Peripheral blood mononuclear cells [PBMC] were isolated by Ficoll density gradient centrifugation [Amersham Pharmacia Biotech, Uppsala, Sweden]. Lamina propria mononuclear cells [LPMCs] were isolated as previously described.25 (link),26 (link) Mesenteric lymph nodes [LNs] were smashed into 70-μm nylon strainers [BD Biosciences] and erythrocytes lysed with red blood cell [RBC] lysis buffer [BD Biosciences].
For characterisation of T helper [Th] cell subsets from PBMCs, LNs, and LPMCs, T cells were stained with a combination of fluorochrome-conjugated antibodies as specified inSupplementary Table 2 and sorted [FACSAria III, BD Biosciences], according to the expression of the following specific surface markers combination: CD4+IL-7R+CD25lowCCR6-CXCR3+ [Th1 cells], CD4+IL-7R+CD25lowCCR6+CXCR3+ [Th1/17 cells] and CD4+IL-7R+CD25lowCCR6+CXCR3- [Th17 cells]. These three subsets were further subdivided according to CCR5 expression.
Human monocytes for antigen-specificity assays and for generation of monocyte-derived dendritic cells [MoDC] were purified from blood samples by Ficoll density gradient separation and by positive selection using CD14+ selection [CD14 Microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany].
For characterisation of T helper [Th] cell subsets from PBMCs, LNs, and LPMCs, T cells were stained with a combination of fluorochrome-conjugated antibodies as specified in
Human monocytes for antigen-specificity assays and for generation of monocyte-derived dendritic cells [MoDC] were purified from blood samples by Ficoll density gradient separation and by positive selection using CD14+ selection [CD14 Microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany].
