Cd4 pe cf594

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.

Peripheral blood mononuclear cells were sorted into subsets at the University of Birmingham. The PBMCs were first thawed as described in the Supplementary Data, http://links.lww.com/HC9/A236. CD14⁺ monocytes were then selected using human CD14 MicroBeads (Miltenyi Biotec, Woking, UK) according to the manufacturer’s instructions; CD14⁺ cell pellets were frozen at −80^°C until RNA extraction. Non-CD14⁺ cells were resuspended in PBS and stained with live/dead marker (Zombie APC-Cy7 at 1:1000; Biolegend, San Diego, CA, USA) for 20 minutes at room temperature. Cells were washed and stained with the following fluorochrome-conjugated antibodies (BD Biosciences, UK): CD3-APC, CD4-PECF594, CD127-PECy7, CD25-BB515, CXCR3-PerCP-Cy5.5, CCR6-BV421, and CD19-PE for fluorescent-associated cell sorting (using BD Aria Fusion, BD, UK) of the immune cell subsets: (1) CD19⁺CD3^– B cells, (2) CD3⁺CD4⁺CD25^highCD127^low T_REG cells, (3) CD3⁺CD4⁺CCR6⁺CXCR3^– T_H17 cells, and (4) CD3⁺CD4⁺CCR6^–CXCR3⁺ T_H1 cells (Fig. S2, http://links.lww.com/HC9/A236). We used an anti-CD3 monoclonal antibody that did not induce T-cell activation (Fig. S3, http://links.lww.com/HC9/A236). All immune cell subsets were sorted into tubes containing RPMI−1640+10% FCS media kept at 4°C during the sorting period. After sorting, cells were pelleted and resuspended in RLT+βME buffer for lysis and RNA extraction.

Splenocytes were isolated under aseptic conditions from all mice groups and layered on Ficoll Hypaque for density gradient separation of the splenic lymphocytes. These splenic lymphocytes were stained for various cell surface and intracellular markers such as CD3, CD4, CD8, CD19, CD11b, F4/80, and RANKL. Briefly, Aliquots of 1 × 10⁶ splenic lymphocytes were washed with ice-cold 1XPBS and fixed with 1% paraformaldehyde for 15 min at 4°C. The cells were washed with FACS buffer (1XPBS + 2% FBS + 0.001% sodium azide) and then labeled with fluorophore tagged antibodies CD3-PB, CD4-PECF594, CD8-PE, CD19-FITC, CD11b-Percp cy5.5 (BD Biosciences, USA), and F4/80 APC-cy7, RANKL-PE (BioLegend, San Diego, USA). Further, the cells were washed with FACS buffer and acquired on FACS Aria (BD Biosciences San Jose, USA). Data analysis was done using FlowJo software (Tree Star, Ashland, USA).