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IMMUNOBIOLOGY
Platelet-derived CD154 enables T-cell priming and protection against Listeria
monocytogenes challenge
Bennett D. Elzey,1,2 Nathan W. Schmidt,3 Scott A. Crist,1 Timothy P. Kresowik,4 John T. Harty,3,5 Bernhard Nieswandt,6,7 and
Timothy L. Ratliff1,8
1Department of Comparative Pathobiology, Purdue University, West Lafayette, IN; 2Department of Urology, Indiana University, Indianapolis; 3Department of
Microbiology, 4Department of Urology, and 5Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City; 6Rudolf Virchow Center, DFG
Research Center for Experimental Biomedicine, and 7Institute of Clinical Biochemistry and Pathobiochemistry, University of Wurzburg, Wurzburg, Germany; and
8Purdue Cancer Center, Purdue University, West Lafayette, IN
Collagen exposure in tissue activates
platelets, initiates wound healing, and
modulates adaptive immunity. In this report, data are presented to demonstrate a
requirement for platelet-derived CD154
for both collagen-induced augmentation
of T-cell immunity and induction of protective immunity to Listeria challenge.
Specifically, we demonstrate that Ad5
encoding the membrane-bound form of
ovalbumin (Ad5-mOVA) delivered in collagen induces higher ovalbumin-specific
cytotoxic T lymphocyte (CTL) activity in a
dose-dependent manner compared with
Ad5-mOVA delivered in PBS. Increased
CTL activity was dependent on the ability
of platelets to respond to collagen and to
express CD154. Furthermore, mice immunized with low-dose Ad5-mOVA in collagen were able to control a challenge of
Listeria monocytogenes recombinant for
ovalbumin expression (Lm-OVA), whereas
mice immunized with low-dose Ad5-mOVA
in PBS were not. These data indicate that
in a physiologic setting that mimics
wounding, platelets perform a sentinel
function when antigen dose is too low to
provoke an efficient immune response,
and can enhance the generation of antigen-specific CD8 T cells that are functionally relevant to the host. (Blood. 2008;111:
3684-3691)
© 2008 by The American Society of Hematology
Introduction
Because pathogens are capable of logarithmic expansion upon host
infection, it is critical that both innate and adaptive immunity are
initiated without delay to ensure survival. An effective adaptive
response is dependent upon efficient activation of innate immunity
since antigen presenting cells (APCs) that are not fully activated
are poor stimulators of T- or B-cell responses.1-3 However, the
initial infection usually involves entry of only a few microbes
resulting in a very low antigen dose unlikely to provoke a prompt
immune response. To compensate, the immune system has been
designed with important activating early signaling components
such as the Toll-like receptor (TLR) family, and costimulatory
molecules such as CD40 ligand (CD40L, CD154), which help
lower the threshold for cellular activation.4,5 CD154 is a molecule
critical to adaptive immunity. It is required for T-dependent
humoral responses including affinity maturation, somatic hypermutation, germinal center (GC) formation, and B-cell memory.6
In addition, there is a role in some models for CD154 in generation
of normal CD8 T-cell memory and cross-presentation of class
I–restricted antigen.7-13 Until recently, functionally relevant expression of CD154 was thought to be restricted to CD4 T cells where its
purpose in cellular immunity is to stimulate APCs that in turn
potently activate T cells2; however, functional CD154, which was
able to generate an inflammatory response from endothelial cells,
has also been reported on platelets.14
The role platelets can play in antimicrobial immunity is well
documented. They are activated by and able to engulf bacteria and
viruses, release antimicrobial and antifungal proteins, release
reactive oxygen species, express TLR, protect against helminth
infections, and play a role in modulating production of inflammatory cytokines in response to bacterial infections.15-17 Because of
their ubiquitous presence in the circulation, platelets are the first
cell type to specifically and intentionally accumulate at sites of
injury where they are activated by many factors, including collagen. While releasing compounds necessary for wound repair,
platelets also release/express molecules that exert potent influence
on innate and adaptive immunity such as TGF-␤, RANTES,
P-selectin, CD154, and several chemoattractants.15-17 Because of
these attributes, platelets are well poised to constitute another early
signaling component in the immune response. Consistent with an
early signaling function of platelets, we reported the ability of
