Subcapsular sinus macrophages in lymph nodes clear
Tobias Junt1, E. Ashley Moseman1, Matteo Iannacone1,3, Steffen Massberg1, Philipp A. Lang4, Marianne Boes2,
Katja Fink5, Sarah E. Henrickson1, Dmitry M. Shayakhmetov6, Nelson C. Di Paolo6, Nico van Rooijen7,
Thorsten R. Mempel1, Sean P. Whelan8& Ulrich H. von Andrian1
Lymph nodes prevent the systemic dissemination of pathogens
such as viruses that infect peripheral tissues after penetrating
the body’s surface barriers. They are also the staging ground of
adaptive immune responses to pathogen-derived antigens1,2. It is
unclear how virus particles are cleared from afferent lymph and
identify a population of CD11b1CD1691MHCII1macrophages
on the floor of the subcapsular sinus (SCS) and in the medulla
of lymph nodes that capture viral particles within minutes after
subcutaneous injection. Macrophages in the SCS translocated
surface-bound viral particles across the SCS floor and presented
them to migrating B cells in the underlying follicles. Selective
depletion of these macrophages compromised local viral reten-
tion, exacerbated viraemia of the host, and impaired local B-cell
activation. These findings indicate that CD1691macrophages
have a dual physiological function. They act as innate ‘flypaper’
by preventing the systemic spread of lymph-borne pathogens and
as critical gatekeepers at the lymph–tissue interface that facilitate
oral immune responses.
We have investigated how virus particles that enter peripheral
tissues are handled within draining lymph nodes. Hind footpads of
mice were injected with fluorescently labelled ultraviolet-inactivated
vesicular stomatitis virus (VSV), a cytopathic rhabdovirus that is
transmissible by insect bites3and elicits T-independent neutralizing
B-cell responses4. Using multiphoton intravital microscopy (MP-
IVM) in popliteal lymph nodes5draining the injected footpad, we
observed that VSV accumulated in discrete patches on the SCS floor
within minutes after subcutaneous injection, whereas the paren-
chyma and roof of the SCS remained free of virus (Fig. 1a and
Supplementary Movie 1). The viral deposits became progressively
denser, forming conspicuous irregular reticular patterns, which
remained fixed in place for hours.
Tocharacterize thetissue originofthepreferredVSVbinding sites
in lymph nodes, we reconstituted irradiated Act(EGFP) mice with
wild-type bone marrow. The resulting C57BL/6RAct(EGFP) chi-
maeras expressed enhanced green fluorescent protein (EGFP) in
non-haematopoietic cells, particularly lymphatic endothelial cells,
on the SCS floor and roof. On injection of fluorescent VSV into
the footpad of C57BL/6RAct(EGFP) chimaeras, viral particles
nal VSV had disappeared, but the SCS floor displayed prominent
patches of VSV that did not colocalize with EGFP1cells, suggesting
that VSV was captured by haematopoietic cells (Fig. 1b and
Supplementary Movie 3). To characterize the putative VSV-
capturing leukocytes, we performed electron microscopy on popli-
teal lymph nodes harvested 5min after injection of VSV (Fig. 1c).
Bullet-shaped, electron-dense VSV particles were selectively bound
to discrete regions on the surface of scattered large cells residing
within the SCS or just below the SCS floor. VSV-binding cells that
were located beneath the SCS floor were typically in contact with the
Ultrastructural studies of lymph nodes have shown that the SCS
contains many macrophages6,7, so we examined whether the VSV-
retaining cells belonged to this population. Indeed, confocal micro-
scopy of frozen lymph node sections obtained 30min after injection
into the footpad showed that VSV colocalized in the SCS with a
macrophage marker, CD169/sialoadhesin (Fig. 1d). Using flow cyto-
metry, we detected CD169 on about 1–2% of mononuclear cells in
lymph nodes, which uniformly expressed CD11b and MHC-II
together, indicating that the VSV-binding cells are indeed macro-
phages (Supplementary Fig. 1). Most CD1691cells also expressed
other macrophage markers, including CD68 and F4/80, but few
expressed CD11c, but at lower levels than CD11chighconventional
dendritic cells. We conclude that intact virions enter the lymph
within minutes after transcutaneous deposition and accumulate
rapidly and selectively on macrophages in the medulla and SCS of
draining lymph nodes.
ing 23108plaque-forming units) was injected into hind footpads,
was no defect in VSV retention in draining lymph nodes of mice
deficient in complement C3 (Fig. 1e). DH-LMP2a mice, which lack
secreted immunoglobulins, had decreased virus titres in spleen
but not in popliteal lymph nodes (Fig. 1f). VSV fixation in lymph
nodes therefore occurs by means of a mechanism distinct from that
used by splenic marginal-zone macrophages, which require C3 and
natural antibodies to capture blood-borne VSV8,9. Conceivably, the
VSV surface glycoprotein may be recognized in lymph nodes by
macrophage-expressed carbohydrate-binding scavenger receptors10,
but the precise mechanism will require further investigation.
