Requirements for T Lymphocyte Migration in Explanted Lymph Nodes

The Journal of Immunology

Requirements for T Lymphocyte Migration in Explanted
Lymph Nodes1

Julie H. Huang,* L. Isabel Cardenas-Navia,† Charles C. Caldwell,‡ Troy J. Plumb,§
´
¶
Caius G. Radu, Paulo N. Rocha, Tuere Wilder,# Jonathan S. Bromberg,**
Bruce N. Cronstein,# Michail Sitkovsky,†† Mark W. Dewhirst,†‡‡ and Michael L. Dustin2*
Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the
requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using
two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We
have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive
T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 m/s and a partial pressure of
O2 (pO2) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles
and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement
for pO2, but also decreased the speed of locomotion in the deep paracortex. Although CCR7 / naive T cells displayed a
small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating
a contribution of G i-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor
knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-
phosphate pathways did not individually alter naive T cell migration. These data implicate pO2, tissue architecture, and
G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs. The Journal of
Immunology, 2007, 178: 7747–7755.

T he adaptive immune response requires matching the Ag MHC is formidable (3). The first step is for DC to gather Ags from
TCR repertoire to peptide-MHC adducts that can enter the the tissues or blood (4, 5). In the second step, the DC migrate to the
body at any location to sense pathogens that enter the lymph nodes (LNs) and form networks in the T cell zones of LNs
body at any location (1, 2). Because each TCR specificity is borne and/or the spleen (6 – 8). Then, T cells enter the LN or spleen from
on a few hundred T cells, the problem of bringing these T cells to the blood and survey the DC networks to detect Ags (9 –11). In-
the dendritic cells (DC)3 (3) bearing the pathogen-derived peptide- sights into the nature of the final step in which T cells scan DC
networks for Ag have been provided by two-photon laser-scanning
microscopy, which allows microscopic examination of fluorescent
*Program in Molecular Pathogenesis, Kimmel Center for Biology and Medicine, T cells and DC interacting in live tissues or animals (12–14).
Skirball Institute, New York University School of Medicine, New York, NY 10016;
†
Department of Biomedical Engineering, Pratt School of Engineering, Duke Univer- The first insights into this repertoire-scanning process were ob-
sity, Durham, NC 27708; ‡Laboratory of Trauma, Sepsis and Inflammation Research, tained by imaging T cell movement in explanted LNs. In these
Department of Surgery, University of Cincinnati, Cincinnati, OH 45267; §Department experiments, fluorescent T cells were transferred into nonfluores-
of Internal Medicine, Section of Nephrology, University of Nebraska Medical Center,
Omaha, NE 68198; ¶Department of Microbiology, Immunology and Molecular Ge- cent recipient mice and allowed to enter LNs via the high endo-
netics, University of California, Los Angeles, CA 90095; Division of Nephrology, thelial venules; the LN was removed without disrupting the cap-
Duke University, Durham Veterans Affairs Medical Centers, Durham, NC 27705;
#
Divisions of Clinical Pharmacology and Rheumatology, Department of Medicine,
sule and perfused with 95% O2 equilibrated medium (partial
New York University School of Medicine, New York, NY 10016; **Department of pressure of O2 (pO2) 95%). Under these conditions, T cells were
Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029; observed to migrate rapidly and randomly with an average velocity
††
Departments of Biology and Pharmaceutical Sciences, Northeastern University,
Boston, MA 02115; and ‡‡Department of Radiation Oncology, Duke University Med- of 10.8 m/min and a motility coefficient for the three-dimensional
ical Center, Duke University, Durham, NC 27710 (3D) random walk of 67 m2/min (13). Similar results were ob-
Received for publication February 12, 2007. Accepted for publication April 4, 2007. tained in vivo suggesting that the high pO2 was compatible with
The costs of publication of this article were defrayed in part by the payment of page physiological migration patterns (15). Recent studies suggest that
charges. This article must therefore be hereby marked advertisement in accordance T cells navigate in the LN by crawling along stromal cell networks
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
that underlay the DC networks and are even more elaborate than
This work was supported by National Institutes of Health Grants AI55037 (to
M.L.D.) and DK38108 (to T.J.P. and P.N.R.) and the Irene Diamond Foundation previously anticipated (16). Nonetheless, the pattern of in vivo
(to M.L.D.). migration can be described as autonomous in that the T cells ap-
2
Address correspondence and reprint requests to Dr. Michael L. Dustin, Program in pear to turn in random fashion as long as they remain in the T cell
Molecular Pathogenesis, Skirball Institute, New York University Medical Center, 540 zones. Although T cell direction appears to be chosen at random in
First Avenue, New York, NY 10016. E-mail address: dustin@saturn.med.nyu.edu
3
the 3D lattice, the signaling processes that motivate the T cells are
Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; pO2, partial
pressure of O2; 3D, three dimensional; PTX, pertussis toxin; WT, wild type; GPCR,
not known.
G protein-coupled receptor; TP, thromboxane-prostanoid receptor; ETYA, 5,8,11,14- Imaging lymphocyte migration in explanted LNs provides an
eicosatetraynoic acid; 2D, two dimensional; A2AR, A2A adenosine receptor; S1P, opportunity to study the molecular and physical requirements in a
shpingosine-1-phospate.
way that would be difficult or impossible in a live mouse. T lym-
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 phocytes in the periphery face a wide range of pO2 from as high as

