JOURNAL OF VIROLOGY, Mar. 2004, p. 3046–3054
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 6
Impaired Lymphoid Chemokine-Mediated Migration due to a Block
on the Chemokine Receptor Switch in Human
Cytomegalovirus-Infected Dendritic Cells
Magdalena Moutaftsi,1,2Paul Brennan,1Stephen A. Spector,2and Zsuzsanna Tabi3*
Section of Infection and Immunity1and Department of Medicine,3University of Wales College of Medicine, Cardiff, United
Kingdom, and Department of Pediatrics, Center for Molecular Genetics and Center for AIDS Research,
University of California, San Diego, La Jolla, California2
Received 30 July 2003/Accepted 24 November 2003
Dendritic cell (DC) migration from the site of infection to the site of T-cell priming is a crucial event in the
generation of antiviral T-cell responses. Here we present to our knowledge the first functional evidence that
human cytomegalovirus (HCMV) blocks the migration of infected monocyte-derived DCs toward lymphoid
chemokines CCL19 and CCL21. DC migration is blocked by viral impairment of the chemokine receptor switch
at the level of the expression of CCR7 molecules. The inhibition occurs with immediate-early–early kinetics,
and viral interference with NF-?B signaling is likely to be at least partially responsible for the lack of CCR7
expression. DCs which migrate from the infected cultures are HCMV antigen negative, and consequently they
do not stimulate HCMV-specific CD8?T cells, while CD4?-T-cell activation is not impaired. Although CD8?
T cells can also be activated by alternative antigen presentation mechanisms, the spatial segregation of naive
T cells and infected DCs seems a potent mechanism of delaying the generation of primary CD8?-T-cell
responses and aiding early viral spread.
Dendritic cells (DCs), the most potent professional antigen
(Ag)-presenting cells, survey peripheral tissues where they take
up Ags. Ag uptake and activation signals induce DCs to mi-
grate to secondary lymphoid organs and interact with Ag-
specific naive T cells (5). This migration is an essential part of
the initiation of an immune response. Activation of virus-spe-
cific cytotoxic T lymphocytes in the draining lymph nodes in a
localized peripheral viral infection occurs soon after infection
(6 to 8 h) and is due to the swift recruitment of DC into the
lymph nodes (26).
DC migration is regulated by changes in the expression of
chemokine receptors on the surface of DCs, a process often
referred to as the chemokine receptor switch. The molecular
events of this chemokine receptor switch involve the down-
regulation of the proinflammatory-type chemokine receptors,
such as CCR5, and the increase of the expression of the lym-
phoid-type chemokine receptors, such as CCR7. Lymph nodes
of CCR7 knockout mice are devoid of T cells, and DCs from
these mice are unable to mount primary T-cell responses in
vivo (14). CCR7 expression during DC maturation enables
DCs to respond to the lymphoid chemokines, CCL19 (ELC or
MIP-3?) and CCL21 (SLC, 6Ckine, or TCA-4), which are
constitutively produced by secondary lymphoid tissues. CCL19
and CCL21 are extremely potent and selective chemoattrac-
tants of mature DCs (9, 12, 20) with overlapping functions (3).
The expression of CCR7 can be induced on DCs by treatment
with alpha/beta interferon (IFN-?/?), interleukin-1? (IL-1?),
tumor necrosis factor alpha (TNF-?) (30), or bacterial cell wall
proteins (19) or upon interaction with apoptotic tumor cells
(17). Augmented expression of CCR7 results in enhanced DC
migratory responses to CCL21 both in vitro and in vivo (38).
The common feature of all these signals is the activation of the
transcription factor NF-?B, which is a key event in DC matu-
ration (4). The most studied pathway enabling DC to migrate
is the stimulation via TNF receptors (TNFRs). DC migration is
suppressed in mice lacking TNFR-p75 (33, 34). Migration is
also impaired in TNF-?-deficient or anti-TNF-? antibody-
treated mice (11).
