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High endothelial venules as traffic control points maintaining lymphocyte

population homeostasis in lymph nodes

Article in Blood · September 2011

DOI: 10.1182/blood-2011-07-367409 · Source: PubMed

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IMMUNOBIOLOGY

High endothelial venules as traffic control points maintaining lymphocyte

population homeostasis in lymph nodes
*Cyril Mionnet,1-3 *Stéphanie L. Sanos,1-3 Isabelle Mondor,1-3 Audrey Jorquera,1-3 Jean-Pierre Laugier,4 Ronald N. Germain,5
and Marc Bajénoff1-3
1Centre d’Immunologie de Marseille Luminy, Aix-Marseille University, Marseille, France; 2Inserm U631, Marseille, France; 3Centre National de la Recherche
Scientifique Unite Mixte de Recherche 6102, Marseille, France; 4Centre Commun de Microscopie Appliquée, Université de Nice-Sophia Antipolis, Nice, France;
and 5Lymphocyte Biology Section, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD

Millions of lymphocytes enter and exit ment in HEVs, finding that HEVs create Thus, rather than being simple entry ports,
mammal lymph nodes (LNs) each day, pockets within which lymphocytes reside HEVs act as gatekeepers able to stack,
accessing the parenchyma via high endo- for several minutes before entering the hold and grant lymphocytes access to LN
thelial venules (HEVs) and egressing via LN proper. The function of these pockets parenchyma in proportion to the rate of
lymphatics. Despite this high rate of cellu- was revealed in experiments performed lymphocyte egress from the LN, enabling
lar flux and the many entry and exit sites under conditions in which lymphocyte the LN to maintain a constant steady-
within a given LN, the number of lympho- egress from the LN was compromised by state cellularity while supporting the ex-
cytes present in a resting LN is extraordi- any of several approaches. Under such tensive cellular trafficking necessary for
nary stable over time, raising the ques- conditions, the HEVs pockets behaved as repertoire scanning. (Blood. 2011;118(23):
tion of how this steady-state is “waiting areas” in which lymphocytes 6115-6122)
maintained. Here we have examined the were held until space was made available
anatomic details of lymphocyte move- to them for entry into the parenchyma.

Introduction
Despite the constant entry and exit of lymphocytes in the nonin- “waiting areas” in which lymphocytes accumulate and are held
flamed state, LNs manage to maintain their cellularity over time, until space is made available to them in the parenchyma as a
indicating the existence of tightly regulated control mechanisms consequence of lymphocytes egress. This simple scheme provides
that balance access and egress of cells.1 Naive lymphocytes enter an elegant way for the LN to maintain a constant cell number while
LNs via HEVs and exit via lymphatic vessels.2 The number of supporting the high flux of cells needed for effective utilization of
HEVs and lymphatic exit sites in an individual LN is quite high, the adaptive immune repertoire.
with these sites showing substantial topographic separation through-
out the LN. This raises the question of how a LN manages
lymphocyte traffic at these many dispersed sites to maintain Methods
homeostasis, a critical issue because LNs provide key survival
signals such as IL-7 and BAFF (B-cell activating factor) to T and Mice
B cells, respectively.3,4 As a resting LN is believed to secrete C57BL/6, RAG2°/°, actin-CFP,7 Ubiquitin-Cre EsR18, and C57BL/6
defined amounts of such survival signals, increases in lymphocyte ubiquitin-GFP mice were purchased from The Jackson Laboratory and
number beyond that properly supported by the survival factors maintained in the CIML and/or Skirball animal facilities. Ubiquitin-Cre
available could lead to cell death and repertoire contraction. EsR1 were crossed with S1P1flox/⫺ mice kindly provided by R. L. Proia.9
For the generation of chimeras, C57BL/6 RAG2°/° ubiquitin-GFP mice
Likewise, too few cells would limit the number of specific cells
were ␥-irradiated (twice with 500 rads) from a cesium source and were
available at any time for antigen-driven activation, diminishing the reconstituted with various mixtures of bone marrow cells indicated in the
efficiency of immune responses. Interestingly, mice grafted with main text (minimum of 2 ⫻ 106 bone marrow cells per mouse). Chimeras
multiple thymi do not harbor massively enlarged LNs despite high were used at 8 weeks after reconstitution. All procedures performed on
numbers of circulating lymphocytes, suggesting that LNs auto- animals in this study have been approved by the ethical committee on
regulate the number of cells they house and nourish.5,6 Altogether, animal research of Marseille (N°1-03112009, France).
these observations suggest that LNs adapt lymphocyte entry and
exit fluxes to constantly host a fixed number of lymphocytes under Antibodies
steady-state conditions. In this study, we investigate this possibility RA3-6B2 antibody specific for B220, 17A2 specific for the CD3 complex,
and demonstrate that, in addition of being passive entry doors for Meca-79 specific for PNAd, 3E2 antibody specific for ICAM-1, MEL-14
lymphocytes, HEVs are traffic control checkpoints able to create specific for CD62L were purchased from BD Biosciences Pharmingen.

