Anti helios

Mice were euthanized and the rostral part (as shown in figure 1C) of both kidneys were cut into small pieces (2–3mm), and incubated in collagenase solution (100U/ml collagenase in Roswell Park Memorial Institute (RPMI) buffer with 2% calf serum, 2mM MgCl₂ and 2mM CaCl₂) for 45 min at 37°C. Samples were homogenized gently with MACS C-tubes and poured through a 70μm filter. A 44%/67% percoll was used to purify the lymphocytes from the kidney. The leukocyte interface was transferred to a new tube and washed with FACS buffer to prepare for cell staining. The single cell kidney suspension was then stained with fluorochrome labeled antibodies: anti-CD8 (Clone 53–6.7 Tonbo Biosciences, CA), anti-CD4 (Clone RM4-5, Biolegend, CA), anti-FoxP3 (Clone FJK-16S eBioscience, CA), anti-Helios (Clone 22F6, Biolegend, CA), anti-T-bet (Clone 4B10, Biolegend, CA) and anti-CD44 (Clone IM7, Biolegend, CA). Spleen and mesenteric lymph nodes were harvested into FACS buffer and a single cell suspension was obtained by mashing though a 70μm filter and stained as described above. Intracellular staining with anti-FoxP3 was performed using the FoxP3 kit as per the manufacturer’s directions (eBioscience). Events collected with LSRFortessa flow cytometer (BD Pharmingen, CA) were analyzed by FlowJo software (Tree Star, San Carlos, CA).

Freshly obtained peripheral blood mononuclear cell (PBMC) were stained with anti-CD4 (-FITC from Biolegend, OKT4, USA), anti-CD25 (-PE from Biolegend, BC96, USA), anti-CD127 (-PECY7 from Biolegend, A019D5, USA), anti-CD226 (-APC from Biolegend, 11A8, USA), anti-TIGIT (-APC from eBioscience, MBSA43, USA). Intracellular detection of Foxp3 with anti-Foxp3 (-Percp-Cy5.5 from eBioscience, 236A/E7, USA) and Helios with anti-Helios (-APC from Biolegend, 22F6, USA) was performed on fixed and permeabilized cells via Foxp3 Staining Buffer Set (eBioscience, USA). Cell fluorescence was acquired on BD LSR Fortessa (BD Biosciences, USA) and analyzed with FlowJo software (version 7.6.5; Tree Star). We usually acquired 10, 000 events in FSC. CD4-FITC positive and SSC gates were used to delineate CD4⁺ cells, then gated with CD25-PE and CD127-PECY7 in these cells, and the acquisition gate was designed on the CD4⁺CD25^highCD127^low/− cells.

RPMI1640 medium containing 10% FCS was used for CD4 naïve T cell cultures. The following antibodies were purchased from BD BioSciences (San Jose, CA): anti-mouse CD3 (145-2C11), anti-mouse CD28 (37.51), anti-mouse IL-2, anti-CD4-PerCP, anti-CD4-PECy7, anti-CD8-FITC, anti-CD25-APC, anti-CD25-PE, anti-CD44-FITC, anti-CD62L-PE, anti-BrdU-FITC. Anti-FoxP3-APC, anti-IFNγ-APC, and anti-IL-17A-PE were from eBioscience (San Diego, CA). Anti-Helios, anti-CD122 and anti-GITR were from Biolegend (San Diego, CA). Recombinant mouse IL-2 and IL-7 were purchased from R&D Systems (Minneapolis, MN) and ovalbumin was from Sigma-Aldrich (St Louis, MO).