Date Published: June 20, 2013
Publisher: Public Library of Science
Author(s): Hassen Kared, Thomas Fabre, Nathalie Bédard, Julie Bruneau, Naglaa H. Shoukry, Michael Bevan.
Loss of CD4 T cell help correlates with virus persistence during acute hepatitis C virus (HCV) infection, but the underlying mechanism(s) remain unknown. We developed a combined proliferation/intracellular cytokine staining assay to monitor expansion of HCV-specific CD4 T cells and helper cytokines expression patterns during acute infections with different outcomes. We demonstrate that acute resolving HCV is characterized by strong Th1/Th17 responses with specific expansion of IL-21-producing CD4 T cells and increased IL-21 levels in plasma. In contrast, viral persistence was associated with lower frequencies of IL-21-producing CD4 T cells, reduced proliferation and increased expression of the inhibitory receptors T cell immunoglobulin and mucin-domain-containing-molecule-3 (Tim-3), programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) on HCV-specific CD8 T cells. Progression to persistent infection was accompanied by increased plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and expansion of Gal-9 expressing regulatory T cells (Tregs). In vitro supplementation of Tim-3high HCV-specific CD8 T cells with IL-21 enhanced their proliferation and prevented Gal-9 induced apoptosis. siRNA-mediated knockdown of Gal-9 in Treg cells rescued IL-21 production by HCV-specific CD4 T cells. We propose that failure of CD4 T cell help during acute HCV is partially due to an imbalance between Th17 and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway may be limited by IL-21 producing Th17 cells or enhanced by Gal-9 producing Tregs.
The outcome of acute hepatitis C virus (HCV) infection towards spontaneous resolution or persistent viremia is dictated by the magnitude, breadth and quality of the virus-specific CD4 and CD8 T cell responses , . The essential role of CD4 helper T cells in mediating spontaneous viral clearance was demonstrated by several observations. First, the loss of CD4 helper T cell proliferative responses during acute HCV was associated with viral recurrence and the development of chronic infection , . Second, broad HCV-specific CD4 T cell responses are induced early in most acutely infected individuals but they undergo progressive loss of IL-2 production and diminished proliferation as infections progress towards viral persistence –. Third, CD4 T cell depletion in the chimpanzee model of HCV infection led to persistent low level viremia, the loss of CD8 function and the development of escape mutations in targeted CD8 cytotoxic T lymphocyte (CTL) epitopes . These observations strongly suggest that CD4 helper T cells are critical in sustaining the functions of HCV-specific CD8 T cells. However, the underlying helper signals and the mechanisms of CD4 T cell failure remain elusive.
In this study, we demonstrated an important role for IL-21 as a major helper cytokine during acute HCV infection by limiting the T cell dysfunction induced by the Gal-9/Tim-3 interaction. We have demonstrated a direct correlation between higher levels of IL-21 production and cell proliferation, as well as cell survival and the inhibition of exhaustion of HCV-specific CD8 T cells. Moreover, our results have identified three different mechanisms of CD4 helper T cell failure during acute HCV infections with chronic evolution. First, the exhaustion of HCV-specific helper T cells may lead to decreased IL-21 production and failure to sustain efficient CTL responses. Second, an imbalance between inflammatory (Th17) and regulatory (Treg) CD4 T cells may have a direct inhibitory effect on HCV-specific CTL responses. Third, we have identified CD39+ Tregs as a potential source of Gal-9 during chronic HCV infection and demonstrated that Gal-9-expressing Tregs can directly inhibit proliferation and IL-21 production by HCV-specific CD4 T cells. These mechanisms combined may limit CD4 T cell help, trigger exhaustion and apoptosis of HCV-specific T cells and favor virus persistence.