Isolation and characterization of T cell subsets

Spleens of recipient rats were collected, grounded, and filtered to obtain cell suspension, which was centrifuged at 1600 rpm at 4°C for 5 mins. The precipitation was resuspended by pre-cooled phosphate buffer solution. Double volume of lymphocyte separation solution purchased from Sigma-Aldrich was added and centrifuged at 2000 RPM for 25 min to obtain cell suspension. CD4⁺ T cells and CD8⁺ T cells were isolated by LS column (Miltenyi Biotec, Germany, #130-042-401) with the method of antibiotic microbeads (Miltenyi Biotec, Germany, # 130-090-319, # 130-090-318) as previously described (37 (link), 38 (link)). Sorted cells were incubated directly with diluted fluorochrome-conjugated monoclonal antibodies as shown below: anti-CD4 (Invitrogen, #11-0040-82, FITC), anti-CD8a (Invitrogen, #11-0084-82, FITC), anti-PD-1 (Proteintech, #65211, Coralite647) and anti-Foxp3 (eBioscience, #12-5773-82, PE).