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Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells.

Halle S, Dujardin HC, Bakocevic N, Fleige H, Danzer H, Willenzon S, Suezer Y, Hämmerling G, Garbi N, Sutter G, Worbs T, Förster R - J. Exp. Med. (2009)

Bottom Line: After intratracheal application, in vitro-differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy.Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance.Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany.

ABSTRACT
Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro-differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

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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.
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fig5: 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.

Mentions: Immunohistological analysis of lung cryosections by epifluorescence microscopy (Fig. 5 A) as well as ex vivo two-photon imaging of BALT within 1–2-mm thick lung slices (Fig. 5 B) revealed that adoptively transferred EGFP+ Ag-DCs as well as TAMRA-labeled OT-I T cells and CMAC-labeled polyclonal T cells were abundantly present in the BALT of MVA-treated WT (Fig. 5, A and B, left) and untreated CCR7−/− (Fig. 5 B, right) recipients. Structural features of BALT, such as adjacent bronchi and blood vessels, were readily identified by their characteristic autofluorescence signals (Fig. 5, A and B), and the second harmonics generation (SHG) signal resulting from two-photon excitation allowed the additional visualization of collagen fibers surrounding the basal surface of bronchial epithelium (Fig. 5 B). To analyze the cellular dynamics within BALT, lung slices were placed in a custom-built incubation chamber and constantly superfused with oxygenated (95% O2/5% CO2) medium at 37°C. Imaging BALT ex vivo 24 and 48 h after the i.t. transfer of Ag-DCs into MVA-treated WT recipients, we found TAMRA-labeled OVA-specific OT-I T cells to interact intensively with EGFP+ Ag-DCs (Fig. 5, C and D; Videos 1 and 2; and not depicted). Interestingly, the observed migration and interaction behavior of OT-I T cells in BALT was reminiscent of the cellular dynamics reported for CD8+ T cells during priming in LNs (Mempel et al., 2004): at 24 h after i.t. DC transfer, long-lasting stable contacts between OT-I T cells and Ag-DCs were predominant, largely confining the migration of interacting OT-I T cells to DC-rich areas of BALT (Fig. 5, C and D; and Videos 1 and 2). At 48 h after i.t. transfer of Ag-DCs, many OT-I T cells were clearly enlarged, exhibiting slow swarming movements in the vicinity of EGFP+ Ag-DCs (unpublished data), probably indicating the start of their proliferative activity (Mempel et al., 2004). In contrast, CMAC-labeled polyclonal CD8+ WT T cells did not engage in stable interactions with Ag-DCs at either time point (Fig. 5 C, Video 1, and not depicted). The quantitative analysis of T cell motility parameters revealed much lower cellular velocities and displacements, and consequently highly reduced motility coefficient values, for OT-I cells when compared with the polyclonal CD8+ T cell population (Fig. 5 E and not depicted), further corroborating the antigen-specific nature of the observed OT-I T cell–DC interaction behavior in BALT.


Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells.

Halle S, Dujardin HC, Bakocevic N, Fleige H, Danzer H, Willenzon S, Suezer Y, Hämmerling G, Garbi N, Sutter G, Worbs T, Förster R - J. Exp. Med. (2009)

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.
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fig5: 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.
Mentions: Immunohistological analysis of lung cryosections by epifluorescence microscopy (Fig. 5 A) as well as ex vivo two-photon imaging of BALT within 1–2-mm thick lung slices (Fig. 5 B) revealed that adoptively transferred EGFP+ Ag-DCs as well as TAMRA-labeled OT-I T cells and CMAC-labeled polyclonal T cells were abundantly present in the BALT of MVA-treated WT (Fig. 5, A and B, left) and untreated CCR7−/− (Fig. 5 B, right) recipients. Structural features of BALT, such as adjacent bronchi and blood vessels, were readily identified by their characteristic autofluorescence signals (Fig. 5, A and B), and the second harmonics generation (SHG) signal resulting from two-photon excitation allowed the additional visualization of collagen fibers surrounding the basal surface of bronchial epithelium (Fig. 5 B). To analyze the cellular dynamics within BALT, lung slices were placed in a custom-built incubation chamber and constantly superfused with oxygenated (95% O2/5% CO2) medium at 37°C. Imaging BALT ex vivo 24 and 48 h after the i.t. transfer of Ag-DCs into MVA-treated WT recipients, we found TAMRA-labeled OVA-specific OT-I T cells to interact intensively with EGFP+ Ag-DCs (Fig. 5, C and D; Videos 1 and 2; and not depicted). Interestingly, the observed migration and interaction behavior of OT-I T cells in BALT was reminiscent of the cellular dynamics reported for CD8+ T cells during priming in LNs (Mempel et al., 2004): at 24 h after i.t. DC transfer, long-lasting stable contacts between OT-I T cells and Ag-DCs were predominant, largely confining the migration of interacting OT-I T cells to DC-rich areas of BALT (Fig. 5, C and D; and Videos 1 and 2). At 48 h after i.t. transfer of Ag-DCs, many OT-I T cells were clearly enlarged, exhibiting slow swarming movements in the vicinity of EGFP+ Ag-DCs (unpublished data), probably indicating the start of their proliferative activity (Mempel et al., 2004). In contrast, CMAC-labeled polyclonal CD8+ WT T cells did not engage in stable interactions with Ag-DCs at either time point (Fig. 5 C, Video 1, and not depicted). The quantitative analysis of T cell motility parameters revealed much lower cellular velocities and displacements, and consequently highly reduced motility coefficient values, for OT-I cells when compared with the polyclonal CD8+ T cell population (Fig. 5 E and not depicted), further corroborating the antigen-specific nature of the observed OT-I T cell–DC interaction behavior in BALT.

Bottom Line: After intratracheal application, in vitro-differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy.Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance.Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany.

ABSTRACT
Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro-differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

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Related in: MedlinePlus