platelet CD154 to stimulate dendritic cells in vitro, a function
corroborated by several other groups.18-21 We also reported the
ability of WT platelets to enhance IgG production, and in concert
with WT CD4 T cells, to enhance GC formation in CD154
knockout mice.18,22 Importantly, we showed that in response to low
doses of antigen, WT mice cannot produce normal levels of IgG in
the absence of platelets. Accordingly, we have proposed that
platelets perform a sentinel function for adaptive and innate
immunity, an observation that has been supported by others.23
To further investigate the role of platelets in a physiologic
setting, we used a previously described wound model24 by subcutaneous injection of low doses of Ad5 encoding the membrane-bound
Submitted May 21, 2007; accepted February 1, 2008. Prepublished online as
Blood First Edition paper, February 6, 2008; DOI 10.1182/blood-200705-091728.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2008 by The American Society of Hematology
3684
BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
form of chicken OVA (Ad5-mOVA) in collagen. This mimics a
wound in that collagen is exposed, and the initial dose of
pathogen is low. Providing further support for our premise that
platelets provide early activating immune signals, we report
here that platelets enhance OVA-specific CD8 T-cell responses
to low doses of Ad5-mOVA that would otherwise fail to
stimulate a detectable immune response. This observation
depended on the ability of platelets to express CD154 and
respond to collagen. Moreover, mice whose OVA-specific CD8
T-cell response was enhanced by platelet-derived CD154 showed
increased clearance of Listeria monocytogenes recombinant for
chicken ovalbumin (Lm-OVA) compared with mice whose
platelets lacked CD154. These data support the hypothesis that
platelet-associated modulation of the initial CD8 antiviral
response results in a biologically significant advantage to the
host in a setting where, without platelet involvement, the viral
dose is too low to prompt effective activation of the adaptive
CD8 T-cell compartment.
Methods
Mice
Normal C57BL/6 (B6) mice were purchased from the National Cancer
Institute (Frederick, MD). CD154 gene knockout mice (CD154⫺/⫺) (B6
background) were purchased from The Jackson Laboratories (Bar Harbor,
ME) and maintained as a breeding colony. All infected mice were housed in
accordance with biosafety regulations, and all animal experiments followed
approved Institutional Animal Care and Use Committee and Institutional
Review Board protocols.
Reagents
The intracellular cytokine staining kit was purchased from PharMingen
(San Diego, CA). All flow cytometry antibodies were purchased from
eBioscience (San Diego, CA). Apyrase and PGE1 were purchased from
Sigma-Aldrich (St Louis, MO). The platelet-depleting antibody (a mixture
of clones p0p3 and p0p4 [p0p3/4]) was purchased from emfret Analytics
(Eibelstadt, Germany). 5,6-Carboxyfluorescein diacetate, succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR). Replication-deficient adenovirus type5-mOVA (Ad5-mOVA) was produced by the
Gene Transfer Vector Core at the University of Iowa. The membrane-bound
form of OVA (mOVA) consists of a fusion protein made up of the first
118 residues of the human transferring receptor, which includes the
cytoplasmic tail and signal/anchor domain and is linked to residues 139-385
from mature OVA. This construct was placed in a shuttle plasmid that was
recombined into Ad5 backbone DNA for packaging into viral particles.25
The collagen used is cross-linked, partially insoluble porcine skin type I
collagen (powdered Surgifoam; Johnson & Johnson, New Brunswick, NJ)
and was suspended at 30 mg/mL in PBS containing appropriate concentrations of virus for immunization.
Isolation of murine platelets
Murine platelets were isolated essentially as previously described.26 Briefly,
mice were anesthetized and bled by cardiac puncture. Blood was collected
into syringes containing 0.5 mL ACD (12.5 g/L Na citrate, 10.0 g/L
D-glucose, 6.85 g/L citric acid), added to 5 mL PIPES (150 mM NaCl,
20 mM PIPES, pH 6.5), and spun at 100g for 15 minutes. The platelet-rich
supernatant was collected, and 1 U/mL apyrase and 1 ␮M PGE1 (final
concentrations) were added and spun at 1000g for 10 minutes. The platelet
pellet was resuspended in Tyrode buffer (134 mM NaCl, 2.9 mM KCL
0.34 mM Na2PO4, 12 mM NaHCO3, 20 mM HEPES, 1 mM MgCl2, 5 mM
glucose, and 0.5 mg/mL BSA [pH to 6.5]), and counted using a Coulter
Particle Counter (Coulter, Miami, FL). All platelet manipulations were
performed at room temperature (RT).
