Anti cd8

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).

The following monoclonal antibodies were used for flow cytometry analysis and cell sorting; anti-CD8 (TONBO Biosciences), anti-CD4 (BD Biosciences), anti-CD45RA (BioLegend), anti-CD45RO (BD Biosciences). LIVE/DEAD fixable yellow dead cell stain kit (Thermo Fisher Scientific). Recombinant human IL-2 was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases). Recombinant human IL-7 and IL-15 were purchased from BioLegend.