PSGL-1 Regulates the Migration and
Proliferation of CD8 + T Cells under
Homeostatic Conditions
This information is current as
of June 18, 2017.
Krystle M. Veerman, Douglas A. Carlow, Iryna Shanina,
John J. Priatel, Marc S. Horwitz and Hermann J. Ziltener
J Immunol 2012; 188:1638-1646; Prepublished online 16
January 2012;
doi: 10.4049/jimmunol.1103026
http://www.jimmunol.org/content/188/4/1638
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References
The Journal of Immunology
PSGL-1 Regulates the Migration and Proliferation of CD8+
T Cells under Homeostatic Conditions
Krystle M. Veerman,*,† Douglas A. Carlow,*,† Iryna Shanina,‡,x John J. Priatel,†,{
Marc S. Horwitz,‡,x and Hermann J. Ziltener*,†
T
cells develop in the thymus and exit upon maturation
as naive T cells (1). Naive T cells travel through the
bloodstream and enter secondary lymphoid organs
(SLOs) including the spleen and lymph nodes. T cells enter lymph
nodes through specialized blood vessels called high endothelial
venules (HEVs) (2) and do so in a controlled process supported by
the sequential engagement of adhesion molecules and chemotactic
signals. Rolling and tethering of T cells on HEVs is initially facilitated by L-selectin on T cells interacting with peripheral node
addressins on HEVs (3, 4). The traction from L-selectin/addressin
interactions dramatically reduces T cell velocity, enabling chemokine CCL21 and CCL19 signaling through CCR7 on T cells.
Chemokine signaling rapidly induces activation of LFA-1 on
lymphocytes during the second step of T cell lymph node entry (5–7), allowing high-affinity LFA-1 binding to ICAM-1 on
HEVs resulting in cell arrest. Once arrested, additional signals
stimulate cell transmigration through the blood vessel wall and
*Biomedical Research Centre, University of British Columbia, Vancouver, British
Columbia V6T 1Z3, Canada; †Department of Pathology and Laboratory Medicine,
University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;
‡
Infection, Inflammation and Immunity Research Group, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; xDepartment of Microbiology and Immunology, Life Sciences Institute, University of
British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; and {Child and
Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada
Received for publication October 21, 2011. Accepted for publication December 13,
2011.
This work was supported by the Canadian Institutes for Health Research (Grant
MOP-111051 to H.J.Z.).
Address correspondence and reprint requests to Dr. Hermann Ziltener, Biomedical
Research Centre and Department of Pathology and Laboratory Medicine, University
of British Columbia, 2222 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
E-mail address: [email protected]
Abbreviations used in this article: DOP, 49-deoxypyridoxine; HEV, high endothelial
venule; LCMV, lymphocytic choriomeningitis virus; PSGL-1, P-selectin glycoprotein
ligand-1; SLO, secondary lymphoid organ; WT, wild-type.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103026
entry into SLOs (8). Entry of naive T cells into SLOs is mainly
believed to occur via this route, whereas memory T cells enter
lymph nodes via both the HEVs and the afferent lymphatics that
drain directly from tissues (9).
Once in the lymph nodes, CCL21 and CCL19 gradients direct
movement of T cells within the various lymph node compartments
(10). Naive T cells encounter and survey APCs for presence of
cognate Ag and also obtain survival signals from homeostatic
cytokines, such as IL-7 produced by fibroblastic reticular cells
(11), which promotes T cell survival via the Bcl-2 family apoptosis pathways (12). IL-15 is also found in lymph nodes and is
necessary for survival of naive and memory T cells (13, 14).
Homeostatic cytokines are constitutively produced within the
lymph nodes and stimulate T cell survival (14); because of ongoing consumption by lymphocytes, these cytokines are normally
present in low concentrations. Under lymphopenic conditions, less
cytokine is used and homeostatic cytokine levels consequently
increase in the lymph nodes to levels sufficient to stimulate both
T cell survival and limited T cell proliferation. Increased exposure
to IL-7, IL-15, IL-2, or IL-4 has been shown to induce such homeostatic expansion of T cells (15). In addition to cytokines, naive
T cells require survival signals through interaction with selfpeptide presented by MHC on APCs. T cells that fail to encounter their cognate Ag in a given lymphoid tissue are guided out of
the lymph nodes into the efferent lymph by an S1P gradient (16)
and re-enter the bloodstream only to repeat the process in another
SLO.
Recently, we discovered that P-selectin glycoprotein ligand-1
(PSGL-1) on resting T cells is required for efficient entry into
SLOs (17). PSGL-1 was found to facilitate lymph node entry
through an interaction with CCL21 and CCL19, independent of its
well-established role in selectin binding. To further elucidate the
significance of this novel chemokine binding function of PSGL-1
on naive T cells, we examined how lack of PSGL-1 affected both
T cell homeostasis in steady state and in mice responding to immune challenge with lymphocytic choriomeningitis virus (LCMV).