Human cytomegalovirus (HCMV) is a significant cause of
morbidity and mortality in immunosuppressed hosts. HCMV
has the capacity to persist asymptomatically in healthy individ-
uals, indicating that it has developed various strategies to avoid
elimination by the immune system. We have previously shown
that HCMV can infect human DCs and inhibits the expression
of major histocompatibility complex class I and class II and
costimulatory molecules (CD80, CD86, CD40) in infected, but
not in bystander, DCs (25). Infected DCs maintain an imma-
ture-like phenotype even upon stimulation with maturation
Under certain circumstances CCR7 can be upregulated on
immature DCs, and these DCs are able to migrate into lymph
nodes (15, 24), where, due to their phenotype, they play a
crucial role in the induction or maintenance of peripheral
tolerance. This study was initiated to investigate whether
HCMV-infected DCs are able to migrate toward gradients of
lymphoid chemokines upon infection or after further stimula-
tion. Our findings indicate that HCMV-infected DCs in fact do
not respond to CCL19 or CCL21 gradients even when stimu-
lated with lipopolysaccharide (LPS). Moreover, migrated DCs,
which represent uninfected, bystander DCs, fail to stimulate
HCMV-specific CD8?T cells.
* Corresponding author. Mailing address: Department of Medicine,
Section of Oncology and Palliative Medicine, University of Wales,
College of Medicine, Velindre Hospital, Whitchurch, Cardiff CF14
2TL, United Kingdom. Phone: 44 2920 196137. Fax: 44 2920 529625.
MATERIALS AND METHODS
Cells. Peripheral blood mononuclear cells (PBMC) were isolated from
HCMV-seropositive and -seronegative healthy laboratory volunteers. Ethical
approval was granted for the study by the Bro Taf Local Ethical Committee.
Informed consent was obtained from the volunteers according to the Declaration
of Helsinki. DCs were generated as previously described (25, 28). In brief,
plastic-adherent or CD14?cells, the latter isolated by a MACS monocyte isola-
tion kit to obtain untouched monocytes (Miltenyi Biotech) according to the
manufacturer’s instructions, were grown in the presence of granulocyte-mac-
rophage colony-stimulating factors (50 ng/ml; Leucomax, Novartis Pharmaceu-
ticals, East Hanover, N.J.) and IL-4 (500 U/ml; BD Pharmingen, San Diego,
Calif.) for 5 to 7 days. Nonadherent and loosely adherent DCs were collected.
More than 90% of these cells were of DC phenotype (CD1a?HLA-DR?CD14?
CD80?) following gating on forward and side scatter parameters to exclude
lymphocytes. DC maturation was induced by adding LPS (1 ?g/ml; Sigma, St.
Louis, Mo.), TNF-? (50 ng/ml; R&D Systems, Minneapolis, Minn.), and IFN-?
(100 ng/ml; PeproTech, Rocky Hill, N.J.) to the culture medium. Human fore-
skin fibroblasts (HFFs) were grown as described previously (25, 31).
Virus. DCs were infected with an endothelial-cell-grown HCMV strain,
TB40/E (kindly provided by Christian Sinzger, Tuebingen, Germany) (27), at a
multiplicity of infection (MOI) of 3 to 10. The virus stock was prepared by
propagating HCMV in HFFs followed by enrichment by ultracentrifugation
(80,000 ? g for 1 h in a Beckman L8-M ultracentrifuge), which removed soluble
factors present in the supernatant of infected fibroblasts. Mock-infected cells
were given the same volume of RPMI medium without the virus.
Flow cytometry. DCs were surface labeled with antibodies specific for
HLA-DR (CyChrome conjugated; BD Pharmingen), CCR5 (phycoerythrin [PE]
conjugated; Serotec, Oxford, United Kingdom), CCR7 (BD Pharmingen), and
TNFR-p55 and TNFR-p75 (both PE conjugated; Caltag Laboratories, Burlin-
game, Calif.) and with a PE-conjugated anti-mouse immunoglobulin G (Sero-
tec). T cells were surface labeled with antibodies specific for CD8 or CD4 (both
CyChrome conjugated; BD Pharmingen). Intracellular staining for IFN-? or for
HCMV pp52 was carried out following fixing either DCs or T cells with 4%
paraformaldehyde and permeabilizing them with 0.025% Triton X-100 (20 min
each step at room temperature). Nonspecific binding was blocked with 2%
mouse serum for 10 min followed by adding fluorescein isothiocyanate (FITC)-
conjugated HCMV pp52-specific (delayed early DNA-binding protein, clone
CCH2; Dako, Carpinteria, Calif.) or IFN-?-specific (BD Pharmingen) antibodies
for 1 h at 37°C. The cells were analyzed on a FACScalibur (BD Biosciences) flow
cytometer using CellQuest software (version 3.1).
