Fastimmune cd28 cd49d

Overnight-rested B-cell-depleted PBMC were seeded in 96-well U bottom plates and stimulated with wild-type SARS-CoV-2 PepTivator (Miltenyi Biotec, 130-127-951/130-126-698) overlapping peptide pools spanning the entire sequences of SARS-CoV-2 S or N proteins, in presence of anti-CD107a-APC (clone H4A3; Biolegend, 328620, diluted 1:40) antibody. One million cells were stimulated per condition and the final concentration of each peptide was 1 µg/ml for both peptide pools. Co-stimulatory antibodies (BD FastImmune™ CD28/CD49d, BD Bioscience, 347690) were added to a final concentration of 1 µg/ml. Stimulation was performed at 37 °C for 6 h. For each sample, an equally treated DMSO-stimulated negative control was included. As a positive control, cells were stimulated with PMA (20 ng/ml) (Sigma-Aldrich, P1585-1MG) and ionomycin (1 μg/ml) (Sigma-Aldrich, I3909-1ML). One hour into stimulation Golgi Stop (BD Bioscience, 554724) and Golgi Plug (BD Bioscience, 555029) were added (final concentration 1 µg/ml) to inhibit vesicular transport and prevent the secretion of the cytokines.

B-cell-depleted PBMC fractions were seeded in 96-well U bottom plates and stimulated with wild-type SARS-CoV-2 PepTivator (Miltenyi Biotec) overlapping peptide pools spanning the entire sequence of spike (S) protein, in presence of anti-CD107a-APC (clone H4A3; Biolegend) antibody. One million cells were stimulated per condition, and the final concentration of each peptide in the stimulation mix was 1 µg/ml. As a co-stimulatory signal, antibodies binding CD28 and CD49d (BD FastImmune™ CD28/CD49d) were added to a final concentration of 1 µg/ml. Stimulation was performed at 37°C for a total of 6 hours. As a negative control, an equally treated DMSO-stimulated sample was included for each biological replicate. As positive control cells stimulated with PMA (20 ng/ml) and ionomycin (1 μg/ml) were used. One hour into stimulation, Golgi Stop and Golgi Plug (BD Bioscience) were added (final concentration 1 µg/ml) to inhibit vesicular transport and prevent the secretion of the cytokines from cells.

B-cell-depleted PBMC fraction was seeded in 96-well U bottom plates and stimulated with two different pools of overlapping peptides: the first covering the mutated regions of the omicron SARS-CoV-2 spike protein (PepTivator® SARS-CoV-2 Prot_S B.1.1.529/BA.1 Mutation Pool, Miltenyi Biotec, 130-129-928) and the second covering conserved regions of the spike (PepTivator® SARS-CoV-2 Prot_S B.1.1.529/BA.1 WT Reference Pool, Miltenyi Biotec, 130-129-927). One million cells were stimulated per condition. The final concentration of each peptide was 1 µg/ml for both peptide pools. Co-stimulatory antibodies (BD FastImmune™ CD28/CD49d, BD Bioscience, 347690) were added to a final concentration of 1 µg/ml. For each sample, an equally treated DMSO-stimulated negative control was included. As a positive control, cells were stimulated with PMA (20 ng/ml) (Sigma-Aldrich, P1585-1MG) and ionomycin (1 μg/ml) (Sigma-Aldrich, I3909-1ML). Stimulation was performed at 37 °C for 6 hours. One hour into stimulation Golgi Stop (BD Bioscience, 554724) and Golgi Plug (BD Bioscience, 555029) were added (final concentration 1 µg/ml) to inhibit vesicular transport and prevent the secretion of the cytokines from cells.