Cd4 percp clone okt4

Microspheres were decapsulated with 5 mM EDTA in HBSS with no Ca²⁺/Mg²⁺ at day seven after encapsulation and 2 million cells prepared for staining in RPMI with 5% foetal bovine serum. To measure the expression of PD-1 in CD4⁺ and CD8⁺ T cells and PD-L1 expression in CD14⁺CD11b⁺ cells, the following antibody panel was used: CD3-PE (clone HIT3a, Biolegend), HLA-DR-PerCP (clone L243, Biolegend), CD4-PerCP (clone OKT4, Biolegend), CD8-APC (clone SK1, Biolegend), CD11b-APC (clone ICRF44, Biolegend), CD45-APC/Cy7 (clone 2D1, Biolegend), CD14-AP/APC (clone HCD14, Biolegend), CD279-BB515 (Clone EH12.1, BD), CD-274-BB515 (Clone MIH1, BD) and True-Stain Monocyte Blocker (Biolegend, USA). Gates were defined using fluorescence minus one control after exclusion of the dead cells using Live/Dead fixable stain (ThermoFisher, UK). Gating strategy is provided in Figure 5—figure supplement 4. Cells were acquired after fixing them in 2% paraformaldehyde in HBSS for 1 hr using FACSAria (Becton Dickinson, UK) and analysed by FACSDiva software (Becton Dickinson) and Flow Jo version 10 (Treestar).

Flow cytometry cell sorting was used for the isolation of CD4⁺CD127^loCD25^hi Treg from PBMCs, unaffected colon tissue and tumor and CD4⁺CD127⁺CD25⁻ conventional T cells from PBMCs and unaffected colon tissue [26 (link)]. Buffy coats from healthy volunteers were pre-enriched for CD4⁺ T cells via negative selection using immunomagnetic sorting with EasySep Human CD4⁺ T cell Isolation (Stemcell). Enriched CD4⁺ T cells were sorted using the following antibodies and reagents: Live/Dead fixable aqua dead cell stain kit from Molecular Probes; CD4-PerCP (clone OKT4) from BioLegend; CD8-BV711 (clone RPA-T8), CD25−BV650 (clone M-A251) and CD127-PE (clone HIL-7R-M21) from BD Biosciences; CD127-FITC (clone eBioRDR5) from eBioscience; and CD39−FITC (clone A1) from AbD Serotec, on a FACS Aria (BD Biosciences) equipped with FACS Diva software (BD Biosciences). FMO and isotype controls were used as reference to determine marker expression. The sorted cells were manually counted with Trypan Blue solution and mean viability of sorted cells was 94% for Treg and 92% for conventional T cells.