Peripheral blood samples were collected from adult CF patients attending the West of Scotland Adult CF Unit, Glasgow. Healthy individuals (controls) had no history of respiratory disease or inter-current illness. Participant characteristics are shown in Table 1 . Patients with CF attending the West of Scotland Adult CF Unit are deemed to be chronically colonized with Pseudomonas aeruginosa if sputum cultures remain positive for the organism after two attempts to clear the organism with combination antibiotic eradication therapy. Intermittent infection is deemed to exist when PA has been isolated and eradicated via antibiotic therapy.
PBMCs were obtained by Ficoll-Paque gradient centrifugation (GE Healthcare). CD14+ monocytes were then magnetically isolated by a positive selection kit (Miltenyi Biotech). Memory CD4+ T cells (purity for CD4+CD45RO+ >98%) were magnetically isolated by a negative selection kit (Miltenyi Biotech). Memory CD4+ T cells were further sorted into CCR6-enriched and CCR6-depleted populations using positive selection microbeads (Stemcell Technologies). Proliferation was measured with CFSE (carboxyfluorescein diacetate succinimidyl ester; Invitrogen) or proliferation dye eFluor®450 (eBioscience) incorporated prior to cell culture.
PBMCs were obtained by Ficoll-Paque gradient centrifugation (GE Healthcare). CD14+ monocytes were then magnetically isolated by a positive selection kit (Miltenyi Biotech). Memory CD4+ T cells (purity for CD4+CD45RO+ >98%) were magnetically isolated by a negative selection kit (Miltenyi Biotech). Memory CD4+ T cells were further sorted into CCR6-enriched and CCR6-depleted populations using positive selection microbeads (Stemcell Technologies). Proliferation was measured with CFSE (carboxyfluorescein diacetate succinimidyl ester; Invitrogen) or proliferation dye eFluor®450 (eBioscience) incorporated prior to cell culture.