Evaluation of the adaptive immune response is critical to the advancement of our basic knowledge and understanding of respiratory syncytial virus (RSV). The cellular composition in the lung following RSV infection is often evaluated using flow cytometry. However, a limitation of this approach has been the inability to readily distinguish cells that are within the lung parenchyma from cells that remain in the pulmonary blood vessels. Herein, we detail a procedure to evaluate the adaptive immune response via flow cytometric analysis that incorporates an in vivo intravascular staining technique. This technique allows for discrimination of immune cells in the lung tissue from cells that remain in the pulmonary vasculature following perfusion. Therefore at any given time point following an RSV infection, the leukocytic populations in the lung parenchyma can be quantified and phenotypically assessed with high resolution. While we focus on the T lymphocyte response in the lung, this technique can be readily adapted to examine various leukocytic cell types in the lung following RSV infection.
Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. CD8 T cells play a critical role in mediating viral clearance following an acute RSV infection. However the role of memory CD8 T cells in providing protection against RSV remains understudied. To generate high magnitude CD8 T cell memory in the absence of CD4 T cell memory and antibodies, we immunized naïve mice with dendritic cells pulsed with an RSV-derived peptide followed by a boost with a recombinant Listeria monocytogenes expressing the same RSV-derived epitope. Memory CD8 T cells significantly reduced viral titers following RSV challenge, but did so at the expense of increased airway dysfunction, weight loss, and mortality compared to controls. Importantly, the severe immunopathology and mortality observed was specific to the context of an RSV infection, as prime-boosted mice challenged with a recombinant influenza virus expressing the same RSV-derived epitope did not exhibit enhanced disease. The induction of a pro-inflammatory cytokine storm mediated by TNF-α and IFN-γ was observed in the serum of prime-boosted mice following RSV challenge. Additionally, RSV-specific memory CD8 T cells produced large amounts of IFN-γ locally within the lung, and adoptive transfer of wild-type but not IFN-γ-deficient memory CD8 T cells resulted in enhanced airway dysfunction and weight loss. Our results indicate that memory CD8 T cells are able to mediate protection against RSV infection. However, memory CD8 T cells acting alone in the absence of antibodies and memory CD4 T cells induce significant immunopathology and mortality through the induction of a systemic pro-inflammatory cytokine storm and local IFN-γ production.
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