PLATELET CONTRIBUTION TO PROTECTIVE IMMUNITY
3685
Flow cytometry
Cells were incubated with pretitrated amounts of appropriate antibodies for
30 minutes at 4°C in the dark, washed 2 ⫻ in flow cytometry buffer
(2% FCS, 0.02% NaN3 in PBS), and resuspended in 2% paraformaldehyde.
Events were collected on a FacScan or FacsCanto (Beckman, San Jose,
CA), and analyzed using Cellquest (Becton Dickinson, San Jose, CA) or
FlowJo software (TreeStar, Ashland, OR).
Bioassay for active murine soluble CD154
Quantitative real time reverse-transcription–polymerase chain reaction
(RT-PCR) was performed to assess induction of MCP-1 mRNA via sCD154
in MS-1 cells, an immortalized pancreatic endothelial cell line that was
obtained from ATCC (Manassas, VA) and routinely cultured in DMEM
with 5% FBS. After 6-hour exposure to platelet-poor plasma or sCD154,
total cellular RNA was isolated using the RNAeasy kit (Qiagen, Valencia,
CA). First-strand synthesis was performed using Superscript III reverse
transcriptase (Invitrogen, Carlsbad, CA) in a reaction using 2 ␮g total RNA
primed with random hexamers following the manufacturer’s instructions.
Of each reverse transcription reaction, 2 ␮L was subjected to real-time
quantitative PCR using proprietary TaqMan primer and probe sets for
mouse MCP-1, and 18s rRNA (Applied Biosystems, Foster City, CA). For
each sample, 3 PCRs were performed. The resulting relative increase in
reporter fluorescent dye emission was monitored by the TaqMan system
(GeneAmp 5700 sequence detection system and software; PerkinElmer,
Waltham, MA). The level of MCP-1 mRNA, relative to 18s rRNA, was
calculated using the formula: relative mRNA expression ⫽ 2 ⫺ (Ct of
MCP-1 ⫺ Ct of 18s rRNA), where Ct is the threshold cycle value.
In vivo CTLs
Splenocytes were harvested from syngeneic naive mice for use as targets.
Half of the targets were loaded with 50 ␮g/mL of the CD8-recognized
SIINFEKL OVA peptide for 1.5 hours at 37°C in RPMI/5% fetal calf serum.
These targets were washed 2 ⫻, and then labeled for 10 minutes at 37°C in
10 ␮M CFSE at 2 ⫻ 106 cells/mL in RPMI/5% fetal calf serum. Splenocytes not SIINFEKL loaded were labeled with 1 ␮M CFSE. All cells were
then washed 2 ⫻, combined, and injected retro-orbitally into mice immunized subcutaneously on the flank 13 days earlier with indicated doses of
Ad5-mOVA in PBS or PBS with 30 mg collagen/mL. Eighteen hours later,
spleens were harvested and flow cytometry was performed to assess the
percentage of OVA-specific lysis of the high CFSE SIINFEKL-loaded
targets compared with low CFSE unloaded.
Detection of OVA-specific CD8 T cells
OVA-specific T cells were detected by surface staining for CD8 together
with intracellular staining for IFN-␥ as previously described27 after a 5-hour
stimulation with 200 nM OVA 257-264 peptide in the presence of brefeldin
A (Biolegend, San Diego, CA) diluted 1:1000. The frequency of OVAspecific CD8 T cells was determined by gating on total CD8⫹ cells, which
were then analyzed for IFN-␥ expression.