We found that PSGL-1 influenced several aspects of general ho-
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P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as
a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into
lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1null mice. Naive CD4+ and CD8+
T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal
levels. Although PSGL-1null T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than
PSGL-1+/+ T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1null CD8+ T cell proliferation was observed
under steady-state conditions and PSGL-1null CD8+ T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4,
and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1null mice exhibited a normal acute response (day 8) to
lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations
demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been
anticipated based on the mild phenotype of PSGL-1null mice. These potentially offsetting effects presumably account for the nearnormal cellularity seen in lymph nodes of PSGL-1null mice. The Journal of Immunology, 2012, 188: 1638–1646.
The Journal of Immunology
meostasis of CD8+ T cells through its effects on migration in and
out of lymph nodes and on responsiveness to the homeostatic
cytokines IL-2, IL-4, and IL-15. Furthermore, although absence of
PSGL-1 did not affect the ability of T cells to mount an acute
immune response against LCMV, it did influence the accumulation
of memory T cells.
Materials and Methods
Mice
Mice were bred and housed in the specific pathogen-free animal unit at the
Biomedical Research Centre. C57BL/6 and congenic C57BL/6 (Thy-1.1 or
CD45.1) and PSGL-12/2 (B6.Cg-Selplgtm1Fur/J stock number: 004201)
backcrossed onto C57BL/6 background were purchased from The Jackson
Laboratory. PSGL-12/2 mice were backcrossed in-house on the C57BL/6.
Thy1.1 background. TCR transgenic mice expressing the male Ag-specific
TCR (18) were maintained in the same facility. Mice used were age- and
sex-matched for experiments and ranged between 6 and 10 wk. LCMVinfected mice were housed at the Wesbrook Animal Unit. Procedures used
in this study were approved by the Animal Care Committee at the University of British Columbia.
Mice were sacrificed using CO2. Tissues used included superficial cervical,
brachial, inguinal, mesenteric, axillary lymph nodes, and spleen. Lymph
nodes and spleens were minced and mechanically dissociated into singlecell suspensions with a stainless-steel sieve in complete RPMI 1640 (containing 10% [v/v] FBS, 2 mM L-glutamine, 50 mM 2-ME, penicillin, and
streptomycin; Life Technologies) and kept at 4˚C. In cases where lymph
node T cell counts were taken, lymph nodes were minced and digested with
0.5 mg/ml collagenase/dispase (Roche) for 2 h at 37˚C in HBSS on
a turning rack with 10 mg/ml DNase (Roche). Peripheral blood was taken
either by perfusion with PBS + 4% FBS + 2.5 mM EDTA or by cardiac
puncture. RBC lysis was performed on all peripheral blood and spleen.
Flow cytometry
Cell suspensions were stained with combinations of CD4-PECy7 (RM4-5;
eBioscience), CD8a-allophycocyanin-Cy7 (53-6.7; eBioscience), CD8bbiotin (H35-17.2; eBioscience), B220-FITC (RA-6B2; hybridoma/ATCC),
CD122/IL-2Rb-biotin (5H4; eBioscience), CD132/gc-PE (TUGm2; BioLegend), CD215/IL-15Ra–allophycocyanin (JM7A4; BioLegend), CD25/
IL-2Ra–PE (PC61; Pharmingen), CD44-PE, -allophycocyanin (IM7.8;
hybridoma/ATCC), CD45.1-allophycocyanin-Cy7, -PE (A20; eBioscience;
hybridoma/ATCC), and Thy1.1-Pacific blue (HIS51; eBioscience) Abs in
FACS buffer (PBS + 2% FBS [v/v] + 2 mM EDTA) for 20 min at 4˚C (on
ice) in the dark. They were washed twice in FACS buffer. Cells stained
with biotinylated Abs were then stained with fluorochrome-conjugated
streptavidin for 10 min at 4˚C (on ice) in the dark. Where indicated,
propidium iodide (PI; 250 ng/ml) was included in the final sample volume
to resolve viable cells. Reference beads (Invitrogen) were included in some
samples to determine absolute cell numbers as previously described (19).
Samples were analyzed on a FACScan, FACSCalibur, or LSRII flow
cytometer (BD Bioscience). Data were analyzed using FlowJo (Tree Star
software).
