CD8 T Cells Utilize TRAIL to Control Influenza Virus Infection¹ Free

Primary infection with influenza virus results in a localized pulmonary infection and inflammation and elicits an influenza-specific CD8⁺ T cell immune response that is necessary for viral clearance (1, 2, 3, 4, 5, 6, 7). These CD8⁺ T cells are thought to control virus infections by killing influenza-infected pulmonary cells using perforin- and Fas-dependent mechanisms (3). When Fas^−/− or perforin^−/− mice were infected with influenza virus in the absence of CD4⁺ T cells, viral titers persisted for an additional 3 days beyond controls, but virus was eventually cleared (3). This result suggests that in the absence of one death-inducing pathway, influenza-specific CD8⁺ T cells will compensate and utilize other cytotoxic mechanisms to eliminate influenza virus-infected cells. In contrast, when perforin^−/− bone marrow-reconstituted Fas^−/− mice were likewise infected with influenza virus, ∼70% of the mice had sustained long-term (i.e., 14 days postinfection (p.i.)³) high viral titers (3), suggesting that influenza-specific CD8⁺ T cell cytotoxicity requires access to either the perforin or Fas cytotoxic pathways to effectively control influenza virus infections. Interestingly, however, ∼30% of the above perforin^−/−Fas^−/− mice were able to reduce pulmonary influenza virus titers, leading to the idea that another cytotoxicity pathway could be involved in viral elimination (3).

CD8⁺ T cells have recently be described to use the TRAIL pathway, in addition to the Fas/FasL and perforin/granzyme (lytic granule) pathways, to kill target cells (8). TRAIL has classically been studied in tumor immunology settings, where it selectively induces apoptosis in transformed cells while leaving nontransformed cells unaffected (9, 10). Beyond a role in tumor surveillance, TRAIL-based immunity is also a component of the immune response during viral infections, including responses to CMV, HIV, and respiratory syncytial virus (11, 12, 13). Moreover, a previous study has shown that the expression of mRNA for TRAIL and its receptor DR5 (TRAIL-R2) are increased in the lungs during influenza virus infections, that TRAIL is expressed by T cells in the lungs of influenza virus-infected mice, and that clearance of influenza virus is delayed by administering a blocking anti-TRAIL mAb during primary infections (14). While these results suggest a role for TRAIL in immunity to influenza virus infections, it remains unknown if the expression of TRAIL by T cells during influenza infections is limited to just influenza-specific T cells, if influenza-specific CD8⁺ T cells utilize TRAIL to kill influenza-infected cells and control virus infection, and how TRAIL deficiency alters the course and magnitude of influenza virus infections. Therefore, we utilized TRAIL^+/+ and TRAIL^−/− mice to determine the contribution of TRAIL to the influenza-specific CD8⁺ T cell immune response during primary influenza virus infections. Our results confirm a role for TRAIL in the primary immune response to influenza virus infection, and they demonstrate that TRAIL-mediated apoptosis is a third mechanism that influenza-specific CD8⁺ T cells can use to eliminate influenza-infected cells and drive recovery from influenza.

C57BL/6 (TRAIL^+/+; H-2^b) mice were purchased from the National Cancer Institute (Frederick, MD). C57BL/6 TRAIL-deficient mice (TRAIL^−/−; H-2^b) were obtained from Amgen (15), and C57BL/6 DR5-deficient mice (DR5^−/−, H-2b) were obtained from Dr. W. S. El-Deiry (University of Pennsylvania) (16). Knockout mice were bred in our own facility at the University of Iowa, according to the Institutional Animal Care and Use Committee (IACUC) guidelines. They are >10 generations backcrossed to C57BL/6. All mice were used at 12–20 wk of age, and all animal experiments followed approved IACUC protocols. The mouse-adapted influenza A virus (A/PuertoRico/8/34 (PR8) H1N1) was grown in the allantoic fluid of 10-day-old embryonated chicken eggs for 2 days at 37°C, as previously described (17, 18). Allantoic fluid was harvested and stored at −80°C. Groups of 24.5–27.5 g TRAIL^+/+ and TRAIL^−/− mice were given a 500 egg infectious units (EIU) dose of mouse-adapted PR8 virus in Iscove’s media intranasally (i.n.) following anesthesia with halothane. The peptides used in this study, nucleocapsid protein (NP)₃₆₆ (ASNENMETM) and acid polymerase (PA)₂₂₄ (SSLENFRAYV), were purchased from Bio-Synthesis and are derived from the amino acid sequence of A/PR/8/34 NP or PA, respectively (19, 20, 21).