Submitted July 13, 2011; accepted August 29, 2011. Prepublished online as Blood The online version of this article contains a data supplement.
First Edition paper, September 21, 2011; DOI 10.1182/blood-2011-07-367409.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
*C.M. and S.L.S. contributed equally to this work. marked ‘‘advertisement’’ in accordance with 18 USC section 1734.

BLOOD, 1 DECEMBER 2011 䡠 VOLUME 118, NUMBER 23 6115

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6116 MIONNET et al BLOOD, 1 DECEMBER 2011 䡠 VOLUME 118, NUMBER 23

ERTR-7 and Desmin antibodies were purchased from Acris Antibodies. Electron microscopy
These antibodies were visualized by direct coupling to Pacific blue,
TEM. PLP-fixed LNs were rinsed in 0.1M phosphate buffer and postfixed
allophycocyanin, Alexa Fluor–488, –568, –647, or through the use of Alexa
for 1 hour in OsO4. LNs were then rinsed in water, dehydrated through
Fluor–488, –568, –647 or -biotin coupled secondary antibodies. Nuclear
graded concentrations of acetone (50, 70, 95, 100% pure, 3 times) before
staining was performed with Sytox 63 (Invitrogen).
incubation for several hours in a 1:1 vol/vol acetone-Epon mixture. LNs
were then incubated overnight in pure Epon before final embedding in
Immunostaining Epon. Eighty millimeter thick LN sections were generated using a LEICA
Ultracut S ultramicrotome. Sections were stained with uranyl acetate
LNs were harvested and fixed in a 0.05M phosphate buffer containing 0.1M followed by lead citrate and were imaged using a CM12 Philips microscope.
L-lysine (pH 7.4), 2 mg/mL NaIO4, and 10 mg/mL paraformaldehyde (PLP) SEM. PLP-fixed LNs were rinsed in 0.1M phosphate buffer and
for 12 hours, then washed in phosphate buffer and dehydrated in 30% immersed for 1 hour in a 20% glycerol solution. LNs were then frozen in
sucrose in phosphate buffer. LNs were snap frozen in Tissue-Tek (Sakura liquid nitrogen and freeze-cracked before being dehydrated through graded
Finetek). Twenty-micrometre frozen sections were cut and then stained with concentrations of ethanol. LNs were desiccated with hexamethyldisilazane
the indicated antibodies as previously described.10 Immunofluorescence and affixed on aluminum stubs with adhesive, coated with gold-palladium,
confocal microscopy was performed with a Leica SP5 confocal microscope. and examined in a SEM (JEOL 6700F, Japan) microscope with an
Separate images were collected for each fluorochrome and overlaid to accelerating voltage of 3 kV.
obtain a multicolor image. Final image processing was performed with 2-P microscopy of lymphocytes immigration in HEVs. The right
Imaris Version 7.2.3 software (Bitplane) and Adobe Photoshop. popliteal LN of anesthetized ubiquitin-RAG2°/°GFP mice reconstituted with
actin-CFP bone marrow cells (2.5% isofluorane in an air/O2 mix) was
Homing assay surgically exposed and imaged with a Zeiss LSM-7 MP system fitted with a
20⫻ water immersion lens (NA ⫽ 1.0, Plan-Apochromat, Zeiss). The 2P
Total cells were purified from the LNs and spleens of WT mice and laser was a Spectra-physics Maï-taï Deepsee tuned to 860 nM. Singles
stained with either CMFDA (0.5␮M) or CMTMR (2␮M; Invitrogen) at slices of shallow HEVs were imaged between 50-120␮M below the capsule
37°C for 15 minutes. The indicated numbers of cells were adoptively and repeated every 4 seconds to create 3D datasets (x, y, and time) that were
transferred intravenously. One hour later, blood, auricular and inguinal then processed with Imaris software (Bitplane) and Adobe After Effects
LNs were harvested. (Adobe).

