Fig 2: Immunohistochemistry of exemplary 4T1 tumors. Representative tumor sections after 7-channel staining of DAPI (blue), Ly-6C (green), F4/80 (yellow), S100A9 (orange), CD8 (red), CD11b (purple) and CD4 (white) with correlating merged images. Sections of implanted 4T1 wt tumors in wt (a) and S100A9 ko (b) mice as well as 4T1 ko tumors in wt (c) and S100A9 ko (d) mice.

Fig 3: Cytotoxic treatments induce alterations in the stromal and immune landscapes and enhance breast cancer cell colonization in lungs.a Gene expression analysis of sorted lung fibroblast (PDGFRa+) following chemotherapy. One dose of doxorubicin (DOX, 5 mg/kg), cisplatin (CIS, 5 mg/kg), or saline (PBS) was administered to naive BALB/c mice by intraperitoneal (i.p) injection. n = 5 (Dox), n = 4 (CIS). b Heatmap of inflammatory and fibrosis-related gene expression. c Flow cytometry analysis of major immune cell populations at lungs following one dose treatment with chemotherapy. Flow cytometry analysis of: d granulocytes (Ly6G+Ly6Cint), e eosinophils (Ly6G-SiglecF+), f T cells (CD3+), g monocytes (Ly6G-Ly6C+). n = 5 mice per group. Data presented are mean percentage of CD45+ cells ± s.e.m; P-values were calculated using one-way ANOVA test. h Lung colonization assay. Naive BALB/c mice were pre-treated with two injections of chemotherapy or PBS as control, and 48 h later 2*105 4T1-luciferase cells (4T1-luc) were injected intravenously (IV) and early lung colonization was monitored using IVIS imaging. i Representative IVIS imaging, 3 and 7 days post-inoculation. j Kinetics of 4T1 lung metastatic growth represented by luciferase bioluminescence intensity following 4T1-luc IV injection. k Quantitative analysis of data shown in (i, j) at day 7. n = 11, 13, and 9 in PBS, DOX, and CIS groups, respectively. Data from two independent experiments are presented as mean ± s.e.m, normalized to PBS group; P-values were calculated using one-tailed Welch’s t-test. Graphical summary was designed using BioRender. Source data are provided as a Source Data file.

Fig 4: Transcriptomic analysis of murine CD8+ T cells. (A) UMAP projection of FACS-sorted CD8+ T cells from peripheral blood of three young and three aged C57BL/6J mice processed by scRNAseq. The cells from mice with the corresponding age were first pooled and labeled with barcoded antibodies. Subsequently, cells from young and aged mice were pooled prior to the scRNAseq. Cells are colored by manual annotations. (B) UMAP projections showing the expression of selected genes. (C) Expression of the signature genes of naïve, ISAGhi, AIMT and effector cells. Color of the dot indicates the average expression, size indicates the percentage of cells with non-zero expression. (D–H) Signature scores showing the expression of different gene modules in naïve, AIMT, AE, and ISAGhi cells. (D) Expression of the module of immune memory genes expressed uniquely in CD8+ memory T lymphocytes (compared with effector or naïve cells), MSigDB # M10845 (19). (E) Expression of the module of genes down-regulated in naïve CD8+ T cells versus effector CD8+ T cells, MSigDB # M3036 (20). (F) Expression of genes from the GO pathway GO:0060338 “regulation of type I interferon-mediated signaling pathway”. (G) Expression of markers of human ISAG+ CD8+ T cells from the pan-cancer single cell T-cell atlas (21). (H) Expression of genes upregulated in murine CD5hi Ly6C+ cells compared to CD5low cells (16). (I) Comparison of the percentage of cells in each scRNAseq cluster in young versus aged mice. (J) Percentage of clonally expanded cells in each scRNAseq cluster. The count indicates the number of cells with the same TCRa CDR3 and TCRß CDR3 nucleotide sequence. Cells with TCR receptors occurring only once in the dataset are not depicted. (K, L) Flow cytometry analysis of ISAGhi and AIMT cells. (K) Quantification (left) and a representative histogram (right) of a flow cytometry analysis of Ly6C on naïve, ISAGhi, AIMT, and AE cells. n = 4 in two independent experiments. (L) Percentage of AIMT cells (top) and ISAGhi cells (bottom) in WT mice and in transgenic mice with monoclonal OT-I and F5 TCRs. See the FMO control in Figure S1M . OT-I: n = 5, F5: n = 5, WT: n = 4. Three independent experiments. Medians are shown. P-value was calculated using the Kruskal-Wallis test.

Fig 5: Role of high-GPR68-expressing monocytes in CKD-induced cardiac inflammation and fibrosis.a Schematic experimental procedure for allogeneic transplantation of monocytes into Splx-5/6Nx mice. Monocytes infected with lentivirus-expressing control or anti-Gpr68 miRNA were intravenously injected into Splx-5/6Nx mice every 3 days for 2 weeks after the 5/6 nephrectomy operation. b Upper panels show immunofluorescence labeling of GPR68 (red) in the cardiac ventricle slices prepared from Splx-5/6Nx mice at 2 weeks after initiating the injection of monocytes infected with lentivirus-expressing control or anti-Gpr68 miRNA. Green fluorescence is derived from the lentiviral GFP reporter. The scale bar indicates 20 µm. Lower panels show the flow cytometry histogram of GFP fluorescence of CD11b+/Ly6C+ cells in the heart. GFP-positive cells were detected only in monocyte transplanted mice. c Serum BNP concentrations in Splx-5/6Nx mice at 2 weeks after initiating the injection of control or GPR68-downregulated monocytes. d Downregulation of GPR68 in monocytes ameliorated CKD-induced cardiac fibrosis. The left panel shows Masson’s trichrome staining of tissue fibrosis in blue. Scale bars indicate 1 mm (upper panel) and 50 µm (lower panel). The right panel shows the quantification of the fibrosis area under light microscopy. e, f The mRNA levels of inflammatory cytokines (Tnf-a and IL-6) and fibrosis-related factors (aSma and Col1a2) in the cardiac ventricle of Splx-5/6Nx mice at 2 weeks after initiating the injection of control or GPR68-downregulated monocytes. g Serum urea nitrogen levels in Splx-5/6Nx mice at 2 weeks after initiating the injection of control or GPR68-downregulated monocytes. For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using one-way ANOVA with Tukey–Kramer post hoc tests (c–g). Numbers and P-values are shown in each graph. Source data are provided as a Source Data file.