Grigorova et. al.

Supplemental materials

Supplementary Methods

Intravital microscopy and two-photon imaging. Briefly, lymph nodes were maintained in 36 °C

RPMI (Cellgro) diffused with 95% O2, 5% CO2 and imaged through the capsule either at the

medullary or the cortical side (Fig. 1c). Inguinal lymph nodes were prepared for intravital

microscopy as described15,38 with some modifications. Mice were anesthetized by i.p. injection of

100 mg of ketamine and 20 mg of xylazine per kg in PBS; maintenance i.m. injections of 40 mg/kg

of ketamine and 8 mg/kg of xylazine were given every 30 min as needed to ensure deep

anesthesia as indicated by regular, shallow respiration and suppression of withdrawal reflexes.

The lower flank was shaved, and a superficial incision was made in the abdominal wall to mobilize

a skin flap containing the inguinal lymph node. The mouse was then positioned on a Biotherm

stage warmer at 37 °C (Biogenics) either prone or supine (for imaging of the cortical or medullary

side, respectively) and the skin flap immobilized on a base consisting of an outer ring of Sylgard

184 silicone elastomer (Fisher) surrounding thermoconductive putty (T-putty 502, Thermagon)

with tissue glue (Vetbond). For cortical imaging a window was made in the skin to expose the

lymph node. Overlying fascia, fat and connective tissue was carefully microdissected to expose

the lymph node capsule leaving blood vessels and lymphatics intact. Anesthetized mice were

given supplemental oxygen via a nose-cone and prepared for two-photon microscopy. The lymph

node was perfused with warm RPMI diffused with 5% CO2, 95% O2. Blood circulation was

confirmed by visible pulsations and blood flow. The width of the subcapsular sinus was used as an

indication of the adequacy of lymphatic flow and when needed intact lymphatic drainage was

confirmed by appearance of R-PE in the lymph node after injection of 30 μl 0.7 mg/ml solution in

PBS (Invitrogen Molecular Probes) in the tail base. The subcapsular sinus was often narrrowed

after 2 or 3 h of imaging, and image acquistion was therefore restricted to a 2–3 h period. 6–9

weeks old C57BL/6 mice from Jackson laboratories were used as recipient mice for two-photon

microscopy. To label LYVE-1+ structures in vivo 12–18 h before intravital microscopy recipient

mice were injected s.c. in the flank and tail base with 20 μg of the mAb LYVE-1 conjugated to

Alexa Fluor 488 or Texas Red to drain to both medial and lateral lobes of the inguinal lymph node.

Nature Immunology: doi:10.1038/ni.1682

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Grigorova et. al. GFP+ T cells (2–4 x 107) either without labeling or labeled with CMTMR were transferred into

recipient mice 12 h before imaging. For comparison between S1P1-deficient and positive T cells,

S1P1-deficient thymocytes (~ 108 cells) and S1P1-positive thymocytes (~ 3 x 108 cells) were

labeled with different colors and adoptively cotransfered (sometimes with CFP+ B cells) into

recipient mice 12 h before imaging.

Deep tissue images were acquired with a custom-built two-photon microscope. A MaiTai

TiSapphire laser (Spectra-Physics) was tuned to provide an excitation wavelength of 810 nm for

imaging Alexa Fluor 488, CFSE, and CMTMR, and capsule in second harmonic or of 870 nm to

image Texas Red, GFP, CMTMR and CFP. Each xy plane spanned 480 400 pixels at a 0.6

μm/pixel resolution and 40-50 xy planes with 2-3 μm z spacings were formed by averaging 10

video frames every 20-25 s. Emission wavelengths of 440-480 nm (second harmonic emission of

collagen fibers), 470-500 nm (CFP), 525-575 nm (CFSE, GFP and Alexa Fluor 488) and 605-675

nm (CMTMR, PE and Texas Red) were collected with photomultiplier tubes (Hamamatsu).

Two-photon image processing and data analysis. Images were acquired with Video Savant (IO

Industries) and maximum intensity time-lapse images generated with MetaMorph (Molecular

Devices). Videos were processed with a low pass noise filter. Cell shape index analysis, cell

tracks, 3-D rotation images and LYVE-1+ structure reconstructions were made with Imaris 5.01

64 (Bitplane). The long and short axis of the cells were measured in a single z plane via the line

segment tool in Imaris software. Cell shape index was then calculated as the ratio of the longer

axis to the shorter axis. Automated tracks of T cells were verified and corrected manually. Using z

plane views and polygon drawing tools in Imaris software, LYVE-1+ structures were subdivided

into regions packed with LYVE-1+ macrophages ( ) if their interior was filled with LYVE-1+

signal or cortical sinuses if they had a well-defined border and no internal meshwork of LYVE-1+

signal (see Fig. 1a, Supplementary Movie 1 and Fig. 4a). T cell localization relative to the

outside, inside and border of the cortical LYVE-1+ sinuses or -filled areas were determined

using Imaris software based on the manual assessment of the relative position and colocalization

of the fluorescent signal between the T cell and LYVE-1+ structure in every z plane containing the

T cell image for every time point. In some cases clear discrimination as to whether a cell was at

Nature Immunology: doi:10.1038/ni.1682

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Grigorova et. al. the border of the sinus or had transmigrated inside was not possible. Those positions were

registered as “arbitrary”. Entry frequency was defined as the ratio of T cell commitments to go

inside the sinus starting at the outer border of the sinus versus total number of times that cells

leave the outer border. Calculation of the frequency of T cell entry was performed both excluding

the “arbitrary” cases from the calculation (Fig. 2c, top) or under a frequency-overestimating

assumption that in all “arbitrary” cases cells did get inside the structure (Fig. 2c, bottom).