In vitro lytic assays
Mice were immunized with Ad5-mOVA and after 10 to 14 days, spleens
were harvested, ground between 2 frosted microscope slides, treated with
ACK lysis buffer, and washed 2 ⫻ to isolate splenocytes. To expand and
detect OVA-specific CTLs generated by Ad5-mOVA immunization, 5 ⫻ 106
splenocytes/well were incubated with 2 ⫻ 105 mitomycin C–treated E.G7OVA lymphoma cells/well in 24-well plates in a humidified 5% CO2 37°C
incubator. E.G7-OVA lymphoma cells (E.G7) are the EL4 lymphoma cell
line transfected with ovalbumin cDNA and express the OVA SIINFEKL
peptide recognized by CD8 T cells.28 Thus, they are useful stimulators and
targets of OVA-specific CTLs. Five days later, viable splenocytes were
recovered and incubated with 51Cr-labeled E.G7 or control EL4 targets in
96-well round-bottom plates. Four hours later, the supernatants were
harvested and counted. Percentage lysis for each sample was calculated
using the formula (sample CPM ⫺ spontaneous release CPM) / (max
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3686
ELZEY et al
release CPM ⫺ spontaneous CPM) ⫻ 100. In experiments using adoptive
transfer of platelets, mice were immunized 4 hours after 3 ⫻ 108 platelets
were injected.
Listeria challenge
Listeria monocytogenes expressing ovalbumin was a gift from Leo
Lefrancois.29 An attenuated version of this strain was generated by inducing
an in-frame deletion in the actA gene as previously described (actA⫺
Lm-OVA).30,31 Bacteria were grown and cultured as previously described.32,33 All mice were infected via the tail vein with 105 actA⫺
Lm-OVA or 2 ⫻ 105 virulent Lm-OVA grown in tryptic soy broth to an
OD600 of approximately 0.1, diluted in pyrogen-free 0.9% sodium chloride
(Abbott Laboratories, North Chicago, IL), and injected intravenously in
0.2 mL per animal. Aliquots were plated onto tryptic soy agar containing
50 ␮g/mL streptomycin to verify the number of colony-forming units
(CFUs) injected and verified by plate counts. Spleens were collected from
mice 24 hours after infection with actA⫺ Lm-OVA or 72 hours after
infection with virulent Lm-OVA, and total CFUs per spleen were determined by homogenizing the spleens in 0.2% IGEPAL (Sigma-Aldrich),
plating 10-fold serial dilutions onto tryptic soy agar containing 50 ␮g/mL
streptomycin, and calculating colony count averages after overnight
incubation at 37°C as previously described.33,34
Results
Previously, we demonstrated that mice injected intratumorally with
canarypox recombinant for IL-2, IL-12, and TNF delivered in a
collagen matrix were able to reject established tumors in a
T cell–dependent manner.35 We also showed that mice immunized
with Ad5-mOVA delivered in collagen have enhanced OVAspecific cytotoxic T lymphocyte (CTL) activity.25 Initially, we
hypothesized that collagen-induced T-cell augmentation occurred
because of a significant improvement in viral transgene expression,
but further studies demonstrated that other noncollagen matrices,
which also improved gene expression, did not augment T-cell
responses (Jennifer M. Sowa, T.P.K., B.D.E., S.A.C., T.L.R.,
manuscript in preparation). Since platelets are highly responsive to
collagen, and wild-type (WT) platelets enhance T-cell activity in
CD154 gene knockout mice (CD154⫺/⫺),18 we hypothesized that
platelets mediated the observed enhancement of T-cell responses in
WT mice. Previous studies demonstrated that platelets contribute to
the T-dependent B-cell response when antigen dose is low, but the
contribution of platelets is circumvented as antigen concentration
increases.22 To determine whether this observation is also true of
the collagen-enhanced CD8 T-cell response, Ad5-mOVA dose
titrations in collagen were performed. Figure 1 demonstrates that
collagen-enhanced in vitro (Figure 1A) and in vivo (Figure 1B)
CTL lytic activities are most apparent at low doses of virus. The
enhancement is most likely due to the increased generation of
OVA-specific CD8 T cells (Figure 1C). Importantly, the low dose
effect is consistent with platelets performing an initial role for the
immune response, where they serve to augment immunity under
conditions of low antigen dose, but are dispensable at higher doses
where their contribution can be overridden. To formally demonstrate that platelets have the ability to modulate CD8 T cells,
C57BL/6 (B6) mice were depleted of platelets and immunized with
Ad5-mOVA 24 hours later. The antiplatelet antibody results in
more than 99% removal of circulating platelets, which begin to
return 4 to 5 days later.36 After 14 days, CD8 T-cell activity was
assessed.35 Figure 2A demonstrates that platelet depletion reduces
in vitro CTL activity. Importantly, immunodepletion of platelets
does not result in the release of active soluble CD154 (sCD154) as
BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
assessed by bioassay using platelet-poor plasma (Figure 2B). Since
platelets appear to be necessary for optimal CD8 T-cell responses to
Ad5-mOVA, further studies were performed in the collagen model
to determine whether collagen-induced enhancement of CD8 T-cell
function was mediated by platelets. Mice whose platelets cannot be
activated by collagen were immunized with Ad5-mOVA in PBS or
collagen. The activating platelet collagen receptor GPVI noncovalently associates with the signal-transducing Fc receptor common gamma chain (FcR␥), which is essential for its expression and
function. Consequently, FcR␥⫺/⫺ mice fail to express GPVI in their
platelets and the cells are unresponsive to collagen.37 In addition in
normal mice, intravenous injection of soluble collagen induces a
rapid and fatal thromboembolism that is entirely dependent upon
the ability of platelets to be activated by collagen through GPVI.