T cell half-life and egress
Wild-type (WT) or PSGL-1null lymph nodes or HY and HY/PSGL-1null
thymi were minced and mechanically dissociated through a stainless-steel
sieve, washed 33 in RPMI 1640 and 10% FCS, and filtered through a 70mm sieve. For experiments using HY TCR transgenic mice, CD8+ singlepositive T cells were enriched by depletion of CD4+ cells using an
EasySep kit as per manufacturer’s instructions (Stem Cell). Isolated cells
were then labeled with either 13 or 2 mM CFSE for 5 min in HBSS (Life
Technologies) at 25˚C, washed with complete media, and resuspended in
HBSS. For T cell half-life experiments, T cells were injected i.v. into WT
mice; peripheral blood was recovered 1, 24, and 48 h later and analyzed by
flow cytometry. For T cell egress experiments, T cells were injected i.v.
into WT mice. Twenty-four hours later, one set of mice was sacrificed,
whereas remaining mice were each injected i.p. with 150 mg each of antia4 and anti-aL integrin Abs (clones PS/2 and TIB217 respectively, produced in-house). Forty-eight hours later, one set of mice was sacrificed,
whereas the remaining mice were again injected with integrin-specific Abs.
Seventy-two hours later, the final set of mice was sacrificed. Lymph nodes
were harvested from mice, dissociated, labeled with Abs, and analyzed by
flow cytometry.
In vivo proliferation assays
Donor lymph node cells were prepared as described earlier, then labeled
with 2 mM CFSE in HBSS for 5 min at 25˚C. Cells were washed twice in
complete RPMI 1640, then resuspended in HBSS at 25 3 106 cells/ml.
Two hundred microliters of cells was injected into each mouse i.v. via the
tail vein. In some experiments, lymphocytes were trapped in lymph nodes
using 49-deoxypyridoxine (DOP; Sigma), by including it at a concentration
of 100 mg/l together with 10 g/l glucose to the drinking water at the time
of injection (20). Water was refreshed weekly. In long-term proliferation
assays, mice were sacrificed after 4–5 wk. Sublethally irradiated WT mice
(350 rad) were sacrificed after 7 and 14 d. Lymphocytes were collected and
analyzed as indicated earlier. In competition assays, donor cells were
distinguished based on congenic marker expression using Abs against
Thy1.1, Thy1.2, CD45.1, and/or CD45.2.
In vitro proliferation assay
Dissociated lymph node cells from WT Thy1.2 and PSGL-1null Thy1.1 mice
were mixed at a 1:1 ratio, simultaneously labeled with 2 mM CFSE in
HBSS, washed, and then added to prewarmed cytokine-enriched media
in 96-well plates to a final density of 1 3 106 cells/ml. Cultures were
maintained at 37˚C with 0.5% CO2. For in vitro experiments with purified
naive and memory T cells, cells were sorted on the basis of CD8b+ and
CD44high and CD44low expression on a FACS Aria (BD Biosciences).
After sorting, cells were counted, combined at a 1:1 ratio, labeled with
CFSE, and added to cytokine-enriched 96-well plate at a concentration of
5 3 105 cells/ml. After 3 d, cells were analyzed on the LSRII with counting beads and PI stain. All analyses were gated for viable PI2 cells.
LCMV infections
Seven-week-old sex-matched mice were injected i.p. with 1 3 105 PFU
LCMV-Armstrong. LCMV-specific MHC class I tetramers were constructed and purified as previously described (21). Spleens were harvested
on either day 8 or 40 postinfection, and stained with PE-labeled H-2DbGP33 or PE-labeled H-2Db-NP396 tetramers in FACS buffer (PBS with
2% FBS [v/v] and 2.5 mM EDTA) for 40 min at 4˚C in the dark. They were
then washed in FACS buffer and subsequently stained with CD8allophycocyanin-Cy7 and CD4-PECy7 for 20 min at 4˚C in the dark.
Samples stained by gp33 tetramer were immediately run on the LSRII.
Samples stained by np396 were first fixed with a 1% (w/v) paraformaldehyde/PBS solution for 15 min, then run on the LSRII. Counting
beads were added to all samples run on the FACS.
Statistics
Data are represented as mean values and error bars are depicted using SD.
Statistical significance was measured using an unpaired, two-tailed Student
t test: *p , 0.05, **p , 0.01, ***p , 0.001. Statistics were calculated
using Microsoft Excel software.
Results
Naive T cell frequency is substantially reduced in the blood of
PSGL-1null mice
While examining the role of PSGL-1 in thymic progenitor homing,
we discovered that PSGL-1null mice have a reduced thymic T cell
output and decreased numbers of CD4+ and CD8+ T cells in the
peripheral blood (22). These findings prompted us to examine how
loss of PSGL-1 affects homeostasis within peripheral lymphocyte
compartments. Lymphocytes from peripheral blood, peripheral
lymph nodes, and spleen were enumerated in PSGL-1null and WT
mice. As previously shown (22), the numbers of CD4+ and CD8+
T cells in the peripheral blood of PSGL-1null mice were found to
be significantly reduced by ∼45% relative to WT mice (Fig. 1A).
Surprisingly, however, CD4+ and CD8+ T cell numbers within
lymph nodes and spleen were comparable between PSGL-1null
mice and WT mice. B220+ B cell numbers in WT versus PSGL1null mice were also comparable in all the tissues analyzed, although a trend for increased B cell numbers in PSGL-1null mice
was noted.
