The magnitude of the T cell response to a clinically significant dose of influenza virus is regulated by TRAIL

Abstract

An immune response of appropriate magnitude should be robust enough to control pathogen spread but not simultaneously lead to immunopathology. Primary infection with influenza A virus (IAV) results in a localized pulmonary infection and inflammation and elicits an IAV-specific CD8 T cell immune response necessary for viral clearance. Clearance of IAV-infected cells, and recovery from infection, is mediated by perforin/granzyme B- and Fas/FasL-mediated mechanisms. We recently reported that TRAIL is another means by which IAV-specific CD8 T cells can kill IAV-infected cells. The current study examined the role of TRAIL in the pulmonary CD8 T cell response to a clinically significant IAV [A/PR/8/34 (PR8; H1N1)] infection (i.e., leads to observable, but limited, morbidity and mortality in wild-type [WT] mice). Compared with WT mice, IAV-infected Trail(-/-) mice experienced increased morbidity and mortality despite similar rates of viral clearance from the lungs. The increased morbidity and mortality in Trail(-/-) mice correlated with increased pulmonary pathology and inflammatory chemokine production. Analysis of lung-infiltrating lymphocytes revealed increased numbers of IAV-specific CD8 T cells in infected Trail(-/-) mice, which correlated with increased pulmonary cytotoxic activity and increased pulmonary expression of MIG and MIP-1α. In addition, there was decreased apoptosis and increased proliferation of IAV-specific CD8 T cells in the lungs of Trail(-/-) mice compared with WT mice. Together, these data suggest that TRAIL regulates the magnitude of the IAV-specific CD8 T cell response during a clinically significant IAV infection to decrease the chance for infection-induced immunopathology.

Figure 1. TRAIL deficiency correlates with increased disease severity during a clinically-significant IAV infection

WT or Trail^−/− C57BL/6 mice (n = 6 mice/group) were infected i.n. with 1500 EIU of A/PR/8/34 and weighed daily to assess morbidity (A) and mortality (B). In A, the values displayed represent the daily weight relative to the weight on day of infection. In B, data represent the percentage of mice surviving on the given day after infection; significantly increased mortality was observed in the Trail^−/− mice. Data are representative of 2 separate experiments. C. Given a clinically-significant IAV infection (1500 EIU of A/PR/8/34), WT and Trail^−/− mice have similar viral titers and clearance. At indicated days after infection, lungs were harvested, and pulmonary viral titers were assessed by determining the TCID₅₀ in Madin-Darby canine kidney cell cultures. No significant difference was observed in the viral titers or the rate of viral clearance at the clinical dose of infection. Data are representative of 2 separate experiments with 3–5 mice per group.

Figure 2. Trail^−/− mice have increased pulmonary cellular infiltration and increased inflammation during a clinically-significant IAV infection

WT or Trail^−/− C57BL/6 mice (n = 6 mice/group) were infected with 1500 EIU of A/PR/8/34. On various days after infection, lungs were harvested and insufflated with 10% buffered formalin. Subsequently, the lung tissue was sectioned, mounted, and stained with Hematoxylin and Eosin (H&E). Images are 10X magnification. The identity of the slides were blinded and slides were evaluated; scores for each time point are indicated in the insert. Averaged results are presented, and statistical comparisons between WT and Trail^−/− mice were done using the Wilcoxon two-sided two-sample exact test (* = p < 0.05).

Figure 3. Trail^−/− mice display enhanced IAV-specific CD8⁺ T cell-mediated in vivo cytotoxicity compared to WT mice, despite similar cytotoxic molecule expression

A. The pulmonary IAV-specific CD8 T cell response in WT or Trail^−/− C57BL/6 mice infected with 1500 EIU of A/PR/8/34 was measured by in vivo cytotoxicity assay on d 8 p.i. Symbols represent killing in individual mice, and bars represent mean killing. Percentage IAV-specific killing was calculated by comparing unpulsed target lysis to IAV peptide-pulsed target lysis. Target cells were verified to be DR5⁺ by flow cytometry (data not shown), and target cell frequencies were normalized to ratios harvested from transfers into naïve mice. B. In contrast to the in vivo cytotoxicity, the in vitro cytotoxic activity of WT and Trail^−/− IAV-specific CD8 T cells was similar. IAV-specific CD8 T cells were MACS-purified from the lungs of WT or Trail^−/− C57BL/6 mice infected with 1500 EIU of A/PR/8/34 on 8 d p.i. The T cells were then cultured with unpulsed or IAV peptide-pulsed ⁵¹Cr-labeled splenocytes at a 50:1 E:T ratio for 18 h. Bars represent the mean (± S.D.) specific lysis measured from triplicate wells. No significant (n.s.) difference was observed between groups containing WT and Trail^−/− effector cells. C–D. Pulmonary T cells from WT and Trail^−/− mice have similar expression of effector molecules. WT or Trail^−/− C57BL/6 mice were infected with 1500 EIU of A/PR/8/34 and then lungs were harvested on d 8 p.i. Isolated cells were stained with anti-CD8α, NP₃₆₆ tetramer or PA₂₂₄ tetramer, anti-CD3ε, anti-granzyme B or isotype control antibody, and anti-FasL or isoptye control antibody. Solid line histograms represent FasL or Granzyme B staining on CD8⁺tetramer⁺ T cells. Gray histograms represent isotype control staining. For TNF and CD107a analysis (D), isolated cells were incubated with NP₃₆₆ or PA₂₂₄ peptides or control media, brefeldin A, and anti-CD107a for 5 h. After incubation, the cells were stained with anti-CD8α, anti-IFNγ or isotype control antibody, and anti-TNF or isotype control antibody. Solid line histograms represent TNF or CD107a expression on CD8⁺IFNγ⁺ cells. Gray histograms represent isotype control staining.

