Fibroblastic Reticular Cells Guide T
Lymphocyte Entry into and Migration within
the Splenic T Cell Zone
This information is current as
of June 18, 2017.
Marc Bajénoff, Nicolas Glaichenhaus and Ronald N.
Germain
J Immunol 2008; 181:3947-3954; ;
doi: 10.4049/jimmunol.181.6.3947
http://www.jimmunol.org/content/181/6/3947
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Supplementary
Material
The Journal of Immunology
Fibroblastic Reticular Cells Guide T Lymphocyte Entry into
and Migration within the Splenic T Cell Zone1
Marc Bajénoff,*†‡ Nicolas Glaichenhaus,† and Ronald N. Germain2*
Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access
the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells
(FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate.
This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T
cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels
(MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site
of entry in the MZ into the PALS, within which they continue to move on the same network. The Journal of Immunology,
2008, 181: 3947–3954.
*Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and †Institut
National de la Santé et de la Recherche Médicale and ‡Centre National de la Recherche
Scientifique, Université de Nice-Sophia Antipolis, Valbonne, France
Received for publication October 30, 2007. Accepted for publication July 14, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported in part by the Intramural Research Program of National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Department
of Health and Human Services, by the Institut de la Santé et de la Recherche Médicale
(INSERM), and by the Centre National de la Recherche Scientifique (CNRS).
blood content (8). This critical function is ensured by specialized
populations of macrophages termed marginal zone macrophages
and marginal zone metallophilic macrophages (MMMs), as well as
by dendritic cells designated marginal zone dendritic cells (9 –11).
Located between the MZ and the WP, the MZ sinus and its sinuslining cells are believed to be the place where recently incoming
lymphocytes can access the neighboring WP (4, 8, 12, 13), although there is little direct experimental support for this model.
Based on histological observation of human spleen sections, an
alternative scheme has been proposed in which specialized fibroblasts guide CD4⫹ T cell entry into the periarteriolar lymphoid
sheath (PALS) (14).
Recently, we demonstrated that a fibroblastic reticular cell
(FRC) network supports and guides T and B cell motility in the T
cell area of LNs (15), dictating the apparent random migratory
behavior of these cells. Lymphocytes adapt their shape to the cell
bodies and processes of these large stellate fibroblastic cells and
follow the supporting fibers of the FRCs during migration within
the paracortical region (T cell zone) that is itself defined by the
extent of this FRC network (15). In the spleen, the only known
function of the FRCs is their ability to create a conduit system that
transports blood-derived material inside the PALS in the same way
that the comparable FRC-based conduit system can transport
lymph content in the paracortex of the LN (16, 17).
In this study, we characterize the exact location and describe additional functions of the FRC network in the spleen. As anticipated
from our previous work, we show that these stromal cells are located
in the splenic PALS and support T cell motility in this region. Surprisingly, we also found that FRCs connect the PALS to the MZ only
where the MZ sinus and MZ macrophages rims are interrupted (i.e.,
the so-called MZ bridging channels (MZBCs)) (18). Using T cell
homing experiments, we show that T cells entering the PALS do not
cross the MZ randomly but only use these FRC-rich bridging channels. Thus, by their unique location, FRCs not only support T cell
motility in the PALS but also provide access roads to this area for T
cells that have recently immigrated into the spleen.
2
Address correspondence and reprint requests to Dr. Ronald N. Germain, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Building
10, Room 11N-311, 10 Center Drive, MSC 1892, Bethesda, MD 20892. E-mail address: [email protected]
Materials and Methods
Mice
3
Abbreviations used in this paper: LN, lymph node; WP, white pulp; RP, red pulp;
MZ, marginal zone; MMM, marginal metallophilic macrophage; PALS, periarteriolar
lymphoid sheath; FRC, fibroblastic reticular cell; MZBC, marginal zone bridging
channel; 2P, two photon.