The observed T cell lymphopenia in the blood of PSGL-1null
mice was explored further by analysis of the naive and memory
T cell subsets to determine whether all subsets were similarly
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Cell preparation
1639
1640
PSGL-1 IMPACTS ON CD8 T CELL HOMEOSTASIS
affected. CD4+ and CD8+ naive T cells, defined as CD44low Lselectinhigh, in the blood of PSGL-1null mice were found to be
reduced by 98 and 95%, respectively, relative to WT (Fig. 1B). By
contrast, naive CD4+ and CD8+ T cells in lymph nodes of PSGL1null mice were reduced by 42 and 54%, respectively, and their
numbers in spleen were not significantly different from WT.
The distribution of other T cell subsets in PSGL-1null mice was
less affected. Frequencies of effector memory T cells, defined as
CD44hi L-selectinlo, were increased in blood and lymph nodes, but
not in spleen. Frequencies of CD4+, but not CD8+ effector T cells,
defined as L-selectinlo CD44lo, were significantly reduced in blood
of PSGL-1null mice, whereas frequencies of CD8+ effector cells
(L-selectinlo CD44lo) and CD8+ effector memory T cells (Lselectinlo CD44hi) were reduced in spleen but not in blood and
lymph nodes.
PSGL-1null T cells are lost in blood but retained in lymph nodes
The absence of naive T cells in blood of PSGL-1null mice was
unexpected as we have previously shown that T cells deficient for
PSGL-1 enter the lymph nodes at approximately half the rate of
WT T cells (17). We therefore expected to find increased numbers
of naive T cells in blood. The lack of naive T cells in blood of
PSGL-1null mice suggested a critical role for PSGL-1 in the
maintenance of naive T cells. To test this hypothesis, we coin-
jected lymph node-derived WT and PSGL-1null T cells, and
measured their blood half-life in recipient mice (Fig. 2). T cells
from PSGL-1null donors disappeared from the blood at a faster rate
than T cells from WT donors. To address the possibility that the
disappearance of PSGL-1null lymph node T cells might have occurred because of a pre-existing deficit in PSGL-1null resident
lymph node T cell integrity, we used male Ag-specific HY TCR
female donors for blood half-life experiments because they have
an abundance of mature CD8+ thymocytes that possess a de facto
naive phenotype. The disappearance of HY/PSGL-1null CD8+
thymocytes relative to HY/PSGL-1+/+ thymocytes was more dramatic than when non-TCR transgenic donor lymph node T cells
were used (Fig. 2) and consistent with a preferential clearance of
CD8+ PSGL-1null T cells with a naive status.
The comparable cellularity of lymph nodes in PSGL-1null mice
relative to WT mice despite T cell lymphopenia in the blood, as
well as the inability of PSGL-1null T cells to efficiently enter
lymph nodes (17), led us to speculate that PSGL-1null T cells
might have a decreased rate of lymph node egress. To test this
hypothesis, we preloaded lymph nodes of recipient mice with
a mixture of donor WT and PSGL-1null lymph node-derived
T cells for 1 d and then used anti-integrin Abs to further block
entry of T cells into lymph nodes. Integrin neutralization used in
this way effectively blocks entry of PBLs into lymph nodes (23,
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FIGURE 1. Naive CD4+ and
CD8+ T cells are decreased in blood
of PSGL-1null mice. Lymphocytes
were isolated from tissues of WT
and PSGL-1null mice, stained with
mAbs, and analyzed by flow cytometry. A, Enumeration of CD4+
and CD8+ T cells, and B220+ B cells
in blood, in pooled peripheral and
mesenteric lymph nodes and in
spleen. B, Enumeration of CD4+ and
CD8+ T cell subsets in blood, lymph
nodes, and spleen. Naive T cells are
defined as L-selectinhiCD44lo, central memory T cells as L-selectinhi
CD44hi, effector T cells as L-selectinloCD44lo, and effector memory
T cells as L-selectinloCD44hi. Data
are shown as absolute numbers in
each tissue and represent a minimum of three independent experiments. Error bars represent SD. The
unpaired Student t test was used to
compare groups. *p , 0.05, **p ,
0.01, ***p , 0.001.
The Journal of Immunology
1641
FIGURE 2. CD8+ PSGL-1null T cells disappear
quicker from blood than WT CD8+ T cells. WT and
PSGL-1null T cells or HY and HY/PSGL-1null CD8+
naive T cells were injected into WT mice and ratios
of injected cells in blood were compared over time.
