Fig 2: IL-6 neutralization and CD40 stimulation plus immune checkpoint blockade synergistically reverses Mϕ-mediated immune suppression and activates GBM-associated T cells.GBM was induced in mice, followed by different treatment and endpoint analyses. a Experimental procedure. b Tumor volume was analyzed pre- and post treatment by bioluminescence imaging. Left, representative images. Right, quantified results (n = 6 mice, mean ± SEM). Statistical analysis by two-way ANOVA with Dunnett’s test. c–f Tumor-derived single-cell suspensions were analyzed by flow cytometry. c, d Cells were probed with c, anti-F4/80 and anti-IL-10, or d anti-CD45 and anti-CD8 antibodies. Left, representative sortings. Right, quantified results (n = 5 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Dunnett’s test. e, f Cells were probed with e anti-CD8 and anti-Ki67, or f anti-CD8 and anti-IFN-γ antibodies. Quantified results are shown (n = 5 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Dunnett’s test. g, h Tumor lysates were subjected to g IL-10 and h TGF-β ELISA analysis (mean ± SEM, n = 4 mice for ICI plus IL-6 Ab treatment group, n = 6 mice for ICIs, CD40 Ab, plus IL-6 Ab treatment group, and n = 5 mice for other groups). Statistical analysis by one-way ANOVA with Dunnett’s test. Source data are provided as a Source data file.

Fig 3: Neutrophilic infiltration increases in the perihematomal region in hyperglycemic ICH mice. (A–G) Ipsilateral striata were dissected and flow cytometry was performed at 1-day post ICH. (A) Gating strategy of identifying resident microglia (7AAD-CD11b+CD45int), neutrophils (7AAD-CD11b+Ly6G+), monocytes/macrophages (7AAD-CD11b+CD45hi), lymphocytes (7AAD-CD45+), and T cells (7AAD-CD3e+CD45+). (B) Representative plots of the infiltrating neutrophils in different group are shown. Quantifications of the percentage of neutrophil (C), resident microglia (D), monocyte/macrophage (E), lymphocyte (F) and T cell counts (G) are shown. (H, I) Immunostaining using antibodies of MPO was performed at 1 and 3 days after ICH. White arrows indicate MPO+ cells. The representative images (H) and quantifications (I) are shown. (J–O) The mRNA was extracted from ipsilateral striata of each group at day 1 after ICH, and real-time RT-PCR was performed to verify the gene expression of cytokines/chemokines. GAPDH was served as the internal control. (C-G, J-O) Two-way ANOVA followed by Sidak's multiple comparisons test. (I) Two-way ANOVA followed Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001 vs corresponding Sham. #p < 0.05, ##p < 0.01 vs Veh + ICH; NS., not significant. Each group contained 5 (C–G), 6 (I), 6 (J–M) and 4 (N, O) animals. Scale bar: (H) 75 µm. Each experiment was repeated at least 3 times independently.

Fig 4: SARS-CoV-2 isolates WA-1/2020 and P.1 (Brazil) induce severe lung vascular endothelial injury and ARDS in K18-hACE2 transgenic mice.(A-B) K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 isolate WA-1/2020 at 1×104 PFU and 1×105 PFU respectively. N=10. The mice were observed for the survival (A) and weight (B). (C-J) The K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 WA-1/2020 at 1×104 PFU. The mice were harvested at Day 7 using non-infected baseline mice as controls. N=4. (C) Macroscopic images of lungs following Evans Blue Albumin (EBA) injection with subsequent perfusion to remove EBA in circulation. Quantification of EBA as a marker of pulmonary transendothelial permeability (D) and of lung wet/dry ratio as a measure of lung edema (E). The representative plots (F) and flow cytometry analysis (G) of CD31+/CD45- lung ECs as a percentage of total lung cells at baseline and day 7 post-inoculation. (H) Quantification of Cdh5 mRNA expression normalized by the housekeeping gene PPIA (peptidylpropyl isomerase A). (I) Macroscopic lung images (1st column), histology images of lung sections with H & E staining (2nd column), and Masson Trichrome (3rd column) and Sirius Red (4th column) staining for collagen deposition at baseline (1st row) and day 7 post-inoculation (2nd row). (J-O) K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 P.1 variant at 1×104 PFU. N=10. The mice were observed to assess survival (J) and weight loss (K). The mice were harvested at Day 6 to evaluate lung vascular leak by EBA assay (L) and lung wet/dry ratio (M). N=4. (N) Real-time quantitative PCR was applied to measure viral loads in the lungs of SARS-CoV-2 P.1 variant at Day 6 post-inoculation and the WA-1/2020 strain at Day 7 post-inoculation. The mRNA expression levels of SARS-CoV-2 Spike (N) and SARS-CoV-2 NSP (O) are shown. Data are presented as mean ± SEM. **: P<0.01, ***: P<0.001, ****: P<0.0001 by Student’s t- test for D, E, G, H, L, and M; Oneway ANOVA for N and O.

Fig 5: Cxcl12-creER+ BMSCs respond to bone marrow ablation-induced osteogenesis.a–c Experimental schemes and procedures of bone marrow ablation model. a Right femurs were operated, while left femurs were unoperated and used as internal control. (b, i–vi): Step-by-step demonstration of surgical procedures. c Timeline for tamoxifen injection, ablation surgery and analysis. d–j Col1(2.3 kb)-GFP; Cxcl12-creER; R26RtdTomato distal femur bone marrow (pulsed at 7 days before surgery) with growth plates on top. Three days (d), 7 days (e), and 14 days after surgery (f, h). h Ablated area, boxed area in (f). Scale bar: 50 µm. g contralateral control side. Gray: DIC. Scale bar: 500 µm. n = 4 (Day 3), n = 3 (Day 7), n = 5 (Day 14), n = 3 (Conrol) mice. i Flow cytometry analysis of CD45/Ter119/CD31neg bone marrow cells. Lower panels: fold change of total tdTomato+ cells (left) or Col1-GFP+tdTomato+cells (right) in ablated side over contralateral control side. Scale bar: 20 µm. n = 6 mice. **p < 0.01, two-tailed, Mann–Whitney’s U test. Data are presented as mean ± s.d. j Regenerated cartilaginous tissue adjacent to growth plate. Gray: DIC. Source data are provided as a Source Data file.