Fig 2: Mucosal exposure of huNSG-A2 mice to EBOV and RESTV.(A) Flow cytometry–based evaluation of the presence of mature human immune cells in skin (lower back area) and lung of huNSG-A2 mice. Gates indicate the percentage of cells expressing human CD45 (h-CD45) in either organ. The gating strategy in the right panels shows the presence of human antigen-presenting cells (APCs) (G1), B cells (G2), CD14+ monocytes (G3), CD16+ monocytes (G4), nonmonocytic APCs (G5), and human DC subsets (G6–G8). (B) Histopathological analysis of huNSG-A2 lung tissue after infection with EBOV or RESTV on the indicated days after infection. White arrowheads indicate the presence of infected cells, showing EBOV NP– and CD45-positive staining. Scale bar: 50 μm (C) Histopathology score (ordinal method, values of 0 to 5) assessing the levels of hCD45 staining in n = 3 lung sections of RESTV- and EBOV-infected and control (Mock) mice. Box-and-whisker plots represent minimum to maximum values. All scoring values are shown.

Fig 3: Mucin and Inulin Induce Enhanced Expression of Immunoregulatory Genes in Tumors(A) qPCR analysis of immune-related genes in subcutaneously transplanted melanoma grown in syngeneic C57BL/6 mice that received a control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation (n = 6).(B) Quantification of CD45.1+ OT-I CD8+ T cells in the tumor-draining lymph nodes (TdLN) and non-draining lymph nodes (ndLN) of C57BL/6 mice (CD45.2+) treated with or without mucin and injected with B16-OVA melanoma cells (TdLN, n = 7; ndLN, n = 8). Right dot plots show gating of CD45.1+ CD8+ cells.(C) Serum cytokine and chemokine levels in naive WT mice treated with or without mucin (n = 10).(D) Serum cytokines in WT mice treated with or without mucin on day 10 after tumor inoculation (n = 10).Data are representative of three independent experiments (A and B) or one experiment (C and D). Graphs show the mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s correction (A) or by two-tailed t test or Mann-Whitney U test (B–D).

Fig 4: Inulin Supplementation Overcomes Melanoma Resistance to MEKi(A) Growth of NRASQ61K mouse melanoma cells (1 × 106) (control, n = 11; mucin, n = 9; inulin, n = 9; MEKi, n = 10; MEKi+mucin, n = 8; MEKi+inulin, n = 10) that were subcutaneously transplanted in syngeneic C57BL/6 mice that received control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation. When tumors reached a volume of 10–20 mm2, mice were administered MEKi (PD325901, 10 mg/kg) once daily by gavage. Tumor volume was assessed every 4 days.(B) Number of tumor-infiltrating effector (CD44hi) CD4+ and CD8+ T cells and total CD45+ cells per tumor weight (in grams) in mice treated as in (A) (MEKi, n = 7; MEKi+mucin, n = 8; MEKi+inulin, n = 8).(C) Number of tumor-infiltrating DCs and DC subsets per tumor weight (in grams) and expression of MHC class I on DCs in mice treated as in (A) (MEKi, n = 8; MEKi+mucin, n = 7; MEKi+inulin, n = 8).(D) Pie chart of taxa enriched in inulin-treated mice and negatively correlated with MaN-RAS tumor size (n = 10).(E) Pie chart of taxa enriched in mucin-treated mice microbiota that negatively correlate with MaN-RAS tumor size (n = 10).(F) Relative abundance of taxa enriched in inulin+MEKi-treated mice and negatively correlated with MaN-RAS tumor size (n = 10).(G) Relative abundance of taxa enriched in mucin+MEKi-treated mice and negatively correlated with MaN-RAS tumor size (n = 10).Data are representative of two independent experiments. Graphs show the mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s correction (B and C) or by two-way ANOVA with Bonferroni’s correction (A).

Fig 5: Ab-retargeting reduces intratumor myeloid cells and supports NK cell and CD8 T-cell infiltration. a MC38-pSia cells were used to establish s.c. tumors in Ad5-vaccinated mice. Tumor-bearing mice received i.v. injections of DE1scFv-pSia or saline, and were sacrificed according to the shown schedule. Tumor tissue was examined for infiltration of different leukocyte subsets via flow cytometry. b Frequencies of NK cells (NK1.1+CD49b+) were calculated as percentage of CD45.2+ leukocytes. NK cell activation was determined by surface expression of CD107a. Macrophages (Gr1+F4/80high) and different fractions of myeloid-derived suppressor cells (MDSCs; P1: CD11b+ Gr1high; P2: CD11b+ Gr1int) are shown in c and d, respectively. Group size was in general n = 5 with following exceptions: n = 7 (NK cells/day 10/DE1scFv-pSia); n = 6 (NK cells/day 17/control and DE1scFv-pSia groups); n = 4 (CD107ahigh – NK cells/control). e Frequencies of CD4+ and CD8+ T cells were measured as percentage of CD90.2-positive lymphocytes, and individual ratios of CD8 to CD4 T cells were calculated. Group size: n = 5 (day 10) and n = 6 (day 17). f Splenocytes were prepared from sacrificed mice, incubated with the mutated MC38 peptide Adpgk-R304M (ASMTNMELM), or an irrelevant control peptide, and responding neoantigen-reactive CD8 T cells were identified by intracellular staining for IFNγ (group size: n = 8). Two-tailed unpaired t test was used to calculate statistics: *p ≤ 0.05; **p ≤ 0.01. Error bars refer to standard deviation (SD). Source data are provided as a source data file