Figure 4.

Induction of Ag-specific CD4 T_RM cells by cognate Ag-based but not by CpG-based RM-pull strategy during the memory phase of T cell responses to parenteral immunization. Mice were immunized parenterally (i.m.) with AdCh68Ag85A. At day 28 post-immunization, mice were administered i.n. with CpG (single dose) or Ag85 complex (two doses 5 days apart) as described in Figure 2A. Mice were then scarified at specified time points (day 5, day 14, and day 28) post-RM-pull, and mononuclear cells from BAL fluids and lungs were subjected to ex vivo stimulation with Ag85A CD4 peptides, CD4 T_RM cell surface marker, or intracellular cytokine immunostaining. (A) Representative dotplots showing frequencies of CD4+IFN-γ+ cells in the airway (BAL). Line graphs showing kinetic changes in numbers of IFN-γ+CD4 T cells in the airway post-RM-pull. (B) Representative dotplots comparing distribution of CD4+IFN-γ+ cells in the lung parenchymal tissue (LPT) and the lung vasculature (LV) of i.m., i.m./CpG, and i.m./Ag85 mice. Bar graph showing absolute number of CD4+IFN-γ+ cells in LPT and LV. (C) Representative dotplots showing frequencies of CD4+IFN-γ+ cells in the LPT. Line graphs showing kinetic changes in numbers of IFN-γ+CD4 T cells in the LPT post-RM-pull. (D) Representative dotplots show frequencies of IFN-γ+CD4+ T cells co-expressing CD69 and CD49a, or CD69 and CD11a in the airway and LPT at day 28 post-RM-pull. Data are expressed as the mean ± S.E.M. of three mice/group/time point, representative of two independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001 compared with CpG group.