The mechanism by which virus-specific CTL lyse target cells may have profound effects on the pathogenesis of disease. The findings in this study highlight the importance of alternative mechanisms of viral clearance in PKO mice. This study shows for the first time that lung T lymphocytes express high levels of surface FasL after primary RSV infection. More importantly, antiviral CTL can lyse target cells by a perforin-independent, FasL-dependent mechanism that can be inhibited by anti-FasL antibody (Fig. B). This complete inhibition of target cell lysis by anti-FasL in our 51Cr release assays strongly suggests that FasL-mediated lysis of RSV-infected cells can act in a compensatory manner in the absence of perforin.
While our study focuses on the CD8+ CTL response to primary RSV infection, it is important to realize that natural killer (NK) cells possess the same cytolytic machinery as CD8+ CTL. Therefore, perforin deficiency may also affect the NK cell response to RSV, which may have importance in initiating clearance and promoting the adaptive immune response. Thus, we cannot exclude the possibility that the delayed illness and exaggerated response are related to an altered NK cell response. Ongoing studies are under way in our lab to address this possibility.
On the other hand, viral clearance can also be mediated in the absence of cell-to-cell contact by the antiviral cytokines IFN-γ and TNF-α and -β (20
). Influenza virus-specific CTL stimulated with peptide produce IFN-γ and TNF-α, which enhance the lysis of influenza virus-infected cells in vitro. Even though anti-FasL antibodies completely inhibited target cell lysis in our 51
Cr release assays, these are short (4- to 8-h) assays. Zheng et al. have shown that TNF-α can mediate mature T-cell receptor-induced apoptosis (44
). In their experiments, apoptosis observed at 24 h could be inhibited by treatment of cells with the Fas-Fc fusion protein but not by TNFR-Fc. However, administration of Fas-Fc or TNFR-Fc at 48 h led to a decrease in apoptosis. Our data show that on day 8 postinfection, IFN-γ and TNF-α levels were significantly elevated in PKO mice (Fig. ). These results suggest the possibility that the antiviral cytokines IFN-γ and TNF-α contributed to the delayed clearance observed in our experiments (Fig. A). Nevertheless, our lab has shown that anti-TNF-α treatment of WT mice during primary RSV infection, despite diminishing illness, has no impact on viral clearance (31
). For this reason, we hypothesize that the elevated and persistent IFN-γ and TNF-α levels in PKO mice are more likely to be responsible for the late illness that was observed (Fig. B). These cytokine levels may have been increased because RSV clearance required a larger T-cell infiltrate in the lungs of PKO mice, which implies that T lymphocytes from PKO mice are probably less efficient at RSV clearance on a per cell basis than WT mice.
Interestingly, this late illness was still seen in PKO mice on and after day 10 postinfection (Fig. ), by which time the virus had been cleared and a dramatic reduction in the number of CD8+ CTL was observed (Fig. A). Additionally, the total number of cells in WT mice fell from an average of 1.88 × 106 cells on day 8 to 0.54 × 106 cells on day 10. In PKO mice, the total cell number declined from 2.65 × 106 on day 8 to 0.45 × 106 on day 10 (data not shown). Therefore, the total number of inflammatory cells in the lungs does not explain the kinetics of illness, supporting the hypothesis that the altered cellular composition and prolonged production of IFN-γ and TNF-α are responsible for the delayed and prolonged illness.
As has been mentioned, the current state of the literature suggests that the Fas/FasL pathway is more important for T-cell homeostasis (8
) and that the perforin/granzyme pathway is more important for clearance of viruses (3
). However, recent data suggest that the perforin pathway may also contribute to T-cell homeostasis (19
). PKO mice infected with LCMV have significantly increased numbers of LCMV-specific CD8+
T cells, to which the inability of PKO mice to downregulate the T-cell response was attributed (28
). Our findings are consistent with this conclusion, as we found increased numbers of lymphocytes in infected lungs by fluorescence-activated cell sorter (FACS) analysis and histopathology on day 8 after RSV infection. PKO mice had greater cellularity focused primarily around the interstitial spaces than did WT controls (Fig. ). In addition, we found a significantly higher percentage of M2-specific CD8+
T cells in PKO mice than in WT controls by use of tetramer analysis (Fig. B).
Overproduction of interleukin 4 (IL-4) is one setting in which RSV infection is known to result in severe or enhanced disease. This has been demonstrated for IL-4-overexpressing mice and by immunization with formulations that promote Th2 responses (10
). It has been postulated that severe primary RSV disease or vaccine-enhanced RSV disease following administration of formalin-inactivated RSV in humans is also related to IL-4 overproduction. The basis for severe disease when IL-4 is overproduced has been assumed to be an exaggerated Th2 CD4 T-cell response with the attendant induction of immunoglobulin E and recruitment of eosinophils. However, our previous work has shown that IL-4 can induce a shift to a more FasL-mediated CTL killing mechanism (1
). We propose that this shift may be a factor in severe RSV disease associated with overproduction of IL-4. In the present study, we have demonstrated that mice deficient in perforin suffer a larger cellular infiltrate in their lungs, possibly with an augmented amount of bystander killing, and increased production of IFN-γ and TNF-α. These events evoke more serious pathology and may potentially explain the FI-RSV vaccine-enhanced disease as a condition of IL-4 overproduction causing a shift to a FasL-dominant CTL killing mechanism. In summary, this study shows that, while perforin is important in the clearance of primary RSV infection, CTL possess alternative mechanisms for the elimination of RSV. Modulating the mechanism of CTL-mediated lysis of RSV-infected cells may be an important factor in the balance of viral clearance and lung immunopathology.