Intravital imaging studies of HIV-1-infected humanized mice by three independent groups revealed the presence of small T cell syncytia. Although these multinucleated entities represent only a small fraction of infected T lymphocytes, they arise at the earliest stages of infection, are highly motile, and they make frequent contact with potential target cells. Importantly, while such transient contacts allow for virus transfer, they typically do not result in fusion and these exclusively T cell-based syncytia thus do not grow beyond the three- or four-nuclei stage. Immunofluorescence microscopy analyses, together with initial multiparameter flow cytometric evaluation and proteomic profiling by our lab and collaborators, identified these small T cell syncytia (henceforth referred to merely as syncytia) as a distinct subpopulation of infected cells. Because they appear as soon as any infected cells are detectable and thus likely contribute to early viral spread, they also trigger innate immune responses, including attacks by cytolytic effectors such as natural killer (NK) cells. Here, we first introduce a newly developed surface split GFP system which allows to unequivocally distinguish syncytia from infected mononucleated cells, and which also enables isolating these entities for in-depth functional analyses. Then, using multiparameter flow cytometry with UMAP clustering and transcriptomic profiling, we document that syncytia represent a subpopulation of infected cells that do indeed clearly differ from both uninfected and infected mononucleated T lymphocytes, particularly also regarding expression of immune-regulatory host factors. Finally, we show that the rate of direct killing by NK cells is substantially higher for syncytia than for infected mononucleated cells, both in 2D suspension co-culture and in a 3D collagen coculture system. While the fitness costs imposed on syncytia by such increased susceptibility to NK cytolysis during early infection might be compensated for by yet to be determined syncytia functions that (directly) enhance virus spread, we note that irrespective of what such virus spread-enhancing functions may be, the increased vulnerability of syncytia could possibly be exploited for the development of novel antiviral strategies.
Girard, J. P., Whitacker, E. E., Frandina, C. P., Veljkovic, P., Carr, M. K., Symeonides, M., Thali, M.
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