T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14+ monocyte/macrophages (M/MØ) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/MØ, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/MØ incubated with HCV-expressing hepatocytes, as well as in primary M/MØ or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/MØ. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.

Tim-3 and PD-1 are powerful immunoinhibitory molecules involved in immune tolerance, autoimmune responses, and antitumor or antiviral immune evasion. A current model for Tim-3 regulation during immune responses suggests a divergent function, such that Tim-3 acts synergistically with TLR signaling pathways in innate immune cells to promote inflammation, yet the same molecule terminates Th1 immunity in adaptive immune cells. To better understand how Tim-3 might be functioning in innate immune responses, we examined the kinetics of Tim-3 expression in human CD14+ M/M(Ф) in relation to expression of IL-12, a key cytokine in the transition of innate to adaptive immunity. Here, we show that Tim-3 is constitutively expressed on unstimulated peripheral blood CD14+ monocytes but decreases rapidly upon TLR stimulation. Conversely, IL-12 expression is low in these cells but increases rapidly in CD14+ M/M(Ф) in correlation with the decrease in Tim-3. Blocking Tim-3 signaling or silencing Tim-3 expression led to a significant increase in TLR-mediated IL-12 production, as well as a decrease in activation-induced up-regulation of the immunoinhibitor, PD-1; TNF-α production was not altered significantly, but IL-10 production was increased. These results suggest that Tim-3 has a role as a regulator of pro- and anti-inflammatory innate immune responses.

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Up-regulation of inhibitory signaling pathways (such as Tim-3) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. While the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3+ Tregs is poorly explored. In this pilot study, we investigated whether and how the Tim-3 pathway alters Foxp3+ Treg development and function in patients with chronic HCV infection. We found that Tim-3 was up-regulated, not only on IL-2-producing CD4+CD25+Foxp3− Teffs, but also on CD4+CD25+Foxp3+ Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared to healthy subjects. Tim-3 expression on Foxp3+ Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3+ Tregs were found to be more resistant to, and Foxp3− Teffs more sensitive to, TCR-activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4+CD25+ T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3+ Tregs/Foxp3− Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control regulatory and effector T cell balance through altering cell proliferation and apoptosis during HCV infection.

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Accumulation of regulatory T cells (Tregs) and up-regulation of inhibitory signaling pathways (such as Tim-3/Gal-9) play pivotal roles in suppressing antiviral effector T cells (Teffs) that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teffs, their role in regulating Tregs is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg development, in this study we provide pilot data showing that HCV-infected hepatocytes express higher levels of Gal-9 and TGF-β, and up-regulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured CD4+ T cells, driving conventional CD4+ T cells into CD25+Foxp3+ Tregs. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4+ T cells into Foxp3+ Tregs in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate Foxp3+ Treg development and function during HCV infection.

SummaryHost immune responses must be tightly regulated by an intricate balance between positive and negative signals while fighting pathogens; persistent pathogens may usurp these regulatory mechanisms to dampen host immunity to facilitate survival in vivo. Here we report that Tim-3, a negative signalling molecule expressed on monocytes and T cells, is up-regulated on natural killer (NK) cells in individuals chronically infected with hepatitis C virus (HCV). Additionally, the transcription factor T-bet was also found to be up-regulated and associated with Tim-3 expression in NK cells during chronic HCV infection. , an miRNA that inhibits signalling proteins involved in immune responses, was down-regulated in NK cells by HCV infection. This Tim-3/T-bet over-expression and miR-155 inhibition were recapitulated in vitro by incubating primary NK cells or NK92 cell line with Huh-7 hepatocytes expressing HCV. Reconstitution of miR-155 in NK cells from HCVinfected patients led to a decrease in T-bet/Tim-3 expression and an increase in interferon-c production. Blocking Tim-3 signalling also enhanced interferon-c production in NK cells by improving signal transducer and activator of transcription-5 phosphorylation. These data indicate that HCV-induced, miR-155-regulated Tim-3 expression regulates NK cell function, suggesting a novel mechanism for balancing immune clearance and immune injury during chronic viral infection.Keywords: hepatitis C virus; interferon-c; microRNA-155; natural killer cells; signal transducer and activator of transcription-5; T-cell immunoglobulin and mucin domain protein-3.

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In this study, we demonstrate that killer cell lectin-like receptor subfamily G member 1 (KLRG1), a transmembrane protein preferentially expressed on T cells, is highly expressed on CD56 ؉ NK cells, which are significantly reduced in their numbers and functions in the peripheral blood of patients with chronic hepatitis C virus (HCV) infection compared to subjects without infection. KLRG1 expression is also upregulated on healthy NK cells exposed to Huh-7 hepatocytes infected with HCV in vitro. Importantly, the expression levels of KLRG1 are inversely associated with the capacity of NK cells to proliferate and to produce gamma interferon (IFN-␥) but positively associated with apoptosis of NK cells in response to inflammatory cytokine stimulation. KLRG1؉ NK cells, including CD56 bright and CD56 dim subsets, exhibit impaired cell activation and IFN-␥ production but increased apoptosis compared to KLRG1؊ NK cells, particularly in HCV-infected individuals. Importantly, blockade of KLRG1 signaling significantly recovered the impaired IFN-␥ production by NK cells from HCV-infected subjects. Blockade of KLRG1 also enhanced the impaired phosphorylation of Akt (Ser473) in NK cells from HCV-infected subjects. Taken together, these results indicate that KLRG1 negatively regulates NK cell numbers and functions via the Akt pathway, thus providing a novel marker and therapeutic target for HCV infection.

Co-infection of hepatitis B virus (HBV) with hepatitis C virus (HCV) is quite common, leading to an increase in morbidity and mortality. As such, HBV vaccination is recommended in HCV-infected individuals. HBV vaccine responses in HCV-infected individuals, however, are often blunted when compared to uninfected populations. The mechanism for this failure of vaccine response in HCV-infected subjects remains unclear. In this study, we investigated the expression and function of an inhibitory receptor, killer cell lectin-like receptor subfamily G member 1 (KLRG1), in regulation of CD4+ T cells and HBV vaccine responses during HCV infection. We demonstrated that KLRG1 was over-expressed on CD4+ T cells from HCV-infected, HBV vaccine non-responders (HBV-NR) compared to those responders (HBV-R). The capacity of CD4+ T cell to proliferate and secrete IL-2 cytokine was inversely associated with the level of KLRG1 expression. Importantly, blocking KLRG1 signaling resulted in a significant improvement of CD4+ T cell proliferation and IL-2 production in HCV-infected, HBV-NR in response to T cell receptor (TCR) stimulation. Moreover, blockade of KLRG1 increased the phosphorylation of Akt (Ser473) and decreased the expression of cell cycle inhibitors p16ink4a and p27kip1, which subsequently enhanced CDK 2 and cyclin E expressions. These results suggest that the KLRG1 pathway impairs CD4+ T cell responses to neo-antigen and induces a state of immune senescence in individuals with HCV infection, raising the possibility that blocking this negative signaling pathway might improve HBV vaccine responses in the setting of chronic viral infection.