Abstract
Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef's dileucine motif LL164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif–dependent manner. Treating HIV-1-infected CD4+ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4+ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4+ T cells.
Highlights
T cells are an essential component of the host immune machinery [1]
PHA/IL-2-activated Peripheral blood mononuclear cells (PBMCs) from healthy donors were infected for 48 h with NL43-enhanced green fluorescent protein (eGFP) or isogenic variants with or without Nef expression and surface levels of four checkpoint receptors: programmed death-1 (PD-1), TIGIT, lymphocyte activation gene-3 (LAG-3), and T-cell immunoglobulin mucin domain-3 (Tim-3), were assessed using flow cytometry (Fig. 1A)
The upregulation of Tim-3 cell surface expression by NL4-3 was partly Nefdependent as a twofold decrease in the fluorescence intensity of Tim-3 was observed in cells infected with a virus failing to express Nef (ΔNef) (Fig. 1, A–C)
Summary
M. Mansour Haeryfar , Andrés Finzi, and Jimmy D. Dikeakos1,* From the 1Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada; 2Centre de Recherche du CHUM, Montreal, Quebec, Canada; 3Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Quebec, Canada; 4Institute of Molecular Virology, Ulm University Medical Center, UIm, Germany; 5Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
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