Buffy coat CD4+ cells were nucleofected with pIRES-H2Kk-PRELI or pIRES-H2Kk vectors. Transfected cells were incubated at 37°C for 16h (2.5x106 cells/ml) after which dead cells were removed and the transfected cells were enriched by MACS. Briefly, dead cells, apoptotic cells and debris were depleted from the cultures by using MACS Dead Cell Removal Microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Subsequently, the remaining cells were incubated with MACSelect Kk MicroBeads coated with H-2Kk-antibody (Miltenyi Biotech) for 15min to magnetically label the transfected cells. The magnetic separation of H-2Kk-positive cells was done with a positive selection column placed in the magnetic field of a MACS separator. After the enrichment, cell viability and purity (H-2Kk-positivity) were determined by flow cytometry. The purified H-2Kk-positive cells were activated.
In PRELI knockdown studies, naive or peripheral blood CD4+ cells, nucleofected with scrambled or PRELI-specific siRNA oligos (1.5μg / 4x106 cells / transfection) were incubated at 37°C for 24h (2x106 cells/ml) and subsequently activated.