Fig 2: Mettl3 promotes the translation of Tirap, CD80, and CD40 mRNA in vitro. a, b Relative luciferase activity of pMIR-REPORT with empty pMIR (Vector), wild-type, or the m6A site mutation of Tirap-3′UTR (Tirap_Wt and Tirap_Mut) (a) or of CD40-3′UTR (CD40_Wt and CD40_Mut) (b) transfected into HEK293T cells. Firefly luciferase activity (Luci) was measured and normalized to Renilla luciferase activity (Ren). c Flag expression in HEK293T cells transfected with either wild-type CD80-CDS (CD80_Wt) or its m6A site mutation (CD80_Mut). Numbers below plot in (a, b) and above plot in c indicate the amount of transfected plasmids. d mRNA level of Firefly luciferase in HEK293T cells transfected with empty pMIR (Vector) or plasmids bearing wild-type (Wt) Tirap 3′-UTR (left) or CD40 3′-UTR (middle) either, or their m6A site mutation 3′UTR (Mut). The results were normalized by Renilla luciferase and presented relative to those transfected with a vector, set as 1. Right: mRNA level of Flag-CD80 CDS in HEK293T cells transfected with either wild-type CD80-CDS (CD80_Wt) or its m6A site mutation (CD80_Mut). Results were normalized by human GAPDH and presented relative to those transfected with CD80 (250 ng), set as 1. Numbers below indicate the amount of plasmids used for transfection. e RIP assay of m6A-modified Tirap, CD80, and CD40 mRNA fragments retrieved by m6A antibody in HEK293T transfected with wild-type or m6A site mutant Tirap-3′UTR, CD80-CDS, and CD40–3′UTR. Data from one representative of three independent experiments (c), and shown as mean ± SEM (a, b, d, e) of three determinants. **P < 0.01, ***P < 0.001, NS, not significant (Student’s t-test, two-tailed)

Fig 3: Dual treatment enhances DC/macrophage activation, promotes tumor-associated antigen cross-priming and boost T cells infiltration. (A and C) CT26-bearing mice were sacrificed to harvest spleen and tumors. (A) Splenocytes from untreated (CTRL) mice or treated with TEM1, RT and dual therapy (TEM1+RT) were stimulated with AH1 peptide and assayed for interferon-gamma (IFN-γ) ELISpot. (C) Quantification by flow cytometry shows percentage of gp70-specific T cells (gp70-Tet.) infiltrating the abscopal tumor (CTRL vs TEM1+RT p=0.009; TEM1 vs TEM1+RT p=0.035; RT vs TEM1+RT p=0.009). Percentage of total CD8+ T cells is reported. Representative flow cytometry plots are shown. (B) In vitro growing CT26 cells or CT26 B2M-deficient cells (CT26 B2M−) were exposed to different doses of ionizing radiation and cocultured with gp70-specific T cells isolated from the spleen of BALB/c (vaccinated against gp70; refer to IgE-gp70-TT vaccine in material and methods) and ELISpot assay was performed to assess IFN-γ production. One-way analysis of variance (ANOVA) tests were performed (p=0.003). (D and E) Mice receiving therapies were sacrificed 4 days after RT to harvest organs. Flow cytometry analysis performed on digested lymph nodes (D) and primary tumors (E) suspensions shows expression of MHCII+CD40+ markers, indicative of DC, macrophage and tumor-associated macrophage (TAM) activation (D, CTRL vs TEM1+RT p=0.002; TEM1 vs TEM1+RT p=0.02; RT vs TEM1+RT p=0.044. E, CTRL vs TEM1+RT p=0.002). (F) ELISpot assay shows ability of CD11c+ DC/macrophage sorted from tumor-draining lymph nodes of treated mice to mediate activation of gp70+ T cells in the presence of AH1 peptide (CTRL vs TEM1 +RT p<0.0001; TEM1 vs TEM1+RT p<0.0001; RT vs TEM1+RT p=0.0063). Means±SEM are shown. Each dot represents a mouse. Tukey’s multiple comparison tests were performed. *p<0.05, **p<0.01, ***p<0.001, n.s. non-significant. DC, dendritic cells; LN, lymph node; RT, radiation therapy; TEM1, tumor endothelial marker 1.

Fig 4: (a) Schematic depiction of the experimental approach for the abscopal effect of the PDM MNs. (b) Photos of primary tumors on day 12. (c) Tumor-growth curves of primary tumors, (d) the relative tumor weight, and (e) the relative fold change of tumor volume. (f) Photos of distal tumors on day 12. (g) Tumor-growth curves of distal tumors, (h) the relative tumor weight, and (i) the relative fold change of tumor volume. (j) The percentage of the matured DCs (CD40+CD11c+MHC II+) in lymph nodes. (k) The percentage of the CD8+ T cells (CD3+ CD8+) in primary tumor tissue. (l) The percentage of the GZMB+ CTLs (GZMB+ CD8+) in primary tumor tissue. (m) The percentage of the CD8+ T cells (CD3+ CD8+) in distal tumor tissue. (n) The percentage of the GZMB+ CTLs (GZMB+ CD8+) in distal tumor tissue. The significance was defined as follows: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig 5: IL-27 Deficiency Suppresses Islet Infiltration of DCs and T Cells(A) The percentages of CD45+ cells among single viable cells in the islets of 4- or 10-week-old NOD and NOD.Il27−/− females.(B) The percentages of CD3+ T cells among CD45+ cells in the islets of 4- or 10-week-old NOD and NOD.Il27−/− females. Cells were gated on viable, single, and CD45+ cells.(C) The percentages of F4/80+ single-positive (SP), F4/80+ CD11c+, and CD11c+ SP among myeloid APCs in the islets of 4- or 10-week-old NOD and NOD.Il27−/− females. Cells were first gated on viable, single, CD45+, and CD3− cells, followed by excluding F4/80− CD11c− cells. For (A)–(C), representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the right panels. Summarized data are representative of two independent experiments. Each symbol represents a mouse. The horizontal line indicates the mean. *p < 0.05 by Mann-Whitney test.(D) Representative flow cytometry plots on the left depict I-Ag7 and CD40 expression among myeloid APC subsets in the islets of 10-week-old NOD or NOD.Il27−/− females. Summarized data on the right show geometric mean fluorescence intensity of I-Ag7 or CD40 among F4/80+ SP, F4/80+ CD11c+, or CD11c+ SP APC subsets in the islet of 10-week-old NOD or NOD.Il27−/− females. Summarized data are representative of two independent experiments. Each symbol represents a mouse. The horizontal bar depicts the mean. *p < 0.05 by Mann-Whitney test.See also Figure S2.