RESULTS Kinetics of CD8+ T cell accumulation, in vivo effector activity, and virus clearance during experimental influenza infection

Infected respiratory epithelial cells are an important target of the immune response because these cell types support productive infection by influenza viruses; therefore, limiting infection of these cells is essential for virus clearance and recovery (Hou and Doherty, 1995; Topham et al., 1997). Because of the importance of these airway lining cells as targets for both the virus and effector CD8+ T cells, we analyzed the kinetics of influenza-specific CD8+ T cell accumulation simultaneously in the airspaces (cells overlaying the respiratory epithelium as collected in the bronchial alveolar lavage [BAL] fluid) and, in parallel, the subepithelial interstitial compartment (pulmonary interstitium) after sublethal experimental A/PR/8/34 influenza infection of BALB/c mice. We quantified the numbers of influenza-specific IFN-–secreting CD8+ T cells in these two lung compartments over time using the in vitro intracellular cytokine staining (ICCS) assay. As Fig. 1 A demonstrates, virus-specific effector CD8+ T cells were first detectable in the respiratory tract between 5 and 6 d postinfection (dpi) and increased rapidly in both the airspaces and interstitium between 6 and 10 dpi, confirming earlier results from several laboratories (Tripp et al., 1995; Crowe et al., 2003; Lawrence and Braciale, 2004). The kinetics of release of the signature effector T cell proinflammatory cytokine, IFN-, into the BAL fluid during infection (Fig. 1 B) reflected the onset of effector T cell accumulation into the infected lungs (ie, at 5 dpi) and reached maximum levels in the BAL fluid at 6 and 7 dpi. IFN- release into the BAL fluid dropped precipitously thereafter, commensurate with the clearance of infectious virus from the infected lungs (Fig. 1 C). In vivo T cell depletion analysis established CD8+ T cells as major contributors to IFN- production (Fig. S1 A) and virus clearance (Fig. S1 B). Comparable results were obtained in the analysis of infected C57BL/6 mice (unpublished data).