Cd3 pe clone hit3a

Cells from ELISpot plate were collected in media supplemented with GolgiStop Protein Transport Inhibitor (BD Biosciences, NJ, USA) and incubated for 6 hr at 37°C. Positive control group was treated with 50 μM PMA (Abcam, UK), 1 mg/mL Ionomycin (Abcam, UK). Cells were then washed, blocked with Fc receptor (Biolegend, CA, USA), and stained with CD3-PE (clone HIT3a, Biolegend), CD4-PE/Cyanine7 (clone RPA-T4, Biolegend), CD8-FITC (clone RPA-T8, Cell Signaling) antibodies for 2 hr at 4°C. Cells were permeabilized for 20 mins at 4°C and then stained overnight with IFN-γ-APC (clone 4S.B3, Biolegend) antibody at 4°C.

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).