Induction of lymphocyte sequestration in LNs on FTY720 and

SEW-2871 treatment
Results
When indicated, mice received either 100 ␮g of FTY720 (Cayman
Chemical Company) intraperitoneally or 1.2 mg SEW-2871 (Cayman Lymphocytes accumulate in “pockets” below endothelial cells
Chemical Company) gavage. Experiments were conducted 36 (FTY-720) or
8 (SEW-2871) hours later, at the peak of lymphopenia. As the entry site for circulating blood lymphocytes, we hypoth-
esized that HEVs may play a key role in regulating resting LNs
cellularity by coupling lymphocyte entry with the rate of exit across
Tamoxifen treatment of Ubi-cre ESR1 ⴛ S1P1flox/ⴚ chimera lymphatic endothelium. To test this hypothesis, we first generated
Animals were treated with 2 mg of 4-hydroxy-tamoxifen intraperitoneally chimeric mice in which HEVs (and other stromal cells) express the
(Sigma-Aldrich) for 3 consecutive days. Mice were used 9 days after the green fluorescent protein (GFP). RAG2°/° ubiquitin promoter-GFP
first injection of tamoxifen. transgenic animals were irradiated and reconstituted with wild-type
bone marrow cells. Chimeric LNs were harvested, stained for
Quantification of lymphocyte densities inside and outside peripheral node addressin (PNAd) and nuclei (Figure 1A) or for
LN HEVs CD3 and B220 (Figure 1B), then analyzed by confocal microscopy.
As expected and previously observed, all PNAd⫹ HEVs were
Immunofluorescence images were segmented into HEVs and the rest of the GFP⫹ and harbored typical cobblestone endothelial shapes with
LN according to PNAd staining. As PNAd is present on the luminal surface numerous embedded lymphocytes.12 Interestingly, a close examina-
of HEV ECs, HEVs areas were defined as 20 ␮m concentric rings centered
tion revealed that T and B cells were frequently packed together
on PNAd labeled structures. The numbers of pixels of each area as well as
into HEV pockets and that these structures were able to house up to
the numbers of adoptively transferred lymphocytes (CMTMR⫹) present in
each area were measured using ImageJ Version 1.44R software (National 4-5 lymphocytes (Figure 1).13,14 Using electron microscopy, we
Institutes of Health). For each LN slice, the densities of CMTMR⫹ confirmed these results and observed that lymphocytes nested in
lymphocytes inside HEVs were divided by the densities of CMTMR⫹ these pockets were separated from the HEV lumen via a very thin
lymphocytes outside HEVs. A small ratio reflects the cells ability to membrane of the HEV endothelial cell (EC; Figure 1C). To
efficiently gain access to the LN parenchyma while an elevated ratio reflects determine whether these cells were localized between 2 ECs or
a delay in this process. For each condition, a minimum of 3 different LNs within a true pocket formed by single ECs, we took advantage of
(⬎ 3 different sections per LN) and 1000 cells were counted per mouse. the asymmetry of HEV ECs. HEVs ECs express molecules such as
P values were calculated using 2-tailed unpaired t test. intercellular adhesion molecule-1 (ICAM-1) on their luminal/
lateral face (Figure 2A).15 If the lymphocytes embedded in these
Morphometric analysis of HEV pockets were localized between 2 ECs, one would expect them to
be entirely surrounded by the ICAM-1 staining of the 2 adjacent
The height of the endothelium of HEVs was measured in LN sections
ECs membranes (Figure 2A right panel). On the other hand, if the
obtained from RAG-2°/°ubiquitin-GFP mice using a protocol previously
described.11 In brief, the outer contour length of GFP⫹ HEVs and the area
lymphocytes present in these pockets were held by a single EC, the
covered by GFP⫹ HEV ECs (including the HEV pockets) were measured inner membrane of this EC should surround them and hence not be
with ImageJ software (National Institutes of Health). Endothelium-height positive for ICAM-1 (Figure 2A left panel). Using such strategy,
was calculated as HEV-surface-area divided by HEV-contour-length, and we determined that lymphocytes nested in these pockets were not
expressed in arbitrary units. localized between 2 HEV ECs (Figure 2B). To distinguish between
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BLOOD, 1 DECEMBER 2011 䡠 VOLUME 118, NUMBER 23 HEV CHECKPOINT CONTROLLING LYMPHOCYTE ENTRY INTO LN 6117

