Protocol for generating human CAR-engineered macrophages by Vpx-containing lentivirus

Human-derived macrophages are notoriously difficult to infect with HIV-1-based lentiviruses, posing a limitation to the advancement of chimeric antigen receptor macrophage (CAR-M) therapy. Here, we present a protocol for generating human chimeric antigen receptor (CAR)-engineered macrophages using the viral protein Vpx (encoded by the Sooty Mangabey simian immunodeficiency virus [SIV] and HIV-2 lineages) incorporated into the lentivirus vector, which enhances infection efficiency. We describe steps for cell cultivation, lentivirus production, concentration, infection procedures, and efficiency assessments. This protocol provides a foundation to study macrophage manipulation, especially with CAR or other immune engagers. For complete details on the use and execution of this protocol, please refer to Gao et al.¹.