Fig 2: Aging causes decreases in dendritic cell (DC) migration and lymphatic pumping and increased leakiness in lymphatic vessels. (A) Representative dot plots of FITC+/CD11c+ DCs from deep cervical lymph nodes (which drain ear skin) after FITC painting of the ear skin. (B) Quantification of FITC+ DCs as a percentage of CD45+ cells from cervical lymph nodes. (C) Quantification total number of FITC+ DCs per lymph node. n = 6 mice per group from two independent experiments. (D) Representative hind limb ICG lymphangiography NIR images showing hind limb collecting lymphatic vessels, dermal backflow, and dye pooling (arrows) in aged mice. (E) Graphical representation of ICG intensity peaks plotted against time in hind limbs of young and aged mice. (F) Quantification of the number of lymphatic pulses per minute in young and aged mice. (G) Quantification of dye pooling, which represents dermal backflow. n = 5–7 mice per group from two independent experiments. (H) Representative confocal images of whole-mounted ear skin from young and aged mice showing FITC-lectin leakage in lymphatic vessels. (I) Quantification of FITC-lectin leakage (total FITC-lectin-containing area). n = 5 mice per group. All quantifications are mean ± SD, unpaired Student’s t test.

Fig 3: CHIME enables deletion of genes without impairing immune homeostasis. a Schematic of chimeric CRISPR-Cas9 system. b CD20 (left), CD64 (middle), or DEC205 (right) expression on B cells, macrophages, or dendritic cells, respectively from chimeric animals following transduction with a non-targeting control sgRNA or targeting sgRNAs to Ms4a1, Fcgr1, or Ly75. c Quantification of CD20, CD64, and DEC205 expression on relevant lineages from (b). d Comparison of frequencies of major immune lineages (of CD45+) in chimera mice (WT: WT stem cells mock transduced, Cas9 + sgRNA: Cas9 stem cells transduced with the Vex sgRNA expression vector) at homeostasis. e, f Chimeric mice were infected with 4 × 106 plaque-forming units (PFU) LCMV Clone 13 and their weight loss (e) and serum viral titer (f) were determined. All experiments had at least three biological replicate animals per group and are representative of two independent experiments. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed among the replicate bone marrow chimeras by one-way ANOVA (c, d), or two-way ANOVA (e, f) (*p < .05, **p < .01, ***p < .001, ****p < .0001). See also Supplementary Fig. 1 and 2. Source data are provided as a source data file

Fig 4: Trained Immunity Shapes the Transcriptional Profile of TANs and Neutrophils from ß-Glucan-Trained Mice Suppress Tumor Growth via ROS Production(A–E) WT mice were treated with ß-glucan or PBS and after 7 days were inoculated with B16-F10 melanoma cells. TANs (CD45+CD11c-CD11b+Ly6c-Ly6g+) were sorted and RNA sequencing analysis was performed (n = 5 mice in the PBS group; n = 4 mice in the ß-glucan group) 14 days after the tumor injection. In (A), (C) and (E), false discovery rate (FDR) = 0.05.(A) Differential gene expression in TANs from mice pre-treated with ß-glucan compared with TANs from PBS-treated mice. Volcano plot showing the distribution of the adjusted p values (-log10(padj)) and fold changes (log2 fold change).(B) GSEA for genes related to a gene set previously implicated in the TAN1 phenotype (Shaul et al., 2016) was used to analyze the transcriptomic effect induced in TANs by ß-glucan administration in mice. Abbreviation is as follows: NES, normalized enrichment score.(C) Top 10 enriched canonical pathways identified by IPA in TANs from ß-glucan-treated mice compared to PBS-treated mice.(D and E ) Shown in (D) is a GSEA for genes related to ROS pathway, and in (E) is a heatmap of genes involved in ROS metabolic process in TANs from ß-glucan-treated mice as compared with TANs from PBS-treated mice.(F) WT mice were treated with ß-glucan or PBS and after 7 days were inoculated with B16-F10 melanoma cells. Staining for ROS in TANs (CD45+CD11b+Ly6g+) was performed by flow cytometry 14 days after the tumor cell injection. Relative mean fluorescence intensity (MFI) is shown. MFI of ROS was measured and expressed in relation to the PBS group, set as 1. Data are presented as mean ± SEM (n = 15 per group).(G) Splenic neutrophils were isolated from mice 7 days after injection with ß-glucan or PBS. Neutrophils were co-cultured with luciferase expressing B16-F10 cells for 24 h. Tumor cell survival was assessed by measuring luminescence (n = 5 per group).(H) Experimental scheme.(I) As indicated in (H), WT or NCF1-deficient mice were injected with ß-glucan or PBS, and after 7 days splenic neutrophils were isolated and were adoptively transferred together with B16-F10 cells into WT recipients. Tumor volume is shown. Data are presented as mean ± SEM (n = 7 mice in the PBS WT group; n = 12 mice in the ß-glucan WT group; n = 11 mice in the PBS NCF1-deficient group; n = 14 mice in the ß-glucan NCF1-deficient group).*p < 0.05, ****p < 0.0001.See also Figure S2.

Fig 5: HFD potentiates T cell recruitment and activation in tumor-seeded eFats. a HFD consumption promotes the peritoneal metastasis of MC-38 cells in nude mice. Six-week-old male nude mice were intraperitoneally injected with MC-38 cells (1.0 × 106 cells in 100 µl PBS) and immediately fed a CD or HFD for 14 days. Peritoneal tumor nodes were collected and weighed (n = 5). b–d HFD consumption enhances T cell infiltration into the eFats. Six-week-old male mice were intraperitoneally injected with MC-38 cells (1.0 × 106 cells in 100 µl PBS) and immediately fed a CD or HFD. Then the stromal cells of the eFats were isolated dynamically. Total T cells (CD45+CD3+CD11b-CD45R-) (b), CD4+ T cells (CD45+CD3+CD11b-CD45R-CD4+CD8-) (c), and CD8+ T cells (CD45+CD3+CD11b-CD45R-CD4-CD8+) (d) were assessed by flow cytometry. Each tested sample was pooled from 4 individual samples (n = 3). e HFD consumption increases the frequencies of adipose tissue T cells in the s.c., in situ and Apcmin+/- tumor-seeded models described in “Methods.” Each tested sample was pooled from 2 individual samples (n = 3). f Frequencies of IFN?+ cells among CD8+ T cells in the eFats of tumor-seeded mice treated with a CD or HFD for 5 days (n = 3). g–i Frequencies of IFN?+ (g), IL17+ (h), and CD25+ (i) cells among CD4+ T cells in the eFats of tumor-seeded mice treated with a CD or HFD for 5 days (n = 3). j Six-week-old male C57BL/6 mice were intraperitoneally injected with MC-38 cells (1.0 × 106 cells in 100 µl PBS) and immediately fed a CD or HFD for 14 days. Each mouse was intraperitoneally treated with antibodies (IgG, anti-CD4, or anti-CD8) 3 times (days 2, 4, and 6) at a dosage of 100 µg day-1. Peritoneal tumor nodes were collected and weighed. (n = 3). All the data are shown as the mean ± s.e.m. The data in b–d were analyzed with two-way ANOVA. The data in other histograms were analyzed with Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.005)