www.jimmunol.org

7748 REQUIREMENTS FOR T CELL MIGRATION

20% in lung alveoli to 1–5% in most peripheral tissues including submerged in Invitrogen Life Technologies Leibovitz’s L-15 medium
secondary lymphoid tissues and 1% in sites of tissue injury or equilibrated with a range of O2:N2 gas mixtures.
The measurement of each LN started from the capsule and the probe was
necrosis (17). Perfusion and high pO2 are the most commonly used
inserted at increments of 50 m. Measurements of pO2 inside the LN were
in vitro conditions, but quantitative studies on requirements for taken at 10 different locations and angles to a maximal depth approximately
either of these parameters have not been performed. Because sec- half the diameter of the LN and pO2 vs depth was plotted. All measure-
ondary lymphoid organs are thought to be typical of most tissues ments were obtained at 35–37°C.
with pO2 in the range of 1–5% (17), it is unclear whether the high
Explanted LN imaging and pO2 measurement
extranodal pO2 conditions are required to achieve a physiological
pO2 within the LN due to the diffusion barrier of the capsule and The setup was composed of a heated flow cell (Bioptechs) with a flow
the depth of the T cell zones or whether the pO2 in the T cell zones channel 1 mm high and 1.3 mm wide and a syringe pump (Harvard Ap-
paratus) to provide a regulated perfusion velocity. The perfusion velocity
is superphysiological under these conditions (15, 18). was defined as the flow rate (volume/time) divided by the cross-sectional
In this study, we investigated the role of O2 and perfusion as area of the flow channel (area) to yield a linear average velocity (distance/
chemokinetic factors for T cell migration in explanted LNs and time). We neglected local changes in velocity that would be expected due
compared the role of O2 to that of signaling through G i-coupled to the parabolic flow in the parallel plate flow cells and the disturbance of
flow by the LN itself, which covers 10% of the area of the flow channel.
receptors classically linked to cell migration. We have also inves- O2:N2 mixes were bubbled with a gas dispersion tube into the medium
tigated the role of a number of specific G protein-coupled receptors reservoir to provide regulated pO2. A special adapter was purchased from
(GPCR) to determine whether these candidates have a nonre- Bioptechs to allow the mounting of the flow cell, which is typically used
dundant role in regulating T cell migration. We focused on one on an inverted microscope, for use on an upright microscope. The O2 level
chemokine receptor, CCR7, as an important candidate upstream in the system is measured with a fiber optic O2 probe that measures lumi-
nescence lifetime and does not consume O2 during the measurement
of G i (19 –21). We found that perfusion above a threshold (Oxymicro; WPI), which allowed for accurate, constant monitoring of pO2.
velocity with pO2 levels 2-fold higher than physiological The fiber optic probe was placed at the outlet of the flow cell such that
measurements is critical for migration of naive T cells in ex- medium pO2 was read throughout the experiment 300 l downstream of
planted LN. Activated T cells migrate more slowly, but with a the Bioptechs flow cell containing the LN.
low pO2 threshold. Surprisingly, no single upstream receptor Two-photon microscopy
could be identified as important in T cell migration in the ex-
planted LNs, but elimination of all G i-mediated signaling pro- We used a custom-built system for our two-photon imaging (24, 25). It
consists of a modified upright Olympus microscope and a modified con-
foundly decrease T cell migration. focal scan head fitted with a 60 water immersion objective (NA 0.9)
and a Spectra-Physics Tsunami femtosecond laser tuned to 780 nm (26). A
Materials and Methods nondescanned photomultiplier tube mounted off the epifluorescence filter
turret was used to detect fluorescence emission of 700 nm (12). Data
Mice acquisition were controlled by the Fluorview software and analyzed using
DO11.10 on the B10.D2 background ( 10 generated backcrossed) and Volocity (Improvision). T lymphocyte position over time was determined
B10.D2 mice ranging from 6 to 12 wk of age were used in all experiments, by acquiring XYZ stacks of 10 –25 XY images of 512 512 pixels (236
except CCR7 / mice (19) were on the B6 or BALB/c background and 236 m) with an axial spacing of 2–3 m. These XYZ stacks were ac-
A2AR / mice (22) were on the B6 background. All mice were bred and quired every 30 s for 15–30 min; X, Y, Z, T data sets were then used to
maintained in accordance with the Institutional Animal Care and Use calculate the instantaneous velocity, turning angle, confinement index, and
Committee guideline of New York University School of Medicine. arrest coefficient. The instantaneous velocity of fixed points in the tissue
BALB/c mice for the CCR7 experiments were purchased from The was not 1.5 m/min when determined by tracking with Volocity. Thus,
Jackson Laboratory. 1.5 m/min was used as a threshold for immobility when calculating the
arrest coefficient.
Cells
Statistics
Naive DO11.10 T lymphocytes were purified by negative selection using
magnetic beads from the pan-T selection kit (Miltenyi Biotec). The cells Comparisons were analyzed for statistical significance using KaleidaGraph
were then labeled with 1 M CFSE at room temperature for 15 min in 2 4.0 software (Synergy). Nonparametric methods such as Wilcoxon-Mann-
mM HEPES, 13.8 mM NaCl, 0.5 mM KCl, 0.07 mM Na2HPO4, and 0.6 Whitney test or ANOVA were used to determine significance.
mMD-glucose (HBS). The concentration of dye was chosen through sys-
tematic preliminary studies balancing fluorescence intensity, viability, and Results
chemotactic responses in vitro. It was important to include 5 mM D-glucose Intravital measurement of pO2 in LNs
during labeling. A total of 5–10 million labeled cells were adoptively trans-
ferred via tail vein injection to the host B10.D2 mice 1 day before imaging. To have a point of reference for ex vivo studies, we first deter-
By flow cytometry, we found the injection of 3 106 cells results in mined the pO2 in the inguinal LN in vivo using an O2 microelec-
0.3– 0.5% CFSE-positive cells in the inguinal LN at day 3. trode. This approach has been extensively used in tumor and mus-
For blast preparation, we mixed 350 g of OVA peptide and 5 g of
LPS for tail vein injection along with the CFSE-labeled naive cells. Im-
cle tissues (27). The probe was calibrated using buffer solutions
aging was performed 24 h after transfer and initiation of activation. The saturated with different gas mixes. The inguinal LN was exposed
activation status was confirmed via FACS analysis. and immobilized using the same surgical approach as used for
Where indicated, mice were injected with 200 g/kg pertussis toxin intravital imaging (28). The physiological pO2 in the LN was be-
(PTX) (Calbiochem) or the same volume of PBS 12–16 h after adoptively tween 0.5 and 6% and varied by location or depth (Fig. 1). The
transferring naive T lymphocytes. Imaging of explanted LNs was per-
formed at 18 –24 h after the treatment. overall mean value for all measurements was 2.2%. The sudden
increase of pO2 in some traces may be due to the probe encoun-
Measurement of LN pO2 tering capillaries. Similar results were obtained from a previous
pO2 was measured with a recessed-tip O2 microelectrode having a tip di-
study focusing on normal spleen (17) and muscle (27).
ameter of 9 –12 m (23). Depth of insertion in the LN was controlled with
a micromanipulator. Electrode current was measured with a minisensor and Measurement of pO2 in explanted LNs
recorded by a computer. For intravital measurements, the inguinal LN was To determine how pO2 within an explanted LN relates to the pO2
exposed and immobilized to allow electrode insertion. For ex vivo analysis,
the mouse was sacrificed and the inguinal LN was removed. After mea- in the perfusing medium, we performed O2 microelectrode exper-
suring the size of the LN, ethyl cyanoacrylate adhesive (Elmer’s Products) iments on excised LNs. We perfused the excised LN in a 37°C bath
was used to fix the LN on nylon mesh in a petri dish, the LN was then that was vigorously bubbled with different O2:N2 gas mixes. The

