Supplemental Data
Supplemental Data for
Bajénoff et al., Immunity 25, pp. 989-1001
- Document S1. Eight Figures
- Movie S1. Three-Dimensional Structure
of the LN Conduit System
An ERTR-7-stained thick section (30 μm) presented in 3D, showing a fibrous and
complex network of interconnected strands.
- Movie S2. A Close-up View of T Cell Interaction with
FRC Fibers
The same T cell (blue) imaged in Figure 1C is shown in 3D, along with the associated FRC fibers stained
with ERTR-7 (green) and desmin (red).
- Movie S3. CFSE Accumulates in the Uropod of Motile T Cells
CFSE-labeled
T cells were transferred i.v. into a wild-type mouse, and their dynamic migratory behavior in the popliteal LN was captured
with intravital 2-P imaging. CFSE intensity is represented with false colors, with the more intense green signal indicating
a higher CFSE content.
- Movie S4. Two-Photon Imaging of the Stromal Network within the LN Paracortex
The
popliteal LN of a chimeric animal was imaged with 2-P microscopy. Note the presence of a thick blood vessel in the FRC network.
- Movie S5. T Cells Migrate along the FRC Network
Dynamic image of T cell (red) migration along
the FRC network (green). The trails of three of the T cells are highlighted in the second part of the movie with colored dots
to help visualize the path taken along the fibers by a given T cell (z stack = 12 μM). The playback speed is 300× in
the first part of the movie and 150× in the second part when the tracks are highlighted.
- Movie
S6. Example of Another T Cell Migrating along the FRC Network
Another image of dynamic T cell (red) migration
along the FRC fiber network (green). z stack = 12 μM, playback speed is 300×.
- Movie S7.
Importance of Hard-to-Visualize FRC Fibers
Comparative sections from a 4D data set demonstrating that, depending
on the thickness of the visualized z stack, thin FRC fiber strands supporting T cell motility can be absent from the represented
volume but still constitute attachment sites for the T cells. The playback speed is 300×.
- Movie
S8. T Cell Dynamic Behavior on the FRC Network
Dynamic image of T cell (red) migration along the FRC network
(green). The trails of two of the T cells are highlighted. Red colored dots highlighted the path of a highly motile cell while
the white dots track the path of a slower motile cell protruding its filapodia on fibers during its displacement (z stack
= 12 μM). The playback speed is 150×.
- Movie S9. T Cells Exit HEVs in Limited Locations
A
single z slice from an intravital 4D data set showing numerous T cells exiting HEV via lucent areas that appear to be gaps
in the FRC sheath (“exit ramps”). The playback speed is 300× for both the main and zoomed image.
- Movie
S10. T Cells Move along the FRC Network after Exiting the HEVs
T cells (red) follow the FRC fibers (green) to
direct the path of their movement as soon as they exit the HEV (z stack = 9 μM). The playback speed is 300×.
- Movie S11. B Cells Move along the FRC Network in the LN Paracortex
In warmed and oxygenated
LN vibratome sections, adoptively transferred B cells (red) follow the ERTR-7+ fibers (green) while moving in the T cell area
(z stack = 12 μM). The playback speed is 150×.
- Movie S12. FRC and FDC Network Dynamics
FRC
and FDC networks present different characteristics in vivo. Note the higher density and less regular shape of the FDC network
as well as its capacity to display movement over time. Arrowheads point out a highly motile region of the FRC network.
- Movie S13. B Cells Track along the FDC Network within the LN Follicles
Dynamic image of B cells
(red) migration along the motile FDC network (green). The trails of two of the B cells are highlighted in the second part
of the movie with colored dots to help visualize the path taken along the fibers by a given B cell (z stack = 12 μM).
The playback speed is 300× in the first part of the movie and 150× in the second part when the tracks are highlighted.