DC migration assay. DC migration was performed in 24-well polycarbonate
transwell culture chambers (Costar, Corning, N.Y.) with 6.5-mm diameter and
5-?m pore size. DCs (0.5 ? 106to 2 ? 106) were placed in 100 ?l of RPMI
medium supplemented with 10% human AB serum into the inserts of transwells.
The bottom chamber contained 600 ?l of medium supplemented as described
above and also containing 100 to 330 ng of CCL19/ml, except for the control
wells. The wells were incubated for 3 to 5 h at 37°C, when cells were collected
from the lower and upper chambers and used in the experiments as indicated.
T-cell stimulation and IFN-? production assay. Nonadherent PBMC of sero-
positive donors were stimulated with a mixture of the following HCMV peptides
at 5 ?g/ml each for 7 days: HLA-A2-restricted peptides IE1 (YVLEETSVML)
and pp65 (ARNLVPMVATVQGON) and HLA-B7-restricted peptides IE1 (F
CRVLCCYVL) and pp65 (TPRVTGGGAM). T cells and peptides were placed
at 2 ? 106PBMC/ml in RPMI medium plus 10% human AB serum in 12-well
trays (2 ml/well) or in upright tissue culture flasks (10 ml). T-cell stimulation was
carried out 7 days later by mixing DC with the prestimulated T cells at a 1:10
ratio, at 1 ? 105to 5 ? 105T cells per group overnight. Golgi-Plug (1 ?l/ml;
Pharmingen) was added to each group 1 h after the initiation of DC–T-cell
cultures. Negative controls contained no DCs or mock-infected DCs, while
positive controls were stimulated with phorbol myristate acetate (10 ng/ml) and
ionomycin (500 ng/ml). IFN-? production by CD3?CD8?and CD3?CD8?cells
was measured by intracellular cytokine staining as described earlier. In the
control experiments, allogeneic PBMC were added to DCs for 5 days at a 10:1
ratio in 96-well tissue culture plates at 105PBMC/well (not shown).
Preparation of nuclear extracts and electrophoretic mobility shift assay. Nu-
clear extracts were generated as described previously (8). Cell treatments were
terminated by adding 5 ml of ice-cold phosphate-buffered saline. Each sample
was centrifuged at 163 ? g in a swing-out rotor and then resuspended in 1 ml of
buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 10 mM
phenylmethylsulfonyl fluoride [PMSF]) and centrifuged at 12,000 ? g for 10 min
in a benchtop microcentrifuge at 4°C. Cells were then lysed by addition to 20 ?l
of buffer A containing 0.1% Nonidet P-40 and left on ice for 10 min, after which
time they were centrifuged at 12,000 ? g for 10 min. The nuclear extract was
prepared by adding 15 ?l of buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl,
1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF) to the supernatant. The mixture
was left on ice for 15 min. It was then centrifuged at 12,000 ? g for 10 min, and
the supernatant was added to 75 ?l of buffer D (10 mM HEPES [pH 7.9], 50 mM
KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM PMSF). The protein in these crude
extracts was determined by the Bradford method. The extracts were assayed
immediately for NF-?B activity or stored at ?70°C until further use. Equivalent
amounts of protein from nuclear extracts were incubated with approximately
10,000 cpm of a32P-labeled oligonucleotide containing the consensus sequence
for NF-?B in binding buffer (40% glycerol, 1 mM EDTA, 10 mM Tris [pH 7.5],
100 mM NaCl, nuclease-free bovine serum albumin [0.1 mg/ml], 50 mM dithio-
threitol) and 2 ?g of poly(dI-dC) at room temperature for 30 min, as described
previously (8). T4 polynucleotide kinase and the 22-bp oligonucleotide contain-
ing the NF-?B consensus sequence (underlined) (5?-AGTTGAGGGGACTTT
CCCAGGC-3?) were from Promega (Madison, Wis.). [?-32P]ATP was from
Amersham International (Amersham, Buckinghamshire, United Kingdom). In-
cubated proteins were subjected to electrophoresis on native 4% polyacrylamide
gels that were subsequently dried and autoradiographed at ?70°C overnight.