Figure 1. HEVs create lymphocyte pockets. (A-B) Confocal images of a LN section from a RAG-2°/°ubiquitin-GFP chimera (green) stained for PNAd (red), sytox 63/nuclei
(blue; A), or CD3 (blue) and B220 (red; B) expression, showing the individual HEV ECs and their “pockets” (arrowheads) containing lymphocytes. (*) ⫽ Perivascular channel.
(C) Representative SEM and TEM pictures of HEV ECs obtained from WT LN sections. Images were pseudocolored to help delineate lymphocytes (L) and endothelial cells
(E.C). Data are representative of 3 different experiments (2 mice per experiment, ⬃ 20 analyzed HEVs per experiment).

a situation in which lymphocytes are retained within ECs (emperip- ously altered in size and location by lymphocyte migration from
olesis) and a situation in which they are nested below ECs, we blood to LN parenchyma. To appreciate such dynamics, we
stained the chimeric LN sections with anti-Desmin and ERTR-7, irradiated RAG2°/° ubiquitin-GFP mice and reconstituted them
2 Abs, respectively specific for myofibroblasts and the conduit with actin-CFP bone marrow cells, allowing us to image the
system secreted and ensheathed by these cells. Confocal images of immigration of endogenous CFP⫹ lymphocytes through the GFP⫹
HEVs revealed that lymphocytes nested in HEV pockets were HEVs. Reconstituted mice were anesthetized and their popliteal
localized between the membrane of GFP⫹ Desmin⫺ EC and the LN surgically exposed for 2-Photon (2P) imaging. Because the
membrane of the GFP⫹ Desmin⫹ myofibroblasts that ensheath the HEV EC membranes forming these pockets are ⬃ 100 nm thick
ERTR-7 conduit system. Altogether, these results suggest that (Figure 1C) and highly motile in X, Y, and Z directions, dynamic
lymphocytes nested in lymphocytes pockets do not accumulate imaging of entire HEV pockets was not possible. Therefore, we
within ECs but below them (Figure 2C). examined highly resolved single “slices” of shallow HEVs to
appreciate the overall dynamics of HEV ECs. In these videos, the
HEVs pockets dynamically modulate HEV shape
continuous ballet of CFP⫹ cells entering the GFP⫹ HEVs revealed
HEVs are characterized by cuboidal cells as opposed to the typical that HEV pockets are highly dynamic structures moving in many
flat endothelial cells found in regular venules.13,14 To determine directions as a result of lymphocyte displacement (supplemental
whether this shape is an inherent property of the HEV EC Video 1, available on the Blood Web site; see the Supplemental
themselves or if it arises as a result of the presence of lymphocytes Materials link at the top of the online article).
in these pockets, we treated chimeric mice with an anti-CD62L HEVs adapt lymphocyte entry to lymphocyte egress in the LN
blocking antibody for 1 hour (Figure 3). Under these conditions,
lymphocyte rolling on HEVs—and hence LN homing—is blocked.16 If HEVs are the site of adjustment of lymphocyte entry into the LN
On such treatment, lymphocyte pockets in HEVs disappeared and to maintain constant cell number in the face of ongoing egress, one
HEVs concomitantly flattened (Figure 3A). Morphometric analysis would expect them to limit LN access to blood-circulating lympho-
demonstrated that the height of LN HEV decreased on anti-CD62L cytes if the cellularity of this LN is maximal, whereas when the
treatment, representing about 68% of the height of untreated HEVs population of a resting LN decreases as a result of lymphocytes
(Figure 3B). Therefore, these data suggest that the typical cobble- egress, HEVs should immediately counterbalance this cellular loss
stone shape of HEV ECs is not intrinsic but rather results from the by allowing the entrance of blood circulating lymphocytes. The
continuous and transient accumulation of lymphocytes within these capacity of HEV EC to generate transient collections of trafficking
pockets. These observations also demonstrate that these holding lymphocytes would provide an elegant solution to this adjustment
sites are not preformed, rigid hollow caverns within which requirement—lymphocytes could be “stored” within the EC, being
lymphocytes accumulate, but rather spaces created by the accumu- held there when the LN was “full” and released if the population
lation of trafficking lymphocytes that have not yet passed into the declined as lymphocytes exited the LNs. Such a system makes
perivascular channel (PVC) on their way into the LN parenchyma. sense in LNs that are densely packed organs with an only slowly
In agreement with this concept, we never observed HEVs in which changeable volume that imposes space limitations. If this concept
pockets were free of lymphocytes. is correct, blood circulating lymphocytes should less efficiently
If HEV pockets are not rigid but adaptable structures, we enter the parenchyma of a LN in which lymphocyte egress is
reasoned that they should be highly dynamic structures continu- blocked and in which cellularity would be maintained at a
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6118 MIONNET et al BLOOD, 1 DECEMBER 2011 䡠 VOLUME 118, NUMBER 23