Pulmonary viral titers were determined via endpoint dilution assay and expressed as 50% tissue culture-infective dose (TCID₅₀). Briefly, 10-fold dilutions of homogenized and clarified lung from influenza virus-infected mice were mixed with 10⁵ Madin-Darby canine kidney cells in DMEM. After 24 h of incubation at 37°C, the inoculum was removed and DMEM media containing 0.0002% l-1-(tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemical) and penicillin (100 U/ml)/streptomycin (100 mg/ml) was added to each well. After 3 days of incubation at 37°C in a humidified atmosphere of 5% CO₂, supernatants were mixed with an equal volume of 0.5% chicken RBC, the agglutination pattern was read, and the TCID₅₀ values were calculated.

Total RNA was harvested from homogenized lungs with TRIzol reagent (Invitrogen). Total RNA (2 mg) was reverse-transcribed using SuperScript II. The quantitative PCR primer/probe sets for mouse TRAIL, DR5, Fas, FasL, perforin, granzyme B, and rRNA were purchased from PE Applied Biosystems. cDNA (250 ng) was used as a template for TaqMan assays for all transcripts and the internal rRNA control. The TaqMan PCR reaction was conducted as described previously (22).

Splenocytes from wild-type DR5^+/+ and DR5^−/− C57BL/6 mice (16) were resuspended in NycoPrep 1.077A (Axis-Shield) and then purified according to the manufacturer’s instructions. NycoPrep-purified splenic mononuclear cells (10⁷/ml) were labeled with either 2 μM CFSE (Invitrogen) at 37°C for 10 min or 2 μM PKH26 (Sigma-Aldrich) at room temperature for 5 min. After labeling, residual non-cell-associated CFSE and PKH26 were neutralized by adding an equal volume of FCS to the cell suspension. CFSE-labeled splenic mononuclear cells (10⁷/ml) were pulsed with 10 μM PA₂₂₄ and NP₃₆₆ peptide for 1 h at 37°C. PKH26⁺ splenic mononuclear cells (10⁷/ml) were similarly incubated without peptide for 1 h at 37°C. The cells were then washed and mixed at a 1:1 ratio, and 10⁷ cells (i.e., 5 × 10⁶ CFSE⁺, 5 × 10⁶ PKH26⁺ cells) were adoptively transferred i.v. into influenza virus-infected TRAIL^+/+ or TRAIL^−/− mice. After 8 h, the lungs were removed, digested, and analyzed by flow cytometry as previously described (18).

Isolated lung cells (10⁶) were stained with: PE, PerCP-CY5.5, or allophycocyanin-conjugated anti-mouse CD8α (53-6.7; BD Biosciences); biotinylated anti-mouse CD178/FasL (MFL3; eBioscience); or biotinylated anti-mouse TRAIL (N2B2; eBioscience). Cells stained with biotinylated mAb were subsequently incubated with strepavidin-PerCP, strepavidin-PE, or strepavidin-allophycocyanin (BD Biosciences). Stained cells were fixed and erythrocytes lysed with FACS lysing solution (BD Biosciences) and subsequently analyzed on a FACSCalibur flow cytometer. NP₃₆₆ and PA₂₂₄ tetramers were obtained from the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility (Germantown, MD).

Isolated lung cells (10⁶) were stained with the FITC-conjugated anti-mouse T1α/podoplanin (8F11; MBL International) or isotype control, and PE-conjugated anti-mouse DR5 (MD5-1; eBioscience) or isotype control. Subsequently, the cells were fixed, permeabilized, and stained with biotinylated anti-NP (H16-L10-4R5; a kind gift from Walter Gerhard, Wistar Institute, University of Pennsylvania). Biotinylated Ab was subsequently revealed with PerCP-CY5.5.