The Journal of Immunology 7749

FIGURE 1. Direct pO2 measurements in LN in vivo. Each line repre-
sents measurements alone one track from the point of microelectrode probe
insertion up to 500 m deep into the LN at 50- m steps. Each point of
measurements is an average of pO2 readout over 10 –30 s. Each track was
FIGURE 3. Perfusion requirements for naive T lymphocyte locomotion
measured from a different location on the LN and the track location was
in explanted LNs. Each column represents one flow velocity set by the
represented on the inguinal LN schematic at the lower right side. The
syringe pump under controlled temperature and pO2. Each dot represents
experiment shown is representative of four LNs.
the average speed of one single cell, which was calculated by dividing the
path length over time. The bar in each column represents the average speed
of all the cells tracked. The medium was equilibrated at 20% pO2. Tracks
bubbling both saturated the medium at a particular pO2 and pro- from two LNs are shown.
vided turbulent mixing around the LN to ensure that the medium
was moving rapidly over the surface of the LN. Then, we mea-
our system, which was sufficient for naive T lymphocyte migration
sured the change of pO2 within the LN at different depths using the
when perfusion was set at 40 m/s (see below). Whether this re-
microelectrode (Fig. 2A). Surprisingly, pO2 was independent of
quirement was due to decreased pO2 in the LN or due to some
depth up to 500 m. Media and LN pO2 were linearly correlated
(Fig. 2B). We conclude that O2 penetrates rapidly into the LN
and that O2 consumption in the tissues did not substantially
reduce the pO2 during this diffusion/transport process. Thus, it
was not necessary to use high-medium pO2 to obtain physio-
logical pO2 at up to 500 m.

