Figure S1: CFSE accumulates in the uropods of motile T cells.
CFSE-labeled T cells were transferred i.v into a wild-type animal. Twelve hours later, the mouse was anesthetized and perfused with PLP fixative. LNs were sectioned and subsequently stained for ICAM-1, an adhesion molecule known to relocate to the uropod of motile cells. Pictures show two polarized T cells with a high CFSE content at one end (A) See also Movie S3. ICAM-1 staining reveals this end to be the uropod (B).
Figure S2: Additional examples of lymphocytes contacting FRCs.
SEM pictures of lymphocytes associated with FRC fibers in the T cell zone. The arrowheads indicate lymphocytes microvilli extensions from the T cell to the FRC fibers. Scale bar: 1 μm.
Figure S3: T cells can be observed probing the FRC network during their displacement.
(A) Several adjacent optical sections from the same time points of 4D datasets show that thin fibers (green) supporting T cell (red) motility can be missed it they are outside of the z slices represented in an image. (B) One example of a migrating T cell extending its filapodia from its leading edge, probing intersecting fibers and following one of the fibers that had been examined. See also Movie S8.
Figure S4: “Non-fluorescent” LNs spaces are not empty spaces for free lymphocyte motility.
(A) SEM of a wild-type LN showing dense packing of leucocytes near HEV. (B) 3 x 105 CMTMR T cells (red) were transferred i.v into a Hu-CD2 GFP mouse. Twelve hours later, LNs were harvested. 10 μm thick sections were prepared and stained with phalloidin (blue) to reveal endogenous T cells (green), as well as other cells. This image emphasizes the many physical contacts a single T cell makes in addition to those made with FRC fibers. Originally imaged with a 63x objective. These images reveal a dense packing of lymphocytes in the interstices between fibers under normal circumstances, suggesting that the turning behavior is not a result of path obstruction due to the mere presence of another physical object, but rather is regulated by the spatial design of the fiber arrays. (C) Experimental protocol used to quantify, from 4D datasets, the correspondence between the turning angle of a migrating lymphocyte and that of the underlying GFP+ fibers.
Figure S5: Additional examples of T cell motility on FRC fibers.
(A) Intravital snapshots of another single T cell (red) moving over time on the cell body and processes of GFP+ FRC (green). (B) Occasionally, elongated T cells are observed “jumping” between fibers. Arrowheads indicate contact points between T cells and the fibers used during the “jump”.
Figure S6: DC and macrophages lie on the FRC network.
LNs from a CX3CR1 GFP+/+ mouse, in which DC, monocytes, and macrophages express GFP (green), were sectioned and stained for ERTR-7 expression (red). Data show confocal pictures of GFP+ cells ensheathing the conduit system. Originally imaged with a 63x objective.
Figure S7: FRCs delineate the T cell zone in the LN.
(A) Confocal image of a chimeric LN section stained for B220 (left panel, blue) or FDC-M2 (right panel, red) expression. S.C: subcapsular sinus. (B) WT mice were either untreated (left panel) or injected with 200 μg of an anti-CD62L blocking Ab (right panel). One day later, LN sections were stained for CD3 (blue), B220 (green), and ERTR-7 (red) expression to highlight the loss of B cells from the terminal peri-follicular ERTR-7+ network when lymphocyte entry into the LN is blocked. Arrowheads indicate numerous B cells close to HEVs while *s indicate the location of the magnified region (inserts). Originally imaged with a 16x and 63x (inserts) objective.
Figure S8: B cells move on the FRC network in the T zone.
(A) Wild-type mice were injected i.v with 10 x 106 CFSE-labeled B cells. Twenty four hours later, mice were perfused with a fixative solution. Representative confocal images of sections from recipients LN showing transferred CFSE-labeled B cell (green) tightly associated with ERTR-7+ (red) and desmin+ (blue) FRC fibers. Originally imaged with a 63x objective. (B) Wild-type mice were injected i.v with 20–30 x 106 SNARF-1-labeled B cells (red). Twenty four hours later, LN were harvested, vibratome sectioned (300μm thick), stained for ERTR-7 (green), incubated in a warm and oxygenated medium and imaged by 2P microscopy. Data show snapshots of a single B cell (red, arrowhead) moving over time on ERTR-7 fibers (green) in a 12 μm thick volume. See also Movie S11. (C) Quantitation of B cells showing turns in dynamic 4D datasets with respect to their location on or off ERTR-7+ fibers.
02: Movie S1
An ERTR-7 stained thick section (30 μm) presented in 3D, showing a fibrous and complex network of interconnected strands.
03: Movie S2
The same T cell (blue) imaged in is shown in 3D, along with the associated FRC fibers stained with ERTR-7 (green) and desmin (red).
04: Movie S3
CFSE-labeled T cells were transferred i.v into a wild-type mouse and their dynamic migratory behavior in the popliteal LN was captured using intravital 2-P imaging. CFSE intensity is represented with false colours, with the more intense green signal indicating a higher CFSE content.
05: Movie S4
The popliteal LN of a chimeric animal was imaged using 2-P microscopy. Note the presence of a thick blood vessel in the FRC network.
06: Movie S5
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 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 300x in the first part of the movie and 150x in the second part when the tracks are highlighted.
07: Movie S6
Another image of dynamic T cell (red) migration along the FRC fiber network (green). z stack = 12 μM, playback speed is 300x.
08: Movie S7
Comparative sections from a 4D dataset 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 300x.
09: Movie S8
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 150x.
10: Movie S9
A single z slice from an intravital 4D dataset showing numerous T cells exiting HEV via lucent areas that appear to be gaps in the FRC sheath (“exit ramps”). The playback speed is 300x for both the main and zoomed image.
11: Movie S10
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 300x.
12: Movie S11
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 150x.
13: Movie S12
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.
14: Movie S13
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 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 300x in the first part of the movie and 150x in the second part when the tracks are highlighted.