For granzyme B, isolated lung cells (10⁶) were surfaced stained with PerCP-CY5.5-conjugated anti-mouse CD8α. Subsequently, the cells were fixed, permeablized, and stained with the PE-conjugated anti-human granzyme B mAb (GB11; Invitrogen) or with isotype control (23). For IFN-γ, cells from mice infected with influenza were cultured at 2 × 10⁶ cells/well in the presence of 1 μM of influenza peptides or media control, FITC-conjugated anti-CD107a (1D45; eBioscience) or isotype control, 400 U/ml recombinant human IL-2, and 1 μg/ml brefeldin A. After 6 h, cells were harvested, stained with PE-conjugated rat anti-mouse CD8α, fixed, permeablized, and stained with allophycocyanin-conjugated rat anti-mouse IFN-γ (XMG1.2; eBioscience) or isotype control (24).

Groups of 24.5–27.5-g TRAIL^+/+ (CD45.2) and TRAIL^−/− (CD45.2) mice were given a 500 EIU dose of mouse-adapted A/PR/8/34 virus in Iscove’s media i.n. following anesthesia with halothane. On day 8 p.i., single-cell suspensions of pulmonary cells from the infected TRAIL^+/+ or TRAIL^−/− mice were incubated with anti-CD8α microbeads and CD8⁺ cells purified according to the manufacturer’s instructions (Miltenyi Biotec). The purified CD8⁺ cells were then transferred i.v. into CD45.1 TRAIL^+/+ mice (obtained from Dr. Robert Cook, University of Iowa) that had previously been infected with a lethal dose (2200 EIU) of mouse-adapted A/PR/8/34. Cell transfer into lethally infected mice occurred on day 5 p.i. Overall morbidity and mortality of the mice were monitored to 21 days after lethal infection.

For each analysis, a normal distribution of data was first verified. To assess the difference between two sets of data with normal distribution, statistical significance was assessed using an unpaired, one-tailed t test or a paired t test for control and experimental data groups that could be paired. If the normality test failed, Mann-Whitney rank sum tests were completed to compare data sets. To assess the differences among multiple sets of data with normal distribution, statistical significance was assessed using an ANOVA analysis of the data sets. If thenormality test failed, a Kruskal-Wallis one-way ANOVA on ranks test was used to determine overall significance with subsequent pairwise comparisons completed using Dunn’s method. To determine differences in survival and viral clearance, Kaplan-Meier survival analysis/log-rank tests were run to determine significant differences between data sets. When appropriate, subsequent pairwise multiple comparisons were completed using the Holm-Sidak method. Differences were considered to be statistically significant at p of ≤0.05.

To rigorously investigate the role that TRAIL plays in the regulation of influenza virus infections, we initially determined the impact of TRAIL deficiency on the severity of influenza virus infections. As shown in Fig. 1,A, TRAIL^−/− mice demonstrated significant (p < 0.01) weight loss (i.e., morbidity) relative to wild-type C57BL/6 (TRAIL^+/+) controls following infection with a low dose of influenza virus. This increase in disease severity correlated with increased pulmonary viral titers (Fig. 1 B). Specifically, while the amount of virus in the lungs of TRAIL^+/+ animals was reduced by ∼1 log between days 4 and 6 p.i., TRAIL^−/− mice showed little change in the amount of infectious virus present in their lungs. Furthermore, while TRAIL^−/− animals were able to eventually reduce pulmonary virus levels by day 8 p.i., the number of TRAIL^−/− mice that had cleared virus below the limit of detection remained significantly reduced relative to TRAIL^+/+ animals. Taken together, these results suggest that the increased morbidity observed in TRAIL^−/− mice might, in part, be tied to an increased and sustained pulmonary viral burden.