HCMV infection inhibits DC migration toward CCL19 and
CCL21. The ability of maturing DCs, which carry processed
Ag, to migrate to secondary lymphoid organs where naive T
cells reside is a crucial step in the generation of primary T-cell
responses. Migration of maturing DCs is controlled by lym-
phoid chemokines CCL19 and CCL21, which are produced by
secondary lymphoid organs. Viruses that infect DCs may in-
terfere with the normal pattern of DC migration. We studied
whether this is the case when HCMV infects DCs. Transwell
experiments were carried out with CCL19 or CCL21, as che-
moattractants, placed in the lower parts of the transwells. DCs,
either infected with an endothelial-cell-grown strain of HCMV
(TB40/E) (27) for 48 h or mock infected, were placed in the
upper parts of migration chambers. The TB40/E strain of
HCMV infected 30 to 70% of DCs in these experiments, as
assessed by intracellular staining with an antibody specific for
the HCMV pp52 Ag. Thus, by detecting the presence of
HCMV-infected cells in the premigrated (Fig. 1a) and postmi-
grated (Fig. 1b to e) populations of DCs following infection
with TB40/E strain at an MOI of 10, we were able to compare
the migratory capacity of infected and uninfected DCs by flow
cytometry. As, regardless of infection, only a few cells migrated
under these conditions (data not shown), additional stimula-
tion was required. Day 1 infected DCs were treated with LPS
in the presence of IFN-? and TNF-? for 24 h. We found that
HCMV Ag-positive DCs were selectively prevented from mi-
grating toward CCL19 or CCL21. Infected DCs remained in
the upper chambers of the migration wells (Fig. 1b and c), and
those which migrated toward CCL21 (Fig. 1d) or CCL19 (Fig.
1e) were uninfected (i.e., HCMV Ag negative [?0.41%]). DCs
before migration (Fig. 1a) seemed to express HLA class II at a
high or medium level, respectively, while HLA class II expres-
sion on cells collected from the migration chambers was more
homogenous (Fig. 1b and c or d and e) and lower than that
before migration. The presence of four subgroups of DCs
before migration was not observed from all donors. The rea-
sons for the changes in HLA class II expression on DCs in the
migration chambers are not known. The level of HLA class II
expression on migrated DCs was similar to or higher than that
on nonmigrated DCs, as seen when comparing Fig. 1d and b
(mean fluorescence intensity [MFI], 1,210 versus 1,272) and
VOL. 78, 2004HCMV INFECTION INHIBITS DC MIGRATION3047
Fig. 1e and c (MFI, 1,961 versus 1,761); furthermore, HLA
class II expression on HCMV Ag-positive cells was lower than
on Ag-negative ones (Fig. 1b [MFI, 905 versus 1,272] and c
[MFI, 1,180 versus 1,761]), confirming previous observations.
The proportion of HCMV-infected DCs was about 100-fold
lower in the migrated population of DCs than that in the
starting population of DCs. The few DCs which were HCMV
pp52 positive in the migrated pool had only very low levels of
pp52 expression, indicating that successful inhibition of DC
migration correlates with a high level of viral replication in
Migrated DCs do not stimulate HCMV-specific T-cell re-
sponses. To determine whether migrated DCs, containing only
a low proportion (?0.41%) of HCMV Ag-positive DCs (Fig.
1d and e), are able to stimulate HCMV-specific memory
CD8?-T-cell responses, DCs from the upper or lower cham-
bers of transwells were mixed with autologous T cells from
seropositive donors. HCMV-specific T cells were enriched in
the responder population of this experiment by stimulating
nonadherent, HLA-A2, B7 PBMCs with HLA-A2- and B7-
restricted HCMV IE1 and pp65 peptides for 7 days. T-cell
activation was measured by flow cytometric detection of IFN-
?-producing CD8?T cells (Fig. 2a to d, upper-right quad-
rants). The background level of CD8?-T-cell activation in the
absence of DCs was low (0.6% [Fig. 2a]), while mock-infected
DCs nonspecifically activated a small proportion of CD8?T
cells (2.7% [Fig. 2b]). Following HCMV infection of DCs,
migrated cells contained only a low proportion of DCs positive
for HCMV pp52 (?0.4%; not shown), and migrated DCs were
only able to stimulate CD8?T cells at levels (3.1% [Fig. 2c])
comparable to those with mock-infected DCs (2.7% [Fig. 2b]).
