Pe selection kit

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the Washington University School of Medicine Animal Care and Use guidelines. We obtained old (i.e., ~18-month-old), female wild-type C57BL/6J mice from the National Institute on Aging (Bethesda, MD) and compared them to strain-matched young (i.e., ~3-month-old), female wild-type C57BL/6J controls. We harvested peritoneal macrophages five days after elicitation with a 2-ml intraperitoneal injection of 4% thioglycollate broth (Sigma-Aldrich, St. Louis, MO). We harvested splenic macrophages by performing positive magnetic cell separation with the PE selection kit (Stem Cell Technologies) and PE anti-F4/80 monoclonal antibody (clone: BM8; eBioscience, Waltham, MA), following manufacturer's instructions. We cultured peritoneal and splenic macrophages in Gibco™ RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin-streptomycin (Thermo Fisher Scientific). When indicated, we treated macrophages with 25 or 50 μg/ml of oxidized LDL (oxLDL; Alfa Aesar, Haverhill, MA) for 24 h prior to further analysis.

CD19⁺ human B cells were separated from PBMC using the Human B cell Enrichment Kit or the Human B cell Isolation Kit (both stemcell, Canada) according to the manufacturer’s instructions. The purity of the separated cells as monitored by flow cytometry was always ≥ 93%. CD19⁺ mouse B cells were separated from spleen cells using the Mouse CD19 Positive Selection Kit II, and CD4⁺ and CD8⁺ T cells were separated after PE staining with the PE Selection Kit (both stemcell, Canada) according to the manufacturer’s instructions.

WT and VISTA KO MDSCs were dye-labeled with either CFSE or Violet CellTrace™ dyes (Thermofisher) at 1 μM concentration in PBS containing 0.1% BSA for 10 min at 37°C and then were washed three times in complete media. WT and KO cells were mixed at a 1:1 ratio. To ensure against dye-specific effects, experiments were repeated with the dye combination switched for WT vs. VISTA KO cells. Mixed dye-labeled WT and KO MDSCs were adoptively transferred i.v. into WT mice bearing tumors >10 mm in diameter. Each recipient mouse received 5 million WT and 5 million KO MDSCs. Twelve hours later, cells were isolated from the tumor with the Miltenyi tumor dissociation kit, and the spleen by homogenization through cell strainers. Tumor cells were enriched for MDSCs by staining with CD11b-PE (Biolegend) followed by enrichment for PE stained cells with a PE selection kit (StemCell Technologies). Finally, spleen and enriched tumor cells were stained for CD45, CD11b, and Gr1 as described above, and analyzed by flow cytometry. FACS analysis was performed to identify WT and KO MDSCs in the spleen vs. the tumor.