www.jimmunol.org
C57BL/6 and C57BL/6 ubiquitin-GFP mice (UBI-GFP/BL6, strain 4353)
were purchased from The Jackson Laboratory and maintained in the
National Institutes of Health animal facilities. Hu-CD2 GFP mice were
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T
he “spleen is quantitatively the most important organ in
the lymphoid system, with more lymphocytes passing
through this organ each day than all the other lymphoid
tissues combined” (1). In lymph nodes (LNs),3 blood-borne lymphocytes enter the paracortex via high endothelial venules, specialized blood vessels that support the rolling, arrest, and diapedesis of T and B lymphocytes across the endothelium into the
surrounding parenchyma (for reviews, see Refs. 2, 3). The molecular events involved in this migration from blood to LN are well
characterized and sequentially involve selectins, chemokines, and
integrins. In the spleen, high endothelial venules are absent (4) and
although a key role for chemokines such as CCL21 and CXCL13
are appreciated in the intrasplenic localization of T (5, 6) and B
cells (7), respectively, there is scant evidence for any special role
of nonhematopoietic structural elements of this organ in guiding
lymphocytes to their sites of accumulation once they have entered
from the vasculature.
The spleen has a complex and well-described microanatomy
(see Fig. 1A). The white pulp (WP), where T and B cell populations segregate, is surrounded by the red pulp (RP), a loose meshwork of reticular fibers and fibroblasts where blood is filtered and
old erythrocytes removed (4). Localized between the WP and the
RP, the marginal zone (MZ) creates a transit area for recently
immigrating blood lymphocytes as well as a filtering zone for
3948
REGULATION OF T CELL MIGRATION IN THE SPLENIC WP
originally a gift from D. Kioussis (Mill Hill, London, U.K.). For the generation of chimeras, C57BL/6 ubiquitin-GFP mice were gamma-irradiated
with a single dose of 950 rads (or twice with 500 rads) from a cesium
source and were reconstituted with 2 ⫻ 106 C57BL/6 bone marrow cells.
At 8 wk after reconstitution, mice were tested for chimerism. Chimeras
were used for subsequent experiments only if analysis of blood leukocytes
showed the presence of less than 2% of CD3⫹ T cells of host origin. All
procedures performed on animals in this study have been approved by the
Animal Care and Use Committee, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Adoptive transfers
T cells were purified from the LNs of wild-type mice with a pan T cell
isolation kit while B cells were purified from their spleens with a pan B cell
isolation kit (Miltenyi Biotec). Cells were stained with either 5-chloromethyl fluorescein diacetate-2 ␮M, carboxy-fluorescein diacetate, succinimidyl ester-2 ␮M, CellTracker red CMTPX (2.5 ␮M), or SNARF-1 (2.5 ␮M)
(Invitrogen) at 37°C for 15 min. The indicated numbers of cells were transferred into host mice by i.v. injection.
ERTR-7 Ab specific for an unknown FRC-secreted molecule and antidesmin serum were purchased from Acris Abs. RA3– 6B2 Ab specific for
B220, 17A2 specific for the CD3 complex, and MECA-89 specific for
MadCAM-1 were from BD Pharmingen. MOMA-1 Ab specific for MMM
was purchased from Cedarlane. A goat polyclonal anti-murine CCL21/
6cKine was purchased from R&D Systems. These Abs were visualized by
direct coupling to biotin; allophycocyanin; Alexa Fluor 488, 568, or 647;
or through the use of Alexa Fluor 488, 568, or 647 coupled secondary Abs
or streptavidin.
Immunostaining
Spleens were harvested and fixed in a 0.05 M phosphate buffer containing
0.1 M L-lysine (pH 7.4), 2 mg/ml NaIO4, and 10 mg/ml paraformaldehyde
(PLP) for 12 h, then washed in phosphate buffer and dehydrated in 30%
sucrose in phosphate buffer. Spleens were snap frozen in Tissue-Tek
(Sakura Finetek). In brief, 10 –30 ␮m frozen sections were cut and then
stained with the indicated Abs as previously described (19). For in situ
fixation experiments, animals were anesthetized with avertin and given an
intracardiac injection of 15 ml PLP fixative. After excision from perfused
animals, spleens were treated using the protocol described above. Immunofluorescence confocal microscopy was performed with a Leica SP5 confocal microscope. Separate images were collected for each fluorochrome
and overlaid to obtain a multicolor image. Quantitative analysis of T/B cell
distribution into different areas of the LN sections was performed using
ImageJ software (National Institutes of Health). For each tissue section, the
number of cells within manually defined regions of interest was calculated
by the image processor. These values were then normalized to the number
of cells present in 0.2 mm2 of each analyzed region. Final image processing
was performed with ImageJ software (National Institutes of Health) and
Adobe Photoshop.