Data were measured by flow cytometry and cells were
gated on CD8 and either Thy1 congenic markers (WT
and PSGL-1null T cells) or on CFSE labeling (HY and
HY/PSGL-1null T cells) to distinguish cell types. Data
are a ratio of PSGL-1null T cells to WT T cells and are
representative of at least three independent experiments. Error bars represent SD. *p , 0.05, ***p ,
0.001 (Student t test).
Adoptively transferred PSGL-1null CD8+ T cells proliferate
spontaneously
Lymphopenia-associated homeostatic proliferation could be another compensatory mechanism to maintain T cell numbers in
lymph nodes of PSGL-1null mice. To determine whether T cell
lymphopenia in PSGL-1null mice induces homeostatic proliferation, we coinjected WT and PSGL-1null CFSE-labeled lymph node
cells at a 1:1 ratio into PSGL-1null and WT recipients; we hypothesized that T cells injected into PSGL-1null recipients would
undergo cell division, whereas T cells injected into WT recipients
would not. PSGL-1null donor CD8+ T cells exhibited more evidence of proliferation compared with WT donor cells 5 wk after
transfer, irrespective of whether cells were transferred into WT or
PSGL-1null recipients (Fig. 4A, 4B). Importantly, the respective
cell division rates for PSGL-1null and WT donor CD8+ T cells
were comparable in PSGL-1null and WT recipients, indicating
that the naive T cell lymphopenia present in PSGL-1null mice was
not sufficient to induce homeostatic proliferation under the con-
ditions used. CD4+ T cells did not show any proliferation after
5 wk (data not shown).
To determine whether PSGL-1null CD8+ T cells responded better
than WT CD8+ T cells in an “induced” homeostatic proliferation
model, we coinjected CFSE-labeled T cells from WT and PSGL1null mice into sublethally irradiated WT recipients. CFSE dilution
was measured in T cells recovered from lymph nodes 7 and
14 d after adoptive transfer. CD8+ T cells from both WT and
PSGL-1null donors divided at a similar rate at day 7, with PSGL1null T cells showing slightly advanced proliferation (Fig. 4C).
After 14 d, there was a larger proportion of PSGL-1null CD8+
T cells that had undergone three or more divisions than coinjected
WT CD8+ T cells. WT CD8+ T cells appeared to slow their
proliferation after 7 d, whereas PSGL-1null CD8+ T cells continued
to divide. CD4+ T cells from both WT and PSGL-1null donors
showed a maximum of one division at both time points and was
equal between both genotypes (data not shown).
Because PSGL-1null T cells have an increased residence time in
lymph nodes (Fig. 3), we considered whether enhanced exposure
to cytokines such as IL-7, IL-15, and IL-2 that are known to
support homeostatic T cell proliferation in lymph nodes (14)
might contribute to the restoration of T cell cellularity in PSGL1null lymph nodes. To address this question, we trapped CFSElabeled WT and PSGL-1null T cells in lymph nodes for 5 wk using
DOP, a drug that blocks T cell egress from lymph nodes by disrupting the sphingosine 1 gradient (20). CFSE dilution analysis of
adoptively transferred cells showed that PSGL-1null CD8+ T cells
maintained a proliferative advantage over WT CD8+ T cells even
when spending equivalent lengths of time in the same lymph node
microenvironment (Fig. 4D). Comparison of the percentage of
divided cells also showed that PSGL-1null CD8+ T cells proliferated more than WT CD8+ T cells (Fig. 4E). Regardless of donor
cell genotype, there was a greater percentage of dividing donor
FIGURE 3. PSGL-1null CD8+ T cells persist in lymph nodes longer than WT T cells. WT and PSGL-1null T cells or HYand HY/PSGL-1null CD8+ naive T cells
were injected into WT recipient mice; 1 d later, lymph nodes were harvested from one set of mice to determine T cell ratio at time 0 h. The remainder of mice was
treated with anti-integrin mAbs to block further T cell entry into lymph nodes, and ratios of CD8 T cells were determined 24 and 48 h after anti-integrin Ab
treatment. Data were measured by flow cytometry and cells were gated based on CFSE labeling. Data are a ratio of PSGL-1null T cells to WT T cells and are
representative of at least four independent experiments. Error bars represent SD. *p , 0.05, **p , 0.01 (Student t test).
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24). Lymph node cells from one cohort of mice were then harvested and donor cells analyzed to determine initial WT/PSGL1null T cell ratios before anti-integrin treatment (0 h). After an
additional 24 and 48 h, recipients were analyzed to determine the
proportion of WT versus PSGL-1null donor T cells remaining in
lymph nodes. The ratio of PSGL-1null CD8+ T cells to WT CD8+
T cells increased over time in integrin mAb-treated mice, indicating that PSGL-1null T cells remained longer in lymph nodes
than WT T cells (Fig. 3). By contrast, untreated mice showed no
increase in ratio of PSGL-1/WT T cells after 24 h (data not
shown). Prolonged lymph node retention of PSGL-1–deficient
CD8+ T cells was also found when lymph node retention was
compared between HY/PSGL-1+/+ and HY/PSGL-1null CD8+
thymocytes (Fig. 3).