What are the consequences of viral capture by macrophages for
virus dissemination and antiviral immunity? To address this ques-
tion, we depleted lymph-node-resident macrophages by injection of
1Immune Disease Institute and Department of Pathology, Harvard Medical School,2Department of Dermatology, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston,
Massachusetts 02115, USA.3Immunopathogenesis of Liver Infections Unit, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.4Institute of Experimental
of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195, USA.7Vrije Universiteit, VUMC, Department of Molecular Cell Biology, Faculty
of Medicine, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.8Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood
Avenue, Boston, Massachusetts 02115, USA.
Vol 450|1 November 2007|doi:10.1038/nature06287
clodronate liposomes (CLL) into the footpad11. At the dose used,
subcutaneously injected CLL selectively eliminated macrophages in
lymph nodes draining the injection site, including the popliteal,
inguinal and para-aortic lymph nodes11, whereas macrophages in
distal lymph nodes and spleen were spared (Supplementary Fig. 2a,
b). Among the different lymph-node-resident CD11b1MHCII1
CLL-treated popliteal lymph nodes had increased B-cell numbers
and enlarged follicles seven days after treatment, but other morpho-
logical parameters, for example demarcation of the T/B border
and the SCS ultrastructure, remained unaltered (Supplementary
In comparison with untreated lymph nodes, we recovered about
tenfold lower viral titres from the draining lymph nodes of CLL-
treated mice (Fig. 1g), suggesting that macrophage depletion ren-
dered lymph filtration inefficient. Indeed, VSV titres were markedly
increased in blood, spleen and non-draining lymph nodes of CLL-
treated mice. Viral dissemination from the injection site to the blood
depended strictly on lymph drainage, because circulating VSV was
undetectable when virus was injected into footpads of mice that
carried an occluding catheter in the thoracic duct, even in CLL-
treated mice. Viral titres were low but detectable in thoracic-duct
lymph fluid of untreated mice, but increased significantly in
CLL-treated animals (Fig. 1h). This indicates that the principal con-
duit for early viral dissemination from peripheral tissues is the
lymph, which is monitored by lymph-node-resident, CLL-sensitive
macrophages that prevent the systemic spread of lymph-borne VSV.
This capture mechanism was not unique to VSV: CD1691SCS
macrophages also retained adenovirus (Supplementary Fig. 3a–c)
and vaccinia virus (Supplementary Fig. 3d), indicating that macro-
phagesactasguardians againstmany structurally distinct pathogens.
In contrast, virus-sized latex beads (200nm diameter) were poorly
retained in the SCS after injection into the footpad (Supplementary
Fig. 3e). SCS macrophages therefore discriminate between lymph-
borne viruses and other particles of similar size. Fluorescent VSV,
adenovirus and vaccinia virus also accumulated in the medulla of
draining lymphnodes,where theywerebound notonlybyCD169low
cells (Fig. 1d) but also by CD1692LYVE-11lymphatic endothelial
cells (Supplementary Fig. 3c, d). This was corroborated in CLL-
treated lymph nodes, in which VSV accumulated exclusively on
medullary LYVE-11cells (Supplementary Fig. 4).
Next we examined how captured VSV is recognized by B cells.
Popliteal lymph nodes contain rare B cells in the SCS lumen
lymphocytes within the SCS on electron micrographs (not shown).
Second harmonic signal
Second harmonic signal
(p.f.u. per organ)]
(p.f.u. per organ)]
(p.f.u. per organ)]
Pop. LNPop. LN
Figure 1 | Capture of lymph-borne VSV by SCS
macrophages. a, MP-IVM micrographs of VSV
indicate minutes after footpad injection. UV-
VSV, ultraviolet-inactivated VSV. Scale bar,
100mm. b, VSV (red) accumulation in a C57BL/
6RAct(EGFP) recipient 3h after injection. Scale
bar, 50mm. c, Electron micrographs of VSV in
lymph node 5min after injection. The middle
micrograph is shown schematically on the left
and at higher magnification on the right.