FIGURE 1. MZBCs directly connect the MZ to the PALS. A, Schematic
representation of the cellular elements of a WP unit and its surrounding MZ
and RP. B, Two adjacent spleen cryostat sections from HuCD2 GFP
(green ⫽ PALS) mice were stained for B220 (blue ⫽ B cell follicles),
Madcam-1 (left panels; red ⫽ MZ sinus), MOMA-1 (right panels; red ⫽
MMM), and imaged using confocal microscopy. This picture is representative of two different experiments.
age stacks are maximum intensity projections and play at 100⫻ or 300⫻
real time.4
Results
FRCs connect the MZ and PALS via the bridging channels
Two photon (2P) microscopy
Freshly isolated T cells were labeled with CellTracker red or SNARF-1 and
injected i.v. into chimeric recipient mice. Twenty-four hours later (unless
otherwise specified), the spleen was removed, fixed on a tissue holder, and
sliced into two nonsymmetric pieces using a vibratome (Leica, VT 1000 S)
in a bath of ice-cold PBS. The holder containing the thickest piece of
spleen was then incubated in a tissue chamber (Bioptechs). Splenic tissue
was perfused with a 37°C RPMI 1640 medium bubbled with a gas mixture
containing 95% O2 and 5% CO2 while being imaged with a Bio-Rad Radiance 2100 MP system attached to a Nikon 600 FN upright microscope
fitted with a 20⫻ water immersion lens (NA ⫽ 0.95, Olympus). The 2P
laser was a Chameleon XR femtosecond pulsed laser (Coherent) tuned to
880 nm. The bandpass filters used to detect GFP and SNARF were 525/50
nm and 620/100 nm, respectively. Three-dimensional images were collected in the PALS, a region characterized by the accumulation of dyelabeled T cells, the presence of a central arteriole, and surrounding FRC
fibers. This volume collection was repeated every 20 –30 s to create 4-D
data sets that were then processed with Imaris software (Bitplane) and
Adobe AfterEffects (Adobe). Supplemental movies created from these im-
In the spleen, the MZ and WP are separated by a rim of
MOMA-1⫹ MZ macrophages underlying the MadCAM-1⫹ MZ
sinus (9). However, this rim is incomplete and interrupted in regions known as MZBCs. Interestingly, these MZBCs are only
present where PALS, but not B cell follicles, abut the MZ (Fig.
1B), suggesting that the PALS may be directly connected to the
MZ at the site of these special bridging corridors.
Given our previous demonstration that FRCs are the substrate
for migration of T cells in the paracortical region (T zone) of LNs
(15), we characterized the exact location of FRCs in the WP as a
first approach to understanding how lymphocytes enter into and
move within the PALS and the possible relationship of such movement to this organization of the splenic microanatomy. Immunostaining of spleen sections revealed that the conduit system and its
4
The online version of this article contains supplemental material.
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Antibodies
The Journal of Immunology
3949
associated FRCs, whose outlines can be respectively delineated by
their reactivity with ERTR-7 and anti-desmin Abs, were present in
the PALS and also in the MZBC (Fig. 2, A and B). A closer analysis indicated that these reticular fibers span across from the MZ to
the PALS exactly where the rim of MMMs and the MZ sinus are
interrupted, that is, the MZBCs (Fig. 2C, movie S1).
FRCs support T cell motility in the PALS
The location of FRCs in the PALS suggested the possibility that
the FRC network might be the substratum for lymphocyte migration in this region. To determine whether naive T cells migrate
along FRC fibers in the PALS, these lymphocytes were visualized
in the spleens of mice using 2P laser scanning microscopy. To
permit simultaneous imaging of the nonhematopoietic stromal cell
populations within the spleen, we generated chimeric mice by using wild-type bone marrow cells to reconstitute irradiated ubiquitin
promoter-GFP transgenic animals, as previously reported (15). To
confirm that GFP-expressing cells within the PALS of chimeric
animals represented the FRC population, splenic sections from
such animals were stained for desmin and ERTR-7 expression and
analyzed using confocal microscopy (Fig. 3A). In the PALS, GFP⫹
cells form a three-dimensional network that surrounds the
ERTR-7⫹ conduit system and overlaps with desmin staining, indicating that this network is indeed formed by FRCs.
