Anti cd28 cd49d

Fresh PBMCs suspension was seed on 96-well flat-bottom plate. Prepare the mixture of complete medium (RPMI-1640 containing 10% fetal calf serum, 100U/ml penicillin, 100ug/ml streptomycin, and 100um HEPES) with or without overlapping peptide pools covering entire sequences of SARS-CoV-2 spike glycoprotein (S, GenScript, Cat No.RP30027), membrane (M, GenScript, Cat No.RP30022), or nucleocapsid (N, GenScript, Cat No.RP30013) proteins respectively. Then costimulatory reagent anti-CD28/CD49d (BD Biosciences) and recombinant interleukin-2 (rIL-2; Hoffmann-La Roche) were added to the cultures. The PBMCs were stimulated for 9 days in vitro at a 37°C and 5% CO₂ incubator, with fresh medium containing IL-2 added twice a week. On day 10, the cells were harvested and tested for intracellular expression after re-stimulation with corresponding peptide pools for 5 hours. Polyclonally activators (PMA, phorbol 12-myristate 13-acetate and Iono, ionomycin), were added as positive controls and those without peptide stimulation as negative controls. Brefeldin A (BFA, eBioscience, Invitrogen™ USA) was added to block cytokine release outside the cell. After re-stimulation, the cells were analyzed by intracellular cytokine staining (ICS) with subsequent flow cytometry.

The following protocol is prepared according to the MIATA (Minimal Information About T cell Assays) recommendations (www.miataproject.org). Briefly, peripheral blood mononuclear cells (PBMCs) were collected from each donor’s whole blood by density gradient centrifugation (GE Healthcare Ficoll-Paque PLUS), resuspended in supplemented RPMI media containing 10% DMSO before cryopreserved in liquid nitrogen. Before use for stimulation, frozen PBMCs were thawed quickly in 37 °C, washed twice with complete RPMI medium, and rested overnight in complete RPMI medium containing 10% fetal bovine serum and 10μg/ml DNase I (Sigma).
PBMCs were stimulated at a density of 1 million per 100 μl of complete RPMI medium in a 96-well U bottom plate using a CMV-pp65 peptide pool containing 138 peptides derived from a peptide scan (15mers with 11 amino acids overlap) of the 65kDa phosphoprotein (Swiss Protein ID: P06725) of human CMV. Cells were incubated with the CMV-pp65 peptide pool (1μg/ml per peptide, PepMix, JPT Peptide Technologies), anti-CD28/CD49d (BD Biosciences), anti-CD107a (BD, clone H4A3), Golgistop (monensin, BD), and Golgiplug (Brefeldin A, BD) for six hours at 37 °C.

Cryopreserved PBMCs were thawed and rested for 3–4 h prior to assay. All experiments were seeded with 2.5 × 10⁵ PBMCs per well together with co-stimulatory anti-CD28/CD49d (347690, BD Biosciences) at 3 μL/mL and stimulated with either spike glycoprotein peptide pool (peptide & elephants, Germany) at 0.5 μg/mL or equivalent volume of DMSO (<1%). The assay was performed according to the manufacturer’s protocol using plates and reagents from a human IFN-γ ELISpot^PRO kit (3420-2APT-2, Mabtech). PBMCs were stimulated for 20 h in assay plates incubated at 37 °C and 5% CO₂. Spots were counted with the IRIS ELISpot reader (Mabtech) and Apex software (Mabtech) using default settings. Visible artifacts were removed by masking. The results are expressed as spot forming units (SFU/10⁶ PBMCs) per 10⁶ cells, calculated by subtracting the mean of negative control wells from the mean of spike stimulation wells. A positive response after subtraction was determined by the mean of negative control wells plus two standard deviations (55 SFU/10⁶ PBMCs). To exclude cross-reactive responses, results were excluded from analysis if the negative control wells had >100 SFU/10⁶ PBMCs PBMCs or if the sample was positive (>55 SFU/10⁶ PBMCs) at baseline.

Fresh DCs and CD4 + and CD8 + LTs were isolated from PBMCs by negative immunoselection using the EasySep Human Pan-DC Pre-Enrichment Kit, EasySep Human CD4 + T Cell Isolation Kit and EasySep Human CD8 + T Cell Isolation Kit, respectively, following the manufacturer's instructions (Stem Cell). After isolation, cells were suspended in 10% RPMI medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin G, 100 µl/ml streptomycin sulphate and 1% l-glutamine). A total of 50,000 DCs were cultured overnight in RPMI 10% without any stimulus (hereafter referred to as "unstimulated condition", US) or with thymosin-alpha-1 (α1Thy) (MyBioSource) at 50 ng/ml in 96-well round-bottom plates at 37 °C/5% CO2. As a positive control, DCs were stimulated with 1 µM CpG-A (ODN 2216; InvivoGen).
For the in vitro co-culture system, after DCs stimulation, autologous CD4 + or CD8 + TLs were added to the α1Thy-stimulated or unstimulated DCs conditions at a DC:TL ratio of 50.000:150.000 for 6 h with 1 µg/ml anti-CD28/CD49d, 0.7 µg/ml monensin (BD Biosciences) and 10 µg/ml brefeldin A (Biolegend) at 37 °C/5% CO2 in the presence or absence of 6 nmoles/peptide of a PepTivator® SARS-CoV-2 Select—premium grade (Miltenyi).