Arrowheads identify VSV particles. Scale bars,
2mm. Mph, macrophage. d, Confocal
micrographs of VSV-draining lymph node
(30min). Wheatgerm agglutinin (WGA, blue)
100mm (left), 15mm (right). e, VSV titres in
popliteal lymph nodes 2h after injection into
wild-type mice (WT), C3-deficient mice or
macrophage-depleted WT mice that received
CLL injections into a footpad (CLL i.fp.). Three
asterisks, P,0.001 (two-way ANOVA,
Bonferroni’s post-test). p.f.u., plaque-forming
units. f, VSV capture in Balb/c (filled squares)
and DH-LMP2a (open squares) mice. Asterisk,
P,0.05 (unpaired t-test). g, VSV titres after
footpad injection in untreated (filled squares)
similar experiments; n53). Left, 2h; right, 6h.
Pop. LN, popliteal lymph node; Prox. LN,
inguinal, para-aortic lymph nodes; Brach. LN,
brachial lymph node. Asterisk, P,0.05; two
asterisks, P,0.01; three asterisks, P,0.001;
n.s., not significant (unpaired t-test). h, Viral
titres in lymph, spleen and blood after
cannulation of the thoracic duct in untreated
(filled squares) and CLL-treated (open squares)
mice. Asterisk, P,0.05 (unpaired t-test).
Horizontal bars in e–h indicate means.
NATURE|Vol 450|1 November 2007
Instead, viral particles were presented to B cells within superficial
follicles by macrophages that extended across the SCS floor. After
injection of either VSV (Fig. 2a) or adenovirus (Supplementary Fig.
5b–e), virions were readily detectable at B-cell–macrophage inter-
faces for at least 4h. This suggested that SCS macrophages shuttle
viral particles across the SCS floor for presentation to B cells.
Transcytosis seemed unlikely because the few vesicles containing
VSV in SCS macrophages showed evidence of viral degradation. In
addition, we did not detect substantial motility of virus-binding
macrophages byMP-IVM, atleastduring thefirst6hafterchallenge.
Viral particles therefore most probably reached the lymph node
parenchyma by moving along the macrophage surface. VSV and
other antigens are also presented to B cells by dendritic cells immig-
rating from peripheral locations12,13, but footpad-derived dendritic
cells are not likely to have a function during these very early events
because their migration into popliteal lymph nodes takes much
longer. We conclude that the SCS floor is not insurmountable for
lymph-borne viruses; CD1691macrophages seem to act as gate-
keepers and facilitators of viral translocation and presentation to B
Next, we explored how naive B cells respond to viral encounter by
NJ) (Supplementary Fig. 6)14. We compared wild-type B cells with B
cells from VI10YEN mice, which express a VSV-IND-specific B cell
receptor that does not bind VSV-NJ15. By contrast, a small fraction
(2–5%) of wild-type B cells bound both serotypes without being
activated. This might reflect low-affinity reactivity with VSV surface
glycoprotein or indirect interactions, for example through comple-
ment16. To assess B cell responses in vivo, differentially labelled wild-
type andVI10YEN B cellswere adoptively transferred andallowed to
home to lymph node follicles. Fluorescent ultraviolet-inactivated
virus was then injected into footpads, and popliteal lymph nodes
nodes (Supplementary Movie 4) or after injection of VSV-NJ
distribution (Fig. 2b, c). In contrast, on injection of VSV-IND,
VI10YEN cells rapidly accumulated below and within the SCS floor
(Supplementary Movie 6). There was no difference in baseline B-cell
motility and distribution between CLL-treated and untreated lymph
nodes, suggesting that VSV-specific B cells are equally likely to probe
the SCS in both conditions (not shown). However, in CLL-treated
lymph nodes, fluorescent virus was not retained in the SCS, and
VI10YEN B cells failed to congregate in that region, indicating that
SCS macrophages are essential for both events (Fig. 2b, Supplemen-
tary Movie 7).
