Pe anti ctla 4

Cell suspensions were incubated with anti-mouse CD16/CD32 (Biolegend, 101,319). For cell surface flow cytometry, cells from spleens, thymuses and LNs were stained with specific antibodies for surface antigens as follows: PE-anti-CD4 (Biolegend, 100,408), Pacific Blue-anti-CD4 (Biolegend, 100,531), Brilliant Violet 510-anti-CD8a (Biolegend, 100,751), APC/Cy7-anti-TCRβ (Biolegend, 109,220), 7-AAD (BD Pharmingen™, 559,925), APC-anti-CD25 (Biolegend, 102,012), PerCP/Cy5.5-anti-CD44 (Biolegend, 103,032), PE/Cy7-anti-CD62L (Biolegend, 104,418), PE-anti-CD278 (ICOS) (Biolegend, 107,705), APC-anti-CD304 (Neuropilin-1, Nrp1) (Biolegend, 145,206), PE/Cy7-anti-CD279 (PD-1) (Biolegend, 109,110). For intracellular staining, cells were fixed and permeabilized with Fixation/Permeabilization Kit (eBioscience, 00–5123, 00–5223), washed with Permeabilization Buffer (eBioscience, 00–8333) and stained with PE-Cy7-anti-ki67 (eBioscience, 25-5698-82), PE-anti-CTLA4 (Biolegend, 106,306), AF488-anti-Foxp3 (Thermo Scientific™, 53-5773-82), PE-anti-Foxp3 (Biolegend, 126,404). For apoptosis analysis, cells were stained with FITC-AnnexinV (Biolegend, 640,906) in AnnexinV Binding Buffer (Biolegend, 422,201). Samples were analyzed by LSRII multicolor flow cytometer (BD Biosciences CA, USA) and data analysis was performed using the FlowJo software (Tree Star, USA).