1642
PSGL-1 IMPACTS ON CD8 T CELL HOMEOSTASIS
cells in DOP-treated recipient mice relative to control untreated
recipients, although this trend did not reach statistical significance.
This observation was consistent with the possibility that lymph
node residence time could be an additional factor contributing to
increased expansion of PSGL-1null CD8+ T cells within the lymph
nodes.
PSGL-null T cells are more sensitive to cytokine-induced
proliferation
FIGURE 4. PSGL-1null CD8+ T cells spontaneously proliferate in vivo.
A, CFSE dilution profiles of WT (solid line) and PSGL-1null (dotted line)
CD8+ T cells after a 35-d period in WT recipients (left panel) and PSGL1null recipients (right panel). B, Comparison of percentages of dividing
CD8+ T cells in individual recipients (d, WT recipient; +, PSGL-1null
recipient). C, CFSE dilution profiles of WT (solid line) and PSGL-1null
(dotted line) CD8+ T cells 7 (left panel) and 14 d (right panel) after injection into WT sublethally irradiated hosts. D, CFSE dilution profiles of
WT (solid line) and PSGL-1null (dotted line) CD8+ T cells after a 35-d
period in WT recipients treated with DOP (right panel) or control (left
panel). E, Comparison of percentages of dividing CD8+ T cells in individual recipients (*, DOP treated; s, untreated). In all experiments, donor
cells were identified using Thy1 and CD45 congenic markers. A, C, and D,
Data are representative of three independent experiments, with a minimum
of three mice per experiment. B and E, Data shown are pooled from three
independent experiments, with at least three mice per experiment.
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The cytokines IL-2, IL-7, and IL-15 have been shown to promote
T cell survival when present at low concentrations and induce
homeostatic proliferation of T cells at high concentrations (25).
To determine whether the spontaneous in vivo PSGL-1null T cell
proliferation, exemplified in Fig. 4, could be caused by altered
responsiveness to homeostatic cytokines, we set up in vitro
cocultures using CFSE-labeled lymph node cells from WT and
PSGL-1null mice that were mixed at a 1:1 ratio and maintained in
the presence of IL-2, IL-4, IL-7, and IL-15. After 4 d in culture,
CD4+ T cells did not show CFSE dilution in response to any of the
stimuli tested (data not shown). CD8+ T cells did not proliferate in
response to IL-7 after 4 d (data not shown) but did proliferate in
response to IL-2, IL-4, and IL-15 starting at day 3 and showed
substantial amounts of dose-dependent proliferation by day 4.
PSGL-1null T cells again exhibited a proliferative advantage in
response to all three cytokines (Fig. 5A). IL-15 stimulated CD8+
T cells most efficiently, as there were ∼4-fold more CD8+ T cells
in cultures supplemented with IL-15 than in cultures with IL-2 or
IL-4 (Fig. 5B).
Memory CD8+ T cells have been shown to express higher levels
of IL-15Ra than naive CD8+ T cells and to proliferate more
rapidly than naive T cells in response to IL-15 (26, 27). To
eliminate the possibility that differences in memory and naive
T cell ratios present in WT and PSGL-1null lymph node preparations might have caused the observed differences in cytokine responsiveness in vitro, we tested IL-15 responsiveness of purified
CD8 memory (CD44hi) and CD8 naive (CD44low) T cells from
WT and PSGL-1null donors. Although our data confirmed that
memory T cells divide more rapidly than naive T cells, PSGL-1
deficiency was associated with increased proliferation in both
naive and memory CD8+ T cells (Fig. 5C). Enhanced responsiveness of PSGL-1null CD8+ T cells was most clearly demonstrated for naive cells stimulated with 10 ng/ml IL-15 where
blasting or cell division was observed in .50% of input cells by
day 3, whereas ∼90% of naive WT CD8+ T cells remained in
a nondividing, nonblasted state.
To examine whether increased proliferation of PSGL-1null
T cells may be caused by differences in expression of gc family
cytokine receptors, we used FACS to compare the cell surface
expression of IL-15Ra, IL-2Ra, IL-2Rb, and gc on CD8+ T cells.