In contrast to CD8?T cells, an ?20-fold increase was observed
in the proportion of CD8?T cells, representing mainly CD4?
T cells (?90% of CD3?CD8?cells are CD3?CD4?; not
shown), which became activated by the migrated DCs (com-
pare Fig. 2c and b, upper left quadrants [2.0 versus 0.1%]).
This result indicates that uptake of viral Ag in the absence of
viral replication by DCs does not impair the migratory and
Ag-presenting capacity of these DCs, and consequently, the
generation of CD4?-T-cell responses is not impaired. When
mixed directly with T cells, infected DCs (from the upper
chambers; 41.4% positive for HCMV pp52) supported strong
IFN-? production by both CD8?and CD8?T cells from an
HCMV-seropositive individual (Fig. 2d). Statistical analysis of
the results from triplicate samples (Fig. 2e and f) reveals that
the slight increase in the proportion of activated CD8?T cells
when the migrated population of HCMV-encountered DCs
were stimulators (Fig. 2e, bar C) did not reach statistical sig-
nificance compared to the proportion of activated CD8?T
cells stimulated with mock-infected DCs (Fig. 2e, bar B). Co-
culture of HCMV-specific T cells with infected DCs from the
upper chambers resulted in high levels of T-cell activation
(bars D of Fig. 2e and f). In contrast to CD8?T cells, a
significant proportion of CD8?T cells was activated by mi-
grated, HCMV-encountered DCs (Fig. 2f, bar C versus bar B).
The impaired ability of migrated DCs, after being exposed to
FIG. 1. HCMV Ag-positive DCs do not migrate toward lymphoid chemokines. DCs, infected with HCMV TB40/E at an MOI of 10 for 24 h
and then matured with LPS/IFN-?/TNF-? for a further 24 h, were placed into transwell migration chambers which contained a 200-ng/ml
concentration of either CCL21 (b and d) or CCL19 (c and e) in the lower chambers. The experiment was run for 4 h, and samples of the input
DCs (a), DCs which did not migrate (b and c), and those which migrated into the lower chambers (d and e) were analyzed for the rate of HCMV
infection by immunofluorescent staining as described in Materials and Methods. The dot plots show the intensity of binding of HCMV pp52-FITC
antibody on the x axes and of the HLA-DR-CyChrome antibody on the y axes by fixed and permeabilized cells. The numbers express the frequency
of HCMV pp52 Ag-positive cells, showing that migration was selective for DCs that remained uninfected (HCMV pp52 Ag-negative DCs). The
results are representative of five experiments.
3048 MOUTAFTSI ET AL.J. VIROL.
HCMV, to stimulate HCMV-specific CD8?T cells above
background level was Ag specific, as DCs that migrated from
either HCMV-infected or mock-infected DC populations were
equally able to stimulate allogeneic T-cell responses (not
HCMV-infected DCs do not express CCR7. Migration of
DCs to lymph nodes requires the expression of CCR7. CCR7
is the receptor for lymphoid chemokines CCL19 and CCL21,
which are produced constitutively by secondary lymphoid or-
gans. Upon receiving stimulatory signals, DCs downregulate
inflammatory chemokine receptors such as CCR5 and upregu-
late CCR7, the process known as chemokine receptor switch.
