Ly6g microbeads

BM cells were extracted from tibiae and femurs of B6, C.B10, CByB6F1, and BALB donor mice, incubated with Ly6G microbeads (Miltenyi Biotec, Auburn, CA), and passed through a magnetic column to collect Ly6G⁺ cells, which were confirmed by flow cytometry to contain 94% to 97% cells with the CD11b⁺Ly6G⁺Ly6C^low phenotype characteristic for G-MDSCs, and these cells suppressed T-cell proliferation in a dose-dependent fashion in vitro (supplemental Figure 1).
In the minor H–mismatched AA model, G-MDSCs enriched from BM of C.B10 donors were infused into C.B10 recipients at 10 × 10⁶ cells/recipient, at the same time of B6 LN cell infusion (prophylaxis) or at day 3 after B6 LN cell infusion (therapy). In a specific study, we enriched G-MDSCs from male C.B10 donors and infused them into female C.B10 recipients at the same time of LN cell injection from female B6 donors. Recipients were assessed at day 14 after LN cell infusion. In a separate experiment, we induced BM suppression in C.B10 mice with 5Gy TBI without LN infusion, IV injected 10 × 10⁶ G-MDSCs to each mouse and evaluated the mice at 14 days after irradiation.
In the MHC-mismatched AA model, we obtained G-MDSCs from B6, BALB, and CByB6F1 donors and infused 10 × 10⁶ G-MDSCs to each CByB6F1 recipient at the time of B6 LN cell infusion. Recipient mice were assessed at day 14 after LN cell infusion.