Anti helios efluor 450

B6 and Rorγ^−/− mice were injected i.p. with ID8A-luc cells (4 × 10⁶ cells per mouse). They were treated with either PBS (200 μl i.p.) or RMP1–14 (5 mg kg⁻¹, BioXcell, 200 μl i.p.) on days 3, 6, 9 and 12. Spleens and ascites from untreated B6 and Rorγ^−/− mice (n=5 per group) were collected at day 35±2 and cells purified. Cells were stained with Fixable Viability Dye-efluor 780, anti-CD3-PE (6 μg ml⁻¹, eBioscience, clone: 145-2C11), anti-CD4-FITC (15 μg ml⁻¹, eBioscience, clone: RM4-4) and anti-PD1-PerCP-Cy5.5 (6 μg ml⁻¹, BioLegend, clone 29F.1A12). The cells were fixed with Foxp3 Fix/Perm Buffer Set and intracellular staining was performed using anti-Foxp3-APC (6 μg ml⁻¹, eBioscience, clone: FJK-16s) and anti-Helios-efluor 450 (3 μl per 100 μl, eBioscience, clone: 22F6). MDSCs were detected with anti-CD11b-efluor 450 (6 μg ml⁻¹, eBioscience, clone: M1/70) and anti-Gr-1-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: RB6-8C5).
In addition, Foxp3 mRNA (QT00138369) expression was analysed by TaqMan assay (LightCycler). The expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase (Gadph) mRNA level and expressed as the relative expression, that is, fold increase (2^−DCT), where ΔCT=CT_{(Target gene)}−CT_(GADPH).

Th17–T_reg subsets (IL-17A⁺Foxp3^neg, IL-17A⁺Foxp3⁺, IL-17A^neg Foxp3^neg and IL-17A^neg Foxp3⁺) were stained with anti-neuropilin-1-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: 3DS304M), anti-Folr4-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: EBio12A5), anti-GARP-eFluor450 (6 μg ml⁻¹, eBioscience, clone: YGIC86), anti-ICOS-eFluor450 (6 μg ml⁻¹, eBioscience, clone: ISA-3), anti-Nrp-1-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: 3DS304M), anti-ST2-PerCP-Cy5.5 (6 μg ml⁻¹, Biolegend, clone: DIH9), anti-TIGIT-PE-Cy7 (6 μg ml⁻¹, Biolegend, clone: 1G9), anti-CD73-PerCP-Cy5.5 (6 μg ml⁻¹, Biolegend, clone: TY/11.8) and anti-Lag3-eFluor710 (6 μg ml⁻¹, eBioscience, clone: eBioC9B7W). The cells were fixed with Foxp3 Fix/Perm Buffer Set and stained intracellularly with anti-Helios-eFluor450 (6 μg ml⁻¹, eBioscience, clone: 22F6). Cells were analysed by flow cytometry (LSRFortessa, BD Biosciences). The fluorescence minus one controls were used to identify the gating boundaries.