To quantify VI10YEN B-cell distribution rigorously, lymph
nodes were harvested 30min after challenge with VSV and analysed
by confocal microscopy. Although the entire follicular VI10YEN
population retained its overall distribution (Fig. 2d), the subset of
cells residing 50mm or less below the SCS shifted towards the SCS in
VSV-IND-containing, but not VSV-NJ-containing, lymph nodes
(Fig. 2e). It seems unlikely that VI10YEN B cells redistributed to
VI10YEN B cells WT B cells
3001020300 10 20300102030
Distance to SCS (µm)
No virus30 min
Figure 2 | Macrophage-mediated transfer of
lymph-borne VSV across the SCS floor alters
virus-specific B cell behaviour. a, Electron
micrographs and schematic drawing (middle)
showing a macrophage (Mph) penetrating the
SCS floor of a popliteal lymph node 30min after
injection of VSV. Scale bars, 10mm (left), 4mm
(right). The arrow indicates a vacuole with
digested VSV; arrowheads indicate virions in the
b, MP-IVM of polyclonal (blue) and VI10YEN B
cells (red) in popliteal lymph nodes. WT, wild
type. Scale bars, 50mm. c, Regional ratios of
VI10YEN B cells/control B cells after VSV
injection. Results are from three movies per
group. Filled squares, subcapsular sinus; filled
triangles, superficial follicle; open squares, deep
of VI10YEN B cells in popliteal lymph nodes
relative to the SCS in the entire follicle (d) and in
(one-way ANOVA with Bonferroni’s post-test);
n.s., not significant; sup., superficial follicle; UV-
VSV, ultraviolet-inactivated VSV. Horizontal
bars in d and e indicate medians.
NATURE|Vol 450|1 November 2007
the SCS because of chemoattractant signals, because unresponsive
polyclonal B cells express the same chemoattractant receptors. It is
more likely that the random contacts of motile VI10YEN cells with
macrophage-bound VSV-IND triggered a B-cell receptor (BCR)-
dependent ‘stopsignal’17:short-termexposure toVSV-IND activates
LFA-1 and/or a4 integrins18on VI10YEN B cells, resulting in adhe-
sion to the respective ligands, intercellular cell-adhesion molecule-1
(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which
are both expressed in the SCS (Supplementary Fig. 7). In addition,
VSV-IND bound to SCS macrophages may provide a substrate for
VI10YEN B-cell adhesion directly by means of the BCR.
To investigate how captured virions are processed on detection by
allowed us to detect endocytosed VSV colocalizing with endosomal
MHC-II as an indicator of B-cell priming19. Within 30min after
injection, VI10YEN3MHCII-EGFP B cells in the superficial follicle
had extensively internalized VSV-IND but not VSV-NJ particles
(Fig. 3a, b, Supplementary Movie 8, and data not shown). Virus-
carrying VSV-specific B cells were infrequent but detectable in deep
cells that carried VSV on their surface (not shown), or they may
correspond to VI10YEN cells that failed to arrest at the SCS after
Although our histological findings show that intact virions are
preferentially detected and acquired by B cells in the SCS and super-
ficial follicle, MP-IVM measurements of B-cell motility revealed
broader antigen dissemination. After injection of VSV-IND,
VI10YEN cells responded with a rapid decrease in velocity through-
out the entire B follicle (Supplementary Fig. 8). This was observed
material was most probably composed of free viral protein, an
inevitable by-product of natural infections. Indeed, purified super-
natant of our VSV stocks induced a potent Ca21flux in VI10YEN B
cells (Supplementary Fig. 6e). Small lymph-borne proteins are
known to diffuse rapidly into follicles and activate cognate B cells20.
Accordingly, injection of viral supernatant suppressed the motility
of follicular VI10YEN B cells without inducing their accumulation
was contained and active within the viral inoculum. This can
explain the macrophage-independent pan-follicular effect of VSV-
To determine the kinetics of VI10YEN B cell activation on viral
encounter, we measured common activation markers (Supplemen-
6h after VSV-IND challenge. CD69 was induced more rapidly, but
also on polyclonal B cells, presumably by pleiotropic interferon-a
signalling21,22. Surface IgM (Fig. 3c, d) was downregulated as early
as 30min after challenge, reaching minimum levels within 2h when
more than 70% of VI10YEN cells were BCRlow/neg. BCR internaliza-
tion therefore provided the earliest specific readout for virus-specific
to downregulate their BCR during the first 2h after subcutaneous
injection of 20mg of VSV-IND (Fig. 3e), indicating that SCS macro-
phages are necessary for efficient early presentation of captured
virions to B cells.