Since our above results suggested that TRAIL played a significant role in the control and resolution of influenza virus infections, we next examined TRAIL and DR5 mRNA expression in influenza-infected lungs. The amount of TRAIL mRNA was increased in total lung homogenates from TRAIL^+/+ mice following i.n. influenza virus infection (as expected, no TRAIL mRNA was detected in the TRAIL^−/− mice; Fig. 2). Moreover, DR5 mRNA expression was similarly up-regulated in the lungs of both TRAIL^+/+ and TRAIL^−/− mice following i.n. influenza virus infection, indicating that the lack of TRAIL expression did not significantly affect DR5 mRNA expression in the TRAIL^−/− mice. Given that the up-regulation of TRAIL and DR5 mRNA (starting at 6–8 days p.i.) in TRAIL^+/+ mice corresponds with the timing of increased disease in TRAIL^−/− mice (Fig. 1 A), these results further support the concept that TRAIL plays a major role in mediating the control and course of influenza virus infection. These results also largely confirm similar data recently described by Yoneyama and colleagues (14); however, unlike their results, we do not observe significant increases in pulmonary TRAIL mRNA expression until 8 days p.i. (as opposed to 4 days p.i.). Furthermore, we observed a more rapid decrease in TRAIL and DR5 mRNA expression from their peak at 8–14 days p.i. These differences may be related to the difference in virus inoculum administered (500 EIU herein vs 25 PFU), as well as the corresponding alterations in the inflammatory cytokines produced.

To verify that the above changes in DR5 mRNA expression were reflected in alterations of DR5 protein expression, we next determined DR5 expression on pulmonary epithelial cells before and during an influenza virus infection. While there was a low amount of DR5 expressed on the surface of alveolar epithelial type I cells (i.e., T1α⁺ cells) (25) from uninfected mice (Fig. 3,A), overall DR5 expression was significantly increased following influenza virus infection. Moreover, the up-regulation of DR5 by epithelial cells correlated with those cells that had been directly infected with influenza virus (i.e., NP⁺ cells), as NP⁺ epithelial cells expressed ∼5-fold more DR5 relative to NP⁻ epithelial cells from the same lungs (Fig. 3, A and B). These data suggest that influenza infection of pulmonary epithelial cells results in selective up-regulation of DR5 on influenza-infected lung epithelial cells, potentially increasing their susceptibility to TRAIL-mediated lysis.

The greatest difference in morbidity between TRAIL^+/+ and TRAIL^−/− mice was seen after 6 days p.i., paralleling the increase in TRAIL and DR5 expression. These kinetics are similar to those described for influenza-specific CD8⁺ T cell recruitment into the lungs (18, 23). Since CD8⁺ T cells mediate the clearance of influenza-infected cells (1, 2, 3, 4, 5), our results suggested that the increased disease severity and viral burden in TRAIL^−/− mice might be linked to altered pulmonary influenza-specific CD8⁺ T cell responses. Therefore, we next examined TRAIL expression on influenza-specific CD8⁺ T cells in the lungs during influenza infections and found that TRAIL was indeed expressed by influenza-specific CD8⁺ T cells in the lungs of TRAIL^+/+ mice (Fig. 3 C). In contrast, the vast majority of non-influenza-specific CD8⁺ T cells within the lungs at the same time did not appear to express TRAIL. The small residual TRAIL⁺ shoulder in the NP₃₆₆- or PA₂₂₄-negative T cell populations is likely attributable to the remaining unstained immunodominant PA₂₂₄- or NP₃₆₆-specific cells, respectively, with potential minor contributions from the other six subdominant epitopes (19, 26). Similar to the correlation with influenza CD8⁺ T cell epitope specificity observed in the lungs, TRAIL was also selectively expressed on influenza-specific CD8⁺ T cells within the lung draining lymph nodes on day 6 p.i. (data not shown). Consistent with a previous report demonstrating TCR stimulus-driven up-regulation of TRAIL on naive and effector CD8 T cells (27), we also observed Ag-specific up-regulation of TRAIL by naive influenza-specific CD8 T cells after 24 h of in vitro culture (data not shown). Taken together, the Ag-specific nature and location of TRAIL expression suggest that TRAIL expression by CD8⁺ T cells likely relates to their initial programming within the draining lymph nodes (27, 28, 29, 30) and could be amplified by interactions with viral-peptide MHC I complexes in the lungs (27).

