Forty mL of peripheral blood samples from six individual volunteers were drawn to heparin-washed tubes and then loaded on Lymphoprep - a Ficoll gradient (07851, STEMCELL Technologies Inc.) and centrifuged at 800xg for 30 min. The peripheral blood mononuclear cells (PBMCs) were collected and washed twice with PBS. Primary CD3+T cells or CD22+ B cells were isolated from PBMCs using MACS microbeads and an MS column (CD3 microbeads 130-050-101, and CD22 microbeads 130-046-401), according to the manufacturer’s instructions.
The purified CD3+ T cells were cultured in a 12-well plate (1x106 cells/1 mL medium/well), pre-coated with 10 μg/mL anti-CD3 (CD3 Monoclonal Antibody (UCHT1), 16-0038-85, eBioscience™) for 4 h at 37°C, in addition to 1 μg/mL anti-CD28 (CD28 Monoclonal Antibody (CD28.2), 16-0289-85, eBioscience™), for 24 h at 37°C. The positively isolated B cells were cultured in a12-well plate (1x106 cells/1 mL medium/well) and activated with 1 µM TLR9 agonist-CpG-ODN (ODN 2006 Class B CpG oligonucleotide, tlrl-2006-1, InvivoGen) and 5 μg/mL CD40L (Recombinant Human CD40 Ligand, 6245-CL, R&D Systems) at 37 °C for 24 h. After 24 h of incubation, 1 μg/mL of P- or T-sema3A or an appropriate amount of a control elution buffer was added for an additional 24 h at 37°C.
The purified CD3+ T cells were cultured in a 12-well plate (1x106 cells/1 mL medium/well), pre-coated with 10 μg/mL anti-CD3 (CD3 Monoclonal Antibody (UCHT1), 16-0038-85, eBioscience™) for 4 h at 37°C, in addition to 1 μg/mL anti-CD28 (CD28 Monoclonal Antibody (CD28.2), 16-0289-85, eBioscience™), for 24 h at 37°C. The positively isolated B cells were cultured in a12-well plate (1x106 cells/1 mL medium/well) and activated with 1 µM TLR9 agonist-CpG-ODN (ODN 2006 Class B CpG oligonucleotide, tlrl-2006-1, InvivoGen) and 5 μg/mL CD40L (Recombinant Human CD40 Ligand, 6245-CL, R&D Systems) at 37 °C for 24 h. After 24 h of incubation, 1 μg/mL of P- or T-sema3A or an appropriate amount of a control elution buffer was added for an additional 24 h at 37°C.