Percp cy5.5 conjugated anti cd4

Flow cytometric analysis was performed using blood samples collected before treatment (weeks 7–8 post-immunization) and at 2, 3, 5, 6, 7, and 8 weeks after treatment (weeks 9–10, 10–11, 13, 14, 15, and 16 post-immunization). All antibodies were purchased from BD Biosciences (San Jose, CA, USA). For analysis of T-cell populations, peripheral blood mononuclear cells (PBMCs) were stained with V500–conjugated anti-CD45 (BD Biosciences), PE-Cy7–conjugated anti-CD3, PerCP-Cy5.5–conjugated anti-CD4, APC-Cy7–conjugated anti-CD8, PE–conjugated anti-CD28, and APC–conjugated anti-CD95 antibodies. For analysis of B-cell populations, PBMCs were stained with V500–conjugated CD45, PE-Cy7–conjugated anti-CD3, FITC–conjugated anti-CD20, V450–conjugated anti-CD27, PerCP-Cy5.5–conjugated anti-CD21, PE–conjugated anti-IgD, and APC–conjugated anti-IgM antibodies. For analysis of regulatory T cells, PBMCs were stained with V500–conjugated anti-CD45, FITC–conjugated anti-CD4, PE-Cy7–conjugated anti-CD25, and PE–conjugated anti-FoxP3 antibodies. Flow cytometric analysis was carried out using a BD LSRFORTESSA cell analyzer (BD Biosciences).

Phycoerythrin (PE)-conjugated anti–interferon-gamma (anti-IFN-γ), APC-Cy7-conjugated anti-CD44, FITC-conjugated anti-CD3, PerCp-Cy5.5-conjugated anti-CD4, and APC-conjugated anti-CD8 were purchased from BD Biosciences (San Jose, CA, USA). For each sample, 1 × 10⁵ events were gated on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For data analysis (Flowjo software; Tree Star, Ashland, OR, USA), positive cells were expressed as a percentage of the respective reference population. The assessment of responses was previously described in more detail [24 (link)]. Briefly, peptide-sensitized PBMCs (1 × 10⁶ cells/mL) stimulated with phytohemagglutinin (PHA, Sigma) and PBMCs pulsed with autologous DCs that were not loaded with any peptide were used as positive and negative controls, respectively. One hour after stimulation, 10 μg of Brefeldin A (Sigma) was added to each well. After 5 additional hours of incubation, PBMCs were washed once with phosphate-buffered saline (PBS) and then incubated in PBS containing 1 mM ethylene-diamine-tetraacetic acid for 10 min. After 2 additional washes with PBS containing 5% FBS, the cells were incubated with fluorescently-labeled monoclonal anti-CD3, anti-CD8, anti-CD4, anti-CD44, and anti–INF-γ antibodies for 15 min on ice in the dark prior to analysis.

For intracellular cytokine staining, cells were treated with 50 ng/mL phorbol-12-myristat-13-acetate (PMA) and 500 ng/mL ionomycin, in the presence of 3 μM goldi-stop (BD bioscience) for 4 h. Flow cytometric analysis of T cells was performed with PerCPcy5.5-conjugated anti-CD4 (GK1.5), allophycocyanin (APC)-conjugated anti-IL17 (TC11-18H10.1), PE-conjugated anti-IL6 receptor alpha (D7715A7), and PE-Cy7-conjugated anti-IFNγ (XMG1.2) from Biolegend (San Diego, CA, USA). eFlour450-conjugated anti-Foxp3 (FJK16S) and PE-conjugated gp130 (KGP130) were from eBioscience (San Diego, CA, USA).
Splenocytes were isolated and stimulated by IL-6 for 30 minutes for phosphorylated STAT3 analysis. These cells were fixed by 4% paraformaldehyde for 10 minutes at room temperature and permeabilized by ice-cold 90% methanol for 30 minutes. Cells were stained by Alexa Fluor-conjugated anti-phosphorylate STAT3 (py705, BD bioscience), PE-conjugated anti-CD25, and PerCPcy5.5-conjugated anti-CD4.