Autophagy-enhancing drugs boost HIV-1 restriction and suppress viral replication ex vivo Alexandra P.M. Cloherty 1,*, Nienke H. van Teijlingen 1, Tracy-Jane T.H.D. Eisden 1,2, John L. van Hamme 1, Anusca G. Rader 1, Teunis B.H. Geijtenbeek 1, Renée R.C.E. Schreurs 1, Carla M.S. Ribeiro 1 1 Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam University Medical Centers, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 2 Department of Medical Oncology, Amsterdam UMC, Vrije Universiteit Amsterdam, Cancer Center Amsterdam, 1081 HV Amsterdam, The Netherlands * Presenting Many human pathologies are associated with autophagy dysregulation, and amongst these is chronic HIV-1 infection and pathogenesis. Although current direct-acting antiviral therapies are highly effective in suppressing HIV-1 replication, HIV-1 persists in long-lived tissue-derived dendritic cells (DCs) and CD4+ T cells of treated patients. In addition, mucosal inflammation undermines current prophylactic antiviral treatment efficacy. Notably, previous research has also shown that HIV-1 infected individuals who remain clinically stable in the absence of antiretroviral therapy have significantly higher levels of autophagy vesicles. We and others have shown that viral components escape or inhibit different stages of the autophagy pathway in human DCs and CD4+ T cells, impeding autophagic destruction of HIV-1. Here, we have investigated the impact of pharmaceutically enhancing autophagy on HIV-1 acquisition and residual viral replication. To this end, we developed a human tissue infection model permitting concurrent analysis of HIV-1 cellular targets ex vivo. Prophylactic treatment with FDA-approved autophagy-enhancing drugs targeting host factors such as mTORC and IP3 boosted HIV-1 restriction in skin-derived CD11c+ DCs and CD4+ T cells. An mTORC inhibitor decreased susceptibility to multiple strains of HIV-1 in skin-derived and vaginal Langerhans cells, which are an epidermal DC subset. Notably, we observed cell-specific effects of therapeutic treatment with autophagy drugs. Only therapeutic treatment with mTORC inhibitors suppressed HIV-1 replication in skin-derived dermal CD11c+ DCs, while all selected drugs limited HIV-1 replication in CD4+ T cells. Strikingly, these results were replicated in primary human intestinal CD4+ T cells. Our findings highlight that manipulation of host autophagy is a relevant target for HIV-1 therapies. Furthermore, this study illustrates that our novel human tissue infection model, which permits discrimination of pharmaceutical effects in distinct immune cell types, is an exciting new option for screening drugs for limitation of mucosal virus replication. We show that that repurposing clinically-approved autophagy drugs can limit mucosal HIV-1 acquisition and suppress ongoing replication in a cell-specific manner. Finally, we propose that combinatory use of autophagy-based and direct-acting antivirals may potentiate the efficacy of direct-acting antivirals by optimizing host protective immune defenses, thereby accelerating treatment efficacy and reducing likelihood of antiviral resistance.