Fig 2: HIF-1a in NKp46+ cells controls susceptibility to the small intestinal damage induced by MTX. (A) NKp46 expression status of EYFP+ cells purified from SI of EYFP-WT and EYFP-HIF-1a KO mice. Top: Gating strategy for EYFP+ cells. Bottom: Representative dot plots of NKp46+ expression by EYFP+ cells together with no NKp46+ staining control purified from SI of EYFP-WT (WT1, WT2) and EYFP-HIF-1a KO (KO1, KO2) mice with corresponding quantitative analysis. FSC, forward scatter; SSC, side scatter. (B) Flow cytometric analysis of alive CD45+ cells, neutrophils (NPs), macrophages (M?), eosinophils (Eos), B cells, total T cells, CD4 cells, CD8 cells, and dendritic cells from small intestinal tissues after MTX treatment on day 1 after treatment from HIF-1a KO and HIF-1a WT mice (n = 4); data are mean values ± SEM; *, P < 0.05 by two-tailed Student’s t test. (C) Ratio of Il22 expression to Ifng expression in small intestinal tissues after MTX treatment from HIF-1a KO and HIF-1a WT mice (pooled data of two independent experiments, n = 6); data are mean values ± SEM; *, P < 0.05 by two-tailed Student’s t test.

Fig 3: Agpat4 silencing in CRC suppresses T cell-dependent tumor progression. a Eight-week-old male NCG mice were intraperitoneally inoculated with sh-Agpat4- or sh-NC-transfected MC-38 cells (4 × 106 cells in 100 µl of PBS per mouse). Twelve days later, the mice were sacrificed, and the tumor nodules were collected and weighed. (n = 6). b Agpat4-silenced MC-38 cells increased the percentage of CD8+ T cells in epididymal fats. Six-week-old mice were intraperitoneally engrafted with sh-Agpat4- or sh-NC-transfected MC-38 cells (4 × 106 cells in 100 µl of PBS per mouse) on day 0 and were sacrificed for the analysis of total T cells, CD4+ T cells and CD8+ T cells in the stromal vascular fraction (SVF) of epididymal fats on days 0, 3, and 5. T cells were marked by the phenotype CD45+CD3+CD11b-. CD4+ T cells were defined by the phenotype CD45+CD3+CD11b-CD8-CD4+. CD8+ T cells were defined by the phenotype CD45+CD3+CD11b-CD4-CD8+. Representative results are shown. c Percentage of IFN-?+ cells in the CD8+ T cells described above in b. (n = 5). d Six-week-old male C57BL/6 mice were intraperitoneally inoculated with MC-38 cells (4 × 106 cells in 100 µl of PBS per mouse) on day 0 and then treated with anti-CD8 antibody or IgG as a control 3 times (days 2, 4, and 6) at a dose of 100 µg/day. Mice were sacrificed for tumor observation on day 14. (n = 5). The data in a–d represent the means ± s.e.ms. (***P < 0.005; Student’s t test)

Fig 4: ScRNA-seq analysis of BCG hydrogel-treated tumors showed enhanced antigen processing and presentation in DC and MF. (A) Scheme of experimental workflow. (B, C) Uniform Manifold Approximation and Projection(UMAP) plot demonstrating clustering and cell type identities obtained for both treatment groups. Cluster annotations: B cell, CD4+ T cell, CD8+ T cell, DC, mDC, pDC, MF and NK cell. (D) Top 40 differentially upregulated genes between BCG hydrogel-treated and PBS hydrogel-treated tumors within identified cell types. Sorted from highest log2 fold change (top) to lowest (bottom), log2 fold change cut-off=0.25 and false discovery rate (FDR)=0.05. (E, F) Gene ontology analysis of biological processes for genes upregulated in BCG hydrogel-treated versus PBS hydrogel-treated tumors. (E) Pie charts demonstrating percentages of biological processes from upregulated genes and (F) net plots showing the relationships between genes associated with selected GO terms. (G) UMAP plot showing distribution of CTSS expression. (H) Violin plot demonstrating CTSS expression level in distinct cell populations. CTSS, Cathepsin S; DC, dendritic cell, GO, gene ontology; mDC, myeloid DC; MF, macrophage; pDC, plasmacytoid DC; scRNA, single-cell RNA.

Fig 5: miR-181a Deficiency Impairs Antigen-Specific CD8 T Cell Expansion and Viral Clearance after LCMV InfectiondLck-Cre+ Rosa26YFP miR-181ab1+/+ (WT) and dLck-Cre+ Rosa26YFP miR-181ab1fl/fl (miR-181a-/-) mice were infected with LCMV-Armstrong; CD8 T cell responses were analyzed on day 8.(A) Representative flow plots of CD44+ splenic CD8 T cells (left) and summary data of the total numbers of YFP+ CD44+ CD62L– CD8 T cells in the spleen from one experiment (right).(B) Representative flow plots of Db LCMV GP33–41 (GP33), Db GP276–286 (GP276), and Db NP396–404 (NP396) tetramer+ cells gated on YFP+ CD8 T cells in the spleen of WT and miR-181a-/- mice.(C and D) YFP+ CD8 T cells specific for the indicated epitopes in the spleen (C) and inguinal lymph nodes and liver (D).(E) Splenocytes were restimulated with GP33, GP276, or NP396 peptides. Stacked bar graphs show the numbers of cytokine-producing YFP+ CD8 T cells (mean ± SEM).(F) Viral titers in the spleen and kidney on day 6 after LCMV infection.Data are representative of three independent experiments with 4–5 mice per group (A–C and E), one experiment with 4–5 mice per group (D), or pooled from two independent experiments with 8 mice per group (F). Unless stated otherwise, data are presented as means. Statistical significance by two-tailed unpaired t test (A, C, D, and F) or two-way ANOVA followed by Tukey’s post-test comparison (E). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S4.