Live dead fixable aqua stain

Human PBMCs collected from likely COVID-19-vaccinated donors between February 2022 and February 2023 were isolated from buffy coats by means of Ficoll density gradient centrifugation and infected for 24 h with 5 MOI of Prime-2-CoV_Beta (SPEk103) or left untreated. Cells were collected by scraping, washed in PBS and incubated for 30 min at 4 °C with the following antibody cocktail in PBS: anti-CD14 APC (BD Biosciences, San Jose, CA, USA, 561383), anti-CD4 BV605 (BD Biosciences, 562843), anti-CD8 APC-H7 (BD Biosciences, 561423), anti-CD19 BV786 (BD Biosciences, 563325), anti-CD3 PE-Cy7 (BD Biosciences, 557749), anti-CD56 PE (BD Biosciences, 556647), anti-CD25 BV421 (BD Biosciences, 567485), LIVE/DEAD™ Fixable Aqua stain (Invitrogen, Waltham, MA, USA, L34957). After three washes in PBS, cells were fixed in 4% formaldehyde for 20 min, washed and finally resuspended in FACS buffer. Data were acquired with an Attune NxT cytometer (Thermo Fisher Scientific). Data analysis was performed using Kaluza software (version 2.1, Beckman Coulter, Brea, CA, USA).

Mononuclear cells isolated from spleens, lymph nodes and tumours were first stained with a dead cell marker (LIVE/DEAD Fixable Aqua stain; Invitrogen) according to the manufacturer's instructions. Cells were then washed and stained for cell surface markers and chemokine receptors. Stained cells were washed, fixed and acquired on a FACS Canto II flow cytometer (BD Biosciences). Data analysis was performed using summit 4.3 software (Dako, Glostrup, Denmark). The antibodies used were as follows: anti-CD4-Pacific Blue antibody (BioLegend, San Diego, CA), goat polyclonal antibody to CCR1 (Santa Cruz Biotechnology, Santa Cruz, CA) followed with phycoerythrin (PE)-conjugated rabbit polyclonal antibody to goat IgG (Abcam, Cambridge, UK), rabbit monoclonal antibody to CCR2 (Abcam) followed with PE-conjugated donkey polyclonal antibody to rabbit IgG (Abcam), anti-CCR4-PE-Cy7 (BioLegend),anti-CCR3-Alexa Fluor 647 (BioLegend), biotin-conjugated anti-CCR5 antibodies followed with streptavidin-PerCP-Cy5.5 (both from eBioscience, San Diego, CA), anti-CXCR3-APC (eBioscience, San Diego, CA), anti-CXCR4-APC (BD Biosciences), rabbit polyclonal antibody to anti-CX₃CR1 (Abcam) followed with PE-conjugated donkey polyclonal antibody to rabbit IgG (Abcam). Foxp3 expression was detected by GFP fluorescence.

Cryopreserved splenocytes were thawed, resuspended in pre-warmed PBS with 0.1% BSA at a final concentration of 1 × 10⁶ cells/mL, and labeled with 750 nM CFSE for 10 min at 37°C, 5% CO₂. The staining was quenched by adding 5 volumes of ice-cold R10 followed by a 5-min incubation on ice. The cells were pelleted, washed, and plated in 96-well round-bottom plates at a concentration of 1 × 10⁶ cells/well. The CFSE-labeled cells were then stimulated for 5 days with 2 μg/mL of peptide, 2.0 μg/mL ionomycin, and 0.5 μg/mL PMA (positive control) or tissue culture media with 1% DMSO (negative control). The cells were stained with a mastermix containing the dead cell marker (LIVE/DEAD Fixable Aqua stain; Invitrogen) and anti-membrane marker mAbs anti-CD4-APC/Cy7 (BioLegend), anti-CD3-PerCPeFluor710, and anti-CD8-eFluor450 (both from eBioscience), fixed and acquired on a BD LSR II flow cytometer. Data analysis was performed using FlowJo software (Tree Star) with gating shown in Figure S6.

For proliferation assays, freshly isolated and/or rested after electroporation CD4⁺ T cells were labeled with 1 μM cell trace violet cell proliferation dye (Invitrogen, Life Technologies) following the manufacturer's recommendations. Labeled cells were stimulated with Dynabeads Human T-Activator CD3/CD28 (Invitrogen, Life Technologies) (1 : 1 or 1 : 10 bead : cell ratio) for 72 hours. Cells were then harvested, stained with Live/Dead Fixable Aqua Stain (Invitrogen, Life Technologies) and processed for FACS analysis using the Attune Focusing Flow Cytometer. PE anti-human CD25 (clone BC96; Biolegend, USA) and PerCP/Cy5.5 anti-human CD4 antibody (clone OKT4; Biolegend, USA) were used to evaluate CD4⁺ T cell activation. All FACS data were analyzed with FlowJo software (Tree Star, Inc., OR, USA).

For cell activation analysis, either complete PBMC cultures or resting CD4+ T cells isolated from HIV-infected cART-suppressed patients were incubated with the tested agents under specified conditions and were stained with relevant antibodies. All antibodies were purchased from BD Biosciences and included Alexa Fluor 700-labeled antibody to CD4, APC-H7-labeled antibody to CD8, PE-Cy7 -labeled antibody to CD19, PE-labeled antibody to CD69, APC-labeled antibody to CD25, and V450-labeled antibody to HLA-DR. Live cells were gated by forward and side scatter and exclusion of the dead cells by Live/Dead Fixable Aqua Stain (Invitrogen). Marker staining was assessed by flow cytometry analysis on a LSR Fortessa with data processing using FlowJo software.

Cryopreserved PBMC were thawed, resuspended in pre-warmed PBS with 0.1% BSA at a final concentration of 10⁶ cells/ml and labelled with 750 nM CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester; Molecular Probes^™) for 10 min at 37°C, 5% CO₂. The staining was quenched by adding 5 volumes of ice-cold R10 followed by a 5-min incubation on ice. The cells were pelleted, washed and plated in 96-well round-bottom plates at a concentration of 1 x 10⁶ cells/well. The CFSE-labelled cells were then stimulated with 1.5 μg/ml of each peptide in personalized pools or 1 μg/ml SEB (positive control) and R10 (negative control) for 5 days, stained with a dead cell marker (LIVE/DEAD Fixable Aqua stain; Invitrogen) and anti-CD4-BV605 (BioLegend), anti-CD3-ECD (Beckman Coulter) and anti-CD8-Aleva Fluor 700 (eBioscience) mAbs, fixed and acquired on a BD LSR II flow cytometer. Data analysis was performed using FlowJo software (Tree Star Inc.) with gating shown in S1 Fig.