JAQ1, a monoclonal ab against GPVI, has been shown to induce
the complete and irreversible down-regulation of GPVI in circulating platelets in vivo resulting in a “GPVI knockout-like” phenotype
and long-term protection from lethal collagen-induced thromboembolism.38 Figure 3A demonstrates that in FcR␥⫺/⫺ mice, collagen
delivery of virus does not result in enhanced CTL activity.
Furthermore, normal B6 mice whose platelets are rendered nonresponsive to collagen by the blocking antibody JAQ1 also do not
have increased CTL lysis after viral delivery in collagen (Figure
3B), demonstrating that collagen-mediated modulation of CTL
activity is platelet-dependent. It is interesting to note that JAQ1
reduces lytic activity of the collagen group in Figure 3B below that
of the PBS group. We have observed that JAQ1 even reduces lytic
activity in mice immunized via PBS, indicating that in routine
subcutaneous immunization the small amount of resultant tissue
disruption/collagen exposure is enough to involve platelets in the
immune response.
Since platelet CD154 was demonstrated to modulate T-cell
responses,18 we investigated the ability of platelet CD154 to
participate in collagen-mediated CTL enhancement. Figure 4A
shows that collagen delivery of Ad5-mOVA in CD154⫺/⫺ mice
does not enhance in vitro CTL activity. To directly demonstrate the
necessity of platelet CD154 in collagen-mediated CTL enhancement, WT B6 platelets were adoptively transferred into CD154⫺/⫺
mice, which was previously demonstrated to provide functional
circulating platelets for up to 4 days.18 Subsequent to platelet
transfer, mice were immunized subcutaneously with Ad5-mOVA
delivered in PBS or collagen. Figure 4B demonstrates that WT
platelets are sufficient to permit collagen-mediated CTL enhancement in CD154⫺/⫺ mice. Consistent with previous studies, immunization with Ad5-mOVA in PBS was also augmented by platelet
transfer,18 but to a lesser degree versus immunization in collagen.
This function can be directly attributed to platelet CD154 since the
WT platelets are the only source of CD154 in this model, although
collagen delivery of Ad5-mOVA in the absence of CD154 also
results in a minor increase in CTL activity. This could be due to
better transgene expression and antigen presentation or other
CD154-independent components. To show that the collagenmediated increase in antigen-specific CTLs has a beneficial biologic consequence, B6 mice immunized with Ad5-mOVA in PBS
or collagen were challenged intravenously 14 days later with
attenuated or virulent Listeria monocytogenes recombinant for
ovalbumin (Lm-OVA). Twenty-four or 72 hours later, respectively,
mice were killed and CFUs/spleen were assessed. Figure 5
demonstrates that mice immunized with Ad5-mOVA in collagen
were better able to control Lm-OVA compared with mice immunized with Ad5-mOVA in PBS.