Effect of perfusion velocity on naive T lymphocyte locomotion
Bousso and Robey (29) noted that moving perfusion is essential
for naive T lymphocyte movement, but did not quantify the re-
quirement. We determined the effect of flow velocity on the mi-
gration speed of naive T lymphocytes measured by two-photon
time-lapse microscopy at 150- m depth in the deep paracortex.
Naive T lymphocytes moved rapidly when the flow velocity was
set at 40 m/s or faster at 20% pO2, and were immobile (speed
lower than 1.5 m/min) at flow velocities of 13 m/s or less,
regardless of pO2 (20 –100%) (Fig. 3). It should be noted that
extranodal pO2 was maintained at over 15% without perfusion in

FIGURE 2. Measurements of pO2 in explanted LN. A, Three lines FIGURE 4. O2 requirements for naive and activated T lymphocyte loco-
represent three independent measurements of pO2 at increments of 50 motion. A, The real-time measurement of naive T lymphocyte locomotion vs
m into the explanted LN, each in medium equilibrated with different environmental pO2. Each graph represents one data set and the black line
gas mixtures. This provides a readout of environment medium pO2 demonstrates the drop of pO2 that was recorded by Oxymicro while constantly
shown as 0 m. The medium (L-15) was vigorously bubbled with O2:N2 purging O2 in the system. The gray line is the average instantaneous velocity
gas mixes and the explanted LN was glued down on a nylon mesh fixed of at least three cells in the filed of image and the x-axis represents time. B,
on a petri dish. The setup was submerged in L-15 while maintaining at Activated T lymphocyte requires less O2 to maintain locomotion. Using same
35–37°C throughout the experiment. B, The correlations between me- type of measurement, the two graphs show that in these two data sets, the
dium pO2 and the average LN pO2 are high (R 0.95). Each dot activated T lymphocytes maintaining locomotion until pO2 drops to 3– 4%.
represents an average of LN pO2 measured at a fixed depth and medium Each panel is a single experiment. A total of three experiments were performed
pO2. Representative of five LNs. for naive and activated T lymphocytes to obtain average values for thresholds.


an A2AR antagonist, or from WT mice transferred with CFSE-
labeled A2AR / T cells. Neither of these conditions revealed any
changes in pO2 sensitivity of naive T cell migration (Fig. 5).

T lymphocyte features in different LN areas, activation states,
and role of G i
We next wanted to ask whether there is any difference in the cell
motility at different depths within a LN using the two-photon im-
FIGURE 5. A2AR / T lymphocytes stopped migrating at the same pO2
as control cells. The real-time measurement of control (left) and A2AR / aging system in shallow and deep regions. We tracked T lympho-
(right) T cell locomotion vs environmental pO2. Each graph represents one cyte movements and then calculated the speed, turning angle, con-
data set and the black line demonstrates the drop of pO2 that was recorded finement index, and arrest coefficient to determine whether naive T
by Oxymicro while constantly purging O2 in the system. Gray line is the lymphocytes had different types of mobility in the shallow and
average instantaneous velocity of at least three cells in the filed of image deep compartments within a LN. The turning angle and confinement
and the x-axis represents time. index quantify the nonlinear nature of T lymphocyte movement. The
higher the turning angle the less linear the cell movement. Con-
finement index is defined here as 1 minus the maximum cell dis-
other effect of perfusion, such as delivering a nutrient or removing placement over total path length; therefore, a higher confinement
a waste product, was not determined. index is representative of greater nonlinearity. The arrest coeffi-
cient is the proportion of time that lymphocytes were moving 1.5
Effect of pO2 on naive T lymphocyte locomotion
m/min (defined as stopped). We define the shallow region in our
We performed real-time pO2 titrations. We started imaging CFSE- system as the subcapsulary sinus, the compartment that is under-
labeled T lymphocytes in the LN perfused at 20% pO2 with a flow neath the capsule and surrounding the cortex and we generally
velocity of 40 m/s and then slowly purged O2 from the medium collect data from a depth of 20 –50 m below the capsule. We
in the reservoir by bubbling with 100% N2, while imaging the LN. visualized the capsule using the second harmonic signal of the
This resulted in a decrease in pO2 of 0.5%/min, which was di- collagen fibers. We define our deep region as the area deeper than
rectly measured with an inline pO2 sensor and plotted against the 150 m from the surface, which corresponds to interfollicular to
instantaneous speed of CFSE-labeled naive T lymphocytes in the deep paracortex T cell zones. These locations were defined in early
3D imaging volume (Fig. 4A). Cell movement stopped at an av- articles (31) and immunostaining of LN by us and other research-
erage of 7.4 1.7% pO2 and was at 50% maximal speed at 10.4 ers (8, 32) is consistent with these definitions.
1.2% pO2. The pO2 required to support T lymphocyte locomotion T lymphocytes moved significantly slower in the shallow com-
in the perfused explanted LNs was greater than the physiological pared with deep regions (median velocity 5.68 vs 9.22 m/min)
pO2 by 2-fold. (Table I). We found that shallow cells were moving with signifi-
cantly less directionality, as the shallow cells had an median turn-
Effect of adenosine receptor A2AR ing angle of 77° while deep cells had an median turning angle of
The result from the O2 experiments led us to investigate whether 47°. The confinement indices for the shallow and deep cells were
the sensitivity of lymphocytes to pO2 reflected a regulatory strat- 0.63 and 0.42, respectively ( p 0.05), although the shallow cells
egy in vivo. When inflammation or tissue damage-associated hyp- had a higher arrest coefficient (0.33) compared with deeper cells
oxia occurs, it is thought that the hypoxia-induced adenosine ac- (0.23), this difference was not statistically significant ( p 0.14).
cumulation triggers increased cytoplasmic cAMP in immune cells Thus, we conclude that T lymphocyte movement was both slower
via A2A adenosine receptors (A2AR), leading to down-regulation and more confined in the shallow subcapsulary region, but slowing
of inflammation (see review in Ref. 30). We hypothesized that this was not attributed to increased cell arrest alone.
pathway may also modulate T lymphocyte migration at low pO2 To address whether activation states could alter the migration
such that antagonism or deficiency of A2AR could result in motility properties of T lymphocytes, we activated the naive DO11.10 T
at lower pO2. lymphocytes by injecting OVA peptide in vivo and then waited
We tested this hypothesis in two ways. We visualized naive T 24 h before imaging migration of the in vivo-activated T lympho-
lymphocyte locomotion in LNs isolated from mice transferred with cytes. At this time, T lymphocytes are resuming migration after
CFSE-labeled wild-type (WT) T cells and treated with ZM241385, early arrest with APC (14, 18, 33). We determined the level of