Confocal microscopy has been successfully used for intravital
imaging of the spleen (20, 21), but useful data can only be obtained
in the most superficial region of the organ with this technology.
Furthermore, because the WP is usually located deep in the mouse
spleen where thin GFP⫹ FRC fibers are difficult to image effectively even by 2P microscopy (⬎200 –300 ␮m under the thick
capsule and surrounded by RP full of RBC that absorb the 2P laser
signal), direct intravital or whole spleen explant imaging was not
adequate for our purpose. Therefore, to analyze the dynamic behavior of T cells in relation to the green fluorescent FRC network,
we developed a different approach. Wild type naive T cells were
labeled with the red fluorescent dye SNARF-1 and injected i.v.
into chimeric animals. One day later, each recipient spleen was
sliced into two nonsymmetric pieces using a vibratome to allow
direct imaging access to the deep WPs of the thickest piece. Bisected spleens were then perfused with warm and oxygenated medium while being imaged by 2P microscopy as previously reported
for LN vibratome sections (15). Using this technique, we found
that T cells maintain their characteristic migratory behavior, moving in the PALS at 8.95 ⫾ 0.9 ␮m/min⫺1, a speed comparable to
that observed in intact LNs (data not shown, movie S2) (22–24).
Analysis of 4-D (x, y, z, and time) datasets suggested that migrating SNARF-1 labeled T cells actively crawled on GFP⫹ FRCs,
following and morphologically adapting to the paths established
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FIGURE 2. FRCs connect the MZ
to the PALS at the MZBC. Spleen cryostat sections from HuCD2 GFP (blue)
mice were stained for ERTR-7
(green ⫽ FRC-derived matrix protein),
MOMA-1 (red, A and C) or desmin (B),
and imaged using confocal microscopy. C,
Inset shows a higher magnification of two
MZBCs. Arrowheads point to the location
of MZBCs while “B” indicates the presence of B cell follicles. This picture is representative of three different experiments.
See also movie S1.
3950
REGULATION OF T CELL MIGRATION IN THE SPLENIC WP
by the cell bodies and extended processes of these nonhematopoietic cells (Fig. 3B, movie S3).
To assess quantitatively whether T cells actively followed the
paths laid out by the FRC network, we used the same approach that
was applied in our prior study of migration of T cells in LNs (15).
We assumed that if the fibers provided guidance for cell movement, then any directional turns made by a T cell should always be
associated with a corresponding turn or branch of a supporting
FRC fiber. Conversely, a lack of correspondence between T cell
directionality and fiber pathways would indicate that spontaneous
turns or physical impediments posed by the many other cells in the
densely packed PALS environment accounted for T cell directional changes. Analysis of T cell turns using this previously described method (15) revealed an 89% correlation (155 of 174 cells)
between changes in T cell direction and the presence of T cellassociated GFP⫹ FRC fibers running at the corresponding angle
(Fig. 3C). Given that this is likely an underestimate of the correspondence between T cell movement and FRC organization due to
the inability to image the thinnest or dimmest FRC fibers, we conclude that as observed in LNs, T cells migrate along FRCs in the
splenic WP.
FRCs support T cell entry in the PALS
Blood circulating lymphocytes enter the spleen in the MZ (8, 20,
21). It is thought that lymphocytes and DCs can enter the WP from
the MZ sinus by passing through a layer of sinus-lining cells that
form a barrier between the MZ and the WP (4) (Fig. 1A). A role for
MZ macrophages in this process has been proposed because of
their unique location in the MZ as well as their ability to bind to
lymphocytes deposited on spleen cryostat sections in a sialoadhesin-dependent manner (25). However, this hypothesized function
is unlikely in light of data showing that lymphocyte homing to the
PALS is unaltered in mice in which MZ macrophages have been
depleted using chlodronate liposomes (26). Because T cells migrate on FRCs in the PALS and because FRCs connect the MZ to
the PALS via the MZBCs, we considered the possibility that FRCs
also support T cell migration from MZs to the PALS, as previously
suggested based on static imaging analyses using human material
(14).
