Dysregulated Tim-3 expression on natural killer cells is associated with increased Galectin-9 levels in HIV-1 infection

Retrovirology Dysregulated Tim-3 expression on natural killer cells is associated with increased Galectin-9 levels in HIV-1 infection Stephanie Jost 0 Uriel Y Moreno-Nieves 0 Wilfredo F Garcia-Beltran 0 Keith Rands 0 Jeff Reardon 0 Ildiko Toth 0 Alicja Piechocka-Trocha 0 Marcus Altfeld 0 1 Marylyn M Addo 0 0 Ragon Institute of MGH, MIT and Harvard , 400 Technology Square, Cambridge, MA 02139 , USA 1 Heinrich-Pette-Institut, Leibniz Institute for Experimental Virology , Hamburg , Germany Background: Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells. Whether HIV-1 infection modulates the expression of Tim-3 on NK cells, or the levels of its ligand Galectin-9 (Gal-9), and how signaling through these molecules affects the NK cell response to HIV-1 remains inadequately understood. Results: We analyzed Tim-3 and Gal-9 expression in a cohort of 85 individuals with early and chronic HIV-1 infection, and in 13 HIV-1 seronegative control subjects. HIV-1 infection was associated with reduced expression of Tim-3 on NK cells, which was normalized by HAART. Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals. Interestingly, Gal-9 expression in immune cells was significantly elevated in early infection, with monocytes and dendritic cells displaying the highest expression levels, which correlated with HIV-1 viral loads. In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation. Conclusions: Our data suggest that high expression levels of Gal-9 during early HIV-1 infection can lead to enhanced NK cell activity, possibly allowing for improved early control of HIV-1. In contrast, persistent Gal-9 production might impair Tim-3 activity and contribute to NK cell dysfunction in chronic HIV-1 infection. Tim-3; Gal-9; HIV-1; Innate immunity; NK cells - Background The human immunodeficiency virus type 1 (HIV-1) affects 34 million adults and children worldwide, and the ongoing spread of the epidemic resulted in about 2.5 million new infections and 1.7 million deaths in 2011 alone [1]. Novel approaches to prevent the transmission of HIV-1 are urgently needed, and whether manipulating innate immune effector cell function could be used as a strategy to enhance HIV-1 vaccine efficiency remains to be determined. Recent data suggest that incorporating components with the potential to harness the antiviral function of natural killer (NK) cells may represent an attractive option to improve future vaccine designs [2-13]. NK cells play an important role in containing viral replication in the very early stages of viral infections, and in shaping the subsequent adaptive immune response by interacting with other immune cells, including dendritic cells (DCs) and CD4+ T cells [14-17]. NK cell function is regulated by the integration of inhibitory and activating signals generated by an arsenal of cell surface receptors, with effector functions taking place when activating signals overcome inhibitory ones [18]. Importantly, mounting evidence suggests that NK cells can mediate antiviral activity in HIV-1-infected humans [2-12]. However, how NK cells recognize and eliminate HIV1infected cells and control HIV1 replication and disease progression remains poorly understood to date. It has recently been described that NK cells constitutively express high levels of T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) [19-21]. Tim-3 is a type I transmembrane receptor that is also expressed on specific subsets of CD4+ and CD8+ T cells, on subpopulations of macrophages and DCs, and on monocytes (reviewed in [22]), albeit to a lesser extent than on NK cells [19]. Tim-3 was originally identified as a marker of terminally differentiated CD4+ Th1 cells [23], and subsequently associated with T-cell exhaustion and impaired virus-specific T-cell responses in HIV-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) infection [24-28]. To date, three ligands have been described for Tim-3, including Galectin-9 (Gal-9), a 40-kD S-type -galactoside-binding lectin [29], cell-surface phosphatidylserine [30,31], andat least in murine modelsthe high-mobility group box 1 (HMGB1) protein [32]. Gal-9 is highly expressed in immune tissues [33], and while engagement of Tim-3 triggers apoptosis in CD4+ Th1 cells [29], T cells and thymocytes [34], this interaction has been suggested to (i) protect activated CD4+ T cells from HIV-1 infection and replication [35], (ii) enhance the production of pro-inflammatory cytokines by immature dendritic cells [36,37] and (iii) regulate the migration of Th2 cells [38]. Therefore, Tim-3 signaling on immune cells can trigger either inhibitory or activating signals, with the outcome depending on the presence of described and potentially as yet undefined ligands for Tim-3 [39]. While Tim-3 has been extensively studied on T cells, much less is known about the impact of Tim-3-mediated signaling on NK cell responses, notably in the context of viral infections. Expression of Tim-3 can be induced on NK cells by various cytokines [20,21], and Tim-3 has recently been proposed to be a marker for mature and fully functional NK cells, with those expressing the highest levels of the receptor displaying the most potent cytotoxic activity or cytokine production [21]. On NK cells, Tim-3 can act as an activating co-receptor, as exposure to Gal-9 enhances IFN- production by Tim-3+ NK cells [20], yet it can also deliver inhibitory signals, given that the ability of NK cells to kill target cells is lost upon Tim-3 crosslinking [21]. In accordance with the latter observation, up-regulation of Tim-3 on NK cells has been associated with reduced anti-viral properties in chronic hepatitis B infection, with NK cell cytolytic function being enhanced upon Tim-3 blockade [40]. Progressive HIV-1 infection leads to significant changes in NK cell phenotype and function, and is associated with an expansion of anergic NK cells [41,42]. However, it is not known whether chronic HIV-1 infection modulates the expression of Tim-3 on NK cells or the levels of its ligand Gal-9, nor is it understood how signaling through these molecules affects the NK cell response to HIV-1. Here, we describe dysregulated expression of Tim-3 on NK cells in HIV-1 infection, with differential responses in early versus chronic HIV-1 infection. Plasma levels of Gal-9 were elevated in HIV-1 -infected individuals and correlated with markers of HIV-1 disease progression. Moreover, we show that stimulation of Tim-3+ NK cells by Gal-9 leads to NK cell activation and Tim-3 downregulation in vitro. Our data suggest that persistent signaling through Tim-3 on NK cells might result in loss of functionality and accumulation of a Tim-3low NK cell population in vivo, thereby contributing to the previously described impaired activity of NK cells in chronic HIV- (...truncated)