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
PLATELET CONTRIBUTION TO PROTECTIVE IMMUNITY
3687
Figure 1. Delivery of Ad5-mOVA in collagen enhances recognition and lysis of OVA-expressing
targets in a dose-dependent manner. (A) Mice were
immunized subcutaneously with the indicated doses of
Ad5-mOVA in PBS or 30 mg/mL collagen in the flank.
After 14 days, spleens were harvested for lytic assays
as described in “Methods.” Data are representative of 2
experiments with 3 mice per group. (B) Mice were
immunized as in panel A, and in vivo CTLs were
initiated 13 days later as described in “Methods.”
SIINFEKL-loaded splenocyte targets were CFSE-high,
and control targets were CFSE-low. Data are representative of 2 experiments of 3 to 5 mice per group.
Percentage lysis is the average of both experiments
plus or minus SEM. (C) Mice were immunized as in
panels A and B, and spleens were harvested after
13 days for 5-hour stimulation with SIINFEKL peptide to
determine levels of IFN␥-expressing OVA-specific CD8
T cells as assessed by flow cytometry. Each panel is
representative of 3 mice. Lysis is the average plus or
minus standard deviation.
Discussion
Naturally acquired infections usually involve small initial doses of
replication-competent pathogens, whereas experimental examina-
tion of immune responses frequently use high doses of replicationlimited or -incompetent microbes for safety reasons or to stimulate
a high number of effector cells that facilitate their study. T-cell
receptor transgenic mice are also used to aid in studying adaptive
immunity; however, the unusually high antigen-specific precursor
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3688
ELZEY et al
Figure 2. Platelet depletion lowers CTL response. (A) Mice were more than 99%
depleted of platelets by intravenous injection of platelet-depleting monoclonal
antibody and immunized with 106 pfu Ad5-mOVA intravenously 24 hours later. After
14 days, spleens were harvested for lytic assays. Data are representative of
4 experiments of 3 mice per group. (B) Immunodepletion of platelets does not release
active sCD154. Mice were depleted of platelets as in panel A and platelet-poor
plasma was harvested 24 hour later, the time at which adenovirus is injected for
initiation of the immune response. Since commercial reagents with sufficient
sensitivity are not available for the detection of murine sCD154 protein, plasma was
bioassayed for activity as described in “Methods.” MS-1 cells were incubated for
6 hours with plasma from normal or depleted mice in the presence of MR1, a potent
blocking monoclonal antibody against full-length or soluble murine CD154, and were
assessed for MCP-1 mRNA content. Values were normalized and are expressed as
stimulation index.
numbers can significantly change the kinetics of the response.39
Investigations aimed at studying the biology of infections and
immunity should use antigen doses close to the limit of immune
detection as well as physiologic levels of antigen-specific precursors to more accurately reflect early immune initiation events. Dose
titrations of replication-incompetent adenovirus demonstrate that
subcutaneous immunization of 106 pfu is a threshold level for
induction of detectable antigen-specific T- and B-cell responses
(data not shown). It is at this dose that we tested the ability of
platelets to contribute to T-cell activation with the objective of
better approximating the biologic setting of initial low antigen dose
near the limit of immune detection. The data reported herein using
collagen to mimic wound healing demonstrate a role for platelets in
modulating early low-dose antigen responses. Platelet-derived
CD154 augmented CD8 T-cell responses against an adenovirus
transgene, which was necessary for induction of a protective
immune response against a subsequent Listeria challenge. We
hypothesize that platelets function to release CD154 early in
infection, modulating APC function. Early maturation of APCs
provides the activation signals to T cells that are required to induce
effective effector cell expansion early in the infection. Due to a lack
of sufficiently sensitive commercial reagents, it is not possible to
distinguish sCD154 activity from membrane bound.
BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
Figure 3. Platelets must be responsive to collagen for collagen-mediated CTL
augmentation. (A) FcR␥⫺/⫺ mice (platelets cannot be activated by collagen) were
immunized subcutaneously on the flank with 106 pfu Ad5-mOVA in PBS or 30 mg/mL
collagen. After 14 days, spleens were harvested for lytic assay. (B) B6 mice were
injected intravenously with 50 ␮g of the GPVI (platelet collagen receptor)–blocking
monoclonal antibody JAQ1. After 24 hours, mice were immunized subcutaneously on
the flank with 106 pfu Ad5-mOVA in PBS or 30 mg/mL collagen. Fourteen days later,
spleens were harvested for lytic assays. Each panel is representative of 2 experiments of 3 mice per group.