Our data show that there were no significant differences in the
expression of these receptors between WT and PSGL-1null CD8+
T cells (Fig. 5D). Furthermore, expression of all receptors increased to a similar degree on both WT and PSGL-1null CD8+
T cells after stimulation with IL-15. After IL-15 stimulation, there
was a small, statistically significant increase in the expression of
The Journal of Immunology
1643
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FIGURE 5. CD8+ PSGL-1null T cells are hyperresponsive to homeostatic cytokines in vitro. A, Percentages of divided CD8+ WT and PSGL-1null T cells
are shown in cultures supplemented with IL-15, IL-2, or IL-4 at days 2, 3, and 4. B, WT and PSGL-1null CD8+ T cells were enumerated after 4 d cultured
with homeostatic cytokines. C, Blasting and cell division of cocultured WT and PSGL-1null CD44hi memory CD8+ T cells and cocultured WT and PSGL1null CD44low naive CD8+ T cells with IL-15. D, Cell surface expression of IL-2 and IL-15 receptor chains on WT and PSGL-1null CD8+ T cells ex vivo
(upper panels) and after stimulation with IL-15 (lower panels). All experiments were done in triplicate and are representative of five (A–C) or three (D)
independent experiments. Error bars represent SD. *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test).
IL-2Rb on CD8+ PSGL-1null versus WT T cells (p = 0.02).
Nevertheless, the difference was small compared with the shift
between T cells that have been stimulated with IL-15 compared
with those that were not.
PSGL-1null mice mount a normal acute response to viral
challenge but exhibit heightened numbers of antiviral memory
T cells
To determine whether altered T cell homeostasis or lymphocyte
trafficking associated with loss of PSGL-1 impacts CD8+ T cell
immune responses in vivo, we challenged PSGL-1null and WT
mice with LCMV-Armstrong. At the peak of the response
8 d postinfection, both WT and PSGL-1null mice showed comparable increases in their splenic CD8+ T cell populations and had
equivalent numbers of anti-LCMV-CD8+ effector T cells as enumerated with MHC class I tetramers specific for the viral immunodominant epitopes H-2Db-GP(33-41) and H-2Db-NP(396-404)
(Fig. 6A). These findings suggest that despite abnormalities in the
distribution of T cell subsets within PSGL-1null mice, they retain
the ability to generate an immune response that is comparable
1644
with WT mice. Next, we analyzed WT and PSGL-1null mice at day
40 postinfection with LCMV. In mice of either genotype, total
CD8+ T cell numbers were reduced to approximately one-fifth of
those at day 8 postinfection (Fig. 6B). However, the numbers of
GP33- and NP396-specific CD8+ memory T cells in the spleens of
PSGL-1null mice were found to be 2.4-fold higher than in WT
mice.
Discussion
PSGL-1 has traditionally been recognized as a key regulator of
leukocyte migration to areas of inflammation. Over the last few
years, recognition of the functional importance of PSGL-1 has been
widened significantly as PSGL-1 has been found to play a role in
other contexts such as hematopoietic stem cell homing to the
bone marrow (28), progenitor homing to thymus (29), and T
cell homing to SLOs through interactions with chemokines (17).
The latter reveals a novel PSGL-1 function associated with its
nonselectin binding form.
Two PSGL-1 knockout mice have been generated and studied.
Numbers of blood lymphocytes were reported to be normal in
PSGL-1null mice generated by the laboratory of Bruce Furie (30),
whereas PSGL-1null mice generated in Rodger McEver’s laboratory (31) were shown to have increased numbers of total lymphocytes in blood. In a follow-up study of these latter PSGL-1null
mice, a decrease in CD4+ and CD8+ T cells but no differences in
the number of total blood lymphocytes were reported (http://www.
functionalglycomics.org/glycomics/publicdata/phenotyping.jsp). Our
detailed analyses of T cell subsets in the Furie PSGL-1null mice
confirmed that PSGL-1 deficiency is associated with a reduction in
blood CD4+ and CD8+ T cells. This decrease in T cell numbers
was only obvious when analysis of B cells and T cell subsets was
performed as the reduction in T cell numbers was balanced by an
increase in B cell numbers. We found that PSGL-1–associated
T cell lymphopenia was compartment specific in that it is manifest
in blood but not in spleen or lymph nodes. T cell lymphopenia
in blood was due to a selective deficit of naive T cells (95–98%
reduced), whereas PSGL-1null memory T cell numbers in blood
were comparable with WT mice. The finding that PSGL-1–associated T cell lymphopenia was not generalized to the whole body
demonstrates that PSGL-1 in its nonselectin binding state is required for the normal distribution of T cells in specific compartments and is crucial for the normal maintenance of circulating
naive T cells.
We have previously found that PSGL-1null thymocytes exit the
thymus at a rate 50% that of WT thymocytes (22). Although this
may partially explain the decrease in naive T cells in the blood,
the near complete lack of naive T cells in blood cannot be
explained by this phenotype alone. Inefficient homing of PSGL1null T cells to SLOs (17) constitutes another factor contributing to
lymphopenia as it has been shown that inefficient SLO entry of
naive T cells results in the decrease of peripheral T cells, likely
because of their reduced exposure to homeostatic cytokines (11).
Memory T cells would be less affected by inefficient SLO entry
via HEV because they can enter lymph nodes through the afferent
lymph and can therefore encounter homeostatic cytokines such as
IL-15 in other organs such as the bone marrow (32).