Figure 2. HEV pockets are plastic structures. (A) The right panel in the diagram represents a situation in which lymphocytes are not engulfed within pockets but simply
migrating between 2 ECs while the left panel represents lymphocytes present in a true structured pocket underneath the HEV. L ⫽ Lymphocyte. (B) Confocal image of a LN
section from a ubiquitin-GFP chimera (green) stained for ICAM-1 (red) and sytox 63/nuclei (blue) Arrowheads indicate the inner membrane of the EC directly in contact with
lymphocytes. (C) Confocal image of a LN section from a ubiquitin-GFP chimera (green) stained for ERTR-7 (red), Desmin (white) and sytox 63/nuclei (blue). Arrows indicate the
membranes of myofibroblasts/pericytes that ensheath the conduit system. Data are representative of 3 different experiments (2 mice per experiment).

maximum. As a first attempt to test this hypothesis, we treated wild the contrary, in drug-treated animals, the great majority of endoge-
type (WT) mice with FTY-720 and SEW-2871, 2 drugs known to nous cells is sequestered within SLOs and therefore no longer
interfere with the sphingosine-1-phosphate (S1P) pathway in- circulates in the blood (supplemental Figure 1A). In addition, a
volved in lymphocytes egress from lymphoid organs.17,18 Such higher fraction of blood circulating T cells is composed of effector/
treatment induces the sequestration of lymphocytes in SLOs and memory CD62Llo cells that no longer compete with the transferred
leads to a concomitant lymphopenia (supplemental Figure 1A). cells in the LN homing assay (supplemental Figure 1B). As a
Drug-treated and control animals were injected intravenously with consequence, if the HEVs of control and treated animals were
WT labeled lymphocytes and euthanized 1 hour later. The LNs supporting the same influx of cells, many more transferred cells
were sectioned, stained for PNAd expression, and the ratio of should have accessed the LN parenchyma of treated mice during
labeled lymphocyte densities in HEVs and LN parenchyma calcu- the 1 hour homing assay. Despite this, we observed that lympho-
lated for each condition. The results showed that many more of the cytes transferred in drug-treated mice were delayed in their
transferred lymphocytes in FTY-720 and SEW-2871 treated mice capacity to access the LN parenchyma.
remained in the HEVs during the homing assay compared with Because ECs express many S1P receptors, including S1P1,
control animals, indicating a reduced capacity to access the LN these drugs are not perfect tools for examining the issue of cellular
parenchyma (Figure 4A-B and supplemental Figure 1C). These trafficking.19 Unfortunately, S1P1°/° mice are not viable and using
results, while consistent with the general expectations of our their fetal livers as a source of progenitors fails to generate
model, are not black and white. However, drug-induced lymphope- chimeric mice with normal levels of lymphocytes.17,20 We therefore
nia is never total and some cells still egress the LNs of treated mice, generated a mouse in which S1P1 deletion can be conditionally
potentially allowing the entry of the same number of cells induced just in lymphocytes. Ubiquitin-Cre ESR1 mice were
according to our hypothesis. In a control animal, the cohort of crossed to S1P1flox/⫺ mice. In the former mice, the cre enzyme is
labeled cells injected in the mouse represents a small percentage of released in the nucleus of all cells on tamoxifen injection while in
the endogenous blood-circulating lymphocyte population. There- the latter, all cells possess a functional S1P1 allele flanked by 2 lox
fore, many more endogenous “black” cells than labeled transferred P sites while the other allele is not functional. In these double
cells enter the untreated LNs during the 1 hour homing assay. On transgenic mice, all cells are S1P1 sufficient but are expected to
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BLOOD, 1 DECEMBER 2011 䡠 VOLUME 118, NUMBER 23 HEV CHECKPOINT CONTROLLING LYMPHOCYTE ENTRY INTO LN 6119

