Fig 2: GMP clusters are clonala–c, Clonality of regenerative GMP clusters: (a) Percent CD45.2+ cells in the peripheral blood (PB) pre- and BM post-5-FU treatment for each of the chimera mice used at the indicated days post-treatment (n = 2–3 mice/group); (b) Representative IF staining showing MPs (red) and CD45.2 (green) expression in 5-FU-treated chimera BM at the indicated days post-treatment; and (c) experimental scheme and representative IF staining showing MPs (red) and CD45.2 (green) expression separately in two independent d12 5-FU-treated chimera BM. Positive (+) clusters have = 75% CD45.2+ cells and negative (-) clusters = 5% CD45.2+ cells. Wks: weeks. d, Clonality of leukemic GMP clusters with experimental scheme and representative IF staining showing MPs (red) and GFP (green) expression from ß-actin-Gfp cells in diseased BA chimera BM. Positive (+) clusters have = 75% GFP+ cells and negative (-) clusters = 5% GFP+ cells. Dotted lines indicate cGMPs.

Fig 3: Global transcription changes in p21Cip1-deficient mice.(A) Differentially expressed genes (DEG) in the liver of p21KO compared to WT mice under ad libitum feeding conditions and upon 24 h fasting. Data were obtained from RNA-Seq analysis of 2–3 male mice per group. Red circles correspond to upregulated and green circles to downregulated genes. Values were considered significant when FDR value was q < 0.05. (B) Heat map of significantly altered pathways in the liver in the same samples as in panel A. Results were obtained from GSEA and considered significant when FDR < 0.25. (C) Representative GSEA plots of immune-related pathways downregulated in fasted p21KO mice compared to fasted controls. FDR < 0.25. (D) Relative amount of CD45+ immune cells in the liver of ad libitum fed and 24 h fasted WT and p21KO mice. Values correspond to average ± s.d. (n = 6 males, 12–16 weeks old). (E) Representative pictures of F4/80 stained liver sections of WT and p21KO mice under feeding conditions, 24 h and 48 h fasting. Bars correspond to 50 µm. Statistical significance was determined by two-way ANOVA and Bonferroni post-hoc test: **p < 0.01. See also Figure S3.

Fig 4: SAT cells and ABCs accumulate in aged injured kidneys with TLTs.Representative images of aged kidneys 45 days after ischemic reperfusion injury (IRI) by (A) periodic acid–Schiff staining and immunofluorescent staining of (B) CD3e and B220 and (C) Ki67 and DAPI. Arrows and arrowheads indicate the localization of TLTs. (D) Expression levels of Cd4 and Cd19 mRNA (n = 4/time point) in aged mouse kidneys following IRI. (E) Immunofluorescence of CD3e and B220 at various time points after 45-minute IRI. (F) Representative FACS plots for identification of SAT cells in young and aged injured kidneys (n = 3/group). (G) Heatmap generated from bulk RNA-Seq analysis depicting the relative expression of selected SAT cell–related genes in PD1–CD153–, PD1+CD153–, and PD1+CD153+CD44hiCD4+ T cells in aged injured kidneys (n = 3/group). (H) Representative FACS plot for the identification of CXCR5+ cells in SAT cells (n = 3/group). (I) Representative FACS plot for the identification of ABCs. (J) Representative histogram and the quantitative mean fluorescence intensity (MFI) of T-bet in CD95–CD11b– cells and ABCs in aged injured kidneys (n = 4/group). Data are presented as mean ± SD. Statistical significance was determined by trend test (D) or unpaired, 2-tailed t test (F and J). **P < 0.01; ***P < 0.001. Scale bars: 300 µm (A and B) and 100 µm (C and E).

Fig 5: Interferon lambda promotes replacement of tissue-resident macrophages with inflammatory monocytes. (A) Flow cytometry gating strategy. Total cell numbers per kidney of CD45+ leukocytes (B), GR1+ neutrophils (C), CD11b+CD11chigh conventional dendritic cells (cDCs) (D) and F4/80 surface expression on CD11b+Gr1-CD11c- monocytes (E,F) were determined by flow cytometry at 6 months after pristane treatment. Each symbol represents an individual sample; bars represent median with interquartile range. For some mice, not enough tissue was available for flow cytometry. Lin = Lineage (CD3, CD5, CD19, Ly6G). Statistical analyses to compare WT + pristane vs. Ifnlr1-/- + pristane were performed using Mann–Whitney U test. ** = p < 0.005; ns = not significant. Values of WT without pristane are shown as reference. Findings were confirmed in at least one other independent experiment.