supernatants were harvested after 24 h; the expression of TNF-α, IL-6, IL-10, and IL-12p70 was assayed by ELISA. IFN-β + M. bovis: BMDCs were treated with exogenous IFN-β (10 ng/mL) and then infected with M. bovis [16]. All data are expressed as mean ± SD, (* p < 0.05; ** p < 0.01; n.s.: no statistical significance).
Fig 4: IFN-? promoted CCL5 expression on macrophages, promoting leukocytes infiltration into the plaque area. (a) Female Apoe-/- mice (n = 15) were put on a Western diet for eight weeks, then the aortic cells were pooled to prepared as described in the methods and stained with an antibody against CD45. Then, CD45+ leukocytes and CD45- non-leukocytes were sorted by FACS, and qPCR was used to analyze Ccl5 mRNA expression. (b and c) Immunofluorescence of the aortic root. The Apoe-/- mice (n = 8) and the Batf3-/- Apoe-/- mice (n = 8) were fed a Western diet for 12 weeks. The cryosections of the aortic root were stained with an antibody against CD45 (red), MAC3 (red), and CCL5 (green). The cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Images were viewed and captured with a Laser Scanning Confocal Microscope. Scale bars: 100 µm, dashed lines indicate the internal elastic lamina, arrows pointing to representative colocalized cells. (d) Primary splenic macrophages were sorted as described in supplementary data, and then treated with100 ng/mL IFN-? for 6 h. Ccl5 mRNA expression was analyzed by qPCR. The Apoe-/- mice (n = 8) and the Batf3-/- Apoe-/- mice (n = 8) were fed a Western diet for 6 weeks, and cryosections of the aortic root were performed. (e) H&E staining. Scale bars: 100 µm. (f) Immunohistochemistry. Representative images of leukocytes (CD45), T cells (CD3), DCs (CD11c), and macrophages (Mac3) in the aortic are shown. Scale bars: 200 µm. Data are presented as mean ± SD. Differences of a P < 0.05 were considered to be statistically significant. **P < 0.01; ***P < 0.001; ns, not significant. Data are representative of three independent experiments.
Fig 5: In Situ Vaccination Significantly Increases the CD8+ T Cell/Treg Ratio and CD11c+ Cells in the SpleenB6 mice were implanted in both flanks with the MC38-luc tumor and sacrificed 1 day after the whole therapy was completed. The spleens were harvested for flow cytometry and an ELISpot assay. (A) Ratio of CD8+ T/Tregs in the spleen. (B) Percentage of CD11c+ in CD45+ splenocytes in four treatment groups. (C) ELISpot assay results using splenocytes stimulated by autologous (MC38-luc) or control (B16) tumor cells. Values represent mean ± SD. One-way ANOVA was used to analyze the statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
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