Figure 5.

Induction of Ag-specific CD8 T_RM cells in the lung by RM-pull strategy requires CD4 T cells. (A) Experimental schema. Mice were immunized i.m. with AdCh68Ag85A. At day 28 post-immunization, mice were administered i.n. with Ag85 complex proteins (two doses 5 days apart). In a group of mice (Ag85/αCD4 mAb), CD4 T cells were depleted i.p. by injection of αCD4 mAb a day before the first dose of Ag85 complex and then a day after the last dose of Ag85 complex. Depletion of CD4 T cells was maintained by repeated αCD4 mAb injections every 7 days until the termination of experiment. Mice were sacrificed 14 days after the final Ag85 dose. Mononuclear cells from BAL and lungs were subjected to ex vivo stimulation with Ag85A CD8 peptides, and Ag85A tetramer and CD8 T_RM surface marker or intracellular cytokine immunostaining. Bar graphs showing frequencies (B) and absolute numbers (C) of tetramer+ cells in the airway (BAL), lung parenchymal tissue (LPT), lung vasculature (LV), and mediastinal lymph nodes (MLN). (D) Representative dotplots and bar graph showing frequencies and numbers of tetramer+ CD8 T cells co-expressing CD69 and CD103, respectively. (E) Representative dotplots and bar graph depicting frequencies and numbers of IFN-γ+ CD8 T cells. Data are presented as the mean ± S.E.M. of three mice/group/time point, representative of two independent experiments. *P < 0.05, **P < 0.01 compared with Ag85 group.