Cd3 apce780

T-cell activation and proliferation were monitored using T-cell activation markers and a T-cell proliferation assay. In parallel to the main experiment, a sample of CellTraceTM Violet (CTV) (1:1000, cat. C34557, Thermo Fisher Scientific, USA) stained T cells (CD4 and CD8) from each donor was performed as per the protocol given by the manufacturer. At each time point a sample of cells was stained with CD69-PE-cy7 (1:200, cat. 557745, BD biosciences, USA), CD226-FITC (1:300, cat. 559788, BD Biosciences, USA) GLUT-1-(1:200, cat. 566580, BD Biosciences, USA) along with CD3-APCe780 (1:300, cat. 47-0032-82, eBioscience, USA), CD4-BV711 (1:400, cat. 563033, BD Bioscience, USA) and CD8-PerCP/Cy5.5 (1:200, cat. 344710, Biolegend, USA). Samples were analyzed using FACS to determine dynamic expression changes. The percentage of CTV- cells were analyzed to determine the T-cell proliferation rate at different time points.

Cells were first stained with LiveDead aqua or blue (Life Technologies). Following FcR-Block (2.4G2), cells were stained using the following monoclonal antibodies (all used at 1:200 dilution): CD3-APCe780, CD4-APC or A700, CD8α-PE/Cy7, CD11c-APC or BV421, MHC-II-PerCP/Cy5.5 or eFlour450, CD11b-BV711 or PE, CD19-e780, CD80-PerCP/Cy5.5, CD40-PE, CD44-BV570, CD69-FITC CD86-AF488, Foxp3-e450, F4/80-PE/Cy7, Gr1-FITC, HuCD2-PE, IgM-APCe780, SiglecF-PE and TCRβ-APCe780 (BD Biosciences, BioLegend or eBioscience). Foxp3 staining was performed with the eBioscience FoxP3 staining kit. Samples were acquired using FACS LSR II or FACS Canto II using BD FACSDiva software and analysed with FlowJo v.9 software (Tree Star). Cytokines were measured in culture supernatants by ELISA using paired monoclonal antibody, and recombinant cytokine standards, or Duosets (eBioscience, BD Biosciences, BioLegend, R&D Systems and Peprotech).