Figure 1: Induction of antigen specific CD8+ T cells in the lungs by L. gasseri expressing targeted mini genes of influenza A subunits. Groups of C57BL/6 mice (n=5/group) were vaccinated with L. gasseri harboring empty vector, FluVac-Ctrl, or FluVac-DC (109cfu/mouse) for four consecutive weeks. One week after the last boost, mice were sacrificed and lymphocytes were isolated from the lungs that were perfused with PBS/Heparin solution (20U of Heparin/1 mL PBS) by collagenase digestion. 1A) Lung lymphocytes were incubated with live/dead stains to identify live cells, Fc block, and then PA and NP tetramers in order to identify NP and PA specific CD8+ T cells. 1B) Cells were subsequently stained for their surface expression of CD3, CD8, and CD44, fixed, permeabilized, and intracellularly stained for IFNγ, TNFα (data not shown), and Granzyme B and analyzed by FACS Canto. Experiments were performed at least three times with similar trends.