Foxp3 apc

For the detection of allogeneically activated Tregs multicolor flow cytometry was performed using a combination of following monoclonal antibodies: anti-human CD4 Brilliant Violet 421 (clone: RPA-T4, biolegend), anti-human CD25 PE-Cy7 (clone: M-A251, biolegend), anti-human GARP PE (clone: ME-9FI, biolegend). Intracellular staining of FOXP3 APC (ebioscience, clone: PCH101) was performed according to manufacturer’s instructions. Subsequent flow cytometry was performed on a LSR II (BD Biosciences). Stimulator PBMCs were identified by CFSE labelling and excluded from the analysis. Tregs were identified as CD4⁺CD25^highFOXP3⁺, activated Tregs were identified by their co-expression of GARP. Frequency of activated Tregs was calculated as ratio of CD4⁺CD25^highFOXP3⁺GARP⁺ to CD4⁺CD25^highFOXP3⁺ (Supplementary Fig. 1).

Frozen PBMC from WNV infected subjects were thawed, rested for 30 minutes and then co-stained with antibodies for surface markers and cytokines of interest, including CD4 PerCP (BD, clone RPA-T4), CD25 PE (BioLegend, clone BC96), CD127 AF488 (Biolegend, clone 8019D5), FoxP3 APC (eBioscience, clone PCH101), CTLA-4 PE-CF594 (BD, clone BN13) and CCR4 BV605 (Biolegend, clone L29IH4). After 20 min at 4 degrees, cells were washed and immediately analyzed by flow cytometry.

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).

The cells were divided into several groups and then stained with fluorophore-conjugated antibodies, including CD11b-FITC or BV421 (eBioscience), Gr-1-PerCP Cy5.5 or APC (eBioscience), CCR5-APC or PE (BioLegend, CA, USA), CD45-AmCyan (BioLegend), CD4-PE (eBioscience), and CD25-FITC (eBioscience) for 30 min at 4°C in staining buffer (PBS with 10% FBS). CCL5-PE/Cyanine7 (BioLegend), Foxp3-APC (eBioscience), and interferon-gamma (IFN-γ)-FITC (eBioscience) were used for intracellular staining after culturing with fixation/permeabilization medium (eBioscience). Data were acquired through FACS AriaIII (BD Biosciences) and analyzed with FlowJo X (BD Biosciences).

Naïve CD4⁺ T cells were isolated from spleens of wild-type C57BL/6 J mice (8–14 weeks; both sexes). In brief, dissected mouse spleens were gently ground through 70 µm cell strainer mesh (Falcon), and naïve CD4⁺/CD25⁺ (regulatory; T_reg) or CD4⁺/CD25^- (effector; T_eff) T cells were isolated and collected using a CD4⁺CD25⁺ T cell isolation kit (Miltenyi Biotec; #130-091-041) and magnetic separation columns (Miltenyi Biotec, #130-042-201 and #130-042-401) according to manufacturer’s protocol. Purity of T cell populations were verified by using flow cytometry (Supplementary Fig. 6a; CD3-PerCP-Cy5.5 1:100, eBioscience, #45-0031-80; CD4-Pacific Blue 1:100, eBioscience, #57-0042-82; and Foxp3-APC 1:100, eBioscience, #17-5773-80; FlowJo v.10.6.2). Isolated CD4⁺ T cells were resuspended in RPMI 1640 containing 10% FBS, 1% PSQ, 1% sodium pyruvate, 1% HEPES, and 1% nonessential amino acids, and plated on poly-L-lysine coated coverslips >1 h before recording. Whole-cell recording of CD4⁺ T cells was carried out within 48 h after isolation.