Table I. Summary table of T lymphocyte motility parameters for experiments determining the importance of depth, activation
state, CCR7, and Gi PCRa

Median Velocity Median Turning
Experiment Cell Type ( m/min) Arrest Coefficient (%) Confinement Index (%) Angle (°)

Depth Deep 9.22 p 0.0001 22.58 2.2 p 0.14 58.21 3.9 p 0.05 47.39 p 0.0001
Shallow 5.68 33.18 6.2 37.32 5.0 77.34
Activation Naive 9.16 p 0.0001 23.08 4.1 p 0.48 60.38 5.2 p 0.07 60.34 p 0.0001
Activated 4.16 31.68 15.3 46.26 2.5 49.95
G -i Control 10.45 p 0.0001 18.64 0.3 p 0.04 60.25 5.6 p 0.01 46.49 p 0.0001
PTX treated 4.1 53.96 13.1 43.83 5.3 84.2
CCR7 Control 11.2 p 0.06 17.96 1.1 p 0.07 54.32 3.1 p 0.11 48.13 p 0.0001
Knockout 8.68 25.68 0.8 43.51 3.1 62.21
a
All the experiments were performed on at least three animals for each condition and two to four movies were collected for each animal. The Wilcoxon
Mann-Whitney test was used to determine significance for median velocity and median turning angle, and the t test was used for average arrest coefficient
and confinement index.


