Live dead fixable dead cell stain

At the end of the 14 day culture, phenotypic analysis of the ex-vivo expanded UCB Treg cells was performed by analysis of surface or intracellular markers, using the following monoclonal antibodies: anti-hCD4/BUV737 (clone SK3, Becton Dickinson, Franklin Lakes, NJ, USA), anti-hCD8/FITC (clone SK1, Becton Dickinson, Franklin Lakes, NJ, USA), anti-hCD25/PE (clone 2A3, Becton Dickinson, Franklin Lakes, NJ, USA), anti-hCD45/APC (clone 2D1, Becton Dickinson, Franklin Lakes, NJ, USA), and anti-mCD45/BV605 (clone 30-F11, Becton Dickinson, Franklin Lakes, NJ, USA) for surface staining; anti-FoxP3/PE-Cy7 (clone 236A/E7, Thermo Fischer Scientific, Waltham, MA, USA), anti-Helios/APC (clone 22F6, Thermo Fischer Scientific, Waltham, MA, USA) for intracellular staining; FOXP3 Fix/Perm buffer set (BioLegend, SanDiego, CA, USA) was used for intracellular staining according to manufacturer’s instructions. LIVE/DEAD™ Fixable Dead Cell Stain (Thermo Fischer Scientific, Waltham, MA, USA) was used as a viability dye for live cell gating. FITC-Labeled Human CD19 (20-291) and Fc Tag (AcroBiosystems, Newark, DE, USA) were used to analyze the chimeric antigen receptor expression of CAR-T cells. Stained cells were acquired on a BD LSR Fortessa X-20 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo^TM software ver.10 (Ashland, OR, USA).

The expression of ACE2/TMPRSS2 was analyzed by using the anti-ACE2 (535919) from R&D, and the anti-TMPRSS2 (EPR3862) from abcam according to our methods (28 (link)–30 (link)). Besides, Live/dead fixable dead cell stain (ThermoFisher) was used to exclude dead cells in flow cytometry. Paraformaldehyde fixed cells were acquired by flow cytometry using a LSRFORTESSA flow cytometer (BD) and analyzed with FlowJo software (version 10).

Healthy donor PBMCs were activated with HSV^GM-CSF overnight at the indicated concentrations, and their ability to kill melanoma cell targets (with or without VPA treatment) stained with Cell Tracker Green (Molecular Probes) was determined using standard 5-h co-culture. Co-cultures were washed and stained for viability using a live-dead fixable dead cell stain (Thermo Fisher Scientific) before analysis using an Attune flow cytometer.

Isolated LP cells were analyzed using FACS Canto II (BD, Franklin Lakes, NJ, USA). Dead cells were excluded using Live/Dead fixable dead cell stain (Thermo Fisher Scientific, Waltham, MA, USA). The following anti-chicken antibodies were used for staining: anti-CD3 (CT-3; Southern Biotech, Birmingham, AL, USA), anti-CD4 (CT-4; Southern Biotech, Birmingham, AL, USA), anti-CD8a (CT-8; Southern Biotech, Birmingham, AL, USA), anti-TCR γδ (TCR-1; Southern Biotech, Birmingham, AL, USA), anti-MHC II (2G11; Southern-Biotech, Birmingham, AL, USA), anti-Bu-1 (AV20; Southern Biotech, Birmingham, AL, USA), and anti-Monocyte/Macrophage (KUL01; Southern Biotech, Birmingham, AL, USA). All antibodies were diluted 1:200 in PBS and incubated for 30 min under dark conditions. Then, all samples were fixed using 4% paraformaldehyde (PFA) and stored at 4 °C until analysis. The analysis was conducted by two panels: (1) MHC II, Bu-1, and monocyte/macrophage for B cells and APCs; (2) CD3, CD4, CD8a, and TCR γδ for T cells. For the detailed gating strategy of flow cytometry analysis, refer to Supplementary Figure S1.