Fig 2: 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.

Fig 3: 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 4: (a) Immunofluorescence staining of CRT (green) in tumor tissue and (b) the relative quantified results. Scale bar =100 µm. (c) Immunofluorescence staining of ß-catenin (green) in tumor tissue and (d) the relative quantified results. Scale bar =100 µm. (e) The CCL4 level in tumor tissue. (f) Immunofluorescence staining of DCs (CD40+CD11c+) in tumor tissue and (g) the relative quantified results. Scale bar: 100 µm. (h) The percentage of the matured DCs (CD40+CD11c+MHC II+) in lymph nodes. (i) The percentage of the CD8+ T cells (CD3+ CD8+) in tumor tissue. (j) The percentage of the GZMB+ CTLs (GZMB+ CD8+) in tumor tissue. (k) Immunofluorescence staining of GZMB+ CTLs (GZMB+ CD8+) in tumor tissue and (l) the relative quantified results. Scale bar: 100 µm. (m) IFN-?, (n) IL-6, and (o) IL-12 levels in sera and tumor tissue of mice. The significance was defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: CCL-34 functions as an immune adjuvant in vivo. (A) The cells with Ag-processing ability were significantly increased in PEC of CCL-34-treated mice. C57BL/6 mice were injected intraperitoneally with DQ-OVA (1 mg/kg) in the presence or absence of CCL-34 (4 mg/kg). Cells were isolated from the spleen, peritoneal cavity, lymph nodes and MLN 24 hours later and analyzed by flow cytometry. The percentages of DQ-OVA green (digested OVA) and DQ-OVA red (digested and accumulated OVA) are shown. #p < 0.05 and *p < 0.05 indicate significant differences of PEC versus MLN, spleen or lymph nodes in vehicle group (n = 4) or CCL-34 group (n = 3). (B–E) C57BL/6 mice were injected intraperitoneally with OVA (100 µg/mouse) in the presence or absence of CCL-34 for 24 hours. (B) Total cell number of PEC in CCL-34/OVA-immunized mice (n = 6) is higher than control group (n = 5). Total cell number of cells isolated from the peritoneal cavity was measured. (C) The percentage of monocytes (SSClow/medCD11b+Ly6C+) in PEC is significantly increased in CCL-34/OVA-injected mice. The PECs were stained and analyzed by flow cytometry. The percentages of macrophages (F4/80+), monocytes (SSClow/medCD11b+Ly6C+), neutrophils (SSChighCD11b+Ly6C+), DCs (CD11c+MHC-IIhigh), T cells (CD45+CD3+) and B cells (CD45+B220+) are shown. (D) The percentages of CD80+ and CD40+ monocytes in PEC are increased in CCL-34/OVA-injected mice. Representative scatter plots and the percentage of monocytes expressing CD40, CD80 or CD86 at 24 hours after injection are shown. (E) The percentage of large peritoneal macrophages is reduced while the percentage of small peritoneal macrophages is increased in PEC of CCL-34/OVA-injected mice. The PECs were stained and analyzed by flow cytometry. The percentages of large peritoneal macrophages (LPM, F4/80++ MHC-IIlow) and small peritoneal macrophages (SPM, F4/80+ MHC-IIhigh) are shown. (F) CCL-34 induces OVA-specific IgG production in CCL-34/OVA-immunized C3H/HeN mice. C3H/HeN (F) or C57BL/6 mice (G,H) mice were immunized with OVA (100 µg/mouse) alone or OVA formulated with Alum (80 mg/kg), vehicle (10% DMSO) or CCL-34 (4 mg/kg) on days 0, 7 and 14. The serum was collected on day 21 and analyzed using ELISA. (F,G) The endpoint (day 21) concentration of OVA-IgG in the serum of immunized mice is shown (the number of mice: PBS = 5, PBS plus OVA = 6, Alum plus OVA = 5, vehicle plus OVA = 6, and CCL-34 plus OVA = 9 for total two trials). (G) CCL-34-induced OVA-specific IgG in CCL-34/OVA-immunized C57BL/6 mice is suppressed by 3-MA treatment. The autophagy inhibitor 3-MA (20 mg/kg) was administered intraperitoneally 30 minutes before immunization. The serum was collected on day 21 and analyzed using ELISA (the number of mice: PBS = 5, PBS plus OVA = 7, Alum plus OVA = 5, vehicle plus OVA = 7, CCL-34 plus OVA = 11, and CCL-34, 3-MA plus OVA = 10 for total two trials). (H) The percentage of CD3+CD4+ T cells is increased in CCL-34/OVA-immunized C57BL/6 mice. Cells isolated from the spleen of immunized mice described in (G) were stained and analyzed by flow cytometry. The percentage of CD4 T cells (CD3+CD4+) is shown (the number of mice: PBS = 5, CCL-34 = 5, PBS plus OVA = 7, Alum plus OVA = 5, vehicle plus OVA = 7, CCL-34 plus OVA = 11, and CCL-34, 3-MA plus OVA = 10 for total two trials). The results were plotted using GraphPad Prism version 8.1.0 (www.graphpad.com). All the data are shown as the mean ± SD, and *p < 0.05 indicates significant difference versus the control. The gating strategies are presented in Supplementary Fig. 7A–C.