Anti cd69 pe

PBMC were isolated from fresh blood using Ficoll and counted using Luna-FL cell counter (Logos Biosystems). Frozen PBMCs were thawed, counted and 1 × 10⁶ PBMCs were stimulated with MVA-Venus (MOI = 1) for 24 h in a total volume of 200 µl of RPMI media with 5% of human serum in a 96 wells plate. Unstimulated cells and PHA (20 µg/ml) were used as negative and positive controls, respectively. Anti-CD107a-APC, Golgi Plug (Brefeldin A) and Golgi Stop (Monesin) were added 4 h before staining. Cells were washed with PBS and stained 15 min at room temperature with Live and dead near IR, anti-CD3-FITC (Biolegend 300440, 1:40), anti-CD4-BV421 (BD 566703, 1:50), anti-CD8-BV605 (Biolegend 301040, 1:100), anti-CD69-PE (BD 555531, 1:10), anti-CCR7-PE/Dazzle 594 (Biolegend 353236, 1:20) and anti-CD45RA-PerCP (Biolegend 304062, 1:20). After washing, cells were permeabilized with Fix/Perm (BD) for 20 min at 4 °C. Cells were washed with Perm/Wash (BD) and intracellular stained with anti-IFNγ-PECy7 for 30 minutes at 4 °C, washed and resuspended in FACS buffer until acquisition. Cells were acquired in Attune NxT cytometer (Thermofisher) and data was analyzed with Flow Jo X software following the gating strategy described in Supplementary Fig. 2. Only two out of five DL3 volunteers had available PBMC samples for the analysis.

Samples of EDTA anticoagulated peripheral blood (5 mL) were collected from three groups’ patients. All samples were tested within 6 h of being obtained. Briefly, CD3⁺/CD4⁺/CD8⁺ T-cell, CD19⁺ B-cell, CD3⁻CD16⁺CD56⁺ NK-cell, CD3⁺CD16⁺CD56⁺ NKT-cell, CD4⁺CD25⁺CD127⁻ Treg-cell, CD3⁺HLA-DR⁺ T-cell and CD4⁺HLA-DR⁺ T-cell, CD8⁺ CD28⁺, and CD16⁺CD56⁺CD69⁺ counts (%) were measured by multiple-color flow cytometry with human monoclonal anti-CD3-FITC, anti-CD4-PE, anti-CD8-APC, anti-CD19-PE, anti-CD16-FITC, anti-CD56-APC, anti-CD25-PE, anti-CD127-BV421, anti-HLA-DR-Alexa700, anti-CD28-BV510 and anti-CD69-PE antibodies (BD Multitest) according to the manufacturer’s instructions. The cells were analyzed by flow cytometry system (Becton Dickinson, USA) according to the operating procedure.

Following cell culture, the following molecules were used to stain cells: Live/Dead fluorescent dye (Life Technologies, L23101) to detect cell viability; CellTrace CFSE (Life Technologies, C34554) or Violet (Life Technologies, C34557) to detect cell proliferation; anti-CD25-PE and anti-CD69-PE (BD Pharmingen, San Diego, CA, cat. no. 555432 and 555531 respectively) to detect T cell activation; anti-HLA-DR-PE and anti-CD54-APC (BD Pharmingen, 347401 and 559771 respectively) to measure APC surface markers on HeLa-CIITA cells. To identify cell cycle stages, we fixed T cells in 70% ethanol on ice and used a combined treatment of RNAse incubation and propidium iodide staining (Sigma Aldrich, cat. no. R6148 and P4864 respectively) as previously reported (Vivar et al., 2009 (link)). To measure the amount of phospho-rpS6 ribosomal protein, cells were fixed in Cytofix/Cytoperm solution (BD Biosciences, 554722), permeabilized with Perm/Wash buffer (BD Biosciences, 554723) and stained with an anti-phospho-rpS6(Ser235/236) antibody (Cell Signaling Technology, Danvers, MA, cat. no. 2211L). All experiments were performed with the MACSQuant flow cytometer (Miltenyi Biotech, Bergish Gladbach, Germany).

Cells were harvested either 2, 4, 6, and 24 h after electroporation or after 48 h of co-culture. The expression of surface markers was determined via flow cytometry using anti-CD215-PE (anti-human IL-15Rα, clone JM7A4) (Biolegend, San Diego, CA, USA) and IgG2b-PE isotype control, anti-CD56-FITC, anti-CD3-APC-Cy7 or anti-CD3-V500, anti-CD69-PE, anti-CD25-BV421 or anti-CD25-PE, and anti-CD54-APC or anti-CD54-PE (all from BD Biosciences, Heidelberg, Germany) as described [39 (link)]. A FACS Canto II flow cytometer (BD Biosciences) and FACSDiva software [40 ] were used to measure immunofluorescence and acquire data, which was evaluated with FCS Express software [41 ].