Ctla 4 percpcy5

Peripheral blood mononuclear cells from HCs and paired PBMCs and SFMCs from RA patients were stained for the presence of T cell co-inhibitory receptors. Non-specific binding was blocked by 50 µg/ml mouse IgG (Jackson), and surface staining was performed using the following antibodies: CD3 V450 (clone: UCHT1, BD), CD4 PE-CF594 (clone: RPA-T4, BD), CD8 BV785 (clone: RPA-T8, BioLegend), CD25 Alexa 700 (clone: BC96, BioLegend), Tim-3 BV711 (clone: F38-2E2, BioLegend), CTLA-4 PerCPCy5.5 (clone: L3D10, BioLegend), Tigit PE-Cy7 (clone: MBSA43, eBioscience), PD-1 APC (clone: MIH4, BD), Live-dead near IR (Thermo Fisher Scientific). Cells were permeabilized by BD Facs lysing solution and BD Facs Perm Solution 2 (both BD bioscience). Intracellular staining was performed using Eomes PE (clone: WD1928 ebioscience). All antibodies were used in the concentration recommended by the manufacturer. Gating was done on lymphocytes, excluding doublets and dead cells. Gates were set using FMOs. CD3⁺ CD4⁺ and CD3⁺ CD8⁺ cells were investigated for their expression of T cell co-inhibitory receptors.

Vehicle-, nivolumab- or ipilimumab-expanded lymphocytes were stained with zombie NIR viability dye, CD8-BV421, CD4-BV510, CD3-FITC, CXCR3-PE, CCR6-APC, CTLA-4-PerCPCy5.5, TIGIT-PE/Cy7, PD-L1-PerCPCy5.5, PD-1-PECy/7, CD25-FITC and FOXP3-BV421 (Biolegend, USA). Intracellular FoxP3 permeabilization and fixation buffer was used for FoxP3 staining (Biolegend, USA). Cells were acquired using the BD FACs Canto II (BD Biosciences) using Diva software and analysed using FlowJoTM v10.7.

Single cells were incubated with Human TruStain FcX (BioLegend) to block Fc receptors. These were then stained with Ghost Dye™ Red 780 Viability Dye (Cell Signaling Technology) to identify dead cells and the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs): CD103-PerCP/Cy5.5 (Ber-ACT8), CTLA-4- PerCP/Cy5.5 (BNI3), Granzyme B-FITC (QA16A02), PD-1-PE (EH12.2H7), CD27- PE/Cy7 (LG.3A10), CD3- PE/Cy7 (HIT3a), CD8a-AF700 (RPA-T8), CD4-APC (OKT4), CD127-BV711 (A019D5), Ki-67-BV605 (Ki-67), CD45RA-BV605 (HI100), CCR7-BV510 (G043H7), CD69-BV421 (FN50), and IFNγ-BV421 (4S.B3) (all from BioLegend). Data were acquired on a LSRFortessa X-20 (BD) and analyzed with FlowJo software.