Anti ccr6

Samples were obtained from healthy donors participating in the Benaroya Research Institute Immune Mediated Disease Registry. Informed consent was obtained from all subjects according to IRB approved protocols at Benaroya Research Institute. CD4⁺CD25⁻ cells were enriched from PBMCs by positive selection with CD4-specific microbeads (Miltenyi Biotec). Memory cell subsets were sorted to over 97% purity as CD4+CD45RA-CD45RO+CD127+CD25− using anti-CD45RA (eBioscience), anti-CD45RO (Biolegend), anti-CD127 (BD Horizon), anti-CD25 (Biolegend) and anti-CD4 (Invitrogen). Antibodies used for sorting of memory cell subsets were: anti-CCR6 (eBioscience); anti-CCR10 (R&D Systems), anti-CCR4 (Biolegend), anti-CXCR3 (BD Pharmingen), anti-IL1R1 (R&D Systems). CD14+ monocytes were isolated from PBMCs by positive selection with CD14-specific microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 1% (vol/vol) nonessential amino acids, 1% (vol/vol) sodium pyruvate, penicillin (50 U/ml), streptomycin (50 µg/ml) (all from Invitrogen) and 5% heat-inactivated human serum. In some experiments, recombinant human IL-1β (10 ng/ml; R&D Systems), IL-12 (10 ng/ml; R&D Systems) or IL-23 (25 ng/ml; eBioscience) were added to the culture.