Anti mouse cd25 clone pc61

FC receptors were blocked with anti-mouse CD16/32 (clone 93, Biolegend) before staining. Dead cells were identified using the Zombie Aqua Fixable Viability Kit (Biolegend). Cells were stained with the following antibody clones: anti-mouse CD8a (clone 53-6.7, Biolegend), anti-mouse/human CD44 (clone IM7, Biolegend), anti-mouse CD62L (clone MEL-14, eBioscience), anti-mouse/human Granzyme B (clone GB11, Biolegend), anti-mouse CTLA4 (clone UC1-4B9, Biolegend), anti-mouse CD25 (clone PC61.5, eBioscience), and anti-mouse IFN-γ (clone XMG1.2, Biolegend). Intracellular staining of Granzyme B, CTLA-4, and IFN-γ was performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). To count cells, 123count eBeads (eBioscience) were added to flow cytometry tubes immediately before flow cytometer acquisition. Data was acquired on a BD LSRFortessa and analyzed in FlowJo. Cells were gated for size, single cells, living cells, and CD8⁺ cells before examination of proliferation curves (Supplementary Fig. 8c). Cells were further gated on proliferation dye⁺ cells to exclude any CD8⁺ T cells in the APC fraction before quantification of division numbers and examination of surface and intracellular proteins. Statistical analyses of results from separate mice were performed using GraphPad Prism software.

Spleens were harvested from the mice and single cell suspensions were generated as described previously.^{9 (link)} One million cells from the spleen were stained with anti-mouse CD11b clone M1/70 (BD Pharmingen), anti-mouse Ly-6G and Ly6C clone RB6-8C5 (BD Pharmingen), or isotype controls for 1 hour at 4°C in the dark. Other splenocytes were stained with anti-mouse CD3e (BD Pharmingen), anti-mouse CD4 (BD Pharmingen), anti-mouse CD25 clone PC61.5 (eBioscience), or isotype controls. These cells were then fixed and permeabilized with the BD CytoFix/CytoPerm™ Fixation/Permeabilization Kit (BD Pharmingen) followed by intracellular staining using an anti-mouse FoxP3 antibody (eBioscience). Flow cytometry was performed on a BD FACS Canto II and analyzed using FlowJo. CD11b+/Gr1+ MDSCs and CD3+/CD4⁺/CD25+/FoxP3⁺ lymphocytes were quantified and averaged as described previously.^{9 (link), 32 (link)} Data are expressed as % of lymphocytes.