Pneumococcal-specific CD4+ T cell Assay

Antigen-specific CD4⁺ T cell responses were measured as previously described.⁸ (link) In brief, non-adherent BAL cells (0.5x10⁶cells/well) suspended in 200 µl of complete media were cultured in 96 well plates and stimulated with pneumococcal cell culture supernatant (8 µg/ml) (prepared from a standard encapsulated type 2 (D39) S. pneumoniae strain as previously described16 , 31 (link)). The cell culture supernatant has been previously utilised to probe pneumococcal-specific T cell immunity,8 (link), 15 (link), 16 , 31 (link) is rich in pneumococcal surface proteins and pneumolysin.³¹ (link) Cells were stained with Violet Viability dye (LIVE/DEAD® Fixable Dead Cell Stain kit, Invitrogen, UK), surface markers (anti-CD3 PE-Cy5, anti-CD4 APC-H7 and anti-CD8 PE-Cy7 antibodies, all from BD Bioscience, UK), and intracellular markers (anti-IFN-γ APC, anti-tumour necrosis factor (TNF) Alexa Fluor 488 and anti-IL17 PE antibodies, all from BD Bioscience, UK). Approximately 50,000 events were acquired in the CD4⁺ gate using a CyAn ADP 9-colour flow cytometer (Beckman Coulter, USA). Flow cytometry data were analysed using FlowJo software (TreeStar, USA).