Primed B cells eventually solicit help from CD41T cells19for class
switch recombination and germinal centre formation. To contact T
Distance of VI10YEN B cells
from the SCS (mm)
VI10YEN × MHCII-EGFP
Time after UV-VSV (min)
of control MFI)
B-cell frequency (%)
– CLL + CLL
Figure 3 | SCS macrophages are required for
early activation of VSV-specific B cells in lymph
(green) colocalization with VSV-IND (red;
30min after injection) in VI10YEN3MHCII-
EGFP B cells at the SCS (arrowhead) but not the
deep follicle (asterisk). Scale bar, 25mm.
b, Distance of VSV-associated and VSV-free
VI10YEN3MHCII-EGFP B cells to the SCS.
Horizontal lines indicate medians; three
on VI10YEN cells (c) and polyclonal B cells
(d) after injection of VSV-IND into the footpad.
In c: dark grey, no virus; blue, 30min; red, 1h;
green, 2h. In d: red, no virus; blue, 2h after
treatment with ultraviolet-inactivated VSV.
e, Expression of BCR on VI10YEN cells in CLL-
treated (open squares) and untreated (filled
squares) popliteal lymph nodes after injection of
VSV-IND (20mg). Mean fluorescence intensities
were normalized to virus-free values (dashed
line). UV-VSV, ultraviolet-inactivated VSV.
Results are means6s.e.m. (three to five mice).
f, g, Confocal micrograph of VI10YEN B cells in
control (f) and CLL-treated popliteal (g) lymph
nodes 6h after injection of VSV-IND (0.4mg).
Scale bar, 125mm. h, Wild-type (WT) and
VI10YEN B-cell frequency at T/B borders and in
folliclesofintact andCLL-depletedlymph nodes,
6h after injection of VSV-IND. Bars depict the
relative frequencies of transferred WT B cells
(upper four bars) and VI10YEN cells (lower
eight bars) in the two follicular compartments.
Colour coding indicates injected doses of
ultraviolet-inactivated VSV into CLL-treated
recipients (light colours) or untreated recipients
(dark colours). Error bars indicate s.e.m. n53–4
follicles per two mice; asterisk, P,0.05; two
asterisks, P,0.01; three asterisks, P,0.001
NATURE|Vol 450|1 November 2007
cells,newlyactivatedBcellsmigrate towards theT/Bborder17,23.This
VI10YEN B cells redistributed to the T/B border within 6h after
injection of as little as 40ng of VSV-IND into the footpad (Fig. 3f,
h, and Supplementary Fig. 10). By contrast, a 100-fold higher viral
dose was needed to elicit full redistribution of VI10YEN B cells in
CLL-treated mice (Fig. 3g, h). By 12h after injection, most VSV-
activated by VSV-derived antigen, although less efficiently.
Thus, we have shown a dual role for CD1691macrophages in
lymph nodes: they capture lymph-borne viruses preventing their
systemic dissemination and they guide captured virions across the
SCS floor for the efficient presentation and activation of follicular
VSV-IND and VSV-NJ virions were purified from culture supernatants of
infected BSRT7 cells and used either unmodified or fluorescently labelled with
Alexa568 (red) or Alexa488 (green). Fluorescent viruses used for tissue imaging
were irradiated with ultraviolet light to prevent the generation of non-fluor-
escent progeny. Fluorescent labelling or ultraviolet irradiation of VSV-IND
particles did not affect their antigenicity or their ability to elicit a Ca21flux in
VI10YEN cells (not shown). After injection of fluorescent virus into footpads,
draining popliteal lymph nodes were harvested for analysis by electron micro-
scopy or to generate frozen sections for immunostaining and confocal micro-
scopy. To image adoptively transferred B cells in lymph nodes, VI10YEN and
wild-type B cells were fluorescently labelled and transferred together by intra-
had homed to B cell follicles, mice were injected with labelled or unlabelled VSV
in the right footpad. At different time intervals thereafter, the draining popliteal
lymph node was observed by MP-IVM or harvested for confocal microscopy or
for flow cytometry to analyse the activation state of virus-specific and control B
cells. In some experiments, macrophages in the popliteal lymph node were
depleted by subcutaneous injections of CLL, and animals were used for experi-
ments seven to ten days later. MP-IVM, electron microscopy, immuno-
histochemistry and flow cytometry for various markers were performed on
lymph nodes with and without previous CLL treatment. Propagation of VSV
from the footpad injection site to the blood and other organs was assessed by
2 or 6h after VSV injection. To measure viral titres, tissues were homogenized
and used in plaque assays. Some viral propagation experiments were performed
after cannulation of the thoracic duct.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 29 July; accepted 21 September 2007.