T-cell responses are dependent on antigen presentation by
dendritic cells (DCs) in draining lymph nodes where the quality of
activation is dependent on the maturation status of the antigenpresenting DCs.1,3 The literature supports a dichotomy of T-cell
activation, one from immature DCs and the other from mature DCs.
In a setting where DCs are quiescent, T cells do not receive
requisite second signals, which results in ignorance or peripheral
tolerance. However, if antigen presenting DCs are licensed by TLR
stimulation and receive maturation signals in the form of CD154,
T cells receive all necessary signals for clonal expansion and full
activation.5 Although DCs are exquisitely sensitive to activation
through TLR, TLR signals do have a threshold that can be
lowered.40 Since TLR9 signaling up-regulates CD40, platelets
could enhance TLR9 signaling resulting in higher DC sensitivity to
CD154, or it may be that the platelets simply serve as a source of
early CD154. Regardless, our data support the hypothesis that
platelets send early signals to DCs before foreign antigen/TLR
agonists are present in sufficient concentration to mature DCs,
which leads to increased T-cell activation. Consistent with this
hypothesis, several reports demonstrate platelet-induced maturation of DCs.18-21 This would be expected to increase activation of
not only CD8 T cells as presented here, but also of CD4 T cells.
Indeed, we have already demonstrated that platelets cooperate with
CD4 T cells to enhance the B-cell GC response. Although the
platelet-CD4 T-cell–B-cell relationship is under current investigation, it seems likely that this would be accomplished via the
follicular DCs.
One of the classic functions of platelets is activation by
collagen resulting in release of procoagulants for the purpose of
hemostasis and wound repair. However, many released components such as CD62P, IL-1␤, and histamine also possess
immunomodulatory function, while others including thrombocidins, TGF-␤, CD154, RANTES, MIP-1␣, MCP-3, and several
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
Figure 4. Platelets must have CD154 for collagen-mediated CTL augmentation.
(A) CD154⫺/⫺ mice were immunized subcutaneously on the flank with 106 pfu
Ad5-mOVA in PBS or 30 mg/mL collagen. After 14 days, spleens were harvested for
lytic assays. (B) CD154⫺/⫺ mice were adoptively transferred with 3 ⫻ 108 B6 platelets
4 hours before subcutaneous immunization with 106 pfu Ad5-mOVA in PBS or
30 mg/mL collagen on the flank. After 14 days, spleens were harvested for lytic
assays. Each panel is representative of 2 experiments of 3 mice per group.
other chemokines have primarily innate/adaptive immune function.17 That platelets can release/produce such a diverse array of
biologic mediators indicates that their function is much broader
than simply facilitating coagulation. Since platelets are the first
cell to respond to injury compromising a vessel wall, they are
uniquely poised to initiate innate and adaptive immunity. We
have recently demonstrated that platelets are able to modulate
adaptive T- and B-cell responses via CD154 expression.18
Others have shown that human platelets from immune thrombocytopenic purpura patients can directly stimulate autologous B
cells to produce antiplatelet antibodies in vitro.41 In addition to
direct stimulation of B cells, platelets, through direct contact
with T cells, release RANTES, creating a feedback loop for
T-cell response amplification.42 Furthermore, adoptively transferred human platelets via CD154 have been shown to cause
immune-mediated rejection of allografts in mice.43 Consistent
with an early signaling function in modulation of adaptive
immunity, human platelets could support graft rejection only if
they were activated at the site of surgery at the time of graft
implantation.43 In our collagen immunization model, we hypothesize that platelets activated at the site of injury express CD154,
which is able to stimulate DC maturation and enhance T-cell
activation. Although we are currently pursuing this question, it
is consistent with our initial report that platelets through CD154
can stimulate DCs in vitro, an observation corroborated by
several others.19,20 Platelets, under conditions of injury, are also
PLATELET CONTRIBUTION TO PROTECTIVE IMMUNITY
3689
Figure 5. Mice immunized with Ad5-mOva delivered in collagen versus PBS
have increased protection against Lm-OVA. (A) B6 mice were immunized
subcutaneously on the flank with 106 pfu Ad5-mOva in PBS or 30 mg/mL collagen.
