Visualization of antigen-specific T cell–DC interactions during T cell priming within BALT. TAMRA-labeled CD8⁺ OT-I T cells and CMAC-labeled polyclonal CD8⁺ WT T cells were injected i.v. into BALT-bearing recipients. 24 h later, SIINFEKL-loaded EGFP⁺ bone marrow–derived DCs (Ag-DCs) were i.t. transferred into the same animals, and lungs were explanted an additional 24 or 48 h later. (A) Cryosections of paraformaldehyde (PFA)-fixated lungs isolated 24 h after i.t. transfer of Ag-DCs into MVA-treated WT mice. (left) EGFP⁺ Ag-DCs and TAMRA⁺ OT-I T cells localize into BALT. (right) Higher magnification image of a BALT structure harboring Ag-DCs, OT-I T cells, and polyclonal CD8⁺ WT T cells. (B) Visualization of BALT within lungs of MVA-treated WT (left) or CCR7^−/− (right) mice by ex vivo two-photon microscopy. Maximum intensity projections of three-dimensional imaging volumes (left: eight Z-slices, 7.5-µm spacing; right: seven Z-slices, 6-µm spacing). Collagen fibers surrounding the basal surface of the bronchial epithelium (dashed line) are visualized by SHG. Asterisks represent the bronchial lumen, and arrowheads indicate a blood vessel. (C) Analysis of T cell–DC interactions within BALT of MVA-treated WT mice by two-photon microscopy (excitation wavelength = 780 nm). 24 h after i.t. transfer of EGFP⁺ Ag-DCs, TAMRA⁺ OT-I T cells exhibit a highly confined migration behavior in the vicinity of Ag-DCs. In contrast, CMAC⁺ polyclonal WT control T cells display a much higher motility (Video 1). (D) The experiment was performed as in C but imaged at 865 nm (Video 2). (E) Motility parameter analysis for OT-I and polyclonal CD8⁺ WT T cells migrating within BALT 24 h after transfer of Ag-DCs. (left) Dots represent individual cell tracks, and bars indicate median average track speed. (middle) Mean displacement plots. (right) Motility coefficient (means + SD). **, P < 0.01. (F) 24 h after i.t. transfer of Ag-DCs into CCR7^−/− recipients, EGFP⁺ DCs were found to enter BALT by directional interstitial migration (arrowhead; Video 3). In contrast, DCs remain largely stationary during Ag presentation within BALT (dashed yellow box; Video 3). Data in A–F are representative of two to four mice per group in two independent experiments. (G) TAMRA⁺ OT-I T cells within BALT display enlarged cell bodies and nuclei 48 h after i.t. transfer of Ag-DCs. The maximum diameters of DAPI-stained nuclei of TAMRA⁺ (OT-I) as well as TAMRA⁻CD3⁺ endogenous T cells within BALT were measured on cryosections. Individual values (dots) and means (red bars) of 140 randomly chosen cells from four sections from two mice are shown. ***, P < 0.001. (H) CSFE profiles of OT-I T cells isolated from the lung, brLNs, or mesenteric LNs 4 d after the i.t. transfer of Ag-DCs in mice treated with FTY720 (initial gavage of 1 mg/kg of body weight on day 0, with drinking water supplemented with 2.5 µg/ml FTY720 afterward; representative data of four mice analyzed in two independent experiments). Bars, 50 µm.