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. 2009 Jan 15;182(2):1174-81.
doi: 10.4049/jimmunol.182.2.1174.

Pulmonary V gamma 4+ gamma delta T cells have proinflammatory and antiviral effects in viral lung disease

Jonathan Dodd  1 , Sabine Riffault, Jayanie S Kodituwakku, Adrian C Hayday, Peter J M Openshaw
Affiliations

Pulmonary V gamma 4+ gamma delta T cells have proinflammatory and antiviral effects in viral lung disease

Jonathan Dodd et al. J Immunol. .

Abstract

Host defenses, while effecting viral clearance, contribute substantially to inflammation and disease. This double action is a substantial obstacle to the development of safe and effective vaccines against many agents, particularly respiratory syncytial virus (RSV). RSV is a common cold virus and the major cause of infantile bronchiolitis worldwide. The role of alphabeta T cells in RSV-driven immunopathology is well studied, but little is known about the role of "unconventional" T cells. During primary RSV challenge of BALB/c mice, some Vgamma7+ gammadelta T cells were present; however, immunization with a live vaccinia vector expressing RSV F protein substantially enhanced Vgamma4+ gammadelta T cell influx after RSV infection. Harvested early, these cells produced IFN-gamma, TNF, and RANTES after ex vivo stimulation. By contrast, those recruited 5 days after challenge made IL-4, IL-5, and IL-10. Depletion of gammadelta T cells in vivo reduced lung inflammation and disease severity and slightly increased peak viral replication but did not prevent viral clearance. These studies demonstrate a novel role for gammadelta T cells in the development of immunopathology and cellular influx into the lungs after immunization and RSV challenge. Though a minor population, gammadelta T cells have a critical influence on disease and are an attractive interventional target in the alleviation of viral lung disease.

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Figures

FIGURE 1
FIGURE 1
γδ T cell response to RSV challenge is enhanced by prior immunization. Mice were scarified with rVV-β-gal, or –F on day 0 and challenged intranasally with RSV on day 14. Weights of β-gal (control) immunized (formula image) and F-primed (■) mice were monitored daily and the percent original body weight calculated (a). The number of TCRδ+CD3+ cells in the lymphoid gate from the BAL (b) or lung (c) are shown. Experiments were performed at least three times containing five mice per group.
FIGURE 2
FIGURE 2
Representative FACS analysis of TCRδ+CD3+ cells from the BAL of mice. Mice were scarified with rVV-β-gal (control), or rVV-F on day 0 and challenged intranasally with RSV on day 14. TCRδ+CD3+ cells in the BAL were identified by flow cytometry. Representative dot plots are shown for mice immunized with rVV-βgal or rVV-F over the time course of RSV infection. The percentage of TCRδ+CD3+ cells in the lymphoid gate (identified by size and granularity) is shown for each plot. Experiments were performed at least three times containing five mice per group.
FIGURE 3
FIGURE 3
Clonotypic analysis of γ-chain expression by γδ T cells. Mice were primed with rVV-F and challenged with RSV on day 14. BAL and lung samples were harvested on day 4 after challenge. BAL and lung cells from infected mice were counted and stained with PE-conjugated anti-γδ TCR and either purified anti-Vγ4, -Vγ5, or -Vγ7 hamster mAbs. Cells were then stained with FITC-conjugated anti-hamster Ab and analyzed. Representative dot plots are shown. Percentage BAL cells that expressed the detected γ-chain are shown. At least 20,000 cells were collected from each sample. Experiments were performed at least three times containing five mice per group.
FIGURE 4
FIGURE 4
Cytokine content of lung γδ T cells. Mice were primed with rVV-F and challenged with RSV on day 14. Lung cells were harvested at various time points, restimulated as described in Materials & Methods, stained with anti-C-γδ-PE, permeabilized, and stained for IFN-γ, RANTES, TNF (a) and IL-4, IL-5, or IL-10 (b). Isotype controls for each Ab were used to determine specific staining. The percentage of lung γδ T cells producing each cytokine is shown. Five mice were analyzed at each time point. C, Representative dot plots (right) and their respective isotype controls (left) from individual mice are shown (day 0 for IFN-γ, day 7 for other cytokines). Experiments were performed at least three times containing five mice per group.
FIGURE 5
FIGURE 5
Effect of γδ T cell depletion on weight loss in RSV-challenged, immunized mice. Mice primed with rVV-F, were after 14 days treated with isotype control (open symbols) or αCγ mAb UC7–13D5 (■) −1 and +2 day relative to RSV challenge. On day 4 after RSV challenge BAL cells were stained for γδ T cells as described in Fig. 1. γδ T cells were present in mice treated with control Ab (a) but not detected in those treated with αCγ mAb (b). Mouse weights are shown as mean ± SEM of starting weights (c). Total RNA was extracted from lungs of immunized, challenged mice at various time points and treated as described in Materials and Methods. The number of viral genome copies was based on detection of the L gene DNA sequence; plasmids (107 to 101 copies) containing the L gene were used as standards and a nontemplate control as the negative (d). Results are shown as mean ± SEM of five mice per group of TCRγδ depleted mice (■) or controls (open symbols). *, p < 0.05; **, p < 0.02; ***, p < 0.001.
FIGURE 6
FIGURE 6
Cytokine levels in bronchial lavage fluid and supernatant of cultured lung cells. Mice were immunized with rVV-F and challenged with RSV with and without in vivo γδ T cell depletion, as described in the legend to Fig. 5. Sequential BAL samples were taken after RSV challenge and tested for IFN-γ (a), IL-4 (b), and RANTES (c) by sandwich ELISA. On day 4 and 10, cells were extracted from whole lungs and stimulated with RSV Ag. IFN-γ (d), IL-4 (e), and RANTES (f) release into supernatants was detected by sandwich ELISA.
FIGURE 7
FIGURE 7
Cell phenotypes and Ab production in the BAL and lungs of immunized, RSV-challenged mice with and without depletion. Total BAL (a) and lung (d) cell recoveries were counted by trypan blue exclusion. BAL cells were stained for CD8 (b), CD4 (e), B220 (c), or DX5 (f) surface markers. BAL supernatants were stored at −70°C before total IgE (g) and IgA (h) detection using biotinylated Abs according to the manufacturer's instructions (as described in Materials and Methods). Results are mean ± SEM of five mice per group of TCRγδ-depleted mice (■) or controls (open symbols). *, p < 0.05; **, p < 0.02.
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