Ova specific cells were transferred i. v. to BALB/c mice followed by infection with 1000 EID50 PR8/Ova. Seven dpi, mice were sacrificed, lungs isolated and stained with antibodies to CD4 and KJ126, followed by staining with NKG2A/C/E and intracellular GrB, or intracellular Tbet and Eomes. A) Shown are representative FACS plots and percentage of WT Ova specific CD4 cells (top) or CD25+/− Ova specific CD4 cells (bottom). Also shown are representative 2 parameter histograms for NKG2ACE and GrB after gating on CD4+/KJ126+ cells. B) Representative overlay histograms of Tbet (top) and Eomes (bottom) after gating on Ova specific cells. The solid line represents expression in WT cells while the shaded histogram is expression in CD25+/− cells. C) Lung cells were also restimulated with Ova peptide or media alone for 4h in vitro followed by staining for CD4, KJ126, CD107a/b and IFN-γ. Shown are 2 parameter histograms for CD107a and IFN-γ after gating on WT Ova specific cells (top) or CD25+/− Ova specific cells (bottom) with or without (media) Ova peptide. The absolute number of Ova specific cells in lungs (D) is shown. GrB MFI (E), percent Tbet+ (F) and percent IFN-γ+ (G) after gating on Ova specific cells are shown. Percent NKG2A/C/E and GrB double positive cells (H), percent Eomes+ (I), percent CD107a/b+ cells (J) and CD107a/b MFI (K) are shown after gating on Ova specific cells. The † represents statistically significant differences where p<0.004 by student’s t-test.