Fig 1: Identification of galectin-3 as a target of mast cell Sirt6.a, b mRNA levels (a, n = 6 per group) in EAT tissues and protein levels (b, n = 6 per group) in plasma for galectin-1, galectin-3 and galectin-9 in WT and KO mice. c Heatmap displaying the ligand-receptor signaling network of the galectin pathway between immune cell types from WT and KO mice. Each cell in the heatmap represents the predicted communication probability from a sender to a receiver cell type. The bar plot above the heatmap quantifies the total outgoing signaling strength of each cell type, while the bar plot to the right reflects the total incoming signaling strength. TCP, total communication probability. d Bone marrow-derived mast cells (BMMCs) were transfected with control siRNA (siCtrl) or Sirt6 siRNA (siSirt6). Successful Sirt6 silencing was confirmed by qPCR and western blotting (n = 6 per group). e–i Lgals3 and Lgals9 mRNA levels (e, n = 6 per group), RELA (p65 subunit of NF-κB) binding to the promoters of Lgals3 and Lgals9 (f, n = 5 per group), Sirt6 binding to the promoters of Lgals3 and Lgasl9 (g, n = 5 per group), enrichment of Ac-H3K9 on the Lgals3 and Lgals9 promoters (h, n = 4 per group), and acetylation of H3K9 (i, n = 3 per group) were compared between control and Sirt6 silenced BMMCs. j, k. Plasma levels of galectin-3 and galectin-9 in human subjects were measured by ELISA (n = 24 for lean, n = 14 for overweight, n = 6 for obese). A scatter plot was created to examine the correlation between mast cell Sirt6 protein levels and plasma galectin-3 and galectin-9 levels. Values are presented as mean ± SD. Unpaired two-tailed t test between the two groups (a, b, d, e, i), one-way ANOVA followed by Sidak’s multiple comparisons analysis (j, k) and two-way ANOVA followed by Bonferroni’s post hoc analysis were conducted for statistical analyses (f–h). Pearson correlation coefficients were calculated between continuous variables in two-sided distributions (j, k). Source data are provided as a Source Data file.
Fig 2: The recruitment and maturation of DC subsets by Ad-LGALS9/PSMA in a subcutaneous tumor model. On day 42 post tumor inoculation, immunized mice (n=5 mice per group) were euthanized, and splenocytes were isolated for flow cytometric analysis. (A) Representative flow cytometry plots showing the percentages of CD11b+CD11c+, CD8+CD11c+ and CD103+CD11c+ DC subsets in the spleen. (B–D) Quantification of the DC subsets shown in (A). (E) Representative flow cytometry plots showing the percentages of CD80+CD11c+, CD86+CD11c+ and MHC-II+CD11c+ mature DCs in the spleen. (F–H) Statistical analysis of CD80+, CD86+, and MHC-II+ DC subsets. (I, J) Serum anti-LGALS9 and anti-PSMA total IgG levels determined by ELISA on day 35 post tumor inoculation. (K) Concentrations of IFN-γ, TNF-α, IL-10, and IL-4 in splenocyte culture supernatants were assessed by ELISA after 48 hours stimulation with PSMA protein (10 µg/mL). Data were presented as the mean±SD. Statistical significance levels were set at *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. DC, dendritic cell; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; ns, not significant; PSMA, prostate-specific membrane antigen; TNF, tumor necrosis factor.
Fig 3: Ad-LGALS9/PSMA vaccination induces antigen-specific CD8+ T cell-mediated antitumor immunity. (A) EdU incorporation assay showing antigen-specific CD8+ T-cell proliferation in splenocytes from each treatment group. (B) Quantification of EdU+ populations within the CD8+ T-cell subset. (C) IFN-γ-producing T cells detected by ELISpot assay. (D, E) CTL killing activity of splenocytes from immunized mice measured by flow cytometry. (F, H) Representative flow cytometry plots showing TNF-α+CD8+, IL-2+CD8+, and IFN-γ+CD8+ T cells in spleens (F) and tumors (H). (G, I) Quantification of cytokine-producing CD8+ T cells in splenocytes (G) and TILs (I). (J, K) The percentages of CD8+ T cells in splenocytes (J) and TILs (K), with representative plots shown (n=5 per group). Data were presented as mean±SD. Statistical significance was indicated as **p<0.01, ***p<0.001, and ****p<0.0001. CTLs, cytotoxic T lymphocytes; ELISpot, enzyme-linked immunospot; IFN, interferon; IL, interleukin; PSMA, prostate-specific membrane antigen; TILs, tumor-infiltrating leukocytes; TNF, tumor necrosis factor.
Fig 4: Preparation and expression of Ad-LGALS9 and Ad-PSMA vaccine. (A) Diagrammatic representation of adenovirus vector assembly and packaging. (B) PCR detection of Ad-LGALS9 and Ad-PSMA in genomic DNA from adenovirus-infected HEK-293 cells. (C) Cytopathic effects in Ad-Ctrl, Ad-LGALS9, and Ad-PSMA groups at day 14 post co-transfection. (D, F) The expression levels of LGALS9 and PSMA in adenovirus-transduced HEK-293T cells were quantitatively assessed using flow cytometric analysis following infection with Ad-LGALS9 and Ad-PSMA constructs, respectively. (E, G) Quantification of LGALS9 and PSMA expression in (D, F). (H, I) LGALS9 and PSMA protein levels were quantified by ELISA in the supernatant of HEK-293T cells transfected with the respective constructs. (J, K) Serum samples from wild-type C57BL/6 mice immunized with Ad-LGALS9, Ad-PSMA, or Ad-LGALS9/PSMA were analyzed by ELISA to assess in vivo antigen expression. Ad-Ctrl denotes an empty-vector adenovirus generated using the same adenoviral backbone and production process as the recombinant vaccines but lacking a transgene insert. Data were presented as the mean±SD. Statistical significance levels were denoted as ***p<0.001 and ****p<0.0001. ns, not significant; PBS, phosphate-buffered saline; PSMA, prostate-specific membrane antigen.
Fig 5: CD8+ T cells are essential for durable antitumor immunity induced by Ad-LGALS9/PSMA. (A, B) Survival analysis of tumor-bearing mice treated with Ad-LGALS9/PSMA following CD4+ or CD8+ T-cell depletion (n=10 per group). (C, D) Flow cytometric quantification of CD8+ T cells and CD8+CD11c+ cells in splenocytes and TILs. (E) Tumor growth curves of Ad-LGALS9/PSMA-cured mice after tumor rechallenge (n=5 per group). (F, G) Survival analysis of rechallenged mice with CD4+ or CD8+ T-cell depletion. (H) Representative flow cytometry plots showing memory CD8+ T-cell populations in splenocytes. (I–K) Quantification of effector memory (CD44+CD62L⁻), central memory (CD44+CD62L+), and naive (CD44⁻CD62L+) CD8+ T-cell subsets. Data were shown as mean±SD. Statistical significance was indicated as **p<0.01, ***p<0.001, and ****p<0.0001. mAb, monoclonal antibody; ns, not significant; PSMA, prostate-specific membrane antigen; TILs, tumor-infiltrating leukocytes.
Supplier Page from Thermo Fisher Scientific for Mouse Galectin-9 ELISA Kit