Although naive T cell numbers were profoundly reduced in the
blood of PSGL-1null mice, they were less strongly reduced in
lymph nodes. New attributes peculiar to PSGL-1null T cells have
emerged in our study that could help reconcile why naive T cell
deficits were much less apparent in lymph nodes than in blood.
First, we found that PSGL-1 deficiency on T cells was associated
with reduced lymph node egress rates. Because directional cues
within the lymph node are normally provided by CCL19 and
CCL21 (10), and absence of PSGL-1 is known to compromise
responsiveness to these chemokines (17), PSGL-1 deficiency
could effectively result in lymphocyte trapping within the lymph
node and thus permit extended exposure to homeostatic cytokines
that are present. Parenthetically, naive T cell numbers were
comparable between WT and PSGL-1null mice in the spleen, an
organ without HEVs and where all T cells enter via the splenic
artery. Indeed, T cell entry into spleen is only weakly reduced by
loss of PSGL-1 (17), and homeostasis of PSGL-1null T cells may
thus be less affected in the spleen. Because CCL21 and CCL19 are
known to guide T cells within the spleen (33), it is, however,
possible that T cell egress from the spleen may also be reduced
for PSGL-1null T cells. Second, enhanced proliferative response
of PSGL-1null CD8+ T cells to homeostatic cytokines likely constitutes an additional mechanism that contributes to restoration of
normal T cell numbers in lymphoid organs. We initially speculated that T cells adoptively transferred into PSGL-1null mice
would homeostatically expand because of the observed T cell
lymphopenia in the blood of these recipient mice. Instead, donor
PSGL-1null CD8+ T cells divided significantly more than WT
CD8+ T cells irrespective of whether they were injected into a WT
or PSGL-1null recipient. Dividing cells were CD44high, a characteristic marker of cells undergoing homeostatic proliferation. In
PSGL-1null mice, we noted a trend for an increase in CD44high
T cells in the blood and lymph nodes, suggesting T cell proliferation may occur naturally within PSGL-1null mice. Enhanced
proliferative responses of PSGL-1null CD8+ T cells to the common
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FIGURE 6. PSGL-1null mice mount a normal immune response against
LCMV but generate more memory T cells. A, Total numbers of CD4+ and
CD8+ T cells (left panel) and CD8+ LCMV-specific T cells (right panel)
8 d after LCMV challenge in WT (black bars) and PSGL-1null (white bars)
mice are shown. B, Total numbers of CD4+ and CD8+ T cells (left panel)
and CD8+ LCMV-specific T cells (right panel) 40 d after LCMV challenge
in WT (black bars) and PSGL-1null (white bars) mice are shown. Minimum
of four mice per group were analyzed. Data are representative of two independent experiments. Error bars represent SD. **p , 0.01, ***p ,
0.001 (Student t test).
PSGL-1 IMPACTS ON CD8 T CELL HOMEOSTASIS
The Journal of Immunology
1null T cells are hyperproliferative to cytokine stimulation and
mitogen stimulation (34), whereas CCR7null T cells were shown to
proliferate less after mitogen stimulation (40). In addition, although PSGL-1null mice had normal levels of effector CD8+
T cells and increased levels of LCMV-specific memory T cells
after LCMV infection, CCR7null mice had approximately half the
levels of LCMV-specific CD8+ T cells both in the acute effector
stage and in the memory stage (41). Therefore, although PSGL-1
and CCR7 are both required for migration of T cells, they appear
to have opposing effects in proliferation and memory T cell production after LCMV infection.
In conclusion, PSGL-1 deficiency appears to influence CD8+
T cell homeostasis in at least three different ways: 1) by interfering with lymph node entry of T cells, thereby limiting access to
homeostatic cytokines and other prosurvival signals in the lymph
node; 2) by prolonging lymph node residence time, thereby extending exposure of T cells to prosurvival signals therein; and
3) by increasing T cell sensitivity to cytokines. Although the
first effect will negatively affect T cell homeostasis, the latter two
effects will be positive. The mild net phenotype seen in PSGL-1null
mice likely results from these potentially offsetting influences.
This study continues to explore the roles of PSGL-1 in its
nonselectin binding state. Our findings that PSGL-1 helps maintain
the distribution of resting T cells to certain compartments, regulates
proliferation of T cells in response to homeostatic cytokines, and
enhances memory T cell formation/survival after LCMV challenge
further extend our understanding of PSGL-1 functionality and
introduce another facet of T cell homeostasis regulation.
Acknowledgments
We are grateful to Michael Williams for technical assistance and to Drs.
Ninan Abraham and Pauline Johnson for helpful discussions throughout
the project.
Disclosures
The authors have no financial conflicts of interest.
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PSGL-1 IMPACTS ON CD8 T CELL HOMEOSTASIS