Silac media

CD66+ MDSC were separated from PBMC as per kit instructions using magnetic bead isolation (EasySep™) of PE-conjugated anti-CD66 (Miltenyi Biotec). CD66− PBMC were cultured at 37°C, 5% CO₂, overnight either alone or with CD66+ MDSC added back at a 1:1 ratio in custom arginine-low media: SILAC media (Sigma Aldrich) plus filter-sterilized L-arginine monohydrochloride (0.115mM, Sigma Aldrich) and filter-sterilized L-lysine hydrochloride (0.2186mM, Sigma Aldrich), 15% human serum (Sigma Aldrich), Penicillin-Streptomycin-Glutamine (0.5mg/ml, Thermo Fisher). Cells were stimulated with 1ug/ml 15mer SIVmac239 Gag peptide pool (NIH AIDS Reagent Program) or 0.4ug/ml superantigen SEB⁷ from Staphylococcus aureus (Sigma Aldrich) for 4 days. Duplicate control un-stimulated wells for each cell mixture (PBMC alone, PBMC with CD66+MDSC) were included for background proliferation subtraction. Proliferation was measured by ICS on day 4 post-stimulation using CD8 clone SK1 PECy7 (Biolegend), Ki67 clone B56 Alexa Flour 488 (Becton Dickinson), CD3 clone SP34-2 APC (Becton Dickinson). CD66+ MDSC-mediated suppression was measured by comparing the proliferation stimulated in the absence of CD66+ cells with the proliferation stimulated in the presence of CD66+ cells. Replicates were included for all PBMC wells, and PBMC + MDSC when MDSC yield allowed, and averaged.