Fourteen days later, mice were challenged intravenously with 105 actA⫺ Lm-OVA, an
attenuated strain. Twenty-four hours later, spleens were harvested and CFUs/spleen
were measured as described in “Methods.” Data are from 3 experiments of 4 to
5 mice per group. Wilcoxon sign-rank 2-tailed P ⫽ .002. (B) Mice were treated as in
panel A, but were challenged with virulent Lm-OVA. Splenic CFU was assessed
72 hours later. LOD indicates limit of detection.
able to mature DCs in vitro.21 Conversely, other reports indicate
that platelets can have inhibitory effects on/through DCs.44-46
The reason for these differences is unclear, although it may be
related to effective platelet/DCs ratios or the mechanism of
platelet activation in each model.
As previously published, the subcutaneous injection of
collagen is a model for studying immune responses in injury.24
Collagen injection mimics a wound with exposed basement
membrane collagen, but importantly allows critical control of
antigen delivery and the amount of collagen that is exposed,
circumstances that would not be possible with an actual wound.
The implantation of collagen for studying immune responses
has previously been thought to be an inert phenomenon.
However, we now demonstrate that collagen exposure can affect
the immune response, an event that is dependent on platelets and
their ability to recognize collagen. Because the Ad5-mOVA
infection is self-limiting, there are no ramifications for the mice
that do not respond to the virus. However, in the normal
physiologic setting of injury, invading pathogens will be able to
replicate and pose a threat to the host. It may be that in this
situation, prompt arousal of innate and adaptive immunity via
platelets serves to limit microbial expansion and damage
compared with what would occur in the absence of platelet
involvement. In our model, Figure 4 demonstrates that platelet
CD154 is required for collagen-mediated augmentation of the
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
3690
BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
ELZEY et al
T-cell response. Because of this, we expect that the primary
mechanism of platelet contribution to adaptive immunity is at
the level of antigen presentation that is currently under investigation. It is well established that T-cell interaction with collagen
via VLA-1 and VLA-2 is important for effector T-cell trafficking
and maintenance of tissue-specific memory T cells.47,48 However, it has also been demonstrated that primary immune
responses or effector T-cell presence in the spleen do not require
these interactions.49 Since our studies probe primary and splenic
secondary T-cell responses, it is unlikely that VLA interactions
with collagen modulate our observations reported herein.
The clearance of Lm-OVA in Figure 5 demonstrates the
effectiveness of collagen-elicited immunity to a low virus dose that
otherwise would not generate a protective response. This could
have clinical significance regarding vaccination of immunocompromised patients. Live vaccines such as oral polio or measles/mumps/
rubella present special problems to HIV patients, or to patients with
certain congenital defects who may not be able to control viral
replication. However, if collagen-delivered vaccines containing
orders of magnitude less virus resulted in effective immunization, it
may be possible to protect these patients from threatening diseases
without exposing them to substantial risk.
Acknowledgments
This work was supported by grants from the Department of
Defense (DAMD17-02-1-0079 [B.D.E.]) and the National Institutes of Health (DK071712 [B.D.E.], T32 A1007511 [N.W.S.],
AI60924 and CA096691 [T.L.R.], others [J.T.H.]).
Authorship
Contribution: B.D.E. designed, directed, and performed research
and drafted the paper; N.W.S., S.A.C., and T.P.K. performed
research; J.T.H. contributed to the integrity and analysis of data;
B.N. contributed vital new reagents and experimental design;
T.L.R. directed research.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Bennett D. Elzey, Wishard Memorial Hospital, Room OPW 320, 1001 W 10th St, Indianapolis, IN 46202;
e-mail: [email protected].
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2008 111: 3684-3691
doi:10.1182/blood-2007-05-091728 originally published
online February 6, 2008
Platelet-derived CD154 enables T-cell priming and protection against
Listeria monocytogenes challenge
Bennett D. Elzey, Nathan W. Schmidt, Scott A. Crist, Timothy P. Kresowik, John T. Harty, Bernhard
Nieswandt and Timothy L. Ratliff
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