CCR7, have important roles in T cell entry into (via both high
endothelial venules and lymph) and positioning (20, 36), so we
transferred CCR7-deficient cells into a WT host, and recorded their
locomotion. It must be noted that the entry of CCR7-deficient
T cells into LNs after transfer was much less efficient than for
WT T cells as previously reported. However, highly pure
CD4 CD62L CD44 naive T cells did enter at levels that al-
lowed tracking, with a small fraction of cells showing central
memory T cell phenotype (Fig. 6A). We found marginally signif-
icant changes in the median velocity (8.68 vs 11.20 m/min, p
0.06), arrest coefficient (0.18 vs 0.26, p 0.07) and confinement
index (0.54 vs 0.44, p 0.11), and with significant increase in
median turning angle compared with WT (62.21° vs 48.13°, p
0.0001) (Table I, Fig. 6B). Thus, CCR7 contributes a small fraction
of signaling for T cell movement within the explanted LNs.
B cell migration in LNs is partially dependent upon G i2 based
on genetic experiments (37). We wanted to determine whether
there is a general requirement of G i activity for T lymphocyte
movement in the LN. PTX is internalized by cells and the catalytic
FIGURE 6. G i protein-coupled receptors, but not CCR7, are re- subunit enters the cytoplasm and ribosylates G i, locking coupled
quired for normal naive T lymphocyte motility. A, Transferred naive GPCR in an inactive state (38 – 40). Because G i is also required
CCR7 / T cells ( ) enter the LN with reduced efficiency, a small for entry into LNs, we took the approach of treating mice with
percentage of central memory T cells (f) were shown. Approximately PTX systemically a day after adoptive transfer of CFSE-labeled T
5 106 purified CFSE-labeled CD4 CD62L T lymphocytes from
lymphocytes, so that the labeled T lymphocytes were already
either CCR7 / or WT were transferred into WT recipients by tail vein
equilibrated in the LN, and then tested T lymphocyte movement
injection. Eighteen hours later, the inguinal LN was harvested and an-
alyzed by flow cytometry. B, Representative dot plot of the velocity of 24 h after PTX treatment. To evaluate the appropriate dose we
CCR7 / and WT T cells in explanted LNs perfused at 40 m/s and used ex vivo transmigration to CCL21. The doses of PTX that are
20% pO2. The horizontal bar indicates the median velocity. C, WT mice typically administered as an adjuvant for induction of autoimmune
were treated with either 40 (1 g) or 200 (5 g) g/kg of PTX (f), or disease or for oral immunization did not inhibit migration of T
equal volume of PBS ( ). Lymphocyte transmigration was abolished lymphocytes (Fig. 6C). Increasing the dose of PTX 5-fold higher
24 hours after treatment with 5 g of PTX, and was 50% reduced after nearly abrogated ex vivo T lymphocyte migration to CCL21 (Fig.
treatment with 1 g of PTX. D, T lymphocyte in vivo motility from the 6C). Naive T lymphocytes in LN from these high-dose PTX-
LN of mice treated with PBS (control) or 200 g/kg PTX (PTX), as
treated mice were 60% slower than the control (Fig. 6D, Table
measured by two-photon imaging. Representative of three experiments.
I) ( p 0.0001) and had significantly higher arrest coefficients
(p 0.04), turning angles ( p 0.0001), and confinement in-
CD69 on the T cells from the explanted LNs after imaging by dices ( p 0.01) compared with the controls (Table I). Thus,
flow microfluorometry expression to confirm the CFSE-labeled G i contributed to a large fraction of signaling for rapid naive
DO11.10 T cells were activated (data not shown). Activated T T lymphocyte migration in LN, but residual, highly confined
lymphocytes moved significantly more slowly (median velocity migration was still detected in the absence of G i function. This
4.16 m/min) compared with naive cells (median velocity 9.16 demonstrates that T cell migration in LNs is mostly directed by
m/min) at this time. Differences between their directionality and GPCR-mediated chemokinetic signals, rather than being an au-
arrest coefficient were not significant (Table I). When perfusion tonomous program.
was maintained at 40 m/s and pO2 decreased, the activated cells Thromboxanes act through a GPCR, the thromboxane-prosta-
were able to migrate at their maximum velocity at lower pO2 than noid (TP) receptor (41), and have been implicated in control of T
naive T lymphocytes (Fig. 4B). Activated T lymphocytes stopped cell-DC interactions via G0 signaling (42) and immune responses
only when pO2 was 3%, the lowest level that we achieved by (43). We tested whether TP functions by influencing cell migration
bubbling with 100% N2 in a vessel open to room air. Thus, acti- in explanted LN by imaging TP / T cells transferred into WT
vated T lymphocytes had lower requirements for pO2 in the ex- mice. We found no difference between TP / and control T cells
planted LN compared to naive T lymphocytes. when comparing speed, arrest coefficient, confinement index, and
Chemokines are responsible for the compartmentalization of im- turning angle (Table II). To more generally test the role of arachi-
mune cell within secondary lymphoid tissues with CCR7 playing donic acid metabolites in T cell migration in the LN, we treated
a major role for T cells (34, 35). CCL21/SLC, the ligands for mice with 250 mg/kg cyclooxygenase/lipoxygenase inhibitor

Table II. Summary table of T lymphocyte motility parameters for experiments determining the importance of TP and G2A, two
GPCRs in the arachidonic acid cascadea

Experiment Cell Type Speed ( m/min) Arrest Coefficient (%) Confinement Index (%) Turning Angle (°)

TP Control 9.71 0.6 p 0.95 20.64 2.1 p 0.28 60.38 5.2 p 0.36 64.23 8.9 p 0.62
Knockout 9.42 0.3 29.02 5.6 52.82 3.4 73.96 7.4
G2A Control 10.11 0.8 p 0.88 21.37 1.2 p 0.44 59.47 2.2 p 0.72 61.34 7.6 p 0.92
Knockout 9.84 1.1 27.19 7.2 56.4 4.5 62.06 9.5
a
These were done using the confocal microscope to collect 2D data sets. All the experiments were performed on at least three animals for each
condition and two to four movies were collected for each animal.


