In this study, we demonstrated that α-GalCer inhibits HBV replication in the liver of HBV transgenic mice. The antiviral effect of α-GalCer is rapid, profound, and specific, and is associated with the induction of IFN-γ and IFN-α/β in the liver. Both of these cytokines mediate the antiviral effect of α-GalCer, since its activity is strongly reduced in mice that are genetically deficient for either IFN-γ or the IFN-α/βR. Since the induction of IFN-γ and IFN-α/β as well as the inhibition of HBV replication occur before a significant number of inflammatory cells are recruited into the organ, it is likely that these cytokines are produced by cells that reside in the liver, specifically NKT cells that are known to produce IFN-γ very rapidly in response to α-GalCer 13
and NK cells that are promptly activated by NKT cells and enhance induction of IFN-γ production 1617
. Importantly, our results also demonstrate that α-GalCer induces IFN-α/β in the liver, a previously unreported effect of this glycolipid.
We have previously shown that IFN-γ (produced in the liver by passively transferred HBV-specific CTLs 23
) and IFN-α/β (produced in the liver after injection of Poly-I/C or during infection with unrelated hepatotropic viruses) can inhibit HBV replication by independent pathways 24
. The results reported herein show that similar antiviral events can be triggered by α-GalCer via its ability to activate intrahepatic NKT cells. Based on these results, it is possible that intrahepatic NKT cells could inhibit HBV replication during natural infection. In keeping with this hypothesis, we recently showed that HBV DNA largely disappears from the liver and the blood of acutely infected chimpanzees concomitant with the appearance of IFN-γ, but before the major influx of CD3+
T cells 28
, suggesting that IFN-γ might have been produced by nonclassical T cells, perhaps by activated NKT cells that are known to express low levels of the CD3–TCR complex and/or by NK cells that are known to be activated by NKT cells. If this is correct, intrahepatic NKT cells might contribute to viral clearance if they become activated during HBV infection.
NKT cells could become activated by direct or indirect mechanisms. Direct mechanisms could include the specific recognition of viral or virus-induced antigen via the TCR. CD1 is known to accommodate highly hydrophobic molecules, such as glycolipids and phospholipids 41424344
. HBV virions and subviral particles contain glycolipids and phospholipids derived from cellular membranes 45
that could be processed by CD1+
cells (professional antigen-presenting cells and hepatocytes) and presented to intrahepatic NKT cells. We doubt that this mechanism is operative during natural HBV infection because we did not observe any difference in the abundance or activation of the NKT cell populations in the livers of HBV transgenic versus nontransgenic mice. Other direct mechanisms include the recognition of putative ligands by non-TCR receptors (such as the NKR-P1 receptor, which is also present on NK cells [46
]) or the recognition of cells that lack surface expression of MHC class I molecules (a mechanism that also induces NK cell activation [46
]). Indirect mechanisms could include activation of NKT cells by virus-induced cytokines 622
. Additional studies will be needed to clarify these alternative scenarios.
NKT cells are resident IHLs, accounting for 20–30% of IHLs 4618
; they have a limited TCR repertoire (Vα14/Vβ8 in mice, Vα24/Vβ11 in humans); and they are restricted by the nonclassical, nonpolymorphic MHC class I–like molecule CD1d. The pertinence of these observations for the treatment of chronic HBV infection derives from the fact that, because of their abundance in the liver, NKT cells do not need to be recruited in order to exert their antiviral effect. Furthermore, because of their limited repertoire, if provided with the appropriate ligand (e.g., α-GalCer) NKT cells can be activated very efficiently in individuals irrespective of their classical MHC class I haplotype. This offers a major advantage over antigen-specific immunotherapy for chronic HBV infection since it eliminates the need for priming and expansion of antigen-specific effector cells and recruitment of these effector cells into the liver. Furthermore, attempts to therapeutically induce HBV antigen–specific T cell responses in chronically HBV-infected patients 48
have not been very successful thus far, probably because of neonatal and/or peripheral tolerance; we have had the same experience in HBV transgenic mice 49
. In contrast, since they probably do not recognize viral peptide antigens, NKT cells are not likely to be deleted in chronically infected patients 5051
, just as they were not in the HBV transgenic mice in this study (not shown). This creates the unique opportunity to use pharmacological approaches to activate the resident intrahepatic NKT cell population and, secondarily, the intrahepatic NK cell population, thereby rapidly and efficiently enhancing the production of potentially curative antiviral cytokines in the liver of infected patients in a quantitatively and temporally controllable manner. Thus, it may be possible to harness the potentially curative antiviral potential of the innate immune response to compensate for the apparent failure of the adaptive immune response in chronically infected patients.
In conclusion, this study demonstrates that α-GalCer activates intrahepatic NKT cells to secrete antiviral cytokines (IFN-γ and IFN-α/β) in the liver. The rapid and strong induction of these cytokines may reflect the relative abundance of NKT cells in the liver 4618
, all of which can be simultaneously activated by the glycolipid. Based on these results, it is conceivable that NKT cells may play a hitherto unsuspected role in the immunobiology of HBV infection. In addition, the results suggest that therapeutic activation of intrahepatic NKT cells may be able to terminate chronic HBV infection.