Figure 3. Blockade of lymphocyte homing to LN induces the disappearance of HEV pockets. (A) Confocal images of LNs section from a RAG-2°/°ubiquitin-GFP chimera
(green) injected or not intravenously for 1 hour with 100 ␮g of anti-CD62L blocking Ab (MEL-14) and stained for CD3 (red) and sytox 63/nuclei (blue). Arrowheads point to HEV
pockets. (B) Height (and standard error) in arbitrary units of LN HEV (see “Morphoretic analysis of HEV”). Data are representative of 2 different experiments (2 mice per
experiment, ⬃ 15 analyzed HEVs for each condition per experiment).

genetically lose S1P1 expression on tamoxifen injection. Because mixture of bone marrow cells isolated from RAG 2°/°WT (80%)
we wanted to generate mice in which only lymphocytes would and ubiquitin-Cre EsR1-S1P1flox/⫺ (20%). These mice will be
show loss of S1P1 expression, we irradiated RAG 2°/° ubiquitin- referred to as S1P1flox/⫺ chimeras. In these chimeric mice, all cells
GFP mice and reconstituted them with either WT bone marrow or a should be S1P1 sufficient before tamoxifen injection but only

Figure 4. S1P1 blockade limits blood lymphocyte access to the LN parenchyma. WT mice were either treated or not with FTY-720 (36 hours) or SEW-2871 (8 hours) and
injected intravenously with 10 millions of CMTMR labeled lymphocytes (red). (A) One hour later, LNs were harvested, sectioned in multiple slices, stained for PNAd (green) and
Sytox63/nuclei (blue) and analyzed by confocal microscopy. The position of each individual cell is highlighted with a red dot. Arrowheads indicate HEVs in which transferred cells
accumulate. (B) For each LN, densities of transferred cells inside and outside PNAd⫹ HEVs were calculated and presented as a ratio. Each symbol represents the averaged ratio
calculated from all the imaged slices of a given LN. Data are representative of 3 different experiments (2 or 3 mice per experiment, minimum of 3 LNs per mouse).
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6120 MIONNET et al BLOOD, 1 DECEMBER 2011 䡠 VOLUME 118, NUMBER 23

Figure 5. Lymphocytes sequestration in LN limits blood lymphocytes access to the LN parenchyma. RAG-2°/°ubiquitin-GFP chimera were irradiated and reconstituted
with either WT bone marrow cells or a mixture of bone marrow cells isolated from RAG-2°/°WT mice (20%) and ubiquitin-Cre Esr1 ⫻ S1P1flox/⫺ mice (80%). Reconstituted
chimeras were treated or not with tamoxifen and injected with 10 millions of CMTMR labeled lymphocytes (green). One hour later, LNs were harvested, sectioned, stained for
PNAd (red) and analyzed by confocal microscopy (A). Arrowheads indicate HEVs in which many transferred cells accumulate. (B) Densities of transferred cells inside and
outside PNAd⫹ HEVs were calculated for each analyzed section and presented as a ratio. (C) A typical HEV imaged in control and tamoxifen-treated ubiquitin-Cre Esr1 ⫻
S1P1flox/⫺ is shown. Data are representative of 3 different experiments (2 or 3 mice per experiment, minimum of 3 LNs per mouse).

