Anti cd4 pb

The total helper T-cell responses were assessed by flow cytometry. MLNs were crushed, and the absolute number of cells was counted with a Guava Personal Cell Analysis flow cytometer (Merck Millipore) using the Guava viability reagent (Merck Millipore 4000-0041). Stimulation of 10⁵ cells was performed with 10^–7 mM phorbol myristate acetate (PMA, Invivogen tlrl-pma), 1 µg/ml ionomycin (Biotrend BN0275) and 2 µM monensin (Sigma-Aldrich M5273) in complete RPMI containing 10% foetal bovine serum (Gibco 10270-106), 10 mM HEPES, 100 units/ml penicillin, 0.1 mg/ml streptomycin and 0.05 beta-mercaptoethanol, for 3 h at 37 °C. Cells were stained with the Aqua fluorescent dead cells stain (Invitrogen, L34957) in flow cytometry buffer (saline with 0.2–0.5 % bovine serum albumin); then with anti-CD4-PB (Biolegend 100428). Intracellular stainings were performed with Foxp3 staining buffer set (Ebioscience 00-5523-00) and with anti-IFNγ-FITC (Biolegend 505806), anti-IL4-PE (Biolegend 504104) and anti-IL13-biotin (Ebioscience 13-7135-85) overnight. Streptavidin-PECy7 (Biolegend 405206) was used to stain anti-IL-13-biotin antibodies. The samples were measured on an LSRII flow cytometer (Becton Dickinson) and analysed with Flowjo software version 9.

PBMC were washed and stained with Live/Dead fixable red dead cell stain (Invitrogen) followed by surface staining with the following antibodies: anti-CD3-AF700, anti-CD4-PB, anti-CD14-PE/Cy7, anti-CD16-AF488, anti-CD19-PE/Cy5, anti-CD25-APC/Cy7, γδ T cell receptor (TCR)-APC, anti-cytotoxic-T-lymphocyte-associated antigen (CTLA)-PE (all from BioLegend), anti-CD8-efluorNC605, and anti-CD127-efluorNC650 (eBioscience). Fluorescence minus one (FMO) controls were used to identify boundaries of gates for CD25, γδTCR, CD127, and CTLA. Samples were acquired on a BD LSR II flow cytometer. Results are presented as percentages of cells after gating out of dead cells and doublets. CD4⁺ and CD8⁺ T cells were identified as CD3⁺ cells, while CD14^+/− and CD16^+/− cells were identified as CD3⁻ and CD19⁻ populations. CTLA⁺ and CD25⁺ CD27⁻ populations were gated on the CD4⁺ cells.

PBMC were counted and plated in 24-well plates at 1 × 10⁶ PBMC/1 ml R10. Plates were incubated for 6 days at 37°C and 5% CO₂ with either 2 μg/ml PPD or 1 μg/ml/peptide of a single pool of antigen 85A peptides. Two wells of PBMC were left unstimulated in R10. After 3 days, PHA was added to one of the unstimulated wells at 0.3 μg/ml. Three days later, the cells were washed in phosphate-buffered saline (PBS) and stained with amine-reactive viability dye (Live/Dead fixable red dead cell stain; Invitrogen). Cells were then permeabilized with Perm/Wash (BD Biosciences) and incubated with monoclonal antibodies: anti-CD3-AF700, anti-CD4-PB, anti-Ki67-phycoerythrin (PE) (BioLegend), and anti-CD8-allophycocyanin (APC)/AF750 (Beckman Coulter). Cells were then washed and acquired on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (Tree Star Inc.). Dead cells were excluded from the analysis, while singlet CD3⁺ T cells, CD3⁺ CD4⁺ T cells, and CD3⁺ CD8⁺ T cells were included. Proliferating cells are presented as the percentage of Ki67⁺ T cells out of the total CD4⁺ or CD8⁺ T cells. Background (unstimulated) values were subtracted from all data.