Cd4 fitc

Antibodies used in the studies are mentioned below:
Anti-mouse: CD3-Pacific Blue, CD4-PE, CD8-APCCy7, CD69-FITC, CD44-FITC, CD62L-APC, IFNγ-APC, IL-17-PECy7, CD11b-APCCy7, CD11c-APC, CD80-FITC, CD86-PerCPCy5.5, CD40-PE, CD4-APC, and CD4-FITC from Biolegend, USA.
Anti-mouse: p38, ph-p38 and β-Actin from Cell Signaling Technology.

Flow cytometry was performed on a Fortessa (BD Biosciences) as previously described methods [2 ]. Anti-mouse CD19-Percp, CD8-PE-Cy7, F4/80-APC, CD3-APC-Cy7, CD11c-PE, CD4-FITC, and CD49b-PE were purchased from Biolegend.

Tumor cells isolated from mice were thawed and stained with LIVE/DEAD Fixable Violet Dead Staining Kit (ThermoFisher). Subsequently, the cells were divided and stained with cocktails of fluorochrome-conjugated monoclonal antibodies: CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD45 BV605, CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C PE, Ly6G APC-Cy7, MHC II FITC, CD80 PE-Cy7 (all from Biolegend) for myeloid cell identification and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, CD25 PE, CD44 PE-Cy7, CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using FACSFortessa flow cytometer with Diva software (Becton Dickinson).

Cryopreserved PBMCs from RA and healthy subjects were cultured at 5 × 10⁶ cells/well in a 48-well plate in RPMI-1640 + 10% HPS with 10 μg/mL of peptide. IL-2 (Novartis) was added at 325 IU/mL on day 6. On day 14, cells were stained for expression of Tmr-PE, CD25 APC (BD Biosciences), and CD4 FITC (BioLegend) (Supplemental Table 8) and then run on a FACSCanto. The data were analyzed by FlowJo software version 10.

The cells were resuspended up to 10⁶ nucleated cells and divided into tubes A and B. In tube A, the cells were incubated with 5 μl CD4-FITC (317407, Biolegend, U.S.A.) and CD185-PE (CXCR5) (356903, BioLegend, U.S.A.) in the dark at RT for 30 min. Then, the tissues were washed twice with PBS for intercellular staining.
Cells in tubes A and B were incubated in 1 ml cold, freshly prepared fixation/permeabilization solution (130-093-142, MACS, GER) in the dark at 4°C for 30 min. After washing twice with permeabilization buffer, the cells were separately stained with Foxp3- Alexa Flour (320013, BioLegend, U.S.A.) and 10 μl Anti-human Aire-APC (130-093-142, MACS, GER) and next incubated for 30 min at RT. The stained cells were detected by flow cytometry immediately.
To guarantee the accuracy of the result, isotype controls were used to determine the gating parameters. FACS Calibur instrument (Becton Dickinson, U.S.A.) was used to conduct flow cytometry.

To detect the expression of Tims in various T-cell subpopulations, PBMCs were surface stained with the following anti-human mAbs: CD3 Pacific Blue, CD4 FITC, CD8 PerCP-Cy5.5, Tim-1 PE and Tim-3 BB515 (BioLegend, San Diego, CA, USA). Dead cells were excluded from analysis by staining with the Fixable Viability Stain 780 (BD, Franklin lakes, NJ, USA) and analysed using FlowJo Software X (Tree Star, Ashland, OR, USA). The gating strategy used for T-cell subsets is shown in Figure S1.

Eight to ten-week old female C57BL/6 mice were subcutaneously inoculated in the right flank with MC38/0 cells (1.1 × 10⁶/0.2 ml/mouse). On the 14th, 15th and 17th day of the experiment, mice were injected i.t. with LVs encoding shRNA against IL-10 (shIL10–3, 2x10⁶TU/50 μl/mouse) or reference LVs encoding scrambled shRNA against human GAPDH (shN). Two days after the third injection, the mice were sacrificed and their tumor nodules were dissected and homogenized. Efficacy of transduction in tumors was measured by flow cytometry as the fluorescence intensity of EGFP among cells isolated from tumors. Concentration of IL-10 was estimated by ELISA in supernatants collected from 24 h culture of 5 mg tumor tissue/ml. Myeloid and lymphocyte populations in tumors were analyzed using LSR Fortessa with Diva software (Becton Dickinson) after staining with fluorochrome-conjugated antibodies: CD45 V500, CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C BV510, Ly6G BV605, MHC II APC-Cy7, for myeloid cell identification (all from Biolegend) and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, (all from BioLegend) for lymphocyte identification.