Flow cytometric analysis of pulmonary lymphocytes from mice infected with respiratory syncytial virus

BALB/c mice were infected intra-nasally with respiratory syncytial virus (RSV) and cells recovered from the lungs by single or repeated bronchoalveolar lavage (BAL). Single BAL gave enough cells (1 - 2 x 10(5) cells/mouse) for morphological study of Giemsa stained cytospin preparations, and allowed the same mouse lungs to be assayed for virus titre or processed for histology. In normal or sham-infected mice the majority of recovered cells were macrophages, with less than 5% lymphocytes. Following infection the proportion of lymphocytes increased to about 20% between days 10 and 16 and decreased thereafter. By contrast, histological changes in the lung peaked at day 7 and resolved by day 9 or 10. Repeated BAL yielded a higher proportion of lymphocytes and provided enough cells for flow cytometric study of cell surface markers. The results of two-colour stains with antibodies to L3T4 (CD4), Lyt2 (CD8), mouse CD3, Thy 1.2 and surface immunoglobulin (SIg) were studied by flow cytometry. Analysis of the small non-granular cells (lymphocytes) showed that fewer than 5% of recovered lymphocytes were SIg+ (B cells), while most bore T cell surface markers. Early during infection (day 3-6), 30-60% of lymphocytes were Thy 1.2-, T3-, CD4-/CD8- and SIg- CD8+ cells outnumbered CD4+ cells 2:1 or 4:1 from day 6 of infection. In conclusion, CD8+ T cells constitute the major subpopulation of lymphocytes recovered from the lungs of mice recovering from RSV infection. BAL provides a useful quantitative method of assessing the cellular immune response in the lung following RSV infection.