Pe conjugated anti foxp3 antibody

First, cells were incubated with Cytofix/Cytoperm (BioLegend) to permeabilize the cell membranes for 20 min at 4°C. Then, cells surfaces were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-Foxp3 antibodies (eBiosciences, CA) according to the instructions. Finally, cytometry was performed with the BD FACS Calibur System (BD Bioscience). The plots were gated for CD4⁺ lymphocytes, and Tregs were identified as CD4⁺Foxp3⁺ T cells.

For intracellular staining, cells were restimulated with 25 ng/mL phorbol 12-myristate 13-acetate and 250 ng/mL ionomycin (both from Sigma-Aldrich) for 4 h in the presence of GolgiStop (BD Biosciences, Sparks, MD, USA). Murine splenocytes were stained with surface PerCP-conjugated anti-CD4 (eBioscience) and APC-conjugated anti-CD25 (BioLegend, San Diego, CA, USA) antibodies. After fixation and permeabilization, cells were stained with FITC-conjugated anti-IL-17 or PE-conjugated anti-Foxp3 antibodies (eBioscience). Events were collected and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Tumor tissues were digested with Liberase TM and Dnase I (Roche, Basel, Switzerland) and filtered through 40 μm cell strainers. Cells were initially incubated with CD16/32 antibody to block Fcγ receptors, then stained with 1) the combination of APC-Cy7-conjugated CD45 antibody (BD Biosciences, Franklin Lakes, NJ USA), FITC-conjugated CD4 antibody and APC-conjugated CD8a antibody (BioLegend, San Diego, CA USA), or 2) the combination of FITC-conjugated CD44 antibody, PE-Cy7-conjugated CD62L antibody and APC-conjugated CD4 or CD8a antibody (BioLegend). For Treg discrimination, cells were further fixed and permeabilized (eBioscience, San Diego, CA USA) according to manufacturer’s instructions, stained with PE-conjugated anti-FOXP3 antibody (eBiosciences), and analyzed with FACSAria cytometer (BD Biosciences).

Before intracellular staining, cells were stimulated with 25 ng/ml phosphomolybdic acid (Sigma-Aldrich), 250 ng/ml ionomycin (Sigma-Aldrich), and Golgi Stop (BD Biosciences, San Diego, CA, USA) in 5% CO₂ at 37 °C for 4 h. Cells were stained with peridinin chlorophyll protein complex–conjugated anti-CD4 and allophycocyanin (APC)-conjugated anti-CD25 antibodies (BD Pharmingen, BD Biosciences), and then with a phycoerythrin (PE)-conjugated anti-Foxp3 antibody (eBioscience, San Diego, CA, USA), followed by fixation and permeabilization using a Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions. The samples were analyzed using a FACSCalibur instrument (BD Pharmingen, BD Biosciences).

As VEGFR-2 expression is reported on Tregs and MDSCs, the effect of vaccination on normal levels of these cells was assessed as a measure of safety. Splenocytes were isolated when mice were sacrificed and the percentage of Tregs and MDSCs were measured by flow cytometry. Regulatory T cells were stained with a FITC-conjugated anti-CD25 antibody, an APC-conjugated CD4 antibody and a PE-conjugated anti-FoxP3 antibody after fixation and permeabilization with commercial buffers (Ebioscience, Vienna, Austria). MDSCs were stained with a FITC-conjugated anti-CD11b antibody and a PE-conjugated anti-Gr1 antibody (Ebioscience). The cells were analyzed with an Accuri C6 flow cytometer and events in the lymphocyte gate were selected for analysis. Color compensation was based on fluorescence minus one (FMO) controls.

hPBMCs were treated with various concentrations of PGRN in the presence or absence of PHA. CD4⁺ T lymphocytes were stimulated with PHA plus PGRN in the presence or absence of Treg inhibitor or TGF-β RI Kinase inhibitor VI. In all experiments the cells were divided into two groups. One set was analyzed to assess cell proliferation and the other was stained with FITC-conjugated anti-CD4 antibody (eBioscience, San Diego, CA, USA) and PE-conjugated anti-Foxp3 antibody (eBioscience, San Diego, CA, USA). Flow cytometry was performed to determine the proportion of CD4⁺Foxp3⁺ Treg cells.

Flow cytometry analysis was performed on splenocytes of MuSK-immunized mouse and of control mouse. Spleen cells were suspended in FACS wash buffer (PBS, 5% BSA) and incubated for 60 min at 4°C in the dark with antibodies to the tested cell surface molecules. Cells were washed and analyzed on a FACScan flow cytometer. The following antibodies were used for flow cytometry: FITC-conjugated anti-mouse CD4 (L3T4) and APC-conjugated anti-mouse CD25 (PC61.5). For intracellular staining, cells were fixed and permeabilized using the fixation/permeabilization kit from e-Bioscience (San Diego, CA) followed by staining with PE-conjugated anti-FoxP3 antibody (e-Bioscience).