Gallios flow cytometer

Cell populations were detected and measured by flow cytometry with the following antibodies: CD31, CD44 (Novus Biologicals, Littleton, CO, USA), CD45 (Biorad, Hercules, CA, USA), CD34 (Genetex, Irvine, CA, USA), CD146 (TermoFisher, Waltham, MA, USA) and CD90 (Abcam, Cambridge, MA, USA). Staining occurred after exclusion of nonviable cells by diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA). Flow cytometry analyses were performed using LSRII (BD, Franklin Lakes, NJ, USA) and Gallios Flow Cytometer, and data were analyzed by FlowJo software (BD, Franklin Lakes, NJ, USA). This analysis was performed on all BMC samples.

Tumor cell lines were seeded in 24-well plates and infected with 100 viral particles per cell (vp/cell) of HDeGFP, cells were incubated with virus at 37 °C 1 h then washed with PBS and appropriate culture medium was replaced. Cells were imaged using a fluorescent microscope (Leica, Wetziar Germany) 24 h post infection, then harvested using trypsin, for flow cytometry analysis. Live/dead discrimination was determined via inclusion of 7-aminoactinomycin D (7AAD; BD Pharmingen, San Diego, CA, USA). Cells were analyzed for GFP expression using Gallios flow cytometer with Kaluza software (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions.

Primed MiHA-specific CD8⁺ T cells were tested for their IFNγ production upon peptide rechallenge. Therefore, cells were restimulated overnight with MiHA peptide (5 µM) in the presence of brefeldin A (1 µg/ml, BD Biosciences). The following day, extracellular staining by MiHA tetramers and antibodies recognizing CD3 and CD8 was performed, followed by intracellular staining for IFNγ. For this, cells were resuspended in 4 % paraformaldehyde (PFA) and incubated for 10 min at RT. Then, cells were incubated in 0.1 % saponin (Sigma) buffer containing 10 % fetal calf serum (Integro, the Netherlands) for 10 min at RT. This was followed by intracellular staining for anti-CD137 (clone 41BB, Biolegend) and anti-IFNγ (clone B27, BD Biosciences) for 30 min at 4 °C after which cells were fixed in 1 % PFA and measured on the Gallios flow cytometer.

T cells were harvested and resuspended at 1 million cells per ml in PBS. CFSE (ThermoFisher C34554) was added to a final concentration of 1.5 μM and LIVE/DEAD^TM Fixable Aqua Dead Cell Stain Kit (ThermoFisher #L34957) to a final concentration of 1 μM. Samples were vortexed gently and let sit for 8 min at room temperature. An equal volume of pre-warmed FBS (100%, filtered) was then added. Cells were incubated at room temperature for 10 min. Cells were centrifuged for 5 min at 400 xg. Supernatants were discarded and the pellet vortexed to obtain a single cell suspension. Cells were washed again using PBS and fixed in 2% paraformaldehyde. Cells were stained at d0 with only CFSE before co-culture and harvested to be stained with live/dead aqua stain kit every 24 h for 4 continuous days. All cell analysis was conducted using a Beckman Coulter Gallios Flow Cytometer (BD Biosciences, San Jose, CA, United States) and data were analyzed by Kaluza Analysis Software. Results were shown as a representative of two independent experiments.