As this is a crucial step in enabling DCs to migrate to lymph
nodes, we analyzed the effect of HCMV infection on DC che-
mokine receptor switch (Fig. 3). CCR5 expression on imma-
ture DCs, infected with HCMV, was compared to that on
mock-infected DCs (Fig. 3, first row). We found a medium
level of CCR5 surface expression on immature DCs (34.6%
[Fig. 3a]), which was downregulated upon HCMV infection
(16.1% [Fig. 3b]) and also upon stimulation with LPS/TNF-?/
IFN-? (24.1% [Fig. 3c]). LPS/TNF-?/IFN-? and HCMV to-
gether acted synergistically, resulting in the complete down-
regulation of CCR5 (0% [Fig. 3d]). Thus, HCMV infection
seems to provide a signal for DCs to undergo the first part of
the chemokine receptor switch, which would enable infected
DCs in vivo to leave the site of infection. The second step of
the receptor switch, the upregulation of CCR7 molecules, was
studied in a similar experiment (Fig. 3, second row). CCR7
expression was low on immature DCs (6.7% [Fig. 3a]), while
LPS/TNF-?/IFN-? treatment was highly efficient in inducing
CCR7 expression (45.9% [Fig. 3c]). However, CCR7 mole-
cules were only slightly upregulated by HCMV (12.5% [Fig.
3b]). When HCMV-infected DCs were stimulated with LPS/
TNF-?/IFN-?, CCR7 expression was lower on HCMV-in-
fected (41%; MFI, 57 [Fig. 3d]) than on mock-infected, stim-
ulated (45.9%; MFI, 111 [Fig. 3c]) DCs. As not all DCs
become infected with HCMV, two-color flow cytometric anal-
ysis was carried out to analyze the level of CCR5 and CCR7
expression separately on pp52 Ag-positive and Ag-negative
DCs (Fig. 3e and f) from the HCMV-infected DC cultures.
Following infection, CCR5 expression was low on both HCMV
Ag-positive and Ag-negative cells even without LPS treatment
(Fig. 3e), although the downregulation was more efficient on
Ag-positive DCs (black histogram). More importantly, CCR7
upregulation following LPS treatment seemed to be the prop-
erty of bystander, HCMV Ag-negative DCs (Fig. 3f, gray line).
FIG. 2. DCs, which migrate toward CCL19, do not stimulate HCMV-specific T cells. HCMV-specific T cells were enriched by incubating the
nonadherent fraction of PBMC from an HCMV-seropositive donor with A2- and B7-restricted pp65 and IE1 peptides for 7 days. These T cells
were then restimulated with the following: medium only (a); mock-infected, nonmigrated DC from the upper chambers of transwells (b); migrated
DC from the lower chambers of HCMV-infected input DC (c); and nonmigrated DC from the upper chambers of HCMV-infected input DC (d).
DC:T ratio was 1:10. T-cell activation following stimulation with phorbol myristate acetate and CaI is not shown (87.9% ? 1.4% for CD8?T cells
and 33.4% ? 3.2% for CD8?T cells). The proportion of IFN-?-producing CD8?(upper right quadrants) and CD8?(upper left quadrants) T cells
from a representative experiment of three repeated ones are shown, as detected by flow cytometry. (e and f) Summary of results and statistical
analysis of CD8?-T-cell (e) and CD8?-T-cell (f) stimulation from triplicate wells. T cells were stimulated as described above with medium only
(bars A), mock-infected nonmigrated DC (bars B), migrated DCs from HCMV-infected input cells (bars C), and nonmigrated DCs of HCMV-
infected input cells (bars D). Statistical analysis was carried out by applying Student’s t test. Significant differences (P ? 0.01) are indicated (?).
NS, not significant; error bars, standard deviations.
VOL. 78, 2004HCMV INFECTION INHIBITS DC MIGRATION 3049
These experiments reveal that the second part of the chemo-
kine receptor switch, the upregulation of surface CCR7 mol-
ecules, is inhibited in HCMV-infected, Ag-positive DCs. This
inhibition is complete in DCs expressing the HCMV early Ag
pp52; thus, it is likely to be an immediate-early or early direct
DCs upregulate CCR7 upon encountering infected cells. We
have shown previously in a cross-presentation model that on
DCs, following coculture with infected fibroblasts, the expres-
sion of major histocompatibility complex class II and CD83
molecules is upregulated (31), indicating bystander maturation
induced by interaction with infected cells. We have now stud-
ied whether DCs, cocultured with infected fibroblasts, are in-
duced to express CCR7 molecules. Compared with immature
DCs (Fig. 4a), maturation agents such as LPS/IFN-?/TNF-?