The observed difference in morbidity between TRAIL^+/+ and TRAIL^−/− mice, combined with the selective expression of TRAIL on influenza-specific CD8⁺ T cells, suggests that vigorous resolution of the infection requires the participation of TRAIL-expressing CD8⁺ T cells. The likely explanation for the increased morbidity in the TRAIL^−/− mice is that the influenza-specific CD8⁺ T cells are unable to kill influenza-infected cells, but it may also be possible that other factors contribute to the pathology, such as a reduction in the number of lung-infiltrating Ag-specific effector CD8⁺ T cells. Thus, we examined the magnitude and phenotype of the pulmonary CD8⁺ T cell response in influenza-infected TRAIL^+/+ and TRAIL^−/− mice. Interestingly, TRAIL deficiency did not alter the magnitude of the NP₃₆₆ or PA₂₂₄ influenza-specific CD8⁺ T cell response in the lungs (Fig. 4, A and B). The level of IFN-γ produced per cell was also similar between both influenza Ag-specific TRAIL^+/+ and TRAIL^−/− CD8⁺ T cells (TRAIL^+/+ NP₃₆₆ mean fluorescence intensity (MFI) of 235; TRAIL^−/− NP₃₆₆ MFI of 303; TRAIL^+/+ PA₂₂₄ MFI of 242; TRAIL^−/− PA₂₂₄ MFI of 423; not significant as determined by Kruskal-Wallis one-way ANOVA on ranks). However, TRAIL deficiency did result in a significant reduction in influenza-specific CD8⁺ T cell-mediated cytotoxicity in vivo (Fig. 4 C). Indeed, while TRAIL^−/− mice have an equal in vivo E:T ratio to that of TRAIL^+/+ mice, the influenza-specific CD8⁺ T cells killed wild-type DR5^+/+ influenza peptide-pulsed targets with significantly reduced (∼40%) efficiency. Furthermore, when influenza peptide-pulsed DR5^−/− targets were adoptively transferred into either influenza infected TRAIL^−/− or TRAIL^+/+ hosts, killing was reduced ∼60% relative to DR5^+/+ targets in TRAIL^+/+ animals.

The reduced cytotoxicity of TRAIL^−/− CD8⁺ T cells or during transfer of DR5^−/− targets was intriguing given the increased viral load and disease severity observed in TRAIL^−/− mice (Fig. 1). Collectively, our results suggest that between 40 and 60% of influenza-specific CD8 T cell-mediated cytotoxicity within the lungs on day 8 p.i. may be TRAIL/DR5 dependent. However, disruption of a single cytotoxicity pathway was not expected to have major pathological consequences, based on the redundant roles of the Fas/FasL and perforin/granzyme pathways in mediating control of influenza virus infections (3). In those studies, mice deficient in either Fas or perforin showed only marginal changes in pulmonary viral load. It took a deficiency in both perforin⁺ T cells and Fas⁺ target cells to sustain viral titers until day 14 p.i., a time point when virus has normally been eliminated from the lungs. Therefore, we next measured the expression of Fas, FasL, perforin, and granzyme B mRNA in the lungs, as well as FasL and granzyme B protein expression and degranulation potential in TRAIL^+/+ and TRAIL^−/− influenza-specific CD8⁺ T cells found within the lungs. Fas, FasL, granzyme B, and perforin mRNA were all up-regulated to similar levels in both TRAIL^+/+ and TRAIL^−/− mice, peaking at day 8 p.i. (Fig. 2). Furthermore, neither NP₃₆₆- or PA₂₂₄-specific TRAIL^−/− CD8⁺ T cells had altered expression of FasL or granzyme B or changes in the ability to degranulate (i.e., surface CD107a expression) relative to TRAIL^+/+ controls (Fig. 5). Therefore, these results collectively suggest that TRAIL/DR5 interactions may play more prominent roles in CD8⁺ T cell-mediated clearance of influenza virus infected cells than has previously been appreciated.