Although we were unable to establish conditions under which
naive lymphocytes could migrate with physiological pO2 in ex-
planted LNs, we were able to obtain high mobility with pO2
much lower than the routinely used 95%. Although there is no
evidence that this high level of pO2 has caused toxic effects or
artifacts, the ability to achieve rapid locomotion at more phys-
iological pO2 is attractive.
During human T lymphocyte activation, metabolism shifts
FIGURE 7. Evaluation of two lipid signaling pathways for effect on from a mixture of oxidative phosphorylation and glycolysis, to
naive T cell migration. A, Blockade of arachidonic acid pathway has no primarily anaerobic glycolysis (51, 52). Mandeville and Wilkin-
effect on T cell migration speed. Confocal microscopy was used to measure son (53) have shown that human lymphoblasts migrate under
the 2D motility. T cell locomotion speed from mice either vehicle treated anaerobic conditions in vitro, but ours is the first study to ad-
(control) or treated with 250 mg/kg ETYA (ETYA) show no significant
dress pO2 in the context of intact lymphoid tissues and with
difference (7.63 vs 7.50 m/min). B, FTY720 treatment does not effect T
cell migration speed in LN. T cell migration speed from mice treated with
naive T lymphocytes. Mempel et al. (18) noted that lymphocyte
vehicle (control) or FTY720 6 mg/mouse for 4 h (4 h) or 24 h (24 h) show motility stopped immediately when blood flow stopped in in-
no significant difference when comparing their 2D motility speed (7.59 vs travital experiments and suggested that this could be due to O2
7.97 vs 6.81 m/min). requirements. However, we would suggest that the rapid de-
crease in motility is more likely due to loss of perfusion, such
that the specific limiting metabolite or signal could not be iden-
5,8,11,14-eicosatetraynoic acid (ETYA) i.p. (44). Treatment with tified in that study. We found that activated T lymphocytes can
this concentration of ETYA for 18 h decreased PGE2 10-fold in migrate under lower pO2 than naive lymphocytes, which is con-
LNs (data not shown). We used confocal microscopy to image sistent with these metabolic differences noted above. Naive T
explanted LNs from mice treated or untreated with ETYA and lymphocytes recirculate and function almost exclusively in sec-
found no significant difference in the two-dimensional (2D) mo- ondary lymphoid tissues, which are well supplied by blood
tility speed (7.63 vs 7.5 m/min) (Fig. 7A). Thus, we found no flow. In contrast, activated T lymphocytes must function in di-
evidence for a role of arachidonic acid metabolites in control of verse peripheral tissue sites, which may include hypoxic re-
basal T cell migration in explanted LNs. gions of tumors or sites of tissue injury that may be hypoxic due
Lipid mediators like shpingosine-1-phospate (S1P) and lyso- to damaged or abnormal vasculature (54). Thus, it is likely to be
phosphatidylcholine acts through GPCR and can mediate che- functionally important that activated T cells can migrate at
motaxis of lymphocytes. It has been demonstrated that the G i lower pO2.
coupled S1P receptor 1 (S1P1) antagonists have no effect on T Hypoxia inducible factor controls the expression levels of sev-
cell migration in T cell zones (45). We found similar results eral essential genes that are required for cell and tissue adaptation
with the S1P1 down-regulating agonist FTY720 (46). Oral ga- to hypoxia including glucose transporters, glycolytic enzymes,
vage with 6 g/kg FTY720 had no effect on T cell migration in vascular endothelial cell growth factor, NO synthase, and eryth-
T cell zones of explanted LN (Fig. 7B). The efficacy of FTY720 ropoietin (55, 56). T cell activation also up-regulates glucose trans-
in this experiment was verified by the dramatic decrease in cir- porters and glycolytic enzymes (57), perhaps to prepare the cells
culating T cells (data not shown). Lysophosphatidylcholine me- for entry into hypoxic environments.
diates T cell chemotaxis through G2A, a GPCR that couples to We established that the pO2 in the explanted LN is similar to
G s, G 13, and G q and has diverse biological effects (47–50). the pO2 of the medium at depths of up to 500 m in contrast to
However, we found that G2A / naive T cells migrated at the studies of ex vivo skeletal muscle tissue, in which the tissue
same speed as control T cells in explanted LNs (Table II). pO2 is always lower than the medium pO2 (27). Diffusion of O2
In summary, perfusion rate, pO2, depth, and G i were found to across tissues is not simply governed by Fick diffusion laws for
be important for controlling T cell migration speed in explanted a homogeneous material because tissues will have regional dif-
LN. Necessary pO2 levels were higher than physiological levels ference in diffusion based on different capsule composition and
measured in vivo, but were lower than standard perfusion condi- integrity (58), the volume fraction of cells and the tortuosity
tions. Ag-activated T cells, in contrast, migrated at physiological factor established by barriers to diffusion in the intercellular
pO2 levels in explanted LNs. We found no essential role of CCR7, spaces of the tissue (59). Thus, it is possible that LNs have
TP, arachidonic acid metabolites, S1P1, or G2A for naive T cell higher O2 permeability than muscle tissue, but its more likely
migration. that higher consumption of O2 in muscle (27) compared with
LNs may be responsible for the difference in apparent O2 pen-
Discussion etration. Although lymphocytes move rapidly in LNs, this
We have developed a closed system for imaging an intact LN with amoeboid movement generates very little contractile force (53)
controlled temperature, pO2, and perfusion velocity to determine and may require relatively little ATP compared with muscle
threshold levels of pO2 and perfusion velocity that support high T contraction. LNs also have an elaborate conduit system that
lymphocyte motility. Surprisingly, the pO2 required to maintain may allow perfusion of the LN interior by medium that is flow-
naive T cell mobility was twice the physiological level, as mea- ing over the LN surface (60).
sured in surgically exposed LN in live animals. This higher re- To further understand the mechanism behind hypoxia in-
quirement of pO2 may reflect limitations in the diffusion of higher duced stopping, we tested the hypothesis that hypoxia hampered
m.w. and slower diffusing nutrients like glucose, thus changing the lymphocyte migration through the adenosine receptor A2AR.
balance of oxidative phosphorylation and glycolysis used to Could modulating A2AR pathway, using antagonist to A2AR,
generate ATP, or a higher requirement for O2-dependent signal lead to sustained motility under hypoxic conditions? To test
generation in the explanted LN. An example of an O2-depen- this, we used a potent antagonist ZM241385 for A2AR and also
dent signal would be the use of O2 to generate leukotrienes and A2AR / mice. These mice develop extensive tissue damage in
PGs, which may have a role in modulating lymphocyte motility. response to concentrations of inflammatory stimuli that would


