No abstract

Key Points HIV-1 Nef reprograms human macrophage migration favoring the mesenchymal mode, which translates in vivo to macrophage tissue accumulation. Nef enhances mesenchymal migration by influencing podosome organization and function via the phagocyte-specific kinase Hck and WASP.

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef’s influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef’s ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor.

Pathogens face varying microenvironments in vivo, but suitable experimental systems and analysis tools to dissect how three-dimensional (3D) tissue environments impact pathogen spread are lacking. Here we develop an Integrative method to Study Pathogen spread by Experiment and Computation within Tissue-like 3D cultures (INSPECT-3D), combining quantification of pathogen replication with imaging to study single-cell and cell population dynamics. We apply INSPECT-3D to analyze HIV-1 spread between primary human CD4 T-lymphocytes using collagen as tissue-like 3D-scaffold. Measurements of virus replication, infectivity, diffusion, cellular motility and interactions are combined by mathematical analyses into an integrated spatial infection model to estimate parameters governing HIV-1 spread. This reveals that environmental restrictions limit infection by cell-free virions but promote cell-associated HIV-1 transmission. Experimental validation identifies cell motility and density as essential determinants of efficacy and mode of HIV-1 spread in 3D. INSPECT-3D represents an adaptable method for quantitative time-resolved analyses of 3D pathogen spread.