In the present study, we have demonstrated that secretion of IFN-γ and TNF-α represents a direct, cytokine-mediated antiviral activity of human CD4+ T cells. Elevated secretion of these cytokines was directed by IL-12; we found no significant contribution, positive or negative, of IFN-α/β. A combination of IFN-γ and TNF-α produced by Th1 cells promotes antiviral responses by two distinct mechanisms. First, IFN-γ and TNF-α can transmit an antiviral signal via a type I interferon-independent pathway, as in the case of HCV infection. In this case, the antiviral activity could be mediated by direct effects of IFN-γ and TNF-α or through the induction of another, non-IFN-α/β cytokine. Alternatively, the activity can be mediated through a cytokine relay network, as in the case of VSV infection, in which type I interferon signaling is required for the antiviral effect.
In agreement with our results, several other groups have shown that CD4+
T cells have the capacity to promote viral clearance in vivo in a “helper-independent” fashion. For instance, clearance of Sendai virus, gammaherpesvirus (γHV68), or influenza A virus can proceed in a CD4+
T cell-dependent fashion in the absence of B cells and CD8+
T cells (40
). Additionally, memory Th cells generated against VSV in CTL-nonresponsive mice provide protection in an antibody-independent manner (64
). In many cases, a deficiency in IFN-γ in vivo abolished the antiviral capacity of CD4+
T cells (42
), and adoptive transfer of an antigen-specific Th1 clone conferred protection from γHV68 infection (45
). However, the target of IFN-γ was undetermined in these studies. Therefore, it was possible that viral clearance could have been mediated by a population of innate cells, such as NK cells, which were activated in the presence of IFN-γ. Here, we definitively demonstrate for the first time that cytokines secreted by Th cells directly impact viral clearance from infected targets.
T cell-mediated control of cytomegalovirus (CMV) in salivary glands requires IFN-γ, but, paradoxically, treatment of virally infected mice with recombinant IFN-γ failed to clear the virus (66
). We have shown that both IFN-γ and TNF-α are required to achieve robust viral inhibition by Th1 cell-secreted factors. Therefore, in vivo treatment of CMV-infected animals with a combination of recombinant IFN-γ and TNF-α could promote viral clearance when neither cytokine alone possessed this activity.
Several groups have reported that TNF-α can induce secretion of IFN-β from target cells and that this IFN-β can synergize with IFN-γ for viral inhibition (33
). However, this effect relied upon pre-treatment of target cells with cytokines for 16–24 hours before in vitro infection. In contrast, we have demonstrated an antiviral activity of IFN-γ and TNF-α which does not require pre-treatment of target cells. Thus, secretion of these cytokines by CD4+
T cells at peripheral sites could have beneficial effects even after cells were already infected.
We found that the antiviral activity of T cell-secreted IFN-γ and TNF-α was independent of type I interferon signaling in the case of HCV infection. Surprisingly, this activity was completely dependent upon the presence of a functional IFNAR in the case of VSV infection. It is currently unclear whether this phenomenon is specific to VSV or represents a more general antiviral mechanism. However, we noted during the course of our experiments that Sendai virus, which blocks type I interferon signaling in infected cells, was also completely resistant to the antiviral effects of T cell conditioned media (K. A. H. and M. G., Jr., unpublished observations).
While the observed antiviral effect of IFN-γ and TNF-α is dependent upon signaling through the IFNAR in the case of VSV, we were unable to detect induction of known type I interferon genes in target cells. This further excludes induction of IFN-β by TNF-α as a mechanism for the observed antiviral effect. Many possible explanations exist for this novel antiviral effect of IFN-γ and TNF-α during VSV infection. For instance, IFN-γ and TNF-α may be inducing expression of a novel type I interferon gene in virally infected target cells. Several new type I interferon genes have been described in recent years (69
); a more extensive search may reveal other, distantly related family members located within or even outside the IFN locus.
Alternatively, IFN-γ and TNF-α may synergize to directly activate IFNAR signaling via a mechanism such as receptor sharing in order to induce type I IFN-like effects in specialized situations. There are many known cases in which two or more unrelated receptors are activated by the same ligand. For instance, glial cell-derived neurotrophic factor (GDNF) signals through both the receptor tyrosine kinase RET and the Ig-domain-containing receptor NCAM (72
). Alternately, a single receptor subunit can be shared among multiple distinct receptors, as in the case of the common gamma chain which is used for cytokine signaling (73
). Consistent with our in vitro studies, it is interesting to note that Müller et. al. demonstrated that the antiviral effects of IFN-γ against VSV were impaired in murine cells lacking IFNAR expression (74
). However, other IFN-γ signaling pathways were unaffected in cells from IFNAR−/−
mice, and IFNγR−/−
mice showed no defect in VSV clearance.
Many viruses encode intracellular or extracellular mechanisms to antagonize antiviral cytokine secretion and signaling by infected host cells. For instance, poxviruses encode soluble, secreted forms of the IFNAR, IFNγR, and TNFR which can neutralize host cytokines (75
). A variety of viruses, including HCV, influenza A virus, and Sendai virus, also inhibit intracellular induction of type I interferon by blockade of the RIG-I pathway (57
). In such cases, exogenously delivered cytokines from Th cells could provide alternative pathways to overcome these blocks and promote pathogen clearance in a noncytopathic manner.
IFN-α is widely used to treat HCV infections, but many patients fail to respond to this therapy. HCV and other flaviviruses, such as West Nile Virus, inhibit IFNAR signal transduction in target cells through inactivation of downstream signaling intermediates (56
). In accordance with previous reports, we demonstrated that IFN-γ possessed substantial antiviral activity against HCV (59
). However, Frese et. al. found no role for TNF-α, either alone or in combination with IFN-γ, in inhibition of HCV replication (60
). In contrast, we observed cooperation between IFN-γ and TNF-α in suppressing HCV NS5A protein expression. Furthermore, our data show that IFN-γ and TNF-α inhibit HCV infection by a type I IFN-independent mechanism. Therefore, Th1 responses generated during infections with these viruses could represent an important alternative mechanism for pathogen clearance when type I IFN is ineffective.