Pd l2 ty25

The individual performing FACS analysis was not blinded to genotype. Anti-mouse Abs CD4 (GK1.5, BD Pharmingen, Franklin Lakes, NJ, USA) and CD8 (53-6.7, BD Pharmingen) were used for the proliferation assay. Anti-mouse CD19 (1D3, BD Pharmingen), CD1d (1B1, BD Pharmingen), CD5 (53-7.3, BD Pharmingen), CD28 (37.51, BD Pharmingen), CD152 (CTLA-4, UC10-4B9), ICOS (C398-4A, BD Pharmingen), PD-L1 (MIH5, eBioscience), and PD-L2 (TY25, eBioscience) were used for this study. Single-cell suspensions were washed with staining medium (PBS containing 0.1% NaN₃ and 2% FCS). After incubation with mAb and washing with staining buffer, propidium iodide (PI) was added to identify dead cells. FACS data acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using isotype control Abs to set quadrants before calculating the percentage of positive cells, using FCS Express (De Novo Software, Los Angeles, CA, USA).

Cultured cells and primary T cells were stained with the indicated Abs. Data were obtained with Gallios (Beckman Coulter) and analyzed using FlowJo (Tree Star). Abs against Bcl6 (K112-91) and CXCR5 (2G8) were purchased from BD bioscience. Abs against CD3 (145-2C11), CD80 (16-10A-1), CD86 (GL-1), and PD-L2 (TY25) were purchased from eBioscience. The Ab against CD8 (5H10) was purchased from Thermo Fisher Scientific. Abs against CD3 (17A2), CD4 (RM4-5), CD28 (37.5), CD45 (30-F11), ICOS (C398.4A), ICOSL (HK5.3), PD-1 (RMP1-30), and PD-L1 (10F.9G2), Brilliant violet 421-conjugated streptavidin, armenian hamster IgG (HTK888), rat IgG2b (RTK4530), and syrian hamster IgG (SHG-1) were purchased from Biolegend. FoxP3 Transcription Factor Staining Buffer Kit (TONBO) was used for the staining of Bcl6.