not elicit responses in WT mice (61). In a lung injury model, movement in the subcapsular zone compared with the deep
oxygenation weakened the A2AR-mediated anti-inflammatory paracortex, in agreement with our findings.
mechanism that was driven by hypoxia, and exacerbates lung Treating the mice with high doses of PTX resulted in decreased
injury (62). However, in A2AR / T cells and T cells treated T cell locomotion speed and increased confinement of T cells in
with antagonists stopped migrating at the same pO2 as control explanted LNs. Chemokine receptors, TP, S1P1, and other re-
cells. Therefore, the anti-inflammatory action of A2AR does not ceptors such as ORG1, DOR1 signal through G i, and they are
involve modulation of naive T cell migration. expressed on T cells. These receptors regulate a range of func-
We found that naive T lymphocyte locomotion in explanted tions including chemotaxis (66), differentiation (67), prolifera-
LNs requires a critical threshold perfusion velocity (Fig. 3). tion, and cytokine production (68). In a recent report, lympho-
Delivery of O2 is likely one function of perfusion, but the per- cytes treated with PTX exhibited only a partial reduction in LN
meability factors in tissues for larger metabolites like glucose egress, though a total block in LN entry (69). Together with our
and lactic acid are 10- to 100-fold lower than for O2 (63). Thus, results, we conclude that G i-coupled receptors largely deter-
it is likely that the perfusion velocity threshold is set by the mine lymphocyte motility within LNs. The confined G i-inde-
need to maintain steep diffusion gradients for glucose into the pendent movement may be mediated by GPCR that use other
LN and waste products of anaerobic metabolism like lactic acid G-proteins. For example, TP couples to G q, G i2, G 11/12/13/16
out of the LN. Alternatively, perfusion above a certain thresh- (70) and has been shown to regulate naive T lymphocyte mi-
old may be required to drive media through the reticular fiber gration (42). However, we examined TP / T cells and the
network as suggested above, serendipitously generating a cir- situation in which ETYA was used to inhibit PG, thromboxane,
culatory system for the explanted LN. Thus, the internal per- and lipoxygenase production and found no decrease in naive T
fusion of explanted LNs may be even more extensive than in cell migration. The failure to detect significant changes in mi-
vivo by capillaries. gration with any single receptor system may indicate either that
We found the naive cells moved slower in the superficial these systems are not involved in stimulation of naive T cell
areas of the LN compared with the deep paracortex. The higher migration or that there are redundant mechanisms such that
confinement suggests that the architecture of the subcapsular elimination of any one of these receptor systems is without
area may contain more obstacles that must be negotiated by effect. Cahalan and colleagues (15) have argued that naive T
cell migration appears to be autonomous. Although this state-
migrating T lymphocytes. A recent article suggested that the
ment can be interpreted in different ways, we feel that naive T
fibroblastic reticular cell network regulates lymphocyte migra-
cell migration is not autonomous in the manner of in vitro mi-
tion, by shaping the microanatomy within the LN (16). Because
gration of cytokine-stimulated T cells, which migrate autono-
pO2 is constant throughout this depth range and metabolite dif-
mously when they have an appropriate substrate like ICAM-1
fusion would seem to favor faster movement near the capsule,
(71). We cannot rule out that the residual migration detected in
it is most likely that the difference is due to tissue architecture.
the presence of PTX may be a more strictly autonomous pro-
For example, DC organization and dynamics are very different
gram mediated by other hormone or cytokine receptors, includ-
in the subcapsular, interfollicular and paracortical areas of the
ing receptor tyrosine kinases (72) and receptors for the JAK-
LN (8). Because DCs are a major interaction partner for T lym-
STAT pathways (73).
phocytes, it is likely that these differences would result in dif-
ferences in movement characteristic unless there were compen-
sating factors. Intravital microscopy studies noted a similar
Acknowledgments
We thank Dr. Wenbiao Gan for setting up the microscope, Dr. Thomas O.
difference in shallow and deep movement in the popliteal LN
Cameron and Teresa Wu for assisting early anoxic experiments, Drs. Rod
(18), but not the inguinal LN (14). These differences could be Braun and Jennifer Lanzen for assisting the in vivo LN pO2 measurement,
due to specific differences in proximity to blood vessels and the Dr. Thomas O. Cameron, V. Kaye Thomas, and Dr. J. C. Rathmell for
specific architecture of the fixed views of the popliteal and in- critical reading of the manuscript, and members of the Dustin laboratory
guinal LNs as presented in intravital microscopy. It is not for thoughtful discussions.
known whether there is any functional significance to these dif-
ferences in motility in different compartments. Disclosures
We found that naive CCR7 / T cells were able to ineffi- The authors have no financial conflict of interest.
ciently traffic to the LN. It has been shown that central memory
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Requirements for T Lymphocyte Migration in Explanted Lymph Nodes

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