Calculations of the T cell entry frequencies, the time periods spent at the outer border or inside

LYVE-1+ sinuses, the median velocities of cells and median velocities of cells over time periods

spent outside, at the outer border or inside the LYVE-1+ structures, as well as reconstruction of

cell trajectories from the same starting point were performed using software programmed in

MatLab (MathWorks). For comparison of frequencies of sinus entry more than 220 T cells of each

type were tracked. Velocity and time at the border of the cortical sinuses were calculated for more

than 70 tracks of each type. Velocities in the medullary region were calculated for 45 tracks of

each type. For time and velocities of wild-type T cells inside the sinuses more than 240 wild-type T

cells were tracked. For comparison of cell propensity to move towards LYVE-1+ sinuses, the

LYVE-1+ structures (both cortical sinuses and filled areas) were approximated by polygons in

each z plane view and the surface of the structures was reconstructed using software

programmed in MatLab. The software calculated minimal distances of cells tracked in Imaris 5.01

64 to the surface of the structures for every time point. Drift of the imaging volume with time was

taken into account. Cell positions were then assigned to zones 1 to 5 for 0–10 μm, 10–20 μm, 20–

30 μm, 30–40 μm and 40–50 μm distances to the surface of the structure, respectively. To

quantify cell propensity to move towards versus away from the structure we quantified the number

of events when cells (that arrived into the given zone from the next more distant zone) moved

forward to the next zone (nf) and when they reversed into the previous zone (nr). Transition

frequency for a given zone was then calculated cumulatively for all cells with continuous tracks

moving through the zone as the ratio of nf to (nf+nr). Transition frequences were calculated based

on 20–25 events on average for each cell type for each zone. Annotation and final movie

compilation was performed in Adobe After Effects 7.0. Video files were converted to mpeg format

with Avi to Mpeg Converter for Windows 1.5 (FlyDragon Software).

Nature Immunology: doi:10.1038/ni.1682

LYVE-1 MOMA-1GFP T cells

200 µm

Supplementary Figure 1. LYVE-1+ cortical sinuses: connections and extension across follicle/T zone interface.

Projection of confocal imagesspanning 16 µm thick sectionof inguinal lymph node withtransferred GFP T cells (green)stained with anti-LYVE-1 (red)and anti-MOMA-1 (blue).

Supplementary Figure 2. Diagram of T cell fate at the border of LYVE-1+ sinus and static reconstruction of the z-image profiles of LYVE-1+ sinuses.

a

Entry frequency = nin + nout

nin

nin

nout

tb

b

c d(a) The diagram represents the possible fates of aT cell once at the outer border of LYVE-1+ sinus. The frequency of entry into the sinus lumen is defined as the ratio of entry events for cells starting at the outer border of the sinus versus the total number of times cells leave the outer border.(b-d) (b) Upper panels: example of consecutive z-plane views (3 µm apart) of the LYVE-1+ cortical structures (red). Arrows indicate examples of a transmigrating cell and cell at the outer border of the cortical sinus. Bottom panels: sinus border delineated via Imaris polygondrawing tool. (c) Volume rendering of the polygons from six z-planes shown in A. (d) Cortical structure volume rendering of the entire z-stack in the imaging volume.

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Supplementary Figures

4Nature Immunology: doi:10.1038/ni.1682

Supplementary Figure 3. Visualization of cell trajectories in the macrophage rich region of LYVE-1+ structures.

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c(a) Three - dimensional reconstruction of the corticalLYVE-1+ structures imaged by two-photon intravital microscopy o f inguinal LN. The structure was subdivided into the macrophage (ΜΦ) -rich sinus area (red) and cortical sinuses (yellow) as described in Methods. Bluearrows show displacement of Edg1+/+ T cells in a region

with flow that is connected to the macrophage�rich region adjacent to the capsule. The LYVE-1+ structures were volumerendered at the mid-point, and there is a small gradual shift in the xy-plane over the imaging period. (b) Fragment of the cortical sinus structure from a. Arrows in dark blue are as in a and arrows in pink and light blue represent displacement of Edg1+/+ T cells inside the regions of LYVE-1+ sinuses connected to a region with flow. Time color-coded (blue to white) are t rajectories of two cells that start in regions without flow and enter (white arrow heads) intothe region with flow. (c) Fragment of macrophage �rich LYVE-1+ region from a. Green tracks represent cells inside themacrophage�rich region.Light blue tracks represent cells that arrive into the macrophagerich region from the cortical structures. White tracks represent cells that come out of the macrophage�rich region into the parenchyma.

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5Nature Immunology: doi:10.1038/ni.1682

Supplementary Figure 4. Multi-step model of T cell egress dynamics from the lymph node.

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A T cell (green) contacts a cortical sinus during random migration and after probing the sinus wall the cell makes the decision to enter in an S1P1-dependent manner (filled arrow) or to migrate away in response to T zone attractive cues (dashed arrow). Entry into a cortical sinus branch lacking flow is followed by continued migration until the cell either returns to the parenchyma (dashed arrow) or enters a branch of the sinus experiencing lymph flow (filled arrow). Movement into a region with flow (yellow arrows) retains the cells within the sinus and the cell is carried to macrophage-containing branches of the sinus that are proximal to the capsule and may often be part of the medullary sinus network. Cells may also enter sinuses directly in a region of flow (not depicted).

In medullary sinuses the T cell may encounter macrophages and become adherent, but usually is carried on with the flow and reaches the subcapsular space and efferent lymph. In some cases, T cells regain migratory activity and return from macrophage-rich sinus areas back to the tissue parenchyma (dashed arrow).

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6Nature Immunology: doi:10.1038/ni.1682