FIG 2.

Effector CD8 T cell response to adjuvanted subunit vaccine in CCR2^−/− mice. Wild-type (WT) or CCR2^−/− mice were immunized intranasally (i.n.) twice (21 days apart) with influenza A H1N1 nucleoprotein (NP) formulated in ADJ (5%) and GLA (5 μg). To distinguish nonvascular cells from vascular cells in the lungs, mice were injected intravenously with fluorescence-labeled anti-CD45.2 antibodies, 3 min prior to euthanasia (CD45.2^+ve, vascular; CD45.2^−ve, nonvascular.) At day 8 postvaccination, single-cell suspensions prepared from the lungs and bronchoalveolar lavage (BAL) fluid were stained with viability dye, followed by D^b/NP366 tetramers in combination with anti-CD4, anti-CD8, anti-CD44, anti-CD69, anti-CD103, anti-T-bet, and anti-TCF-1. (A and B) Lung cells were stained for innate immune cell markers as described in the legend to Fig. 1. Data are percentages of monocytes in the lungs of vaccinated WT and CCR2^−/− mice. (C and D) FACS plots show percentages of NP366 tetramer-binding cells among CD8 T cells. (E) Percentages of vascular and nonvascular cells among NP366-specific CD8 T cells. (F to I) FACS plots are gated on D^b/NP366 tetramer-binding CD8 T cells, and numbers are percentages of CD69⁺/CD103⁺ (F and G) and T-bet⁺ and TCF-1⁺ (H and I) cells in the respective gates or quadrants. (I) MFI of TCF-1 and T-bet in NP366-specific CD8 cells in lungs and ratio of TCF-1 to T-bet MFI in NP366-specific CD8 cells in lungs. Data are pooled from three independent experiments (C and D) or represent one of three independent experiments (A, B, and F to I). Mann-Whitney U test; *, **, and *** indicate significance at P values of <0.05, <0.01, and <0.001, respectively.