Published online 14 October 2007.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank G. Cheng, M. Flynn and D. Baumjohann for
technical support; R. M. Zinkernagel and H. Hengartner for providing VI10YEN
mice; A. Wagers for providing Act(EGFP) mice; M. Ericsson and E. Benecchi for
expert support in electron microscopy studies; S. Behnke for
This work was supported by grants from the NIH-NIAID (to U.H.v.A.), a Pilot and
Feasibility Grant from the Harvard Skin Disease Research Center (to T.J. and
T32 Training Grant in Hematology (to E.A.M.).
Author Contributions T.J. and U.H.v.A. designed the study; T.J., E.A.M., M.I., S.M.
and P.A.L. performed experiments; T.J., E.A.M., M.I. and S.M. collected and
mice; T.J., E.A.M., M.I. and U.H.v.A. wrote the manuscript; S.M., K.F., S.E.H., T.M.
and S.P.W. gave technical support and conceptual advice.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. Correspondence and requests for materials should be
addressed to U.V.A. (email@example.com).
NATURE|Vol 450|1 November 2007
Mice and antibodies. C57BL/6 and BALB/c mice were purchased from Taconic
Farms. VI10YEN15, C32/2(ref. 24), MHCII-EGFP25, Act(EGFP)26and DH-
LMP2A mice27were bred in barrier animal facilities at Harvard Medical
School and the Immune Disease Institute (IDI). Radiation chimaeras were gen-
erated by irradiation of Act(EGFP) mice with two doses of 650rad and recon-
stitution with C57BL/6 bone marrow,and were allowed to reconstitute for eight
weeks before use. In some experiments, SCS macrophages were depleted by
footpad injections of 30ml clodronate liposomes(CLL), seven to ten days before
the experiment. Clodronate was a gift from Roche Diagnostics GmbH. Other
reagents for preparation of liposomes were phosphatidylcholine (LIPOID E PC;
Lipoid GmbH) and cholesterol (Sigma-Aldrich). Mice were housed under spe-
cific pathogen-free and antiviral antibody-free conditions in accordance with
National Institutes of Health guidelines. All experimental animal procedures
were approved by the Institutional Animal Committees of Harvard Medical
School and the IDI. Antibodies were purchased from BD Biosciences, except
anti-B220-Alexa647 (Invitrogen-Caltag), anti-LYVE-1 (Millipore-Upstate), goat
anti-rabbit–APC (Invitrogen), goat anti-GFP–FITC (Rockland), anti-FITC-
Alexa488 (Invitrogen) and Fab anti-IgM-FITC (Jackson Immunoresearch). The
following antibodies were purchased from AbD-Serotec: anti-CD68-Alexa647,
anti-CD11b-Alexa647, F4/80-Alexa647 and anti-CD169-FITC (3D6). The anti-
idiotypic antibody 35.61 for detection of the VI10 BCR in VI10YEN mice15was
produced from hybridoma supernatants in accordance with standard methods.
retro-orbital phlebotomy of mice and lysis of erythrocytes with ACK buffer
(0.15M NH4Cl, 1mM KHCO3, 0.1mM EDTA (disodium salt), pH7.2).
Single-cell suspensions of lymph nodes and spleens for flow cytometry were
generated by careful mincing of tissues and subsequent digestion at 37uC for
40min in DMEM (Invitrogen-Gibco) in the presence of 250mgml21liberase
CI (Roche) plus 50mgml21DNase I (Roche). After 20min of digestion, samples
were passed vigorously through an 18-gauge needle to ensure complete organ
were labelled for 90min at 37uC with 4mM Fluo-LOJO (Teflabs) in DMEM
containing 10% FCS. Cells were spun through FCS and used immediately.
Viruses and VSV plaque assay. VSV, serotypes Indiana (VSV-IND; Mudd-
Summers-derived clone, in vitro rescued28and plaque purified) or New Jersey
(VSV-NJ; Pringle Isolate, plaque purified) were propagated at a multiplicity of
infection (m.o.i.) of 0.01 on BSRT7 cells. Supernatants of infected cells were
cleared from cell debris by centrifugation at 2,000g, filtered through 0.45-mm
sterile filters and subjected to ultracentrifugation at 40,000g for 90min. Pellets
wereresuspendedinPBS andpurified byultracentrifugation (157,000g, 60min)
through a cushion of 10% sucrose in NTE buffer (0.5mM NaCl, 10mM Tris-
HCl pH7.5, 5mM EDTA pH8). After resuspension overnight in PBS, virus
protein was quantified by bicinchoninic acid assay (Pierce), and infectivity
was quantified by plaque assay. Some batches were labelled with carboxylic acid
Probes) at a 104–105-fold molar excess of Alexa dye over virus particles.
