Fig 2: Normal germinal center (GC) size, but abnormal GC number in Ptenfl/fl Cγ1Cre/+ mice. (A) Immunofluorescence analysis of spleen sections from immunized Pten+/+ Cγ1Cre/+ and Ptenfl/fl Cγ1Cre/+ mice at day 7 after sheep red blood cell immunization. Antibodies detecting B220 (expressed on B cells), FAS (expressed by GCBs), and CD3ε (expressed on T cells) were utilized. Data were given from one representative of at least three independent experiments. The scale bar represents 74.4 µm. (B) The statistical quantification of the GC size (left) measured in terms of area or the GC number (right) measured in terms of the number of GCs per each spleen section in Pten+/+ Cγ1Cre/+ and Ptenfl/fl Cγ1Cre/+ mice as described in (A). The bars represent the mean ± SD from one representative of at least three independent experiments. Two-tailed t tests were performed for statistical comparisons.

Fig 3: Knockout of Mettl3 does not affect the pro-fibrogenic activity of B cells in liver fibrosis. (a) Quantitation of mRNA expression level of Mettl3 in B cells isolated from mouse livers treated with CCl4 or Oil (control) for 6 weeks. Results were retrieved from published microarray data [47]. (b) Schematic diagram of CCl4-induced mouse liver fibrosis. (c, d) Quantification of flow cytometry analysis for B cell activation marker CD86 and CD95 in the spleen (c) and liver tissues (d) of WT control and Mettl3 cKO mice after fibrosis induction. (e) Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), and alkaline phosphatase (ALP) levels in indicated groups 24 h after the last CCl4 treatment (n = 5 for WT control and n = 6 mice for Mettl3 cKO). (f) RT-qPCR for profibrogenic genes of liver tissues in indicated groups (n = 5 for WT control and n = 6 for Mettl3 cKO). (g) Representative western blot (left) and quantification (right) for profibrogenic genes in indicated groups (n = 5/group). Gapdh was used as a loading control. (h) Representative photographs of H&E staining (left), αSMA immunohistochemistry staining (middle), and picrosirius red (PSR) staining (right) of fibrotic liver sections from WT control and Mettl3 cKO mice. (i, j) Quantification of the aSMA positive area (i) and PSR staining positive area (j) in indicated groups (n = 5 for WT control and n = 6 for Mettl3 cKO). Data in (a), (c)–(f), and (i, j) were presented as means ± SEM with the indicated significance (∗P < 0.05; student's t-test).

Fig 4: MnJ promotes antigen presentation and specific T-cell responses. a, b WT mice were immunized subcutaneously with PBS, phycoerythrin (PE, 10 μg), PE (10 μg) + MnJ (20 μg), or PE (10 μg) + Imject Alum (200 μg). Inguinal lymph nodes were collected 12 or 24 h later (n = 3). The ratio and number of PE+ APCs (a) and Mo-DCs (b) among dLN cells were analyzed by FACS. Live cells were identified by DAPI staining. Among live singlet cells, APCs were identified as the cell subset single or double positive for CD11c and F4/80. Among APCs, Mo-DCs were identified as CD8a+ CD64+ cells. c BMDCs were treated with MnCl2 (20 μg/ml), MnJ (20 μg/ml), or Imject Alum (20 μg/ml) for 20 h. CD86 expression was analyzed by FACS. d CD45.1+ OT-I CD8+ or OT-II CD4+ T cells were labeled with CFSE and transferred to CD45.2+ WT mice. These mice were then immunized with OVA (1 μg), OVA (1 μg) + MnJ (10 μg), or OVA (1 μg) + Imject Alum (1320 μg). After 3 days, T-cell proliferation was analyzed by FACS (n = 3). e WT mice were immunized intramuscularly with OVA (10 μg), OVA (10 μg) + MnJ (10 μg), or OVA (10 μg) + Imject Alum (1320 μg) on days 0, 7, and 14. Splenocytes were collected on day 21 and stimulated with OVA (100 μg/ml). IL-2, IL-4, IFNγ, and IL-10 secretion by T cells was measured by ELISA (n = 3). f OVA-specific cytotoxicity was measured on day 21 in an in vivo killing assay (mice were immunized on days 0, 7, and 14, n = 4). g Numbers of Tfh or GC B cells in dLNs from WT mice were analyzed by FACS. Live cells were identified by DAPI staining. Among live singlet cells, CD4+ T cells were identified as the cell subset double positive for CD3 and CD4. Among CD4+ T cells, Tfh cells were identified as PD1+ CXCR5+ cells. B cells were identified as the cell subset double positive for CD45 and B220. Among B cells, GC B cells were identified as Fas+ GL7+ cells (n = 3). One representative experiment of at least three independent experiments is shown, and each was performed in triplicate. Error bars represent the SEM; data were analyzed by an unpaired t-test. ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 5: IL23R antibody treatment combined with GITR activation to promote tumor immunity. (A) Tumor growth curves in C57BL/6 mice treated intraperitoneally with anti-IL23R (100 μg/treatment), anti-GITR (100 μg/treatment), isotype controls (100 μg/treatment), or a combination of both IL23R and GITR antibodies (50 μg each/treatment), as in Fig. 4B. (B) Tumor inhibition of each antibody monotherapy and combination therapy, shown as a percentage of isotype-treated mice, where 0% inhibition indicates tumors that grew equal to isotype treatment and 100% inhibition indicates no tumor growth. (C) Tumor growth curves as in (A) using a higher dose (5 × 105) of MC38. (D) Survival analysis of high-dose–challenged mice as in (C). (E–H) Flow cytometry analysis of tumor-infiltrating Treg high-dose challenged mice including those with (E) Treg IFNγ expression, (F) Treg IL12Rβ2 expression, (G) CD8 T-cell IFNγ expression, and (H) CD8 T-cell Fas expression. Data are pooled from three independent experiments (n = 14 to 15/group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, non significant. Iso, isotype.