Since our results suggested that influenza-specific CD8⁺ T cells utilize TRAIL to eliminate virally infected pulmonary epithelial cells and therein control virus infections, we tested the ability of TRAIL^+/+ and TRAIL^−/− influenza-specific effector CD8⁺ T cells to control and resolve an ongoing lethal dose influenza virus infection in TRAIL^+/+ mice. During such infections, endogenous pulmonary influenza-specific CD8⁺ T cells and virus control are limited due to a previously described elimination of effector CD8⁺ T cells during their development in the lymph nodes (18). However, this lethality can be overcome when normal T cell numbers are restored to the lungs (18). When we i.v. adoptively transferred TRAIL^+/+ pulmonary effector CD8⁺ T cells 5 days p.i into lethal dose influenza-infected mice, 83.3% of the mice survived and recovered from the high-dose influenza infection (Fig. 6). In contrast, while the donor TRAIL^−/− effector T cells migrated into the lungs at equivalent numbers to TRAIL^+/+ effector T cells (data not shown), the TRAIL^−/− effector T cells were only able to protect 33.3% of the lethally infected mice, a percentage that was not statistically different from the non-T cell transferred controls. Since no differences in IFN-γ, FasL, granzyme B, or degranulation were observed in the NP₃₆₆ and PA₂₂₄ effector CD8 T cell populations (Fig. 5) and these T cells arrived in the lungs in equivalent numbers upon adoptive transfer, our results suggest that the TRAIL expression or deficiency alone is responsible for the differential ability to protect these mice from lethal dose influenza virus infections.

TRAIL-expressing CD8⁺ T cells, NK cells, and plasmacytoid DC have all been implicated in cytotoxicity and control of viral infections (31, 32, 33). The results presented herein suggest that the differences observed during influenza virus infection of TRAIL^+/+ and TRAIL^−/− mice are mainly due to altered CD8⁺ T cells responses. First, while some minor increases in morbidity were observed within the first 3 days following influenza virus infection (i.e., the window of time normally ascribed to innate (NK cell/pDC) control of infection), significant increases were not seen until after influenza-specific CD8⁺ T cells had arrived in the lungs (i.e., about day 4+ p.i.) (18, 23, 34). Furthermore, the kinetics of TRAIL and DR5 expression within the lungs correspond with the appearance of influenza-specific CD8⁺ T cells in the lungs. TRAIL expression by pulmonary CD8⁺ T cells appears to be directly linked to influenza virus specificity, and adoptive transfer of TRAIL^+/+, but not TRAIL^−/−, T cells can mediate protection from ongoing lethal dose influenza virus infections. Finally, our in vivo cytotoxicity experiments directly show that TRAIL deficiency on T cells or DR5 deficiency on target cells results in significantly (p < 0.029) reduced CD8⁺ T cell cytotoxicity of influenza peptide-pulsed target cells despite the presence of equal numbers of pulmonary influenza-specific CD8⁺ T cells and identical levels of IFN-γ, FasL, granzyme B, and degranulation by influenza-specific TRAIL^+/+ and TRAIL^−/− CD8 effectors.

Of note, our results show that TRAIL expression appears to selectively correlate only with those pulmonary CD8⁺ T cells that are specific for influenza. Furthermore, up-regulation of DR5, the receptor for TRAIL, to high levels on pulmonary epithelial cells is linked to direct infection of the cells with influenza virus. Taken together, these results suggest that elimination of virus-infected cells by influenza-specific CD8⁺ T cells could be specific not only at the level of recognition of virus peptide/MHC I complexes, but also by the high level of DR5 expression. In this manner high expression of DR5 may permit T cell-mediated elimination of virally infected cells and allow survival of any surrounding noninfected epithelial cells or even pulmonary APC that carry viral peptide MHC complexes but have not up-regulated DR5 due to direct infection, an idea that would be consistent with previously described selective TRAIL cytotoxicity within tumor systems (35). Importantly, the amount of DR5 expressed on a cell’s surface is not the only point that determines TRAIL susceptibility. The events that are required for TRAIL-resistant cells to become susceptible to TRAIL are complicated and not well understood. Many viruses significantly alter host cell metabolism, such that it might be predicted that cells infected with viruses acquire sensitivity to death-inducing ligands (including TRAIL). Normal cells infected with respiratory syncytial virus, human CMV, or encephalomyocarditis virus become susceptible to TRAIL-mediated killing (11, 12, 36, 37), and we have also found the TRAIL-resistant human lung adenocarcinoma cell line A549 can be sensitized to TRAIL following influenza virus infection (E. L. Brincks, T. S. Griffith, and K. L. Legge, unpublished data). While it is beyond the scope of this report, we are actively investigating the mechanism(s) that regulate TRAIL susceptibility in influenza virus-infected cells.