Unconjugated dye was removed by ultracentrifugation through 10% sucrose
in NTE buffer; pellets were resuspended in PBS and stored frozen. Infectivity
of VSV preparations was quantified by plaque assay on green monkey kidney
cells (Vero). VSV titres from organs of infected mice were determined similarly,
necessary, during viral preparation, the roughly 4-ml supernatants from the
157,000g ultracentrifugation were collected and concentrated with a 10-kDa
molecular mass cut-off Amicon Ultra (Millipore). To account for residual
infectivity in concentrated supernatants, VSV stocks were diluted to levels of
infectivity equal to that of the concentrated supernatants, and the Ca21flux in
VI10YEN B cells was compared over further 100-fold dilutions of VSV and
supernatant. Ultraviolet-inactivated, AlexaFluor-568-labelled adenovirus 5
was generated in accordance with standard procedures29. All work on infectious
materials was performed in designated BL21 workspaces, in accordance
with institutional guidelines, and approved by the Harvard Committee on
in MEM containing 2% FCS. Serial twofold dilutions were mixed with equal
volumes of VSV (500 plaque-forming unitsml21) and incubated for 90min at
37uC in 5% CO2. Serum–virus mixture (100ml) was transferred to Vero cell
monolayers in 96-well plates and incubated for 1h at 37uC. The monolayers
were overlaid with 100ml of DMEM containing 1% methylcellulose and incu-
bated for 24h at 37uC. Subsequently, the overlay was discarded and the mono-
IgG titres, undiluted serum was pretreated with an equal volume of 0.1mM
2-mercaptoethanol in saline solution.
recombinant murine VCAM-1-Fc or ICAM-1-Fc (R&D Systems) or with puri-
fied VSV-IND in PBS in triplicate. Negative control wells were coated with 4%
BSA, positive control wells were coated with 1 mgml21poly-(L-lysine). Plates
were blocked for 1–2h at 4uC with HBSS/1% BSA and washed. Naive B cells
from VI10YEN or C57BL/6 mice were negatively selected by magnetic cell sepa-
ration with CD43 magnetic beads (Miltenyi) and added to the plates at 33105
per well in HBSS with 1% BSA, 1mM Ca21and 1mM Mg21in the presence or
absence of ultraviolet-inactivated VSV-IND (m.o.i. 1,000) for 30min at 37uC.
After gentle washing three, times in HBSS with 1% BSA), plates were fixed
for 10min with PBS/10% glutaraldehyde, stained for 45min with 0.5% crystal
violet/20% methanol and washed in water. The dye was eluted by addition of
1% SDS and the absorbance at 570nm was determined spectrophotometrically
Devices Corporation) after 30min.
Confocal microscopy. For some analyses, both hind footpads of C57BL/6 mice
were injected with 20mg of AlexaFluor-568-labelled or AlexaFluor-488-labelled
cells from VI10YEN3MHCII-EGFP mice one day before the experiment. At
predetermined time points, popliteal lymph nodes were fixed in situ by footpad
injections of phosphate-buffered L-lysine with 1% paraformaldehyde/periodate
(PLP). After removal of popliteal lymph nodes and incubation for 3–5h in PLP
at 4uC, popliteal lymph nodes were washed in 0.1M PBS pH7.2 and cryopro-
snap-frozen in TBS tissue-freezing liquid (Triangle Biomedical Sciences) and
stored at 280uC. Sections of 40mm thickness were mounted on Superfrost Plus
slides (Fisherbrand) and stained with fluorescent antibodies in a humidified
chamber after Fc receptor blockade with 1mgml21antibody 2.4G2 (BD
Pharmingen). Samples were mounted in FluorSave reagent solution (EMD-
Calbiochem) and stored at 4uC until analysis. Images were collected with a
Bio-Rad confocal microscopy system using an Olympus BX50WI microscope
and 103/0.4 numerical aperture or 603/1.2 numerical aperture water-
immersion objective lenses. Images were analysed withLaserSharp2000software
(Bio-Rad Cell Science) and Photoshop CS (Adobe). Quantification of B cells
localized at the T/B border was done by counting cells that were within 50mm
of the T/B border, as denoted by B220 counterstain; any cells localized in more
central regions were considered follicular.
of 2% formaldehyde and 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer
pH7.4. The lymph nodeswere excisedand immersed inthe same buffer overnight
potassium ferrocyanide (in water) for 1h at 18–22uC in the dark. After being
in dilutions of ethanol in water (70%, 90% and 100%), incubated in propylene
oxide for 1h and transferred into Epon mixed 1:1 with propylene oxide at room
temperature overnight. Samples were moved to an embedding mould filled with
freshly mixed Epon, and heated for 24–48h at 60uC for polymerization. Samples
Medical School electron microscope facility.
