von Andrian Lab Videos
 

 

T CELL PRIMING BY DENDRITIC CELLS IN LYMPH NODES

Nature Supplementary Movie 1A.mp4

Inhomogeneous distribution of Ag-loaded DCs and T cells in LNs early after LN entry. A 30 min time-lapse sequence recorded 4-5 hours after transfer of T cells (green), showing clustering of Ag-bearing, LPS-activated DCs (red) and T cells in a LN T cell area (corresponding to supplemental figure S1b). The reticular network is visualized through second harmonic generation of collagen fibers (blue). No anti-L-selectin was injected in this experiment. Images are a rendition of 3 optical sections spaced 5 mm apart in z corresponding to a 10 mm thick volume of the LN. Scale bar = 25 mm. Time is shown in minutes and seconds. Events are accelerated 225x over real-time.


Nature Supplementary Movie 1B.mp4

DCs cluster around HEV early after LN entry. A 1 min sequence from the same experiment as in video 1A, recorded during i.v.-injection of FITC-dextran (yellow-green) to delineate blood vessels. Rapid filling of all vessels indicates adequate perfusion of the LN preparation. Note that DCs (red) preferentially localized along the branched HEV in the center of the field of view (blood flow from top to bottom). The reticular network is visualized through second harmonic generation of collagen fibers (blue). Images represent one optical section through the LN. Scale bar = 50 mm. Time is shown in seconds. Events are accelerated 9x over real-time.


Nature Supplementary Movie 2.mp4

Homogenous distribution of DCs and rapid T cell migration in LNs late after LN entry. Low magnification view of a LN T cell area 44h after transfer of T cells (green). The reticular network is visualized through second harmonic generation of collagen fibers (blue). Many Ag-loaded DCs (red) appear to attach to fibers. While DCs have decreased in motility at this time-point, activated T cells migrate at high velocities. Images represent volume renditions of six optical z-section spaced 6 µm apart (total thickness 30 mm). Scale bar = 50 mm. Time is shown in minutes and seconds. Events are accelerated 225x over real-time.


Nature Supplementary Movie s3.mp4

DC migration and shape change in LNs. Migrated DCs (red) without antigen in a LN T cell area 2 hours after injection of T cells (T cells are not shown for better visual clarity). The imaged volume is 30 mm thick. Scale bar = 25 mm. Time is shown in minutes and seconds. Events are accelerated 225x over real-time.


Nature Supplementary Movie s4.mp4

Lack of blood flow leads to rapid cessation of T cell and DC migration. For this movie respiratory and cardiovascular arrest was induced by an i.v. overdose of anaesthetic at timepoint 30:00 min. during acquisition of a time-lapse sequence of migrating T cells (green) and DCs (red). The reticular network was visualized through second harmonic generation of collagen fibers (blue). A few minutes after induction of cardiovascular arrest, both DCs and T cells stopped migrating and partly lost their polarized shape. The imaged volume is 30 mm thick. Scale bar = 25 mm. Time is shown in minutes and seconds. Events are accelerated 450x over real-time.

 


Nature Supplementary Movie s5.mp4

T cells cover large territories in LNs. This scene corresponds to fig. 2a. T cells (green) are shown migrating in the LN paracortex in the absence of injected DCs. To provide an anatomical landmark, TRITC-dextran (red) was injected i.v. to delineate capillaries. The imaged volume is 25 mm thick. Scale bar = 25 mm. Time is shown in minutes and seconds. Events are accelerated 225x over real-time.


Nature Supplementary Movie s6.mp4

Short, serial T cell-DC contacts predominate during phase one. This scene shows T cells (green) and antigen-loaded DCs (red) migrating in the vicinity of a venular tree, which was made visible by i.v.-injection of FITC-dextran (green, blood flow from bottom to top). 3.5 hours after T cell entry into the LN (1.5 hours after anti-L-selectin injection), T cells interact only briefly with DCs in the vicinity of HEV. The imaged volume is 30 mm thick. Scale bars = 25 mm. Time is shown in minutes and seconds. Events are accelerated 225x over real-time.

 


Nature Supplementary Movie s7.mp4

Stable T cell-DC conjugates predominate during phase two. At 10 hours after T cell transfer (8 hours after anti-L-selectin injection), stable conjugates of Ag-loaded DCs (red) with one or several T cells (green) predominate. T cells attach either to the DC’s body or to their dendrites. The reticular network is visualized through second harmonic generation of collagen fibers (blue). The imaged volume is 30 mm thick. Scale bar = 10 mm. Time is shown in minutes and seconds. Events are accelerated 225x over real-time.

 


Nature Supplementary Movie s8.mp4

DCs (red) can undergo stable and dynamic interactions with T cells (green) simultaneously. During the transition from phase one to phase two (6h – 7h after T cell transfer), one T cell (*) is stably attached to a DC and is dragged along with the migrating DC, while a second T cell (**) makes two successive short contacts with the same DC (indicated by circles). The imaged volume is 30 mm thick. Scale bar = 10 mm. Time is shown in minutes and seconds. Events are accelerated 225x over real-time.



For details on experimental methods & techniques see:
Mempel, T.R., Henrickson, S.E. and von Andrian, U.H. T cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427: 154-159, 2004.[Full Text PDF]