Imaging and Defining Emergent Behaviors of the Immune Response

The Immune System in the Lung

Asthma Studies:

The Immune system is always engaged in surveilling the lung. This involves ongoing ‘sampling’ of the airway contents, as we reveal, by a subset of phagocytic cells called dendritic cells. These cells compete with another set of cells, called alveolar macrophages, which also can ingest materials that are breathed in. In Allergic Airway diseases (of which Asthma is an example), direct imaging reveals a bottleneck that forms adjacent to the airway; a meeting site is formed where inhaled particle appears to be ‘presented’ to instigating T cells. This site will be a focus for our ongoing work, in an effort to understand how immunity and tolerance are achieved at this mucosal surface.


How do immune cells interact with a primary tumor?

 Live slice imaging of T cell interactions with tumor and with tumor-associate myeloid cells. In this movie, blue T cells are seen to interact with myeloid immune cells that have been programmed to turn them ‘off’ (green, yellow and orange cells, so-called Tumor Associated Macrophages or TAMs) as well as with cells that can turn T cells ‘on’ (deep red cells, Dendritic Cells). Note that the TAMs get the priveledged position, just next to the tumor (tumor is in the black space in the center).  Details: Live tumor slice movie from PyMTchOVA x Cx3cr1-eGFP x Cd11c-mcherry with Day 4 adoptively transferred Actin-CFP OT-1 CD8+ T cells (Blue). DC1/2 populations labeled in red, TAM1 in green and, TAM2 in yellow. Movie spans a 55-minute imaging window. First sequence shows whole view (scale bar = 30um) for duration of time, then movie zooms (scale bar = 10um) to highlight T cell interactions with green TAM1 and yellow TAM2 cells, then subsequently red DC1/2 cells.


Real time bright field imaging of a large airway in a lung section two hours after sectioning shows ciliary movement on the surface of a large airway. 
Movie plays at the speed that it was acquired (i.e. real-time).  From Thornton et al. 2012.

The surveillance of the lung by lung dendritic cells.
Lung section of CD11c-EYFP (green) Actin-CFP (blue) shows DC localization just below the surface of the airway and between alveoli.  Timelapse shows center-of-mass motility of DCs along the surface of the airway with little center-of-mass motility of alveolar DCs. Note that timelapse repeats 2 times. Timestamp indicates elapsed time. From Thornton et al. 2012.

Ongoing surveillance of the surfactant/airspace in the atria of alveoli.
Zoom-in and timelapse of an OVA allergen challenged CD11c-EYFP (green) lung stained with Hoechst (blue) shows alveolar DCs sending processes along the alveolar epithelium. Note that timelapse repeats 3 times after the zoom. Timestamp indicates elapsed time. From Thornton et al. 2012.

Dendritic cells in alveoli actively ingest inhaled particulates.
Zoom-in and timelapse of an OVA allergen challenged c-fms-EGFP (green) Actin-CFP (blue) lung after inhalation of red polystyrene beads (red) shows direct uptake of a bead by an alveolar DC. Note that timelapse repeats 3 times after the zoom. Timestamp indicates elapsed time. From Thornton et al. 2012.

Dendritic cells move to and accumulate near the airways in asthma where they interact with T cells.
Zoom-in and timelapse of an OVA allergen challenged CD11c-EYFP (green) Actin-CFP (blue) that received CD2-RFP OTII T cells (red) before sensitization shows sustained T-DC interactions near an airway.  Note that timelapse repeats 3 times after the zoom. Timestamp indicates elapsed time. From Thornton et al. 2012.

Understanding the origins and nature of lung injury:

Exposure of the lungs to overinflation, as might happen in ventilator-induced lung injury, or to lipid products from bacteria are associated with clinical lung-injury.

Dynamics of Fluid Leakage into Lung in response to bacterial products.
Two-photon video of lung tissue marked with actin-CFP (blue) showing dextran leak (red) into the extravascular space 50 min post-LPS treatment (5 mg/kg, i.t., 40 mm z stack). From Looney et al. Nature Methods 2011.

 

 

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