Cd4 pe cy7

The cultured cells or isolated PBMCs were stimulated with 0.5 μg/ml concentration of the peptide pool. After 1 h of culture (37°C, 5% CO₂), the cells were supplemented with brefeldin A solution (BioLegend) and cultured for 4 h. The samples stimulated with the peptide solvent alone (20% DMSO in PBS) were used as unstimulated controls. The cells were transferred to a V‐bottom 96‐well plate (Nalgene) and stained as described¹⁴ with live/dead fixable stain and the following antibodies: CD4‐PE‐Cy7 and CD8‐Alexa Fluor 700 (Exbio), CD3‐PerCP‐Cy5.5, TNF‐α‐APC, IFNγ‐PE (Becton Dickinson). For T cell phenotype analyses, the cells were stained with live/dead fixable stain and the following antibodies: CD4‐PE‐Cy7 and CD8‐Alexa Fluor 700 (Exbio), CD3‐PerCP‐Cy5.5 and TNF‐α‐APC (Becton Dickinson), and CD62L‐FITC and CD45RO‐PE (Exbio). The cells were analyzed by FACSAria II (Becton Dickinson) and the data were processed by FlowJo software (Tree Star). The frequency of reactive T cells was calculated as the difference between the frequency of the cytokine‐producing T cells of the vehicle‐stimulated sample and the peptide pool‐stimulated sample of the same donor.

Brain tissues were immediately minced with a lancet, shaken for 30 min on a petri dish in a phosphate-buffered saline solution with 2 mM ethylenediaminetetraacetic acid to prevent aggregation, filtered through a 100 μm cell strainer (Biologix Group Limited, Shandong, China) and pelleted by centrifugation. Blood and CSF samples were immediately processed for antibody staining according to the routine protocols. Lymphocyte subpopulations were evaluated using the following antibody mixtures: 1) CD3 FITC, HLADR PE, CD45 PerCP, CD4 PE-Cy7, CD19 APC, CD8 APC-Cy7, CD14 PB and 2) CD3 FITC, CD16 + CD56 PE, CD45 PerCP-Cy5.5, CD4 PE-Cy7, CD19 APC, CD8 APC-Cy7, HLADR PB (both Exbio, Prague, Czech Republic). Samples were measured with one of the following flow cytometers: BD LSRII, BD FACSLyric (BD Biosciences, San Jose, CA, USA), Cyan ADP Flow Cytometer (Dako, Glostrup, Denmark). The data were analyzed in FlowJo software, version 8.8.7 (FlowJo, LLC, Ashland, OR). The distributions of CD19⁺, CD3⁺, CD4⁺ and CD8⁺ cells are expressed as percentages from the lymphocytic gate (CD45⁺⁺ cells and the side scatter corresponding to lymphocytes), and activation is expressed as a percentage of HLADR⁺ cells among CD4⁺ or CD8⁺ T cells (HLADR⁺/CD3⁺CD4⁺, HLADR⁺/CD3⁺CD8⁺).

The investigation of the PD-1 expression on CD4 and CD8 T-cells was performed by multiparametric-polychromatic flow cytometry (quintuple fluorescence protocol). The following monoclonal antibodies bound to the appropriate fluorochromes were used: CD3 FITC, CD4 PE-Cy7, CD8 APC-Cy7, CD45 PerCP-Cy5.5, CD279 PE and the appropriate isotype matched control antibody PE to determine PD1.
All monoclonal antibodies were purchased from EXBIO (Praha Czech Republic).
Sample preparation was performed according to standard protocols and acquisition of 50.000 events was performed within 30 minutes on MINDRAY flow cytometry analyser. Lymphocytes were identified by their forward (FSC) and side scatter (SSC) characteristics combined with the CD45 expression, whereas T-cells, T-helper (CD4) and T-cytotoxic (CD8) cells were identified by the expression of the specific markers anti-CD3, anti-CD4 and anti-CD8, respectively.