Figure 4. Trail^−/− mice given a clinically-significant IAV infection show enhanced pulmonary T cell recruitment compared to WT mice

WT or Trail^−/− C57BL/6 mice were infected with 1500 EIU of A/PR/8/34. A. On d 6, 8, and 10 p.i., lungs were harvested and homogenized, and the isolated cells were stained with anti-CD3ε, anti-CD8α, and NP₃₆₆ tetramer or PA₂₂₄ tetramer. The number of CD8⁺tetramer⁺ T cells from the infected WT or Trail^−/− mice was enumerated using total pulmonary cell counts and flow cytometry. Data are averaged from 5 mice per group. B. On d 8 p.i., lungs were harvested and homogenized, and the isolated cells were incubated in vitro with NP₃₃₆ or PA₂₂₄ peptide for 6 h. The number of Ag-specific CD8⁺ T cells, based on IFNγ production after restimulation, from the infected WT or Trail^−/− mice was enumerated using total pulmonary cell counts and flow cytometry. Data are averaged from 5 mice per group.

Figure 5. Trail^−/− mice have increased pulmonary chemokine expression during a clinically-significant IAV infection compared to WT mice

WT or Trail^−/− C57BL/6 mice were infected with 1500 EIU of A/PR/8/34. A. Lungs were harvested on d 6 p.i. and homogenized in 3 ml of DMEM. Subsequently, the pulmonary chemokine expression was determined using multiplex analysis. Data presented are the average chemokine concentration measured from 4 WT or Trail^−/− mice, and are representative of two independent experiments. B. Lungs were harvested on d 8 p.i., and the isolated cells were stained with anti-CD8α, NP₃₆₆ tetramer, PA₂₂₄ tetramer, anti-CD3ε, anti-CXCR3, anti-CCR5, or isotype control. Histograms show CXCR3 and CCR5 expression on CD3⁺CD8⁺ tetramer⁺ T cells from WT (solid line) and Trail^−/− (dashed line) mice, or the isotype control (shaded histogram). Data are representative of 5 mice from 2 independent experiments. C. Abrogation of chemokine signals to T cells blocks their migration to the lung after IAV infection. Details of the experimental design are presented in Supplemental Figure 4.

Figure 6. Decreased apoptosis of IAV-specific CD8 T cells in the lungs of Trail^−/− mice given a clinically-significant IAV infection compared to WT mice

WT or Trail^−/− C57BL/6 mice were infected with 1500 EIU of A/PR/8/34. On d 6, 8, and 10 p.i., lungs were harvested and the isolated cells were analyzed by flow cytometry for apoptosis of IAV-specific CD8 T cells as measured by the frequency of active caspase3/7⁺ cells of the CD3⁺CD8⁺NP₃₆₆ or PA₂₂₄ tetramer⁺ cells. Data are averaged from 4 mice/group/timepoint.

Figure 7. Increased number of actively proliferating IAV-specific CD8 T cells in Trail^−/− mice given a clinically-significant IAV infection compared to WT mice

WT or Trail^−/− B6 mice were infected with 1500 EIU of PR8. On d 7 p.i., mice were given CFSE i.n., followed by BrdU i.n. 2 h later. Lungs were harvested 4 h later, homogenized, and the isolated cells were analyzed by flow cytometry for proliferation of IAV-specific CD8 T cells as measured by the frequency of BrdU⁺ cells of the CFSE⁺CD8⁺NP₃₆₆ or PA₂₂₄ tetramer⁺ cells. The gating strategy, representative plots gated on CFSE⁺CD8⁺ cells (A), and averaged data (B) based on 4 mice/group are shown.