Pe anti ki67

The following anti-mouse antibodies were used for detection and enrichment of immune cells: APC anti-CD3 (17A2), PE anti-TCRβ (H57–597), APC-Cy7 anti-CD4 (GK1.5), Alexa Fluor^® 488 anti-Foxp3 (R16–715), and PE-Cy7 anti-CD8a (53–6.7) (BD Pharmingen); PE anti-CTLA-4 (UC10–4B9) and PE anti-ICOS (7E.17G9) (eBioscience). PE anti-Ki67 (Biolegend), Anti-EGFR antibody (ab30) (Abcam). For cell surface staining, cells were incubated with corresponding antibodies in PBS for 15 min on ice before analysis on a FACSAria™ III cell sorter (FACSDiva software, BD). Dead cells that stained by propidium iodide (2 μg/ml) were excluded. For intracellular cytokine staining, cells were fixed and permeabilized with BD Cytofix/perm and Perm/wash buffer according to the manufacturer's protocol. Then cells were stained at room temperature for 30 min with Alexa Fluor^® 488 anti-IFN-γ (XMG1.2, Biolegend), Alexa Fluor^® 647 anti-TNF-α (MP6-XT22, Biolegend), APC anti-perforin (eBioOMAK-D, eBioscience) and PE anti-granzyme B (NGZB, eBioscience) respectively before analysis on a BD LSRII flow cytometer. For Foxp3 staining, Foxp3 fix/perm buffer (Biolegend) set was used according to the manufacturer's instruction. All flow cytometry data was analyzed with Flowjo 7.6.1 software. Cell sorting was performed on a FACSAria™ III cell sorter (FACSDiva software, BD) based on cell surface marker staining.

Single-cell suspensions prepared from BM, peripheral blood (PB), and spleen were incubated for 30 min at 4 °C with fluorochrome–antibodies (BioLegend). Dead cells were excluded by FVD eFluor® 455UV (eBioscience) staining. Flow cytometry was performed using CytoFLEX (Beckman), LSR Fortessa, or FACSAria II (BD) flow cytometer. Antibodies used in this study are available in Supplementary Methods.
For cell cycle or quiescence analysis of HSCs, the BM cells were incubated with fluorochrome–antibodies, fixed and permeabilized with Transcription Factor Buffer Set (BD), and stained with 5 μg/ml DAPI (Sigma) or PE-anti-Ki67 (BioLegend).
For Akt phosphorylation assay, live Lin⁻ BM cells were sorted, starved in 2% FBS/IMDM (Gibco) for 1 h, and then treated with 5 ng/ml GM-CSF (Peprotech) at 37 °C for indicated time. After stimulation, the cells were fixed, permeabilized, and stained with APC-Cy7-anti-CD117, PerCP-Cy5.5-anti-Sca1, anti-p-Akt (Cell Signaling), and PE-anti-IgG (eBioscience).

PBLs without treatment, PBLs stimulated with pamidronate, and PBLs cocultured with auto- or allo- DCs^pami+ were treated with phorbol myristate acetate (PMA) and ionomycin Cocktail (Thermo Fisher Scientific) at 37°C for 4 h. Cells were collected, fixed and permeabilized with the FIX&PERM kit (MultiSciences Biotech, China) according to the manufacturer's instructions, and stained with PE anti-ki67, BV421 anti-IFN-γ, and PerCP anti-TNF-α antibodies (Biolegend, USA).