We first attempted to use our vibratome cut method to address
this issue but found that using this procedure, the structure of the
MZ loosely attached to the WP becomes compromised, inducing
lymphocytes to leak out of the MZ over time, a phenomenon we
never observed in the more rigid and compact WP (data not
shown). As a consequence, we used an alternative approach. Naive
polyclonal T cells were labeled with 5-chloromethyl fluorescein
diacetate-2 ␮M or CFSE and injected i.v. into recipients that were
euthanized 10, 20, 30, and 180 min later. Spleens were harvested
and used to prepare fixed sections that were stained with B220 to
locate B cell follicles, as well as with ERTR-7 to highlight the
PALS and the MZBC that connect the MZ to the PALS (Fig. 4A).
We reasoned that if T cells are not constrained to enter the WP via
FRCs, we should observe T cells initially accessing the PALS not
only adjacent to the MZBCs but everywhere through the WP. At
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FIGURE 3. T cells crawl on the
splenic FRC network. A, Twenty micrometer thick spleen cryostat sections
from GFP (green) chimeric mice were
examined using confocal microscopy
after staining for desmin (red) and
ERTR-7 (blue). C.A., Central arteriole.
M.Z, Marginal zone. B, Five ⫻ 106
SNARF-1 labeled T cells were injected
i.v into chimeric mice. One day later,
spleens were sectioned using a vibratome, perfused with warm, oxygenated medium, and imaged using 2P
microscopy. Data show intravital snapshots of a single T cell (red) moving
over time on FRC fibers (green) in a 12
␮m thick volume. C, Quantification of
T cells showing turns in 4D datasets
with respect to their location on or off
GFP-marked stromal fibers in chimeric
animals. Data are representative of at
least three experiments.
The Journal of Immunology
3951
later times, T cells would eventually gather in the PALS. Alternatively, if T cells enter the WP using the FRCs located in the
MZBCs, they should be observed entering the WP only where
these structures connect to the PALS.
Using this sequential static imaging approach, T cells were
found throughout the MZ surrounding the WP 10 min after the
transfer. Starting 20 –30 min after the transfer, T cells started to
enter the WP. Strikingly, T cells only entered the WP at the
MZBCs (Fig. 4A). Three hours after the transfer, T cells were
localized in the PALS. To quantify this phenomenon, we counted
the numbers of T cells present at 10, 20, and 30 min (i.e., when
they were in the process of entering the WP) in both ERTR-7⫺ and
ERTR-7⫹ regions of the WP and normalized these numbers to the
surface areas of the respective regions. The analysis revealed that
T cell entry into the WP almost exclusively occurred in MZBC
regions where FRCs connect the PALS to the MZ (Fig. 4B).
B cells move across MZBCs and on FRCs outside the follicles
B cells reside in WP follicles that lack an FRC network, leaving
open the question of how this lymphocyte subset accesses this
region. Forster et al. (6) observed that before accessing the adjacent follicles, B cells are transiently retained in the PALS in a
CCR7-dependent manner, indicating that B cells do not directly
cross the MZ/WP border to access the follicles but rather follow a
path similar to T cells. To assess whether B cells enter the WP
using the MZBC and to quantify this phenomenon, we adoptively
transferred CFSE-labeled B cells into WT recipients and counted
the numbers of labeled B cells present at 30 min, 1, 2, 3, and 8 h
in three different regions of the WP: the B cell follicles (B220⫹
ERTR-7⫺), the T cell zone (B220⫺ ERTR-7⫹), and the T/B interface (B220⫹ ERTR-7⫹). These numbers were then normalized
to the surface areas of the respective regions (Fig. 5, A and B). Like
T cells, B cells were observed entering the WP via the MZBC over
time. Interestingly, B cells seemed to preferentially use the external part of the corridor corresponding to the T/B interface (the
equivalent of the cortical ridge present in the LN (27)) to gain
access to the follicles. Upon closer examination of B cells within
this region using a previously described fixative-perfusion technique (15), we found that the vast majority (48 of 55) of elongated
and polarized B cells were touching an ERTR-7⫹ desmin⫹ FRC
fiber (Fig. 5C). These data suggest that B cells migrate on the FRC
network as they transition between PALS and B follicle regions of
the WP. It is important to point out that a small number of B cells
were observed in the follicles at the earliest time point examined.
