Cd4 v500

Flow cytometry was used to assess Treg cells following isolation from leukapheresis product and following expansion protocols. Antibodies (anti-human) against the following targets were used in the staining and analysis: CD3 Alexa Fluor 700 (Invitrogen), CD4 V500 (BD Biosciences), CD8 eFluor 450 (Invitrogen), CD25 PerCP-Cy 5.5 (BD Biosciences), FOXP3 Alexa Fluor 488 (Invitrogen). Appropriate isotype controls were set for gating schemes and to establish background parameters. Live/Dead fixable blue dead cell UV stain kit was used to assess viability of the cells. For intracellular FOXP3 staining, cells were fixed and permeabilized using the FOXP3/Transcription Factor Staining Buffer Set (BD Biosciences) prior to staining. Cells were subsequently analyzed using a BD LSRII flow cytometer with BD FACSDIVA software.

Activated Tfh cells in the spleen were labeled with FVS-440UV, CD45-BV786, CD3-FITC (Clone 145-2C11, BD), CD4-V500, CD8-BV605, and CXCR5-PE (Clone 2G8, BD) following surface antigen staining procedure. Subsequently, cell pallets were suspended in 1 ml fixation buffer (Cat. 554655, BD) for 15 min under room temperature and washed with 1 ml perm/wash buffer (Cat. 557885, BD) twice. Anti-mouse Bcl-6-Alexa Fluor 647 (Clone K112-91, BD) was added at 1 : 50 ratio and incubated in the dark for 30 min at 4°C. Cells were then washed twice and resuspended in cold PBS for flow cytometry assay. Plasma cells in peripheral blood cells (PBMCs) were labeled with FVS-440UV, CD45-BV786, CD3-FITC, B220-PE-Cy7, CD19-APC-cy7 (Clone 1D3, BD), and CD138-BV421 (Clone 281-2, BD) following the procedure described earlier.

WNV-specific T cells were expanded by stimulating purified CD4⁺CD45RA^- cells with selected WNV peptides in vitro for 14 days in the presence of autologous adherent cells as antigen presenting cells. These cells were rested for 3 days and then stained with PE-conjugated tetramers for 30 min at 37°C and stimulated with 25 ng/mL PMA and 1 μg/mL ionomycin at 37°C. Brefelden A was then added and the cells incubated for 3–4 hours at 37°C, 5% CO2. For cytokine staining, surface staining was performed first, followed by fixation/permeabilization as per the manufacturer’s protocol (eBioscience). Cells were then co-stained with antibodies for surface markers and cytokines of interest, including CD3 PE-Cy5 (BioLegend, clone HIT3a), CD4 V500 (BD, clone RPA-T4), IFN-γ AF700 (BioLegend, clone 4S.B3), IL-4 fluorescein isothiocyanate (FITC) (eBioscience, clone 8D4-8), IL-10 PE-Cy7 (Biolegend, clone JE53-9D7), and IL-17 APC-Cy7 (Biolegend, clone BL168), or alternatively with CD4 PerCP-Cy5.5 (BD, clone RPA-T4) and IL-17 APC (Biolegend, clone BL168). ViViD fixable violet dead cell stain was then added to remove dead cells from the analysis. After 20 min at room temperature, cells were washed and immediately analyzed by flow cytometry.

For surface staining, cells were incubated at room temperature with human Fc block (BD Biosciences, San Diego, CA, USA) and followed by staining with directly conjugated mAbs for 30 min at 4 °C. The mAbs used were anti-human CD3-BV605, CD4-V500, CD8-APC-H7, CD45RA-BV421, CCR7-PerCp-Cy5.5, PD-1-PE-Cy7, CD160-Alexa Fluor 488 (BD Biosciences), CD4-FITC, TIM-3-BV421, 2B4-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA), and TIGIT-APC (eBioscience, San Diego, CA, USA). Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences). FlowJo Software (Tree Star, Ashland, OR, USA) was used in data analysis.

PBMCs were phenotyped using the following antibodies from BD Biosciences: CD4-V500, CD8-PE, CCR7-FITC, CD45RA-PECy7, CD31-FITC, CD39-APC, CD25-PE, CD127-V450, FoxP3-APC. Intranuclear staining was performed using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience). Data were acquired on a Canto II and analysed using FlowJo v7.6.5 (Tree Star Inc).