Pe anti ctla 4

Anti-CD14 conjugated to violet blue was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). FITC-anti-CD4, PE Cy5-anti-CD11c, PE-anti-CD25, BV650-anti-CD25, APC-anti-CD45RA, BV711-anti-CD15s, PE-anti-CD45RA, PC5-anti-CD28, and PE anti-CTLA-4 were obtained from BD Biosciences (Mountain View, CA, USA). PE CY7 anti-CD127, BV421-anti CCR7, PE-anti-CD59, and BV510-anti-CD27 were purchased from Sony Biotechnology (San Jose, CA, USA); PE-FOXP3, APC-FOXP3, and eF450-Helios were supplied by eBiosciences (San Diego, CA, USA); PC5 anti-ICOS, APC-anti-PD1, BV421-anti-CD39, APC-anti-GITR, and BV421-anti LAG3 were purchased from Biolegend (San Diego, CA, USA); and staphylococcal enterotoxin E (SEE) was purchased from Toxin Technology Inc.; and CellTrace carboxyfluorescein succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR, USA). The anti-CD3 (OKT3) used for some proliferation assays was purchased from Biolegend (San Diego, CA, USA).

Surface and intracellular staining for flow cytometry analysis were performed as described previously (23 (link), 26 (link)). The antibodies used for surface staining included APC-Cy7-anti-CD4, Pacific Blue-anti-CD8a, APC-anti-CD44, APC-anti-PD-1, PerCP-Cy5.5-anti-CD43, PE-Cy7-anti-CD62L, PE-anti-CTLA-4, PE-anti-LAG-3 (all from BD Biosciences), PE-anti-CD45.1, PE-anti-CD45.2, and PE-Cy7-anti-CD45.2 (all from BioLegend). For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (Invitrogen) and were stained with APC-anti-interferon (IFN)γ, PerCP-Cy5.5-anti-interleukin (IL)-2, FITC-anti-tumor necrosis factor (TNFα) (from BD Bioscience). For Ki67 staining, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Biolegend) and were stained with BV421-anti-Ki-67 (Biolegend). Freshly isolated cells were used for all assays and about 20,000–40,000 T cells were acquired for each sample using BD FACSCanto II flow cytometer. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506 (eBioscience).