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.

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.

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