Intravital MP-IVM of the popliteal lymph node. Naive B cells were negatively
selected by magnetic isolation with CD43 beads (Miltenyi). VI10YEN B cells
were labelled for 20min at 37uC with 10mM 5-(and 6-)-(((4-chloromethyl)
benzoyl)amino)tetramethylrhodamine (CMTMR; Invitrogen), C57BL/6 B cells
werelabelledfor25minat 37uC with10mM 7-amino-4-chloromethylcoumarin
(CMAC; Invitrogen). In some experiments, labels were swapped between wild-
type and VI10YEN B cells to exclude unspecific dye effects (data not shown). B
cells ((5–6)3106) from eachpopulationwere mixed andadoptively transferred
by tail-vein injection into C57BL/6 recipient mice one day before analysis. In
some experiments, recipient C57BL/6 mice had received an injection of 30ml of
CLL into the hind footpad 7–10 days before the experiment to eliminate SCS
macrophages11. At 18h after adoptive B-cell transfer, recipient mice were anaes-
thetized by intraperitoneal injection of ketamine (50mgkg21) and xylazine
(10mgkg21). The right popliteal lymph node was prepared microsurgically
for MP-IVM and positioned on a custom-built microscope stage as described5.
Care was taken to spare blood vessels and afferent lymph vessels. The exposed
lymph node was submerged in normal saline and covered with a glass coverslip.
A thermocouple was placed next to the lymph node to monitor local temper-
2100MP system at an excitation wavelength of 800nm, from a tunable MaiTai
Ti:sapphire laser (Spectra-Physics). Fluorescently labelled VSV (20mg in 20ml)
was injected through a 31-gauge needle into the right hind footpad of recipient
mice concomitant to observation. For four-dimensional offline analysis of cell
migration, stacks of 11 optical x–y sections with 4-mm z spacing were acquired
every 15s with electronic zooming to 31.8–3.0 through a 203/0.95 numerical
aperture water-immersion objective lens (Olympus). Emitted fluorescence
and second-harmonic signals were detected through 400/40nm, 450/80nm,
525/50nm and 630/120nm bandpass filters with non-descanned detectors to
generate three-colour images. Sequences of image stacks were transformed into
volume-rendered, four-dimensional time-lapse movies using Volocity software
(Improvision). Three-dimensional instantaneous velocities were determined
by semi-automated cell tracking with Volocity and computational analysis by
Matlab (Mathworks). Accumulation of cells at the SCS was determined by
manual movie analysis performed by blinded observers. Every 2min, the
follicle (less than 50mm distance from the SCS) and the deep follicle (more than
50mm distance from the SCS), and ratios of VI10YEN/polyclonal B cells were
expressed for each compartment in the entire 30-min movie.
of olive oil by gavage 30min before cannulation to facilitate visualization of the
lymph vessels. Animals were then anaesthetized with xylazine (10mgkg21) and
ketamine HCl (50mgkg21). A polyethylene catheter (PE-10) was inserted into
the right jugular vein for continuous infusion (2mlh21) of Ringer’s lactate
(Abbott Laboratories) containing 1Uml21heparin (American Pharmaceutical
through a left subcostal incision. Silastic silicon tubing (0.012inch internal dia-
meter; Dow Corning) was flushed with heparinized (50Uml21) PBS (DPBS;
Mediatech), inserted into the cisterna chyli through a roughly 0.3-mm incision
and fixed with isobutyl cyanoacrylate monomer (Nexaband; Abbott
Laboratories). The remaining part of the tubing was drawn to the exterior
through the posterior abdominal wall. Subsequently, the abdominal incision
was closed with a 6-0 nonabsorbable running suture (Sofsilk; Tyco Healthcare
collected on ice for 6h. Blood and organs were taken after 6h of collection of
thoracic duct lymph and plaque-assayed as described above. Lymph and organs
were plaque-assayed as described above. In some experiments the draining pop-
liteal and para-aortic lymph nodes were surgically excised and the surrounding
lymph vessels were cauterized to prevent lymph-borne viral access to the blood.
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