Elucidating whether these cells directly crossed the MZ sinus or
alternatively, rapidly homed to the follicles after entering the
PALS, is difficult but this observation may indicate that some B
cells may follow a path other than through the MZBCs to enter
the WP.
Discussion
In this study, we present evidence that FRCs support T cell access
to the splenic WP by creating physical roads for T cell migration.
These FRC pathways connect the MZ to the PALS via breaks in
the shell of MZ macrophages and the MZ sinus, regions called
MZBCs. After accessing the PALS along these guides, the T cells
migrate on the FRC fibers within the T zone of the spleen, as
previously observed in the paracortical region of LNs (15). Interestingly, like their LN counterparts, splenic FRCs secrete and are
tightly associated with CCL21 (Ref. 28 and Fig. 6A), a chemokine
that is an important regulator of T cell motility and positioning in
the PALS and that has been shown to provide an important chemokinetic stimulus to T cells in the LN paracortex (29 –31). In
Plt/Plt mice that lack expression of CCL19/CCL21 (ELC/SLC),
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FIGURE 4. T cells access the PALS
using MZBCs. A, Ten ⫻ 106 CFSE labeled polyclonal T cells (green) were
transferred in recipient mice. At 10, 20,
30, and 180 min later, spleen cryostat
sections were stained for B220 (blue)
and ERTR-7 (red) and imaged using
confocal microscopy to identify
where T cells enter the WP. Arrowheads point to the location where
MZBC connect the MZ. B, Quantification of T cells entering the WP in
ERTR-7⫺ and ERTR-7⫹ areas of the
WP over time. Data are representative
of three different experiments.
3952
REGULATION OF T CELL MIGRATION IN THE SPLENIC WP
naive T cells do not enter the PALS efficiently (Ref. 5 and Fig. 6B).
Similarly, in mice deficient for CCR7, the cognate receptor for
these chemokines, naive T cells cannot enter the PALS, indicating
the crucial role of this molecular interaction in regulating naive T
cell access to this region (6). Interestingly, in the MZ, a subset of
DCs called MZ DCs is localized at the border of MZBC (10).
Upon LPS activation, these DCs migrate to the PALS and this
migration is correlated with increased expression of CCR7 by the
DCs (10, 32, 33). It is therefore likely that both MZ DCs and naive
T cells access the PALS by using CCL19/CCL21 present on FRCs
to guide their migration. In addition, large heavily carbon-laden
macrophages located only in the RP at 30 min after carbon injection start to appear along MZBCs 1– 6 h later (34). Taken together,
these observations suggest that FRC-rich MZBCs are the entry
door to the PALS for several different cell types. MZBCs have
previously been suggested to be the place where lymphocytes exit
(but do not enter) the WP (18, 35). Therefore, it is possible that
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FIGURE 5. B cells access the PALS
using MZBCs. Twenty ⫻ 106 CFSE labeled polyclonal B cells (red) were
transferred i.v. into recipient mice. At
0.5, 1, 2, 3, and 8 h later, spleens were
harvested and sectioned using a cryostat. A, Sections were stained for
ERTR-7 (green) and B220 (blue) to
identify by confocal microscopy the
MZBCs, the B cell zone (B220⫹
ERTR-7⫺) and the T cell zone separated into the deep T zone (B220⫺
ERTR-7⫹), and the superficial T zone
(or T/B interface) where T and B cells
intermingle (B220⫹ ERTR-7⫹). At t ⫽
1 h, the inset provides a magnified view
of an MZBC where B cells gain access
to the WP. B, Quantification of B cells
entering the WP in the B zone, deep T
zone, and superficial T zone of the WP
over time. Data are representative of
two different experiments. C, Three
hours after B cell adoptive transfer,
mice were perfused with a fixative solution and spleens were harvested,
sectioned using a cryostat, and
stained for desmin (red) and ERTR-7
(blue). Three examples of CFSE-labeled B cell (green) interactions with
FRC fibers are shown. Left panels,
FRC fiber staining alone. Right panels, Overlay with B cells. Scale bar,
10 ␮m. Data are representative of two
different experiments.
these corridors govern cell exchanges between the RP/MZ and the
WP in both directions.