In the present study, TRAIL expression by CD8⁺ T cells correlates with reduced viral loads and disease severity; however, increased TRAIL expression may also lead to increased disease during some influenza virus infections (38). H5N1 influenza virus-infected human monocyte-derived macrophages express TRAIL at levels that are able to kill T cells, an outcome that could be inhibited by introduction of anti-TRAIL-R2-blocking Abs (38). In this manner the TRAIL-expressing macrophages are thought to help drive the T cell lymphopenia observed with H5N1 influenza infections (38, 39). Influenza infection commonly induces the production of type I and type II IFN as part of the innate immune response (40, 41, 42). Both types of IFN are potent inducers of TRAIL expression on many cells in the immune system (43, 44), and monocytes/macrophages are exquisitely sensitive to IFN-induced TRAIL expression (45). Thus, while direct H5N1 influenza infection can induce TRAIL expression, the breadth and amount of TRAIL expressed by cells of the immune system may be further enhanced by IFN-mediated events. Therefore, our data and the results from the above studies would collectively suggest that TRAIL expression during influenza infections normally has a beneficial effect on viral control (e.g., CD8⁺ T cell-mediated elimination of infected pulmonary epithelial cells), but that TRAIL may also serve to enhance the virulence of some influenza virus infections. The factors that regulate the beneficial vs deleterious effects, as well as possibly distinct cellular expression patterns of TRAIL during influenza infections, await further study.

Our observation that TRAIL^−/− mice do not significantly reduce viral titers as substantially from day 4 to 6 p.i. when compared with TRAIL^+/+ mice (Fig. 1 B) suggests that TRAIL-mediated cytotoxicity by CD8⁺ T cells may be more important than the Fas and perforin pathways of cytotoxicity during the early stages of influenza infections. This increased dependence on TRAIL at these early times might relate to the low numbers of influenza-specific CD8⁺ T cells present (18, 23, 34), and hence low functional in vivo E:T ratios in the lungs, an idea that would be consistent with TRAIL-dependent killing of some target cell lines in vitro (46). However, at later stages of infection (i.e., days 6–8 p.i.) when the number of effector CD8⁺ T cells has significantly expanded (18, 23, 34), the loss of TRAIL may be compensated for by the other cytotoxicity pathways, resulting in redundant and overlapping mechanisms of viral control and the 1 log reduction in pulmonary virus levels. Regardless, our results importantly show that a third pathway (i.e., TRAIL/DR5) of cytotoxicity is used along with the previously described Fas- and perforin-dependent killing pathways to eliminate and control influenza virus infection.

In conclusion, the results presented herein show that TRAIL plays a role in the regulation and control of influenza virus infections. Specifically, the early adaptive influenza-specific CD8⁺ T cell response appears to utilize TRAIL-mediated lysis of DR5⁺ (i.e., TRAIL receptor) influenza-infected cells in addition to FasL/Fas- and perforin/granzyme-dependent cytotoxicity pathways to control influenza virus infection. Furthermore, our results show that TRAIL and DR5 up-regulation by CD8⁺ T cells and pulmonary epithelial cells is closely linked to either the Ag specificity of the T cells or the infection status of the epithelial cells, respectively. This suggests that TRAIL/DR5-specific interactions may partner with TCR/viral peptide-MHCI interactions to allow the targeted elimination of only influenza virus-infected cells.

We thank Drs. Jonathan W. Heusel and Thomas J. Waldschmidt for critical reading of the manuscript.

The authors have no financial conflicts of interest.