Anti cd4

Basal expression of the different molecules was determined in peripheral blood obtained from seven RA patients, as described above. The effect of IL-15 on the expression of these molecules was studied in PBMCs obtained from 10 RA patients and cultured in medium alone or in the presence of IL-15 (50 ng/mL) (Peprotech INC, Rockyhill, NJ, USA) for 18 h. Overnight treatment with IL-15 has been seen to be the appropriate frame time for protein expression detection in previous studies [8 (link),16 (link)]. The surface stain in both cases was made with anti-CD4 (APC) or anti-CD4 (PECy7), anti CD45RA (APCFire), anti-CD8 (PB), anti-CD28 (BV), anti-CD44 (FITC), anti-CX3CR1 (PE), anti-CCR5 (PECy7), and anti-CD11a (FITC) (Biolegend, San Diego, CA, USA), as well as anti-CD3 (PerCP) and anti-CD49d (APC) (BD Bioscience). Cells from whole blood were stained as described above. PBMCs were stained for 30 min at 4 °C. Then, cells were washed and resuspended in PBS until they were acquired in a Navios flow cytometer and analysed with Kaluza software (Beckman Coulter Life Science, Brea, CA, USA). The cytometer compensation was carried out using the VersaComp Antibody Capture Bead Kit (Beckman Coulter). The marker settings for determining the negative/positive cell populations were established using the FMO strategy.

In the peak phase of EAE in mice, splenic MNCs and brain cells were harvested as described previously (29 (link)). For intracellular cytokine staining, splenic MNCs were restimulated in complete 1640 medium with a cell activation cocktail containing Brefeldin A (BioLegend) for 6 h. The cells were surface-stained with an anti-CD4 antibody for 30 min. After washing and fixation, the cells were stained with anti-IFN-γ (BioLegend) for Th1 cells, anti-IL-4 (BioLegend) antibody for Th2 cells, and anti-IL-17 (BioLegend) for Th17 cells. To quantify the Treg cells, the cells were surface-stained with anti-CD4 (BioLegend) and anti-CD25 (BioLegend) without the stimulation protocol. After fixation, permeabilization buffer was used as a washing solution (BioLegend), and the cells were then stained with anti-Foxp3 antibodies. The antibodies were tagged with phycoerythrin, allophycocyanin, or fluorescein isothiocyanate. Data were acquired on a FACSAria flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (Ashland, OR, USA).

Leukocytes were isolated from liver as described previously^{52 (link)}. For flow cytometry, cell preparations were incubated with purified anti-CD16/CD32 antibody (BioLegend, San Diego, CA) for 15 min at 4 °C, and then stained with the following antibodies conjugated to fluorescent labels: anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, anti-CD19, anti-CD44, anti-CD62L, LAG3 and 2B4 (BioLegend).
For intracellular cytokine staining, approximately 10⁶ liver mononuclear cells were stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience, California, USA) for 4 h, or EmP (5 μg/ml) for 10 h (37 °C, 5% CO2) as previously described^{52 (link)}. The cells were stained for surface anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, LAG3 and 2B4, and then stained for intracellular anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α, anti-IL-10 and granzyme B (Biolegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). ALSRFortessa flow cytometry (BD Immunocytometry Systems, San Jose, CA) was used to acquire data, which were analyzed with Flowjo software (Treestar, Inc., Ashland, OR).