Flow cytometric detection of lymphocyte proliferation in eyes with immunogenic inflammation

Identification and analysis of antigen-activated lymphocytes from sites of ocular inflammation is important to understanding of the role of infiltrating immune effectors in ocular inflammatory disease. Unfortunately, few assays can distinguish activated lymphocytes from the antigen-irrelevant lymphocytes that randomly migrate to sites of inflammation. The authors describe a method that allows the quantitative recovery and identification of activated lymphocytes from mouse eyes. Recovered subconjunctival lymphocytes were simultaneously stained for specific cell-surface markers (with fluorescein-labeled antibodies) and for DNA content (with propidium iodide), then analyzed by flow cytometry. For any subpopulation of lymphocytes, the relative percentage and absolute number of cells in each phase of the cell cycle was determined. The activated subset of T and B cells was quantitated by determining the number of proliferating cells (ie, the number of cells in the S + G2 + M phases of the cell cycle). Using this method, T cell proliferation was found to be a specific component of the subconjunctival immunogenic inflammatory response induced by a local graft-versus-host reaction and by subconjunctival delayed hypersensitivity reactions. In addition, proliferating T cells could be distinguished from nonproliferating B cells produced by the subconjunctival inoculation of a T cell tumor.