Although our present data together with past results point to a
clear role for CCL21 and FRC fibers in guiding T cell migration
into and within the PALS, the mechanisms underlying T cell
migration in the MZ itself remain unclear. In the MZ, MMM
location is regulated by CCL19/CCL21 because Plt/Plt mice
show a reduction in MMMs colonizing the MZ (36). Because,
as this result indicates, these chemokines are present and functional in the MZ, T cell migration in this region of the spleen
might also be regulated by CCL19/CCL21 until the presence of
FRCs at the MZBCs provides a presumably more attractive path
for the T cells to follow. Alternatively, because the capillary
meshwork discharges blood in the MZ, this specific area is continuously perfused (20, 21). In the MZ, a dense network of
reticular fibers creates a maze that may slow down lymphocytes
(Fig. 2). As a consequence, it is possible that, while flowing in
The Journal of Immunology
3953
the MZ, lymphocytes that can “catch” an FRC present at the
MZ/PALS border in the MZBC will begin their journey to the
underlying PALS, while those that do not successfully interact
with these FRCs will continue their movement and either enter
a downstream PALS or remain in the RP.
A central question remains: which molecules beside CCR7
ligands, if any, govern T cell locomotion on FRCs in the spleen
and LNs? So far, CCR7 ligands are the only molecules that have
been shown experimentally to have a direct role in controlling
the naive T cell movement in LNs in vivo in the steady state
(29 –31), but eliminating expression of this chemokine or its
receptor only fractionally reduces the velocity of the T cells; it
does not prevent their movement. Recently, Woolf et al. (37)
demonstrated that, in a shear-free environment, immobilized
but not soluble CCR7 ligands are chemokinetic agents for T
cells and that neither LFA-1 nor VLA-4 are required for T cell
motility in LNs. FRCs wrap around collagen fibers, preventing
lymphocytes from directly contacting these ECM molecules
(17, 38). However, in absence of chemokines, lymphocytes
move in vitro on collagen lattices but fail to do so on 2-D
collagen coated surfaces (37, 39). This observation raises the
possibility that T cells can crawl on any 3-D (but not 2-D)
substratum that provides a sufficient grip. This adhesiveness
may be regulated by integrins or other adhesions molecules that
remain to be identified but it is also possible that nonspecific
physical interactions such as those mediated by charges or Van
der Waals forces may be involved. In addition, our previous
observation that lymphocytes maintain numerous microvilli
while moving in the LN environment raises the possibility that
T cells are using these protrusions to exert the mandatory traction forces required for their migration (15).
Overall, the data we present in this study reinforce the conclusion we reached from our prior study of lymphocyte migration in
the LN (15). Although at the gross level in the absence of inflammation, T cell migration appears random in secondary lymphoid
tissues (40, 41), when examined closely, it is clear that the movement of these lymphocytes occurs along preformed paths. In the
LN, these paths are studded with DCs, thus facilitating the interaction of these two key cell types. As noted in this study, activated
DCs traffic use similar chemokine signals as the T cells to move
into the PALS and it is likely they occupy places on the same FRC
network trafficked by these lymphocytes. Thus, it seems that
throughout lymphoid tissues, initiation of adaptive immune responses is left less to chance than might be imagined.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Splenic FRCs secrete and
are covered with CCL21. A, Twenty micrometer thick splenic cryostat sections
from chimeric (green) mice were examined using confocal microscopy after
staining for CD3 (blue) and CCL21 (red).
C.A., Central arteriole. B, Ten ⫻ 106
CFSE labeled T cells were injected i.v into
wild type and plt/plt mice. Three hours
later, splenic cryostat sections were examined using confocal microscopy after
staining with ERTR-7 (red) and B220
(blue). T cells enter the PALS in wild
type but not plt/plt mice, as previously
described (5). Scale bar, 60 ␮m. Data
are representative of three different
experiments.
3954
Acknowledgments
We thank Hai Qi and Jackson G. Egen for suggestions on the manuscript.
Disclosures
The authors have no financial conflict of interest.
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REGULATION OF T CELL MIGRATION IN THE SPLENIC WP