lymphocytes (and 20% of the remaining hematopoietic cells) outside of the PVC until needed to replace exiting lymphocytes,
would become S1P1 deficient on tamoxifen injection. Reconsti- HEVs behave as traffic control checkpoints that flexibly adapt to
tuted chimeras were treated or not with tamoxifen and 1 week later, the needs of the resting LN as it seeks to balance lymphocyte entry
their lymphopenia was evaluated as a result of lymphocytes and exit rates. This in turn allows the LN to constantly function at
sequestration in SLOs (supplemental Figure 2). In tamoxifen- its maximal capacity at steady state.
treated S1P1flox/⫺ chimeras, we observed a 70% reduction of blood Given the extreme cellular density of a LN, one could wonder
lymphocytes, indicating that S1P1 deficiency on lymphocytes is how the space freed by cells leaving in the medulla would
sufficient to induce their sequestration in LNs. We then adoptively immediately be available to cells present in HEVs located hundreds
transferred a cohort of labeled WT lymphocytes in the various microns away. Importantly, lymphocytes do not physically exit the
groups of mice and after 1 hour stained LNs sections for PNAd parenchyma from LN medullary sinuses but from thinner cortical
expression (Figure 5A-B). The data show that lymphocytes ac- sinuses located nearby HEVs.21-23 Therefore, the juxtaposition of
cessed the LN parenchyma of untreated S1P1flox/⫺ chimeras and entry and exit sites would confine the regulation of entry/exit fluxes
WT chimeras treated with tamoxifen equally. In contrast, in to very restricted regions of LNs. By itself, this juxtaposition is not
tamoxifen-treated S1P1flox/⫺ chimeras, we observed that the lympho- sufficient to explain how the cellular density of the parenchyma is
cytes were impaired in this process, indicating that lymphocyte constantly sensed by the ECs and/or the nested lymphocytes.
sequestration in LNs controls the influx of blood circulating However, when lymphocytes exit HEV pockets, they enter the
lymphocytes. We then focused our attention on the GFP⫹ PNAd⫹ PVC and adapt a typical elongated shape likely imposed by the
HEVs of these different groups of mice, wishing to investigate if small diameter of the PVC.24 Lymphocytes further progress into
the lymphocytes were specifically held in the LN HEV pockets of this narrow corridor until they find an exit point (flap) formed by
S1P1flox/⫺ chimeras treated with tamoxifen. As presented in the membranes of 2 adjacent FRCs through which they squeeze out
Figure 5A and C, most transferred lymphocytes had already left the in the parenchyma.12 In a fully loaded LN, the cellular pressure in
HEVs of controlled LNs during the 1 hour homing assay. On the the parenchyma may narrow the diameter of the PVC and
contrary, HEVs of the tamoxifen-treated S1P1flox/⫺ chimera con- concomitantly increase the resistance of the exit flaps. In this
tained numerous labeled lymphocytes confined to these pockets, situation, the lymphocytes would be retained for a longer period of
indicating that these structures that only transiently hold lympho- time in the pockets until lymphocytes exit in the cortical sinuses
cytes in a normal situation were still occupied by lymphocytes in and release some pressure on the flaps and the PVC.
overloaded LNs. While hypothetical, this model resolves another logistical
issue. When a few lymphocytes exit the parenchyma via a given
cortical sinus, our model suggests that the nearby HEVs should
Discussion counterbalance this cellular loss. As an HEV is formed by
hundreds of ECs able to retain lymphocytes, how can each of
Overall, these data suggest that HEVs are more than passive these ECs know the number of cells they should individually
transfer points for lymphocytes from blood to LN parenchyma. By release from their pockets? As ECs constantly replace the
accommodating lymphocytes in pockets where they can be held lymphocyte content of the underlying PVC, it is likely that any
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BLOOD, 1 DECEMBER 2011 䡠 VOLUME 118, NUMBER 23 HEV CHECKPOINT CONTROLLING LYMPHOCYTE ENTRY INTO LN 6121

