Figure 7.

Involvement of TNF-α in induction of CD8 T_RM cells in the lung by RM-pull strategy. (A) Bar graph demonstrating levels of IFN-γ and TNF-α in BAL of i.m.-immunized animals at day 5 following RM-pull with Ag85 complex proteins without (Ag85) and with CD4 T cell depletion (Ag85/αCD4 mAb). (B) Experimental schema. Mice were immunized i.m. with AdCh68Ag85A. At day 28 post-immunization, mice were administered i.n. with Ag85 complex (two doses 5 days apart). The mice were depleted of TNF-α by i.n. delivery of an isotype Ab or TNF-α mAb every other day starting at day 1 post-RM-pull. At day 5 post-RM-pull, mononuclear cells from BAL, LPT, and MLN were ex vivo stimulated with Ag85A CD4 or CD8 peptides, and immunostained for tetramer, T_RM surface markers, and intracellular cytokines. (C) Bar graph demonstrating numbers of tetramer+ CD8 T cells in BAL, LPT, and MLN. (D) Bar graph showing numbers of IFN-γ+ CD8 T cells in LPT. (E) Bar graph showing numbers of tetramer+ CD8 T cells co-expressing CD69 and CD103, or CD69 and CD49a in LPT. (F) Bar graph showing numbers of IFN-γ+ CD4 T cells in BAL and LPT. (G) Bar graph showing numbers of IFN-γ+ CD4 T cells co-expressing CD69 and CD49a, or CD69 and CD11a in BAL. Data are presented as the mean ± S.E.M. of three mice/group, representative of two independent experiments. *P < 0.05, ***P < 0.001 compared with Ag85A group.