lymphocyte exiting the PVC would be immediately replaced by down.”37,38 Instead of creating a bottleneck situation as one would
lymphocytes nested in the closest EC sitting on top of this portion of the expect from a simple application of our pressure-gated access
PVC, thus linking local pressure relief to trafficking and flux through the model, this combination of events induces a tremendous LN
HEV pockets. swelling because of a massive increase in total cell numbers,
One question that arises from our observations is how lympho- despite the presumed structural limitation of the surrounding
cytes nested in HEV pockets exit from these structures to access the capsule. Given the results presented here, we suggest that there
PVC. To date, the most important directional cues known to attract must be mechanisms that relieve the physical constraint of pressure
and retain naive lymphocytes are chemokines such as CCL21 or build-up because of excess cell entry and subsequent proliferation
CXCL13.25-29 Interestingly, in 1976, Anderson et al observed that (eg, tension release within the structure of the capsule) and/or
on a subcutaneous injection of horseradish peroxidase, this small change the gating behavior we have described, mechanisms whose
tracer “crossed the reticular sheath, penetrated spaces between unraveling should prove valuable for better understanding lym-
endothelial cells where it surrounded emigrating lymphocytes, and phoid tissue function in health and disease.
then entered the venular lumen.”13 In 2000, the same group
described the function of the conduit system and demonstrated that
this network of reticular fibers was involved in the transport of
lymph-borne chemokines to the abluminal and luminal surfaces of Acknowledgments
HEVs.30 Altogether, these results suggest that lymph-borne chemo-
The authors thank B. Bréart, S. Schwab, and R. Proia for providing
kines are transported from the conduit system to the basal
the bone marrow cells of Ubi-cre ESR-1 ⫻ S1P1flox/⫺ mice and
membrane of the HEV before their presentation on the luminal side
constructive comments on the manuscript.
of the HEV, possibly creating a local gradient of chemokines that
This research was supported in part by CNRS, Inserm, the
lymphocytes nested in the HEV pockets may sense to find the exit
Agence Nationale de la Recherche, the Association pour la
site toward the PVC.
Recherche sur le Cancer, the Institut National du Cancer, and the
One question that still animates numerous debates in the field is
Intramural Research Program of NIAID, National Institutes of
the exact mode of lymphocyte migration across HEV ECs. While
Health.
some of these studies suggested that lymphocytes migrate between
2 adjacent ECs (paracellular migration),13,31,32 others have claimed
that lymphocytes migrate within the EC (transcellular migration),
possibly via the formation of transendothelial channels.33-36 Our Authorship
data do not support or reject any of these models and the precise
cascade of events leading to the entry of lymphocytes into the HEV Contribution: S.L.S., C.M., R.N.G. and M.B. designed research;
pockets remains to be definitely determined. S.L.S., C.M., I.M., A.J. and J.P.L. performed the experiments; and
In summary, our data provide new insight into how HEVs adjust R.N.G. and M.B. wrote the manuscript.
lymphocyte entry fluxes to balance lymphocyte exit fluxes in Conflict-of-interest disclosure: The authors declare no compet-
noninflamed LNs. Such control may of course be switched off or ing financial interests.
overridden during an active immune response. After inflammation, Correspondence: Marc Bajénoff, Centre d’Immunologie de
HEVs located in the draining LN increase their capacity to recruit Marseille Luminy, Parc Scientifique et Technologique de Marseille
naive lymphocytes and, at the same moment, lymphocytes egress is Luminy, Case 906, 13288 Marseille, France; e-mail: bajenoff@
transiently blocked, a phenomenon known as the “LN shut ciml.univ-mrs.fr.

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 From www.bloodjournal.org by guest on October 29, 2015. For personal use only.

2011 118: 6115-6122

doi:10.1182/blood-2011-07-367409 originally published
online September 21, 2011

High endothelial venules as traffic control points maintaining

lymphocyte population homeostasis in lymph nodes
Cyril Mionnet, Stéphanie L. Sanos, Isabelle Mondor, Audrey Jorquera, Jean-Pierre Laugier, Ronald
N. Germain and Marc Bajénoff

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