(Fig. 4b) or coculture with TB40/E-infected fibroblasts (Fig. 4f)
indeed increased the expression rate of CCR7 on DCs, while
infection with TB40/E (without further treatment) (Fig. 4d)
also caused bystander DC maturation, although to a lesser
extent. In this case, DCs which became infected with TB40/E
(pp52 Ag positive) remained negative for CCR7, again dem-
onstrating the viral inhibition of CCR7 expression (Fig. 4d,
right panel). The difference in bystander DC activation seems
to depend upon the type of infected cells in the cocultures, as
infected HFFs were more efficient than infected DCs in induc-
ing CCR7 upregulation. Laboratory HCMV strain AD169,
which does not infect DCs (Fig. 4c), and coculture of DCs with
uninfected fibroblasts (Fig. 4e) were relatively inefficient in
upregulating CCR7. This experiment indicates that although
directly infected DCs do not express receptors for lymphoid
chemokines, DCs which encountered infected HFFs express
lymphoid chemokine receptors and are thus likely to be able to
migrate into the lymph nodes. Further work is in progress to
identify whether migrated bystander DCs are involved and are
efficient in HCMV Ag cross-presentation for CD8?T cells.
TNFR-p75 expression is not impaired following HCMV in-
fection of DCs. TNF-? is necessary for optimal DC migration
in vivo (11, 38), and CCR7 mRNA is induced upon treating
DCs with TNF-? in vitro (13, 32, 38). Thus, to reveal why
HCMV-infected DCs were unable to migrate toward lymphoid
chemokines, we analyzed the ability of HCMV-infected DCs to
produce TNF-? and to express TNFRs. We showed earlier that
HCMV-infected DCs produce low but significant levels of
TNF-? upon infection and upon further stimulation with LPS
(25). Furthermore, in the experiments described here, the ex-
ogenous concentration of TNF-? was identical for both Ag-
positive and Ag-negative DCs as they were in the same culture
wells. Thus, lack of TNF-? is unlikely to be responsible for the
selective inhibition of the migration of HCMV-infected DCs.
Next we studied the expression of TNFRs on DCs following
HCMV infection (Fig. 5). TNFR-p75 and TNFR-p55 were
expressed at medium levels on both mock-infected (Fig. 5a)
and HCMV-infected (Fig. 5b) DCs. Lower levels of both TNF
receptors, but especially that of TNFR-p55, were observed on
DCs following stimulation with LPS/TNF-?/IFN-?, compared
to untreated DCs (Fig. 5c versus a). This downregulation was
FIG. 3. CCR5 and CCR7 expression following HCMV infection of DCs. DCs were either mock infected (a and c) or infected with HCMV for
24 h (b and d). DCs then were either left untreated (a and b) (immature DCs) or treated with LPS/TNF-?/IFN-? for a further 24 h (c and d)
(mature DCs). Surface expression of CCR5 or CCR7 was tested by flow cytometry. The broken lines represent the binding of an irrelevant
antibody, while the continuous lines represent the binding of CCR5 or CCR7 antibodies, respectively. The numbers represent the percentage of
chemokine receptor-expressing cells above background (irrelevant antibody). The results are representative of three experiments. (e and f) CCR5
and CCR7 expression in HCMV-infected cultures on HCMV Ag-positive DCs (black histograms), compared to Ag-negative DCs (gray lines).
Two-color flow cytometric analysis of cell surface expression of chemokine receptors and intracellular detection of HCMV pp52 Ag are shown.
(e) Expression of CCR5 following HCMV infection of immature DCs. (f) Upregulation of CCR7 by LPS/TNF-?/IFN-? on TB40/E-infected DCs.
3050 MOUTAFTSI ET AL.J. VIROL.
also observed when DCs were infected with HCMV before
stimulation (Fig. 5d versus a). Two-color flow cytometric anal-
ysis to detect TNF receptor expression on HCMV-infected
DCs confirmed that HCMV Ag-positive DCs expressed levels
of TNFR-p75 similar to those expressed by HCMV Ag-nega-
tive DCs, while expression of TNFR-p55 was slightly lower in
Ag-positive DCs (not shown). Thus, as the expression of
TNFR-p75 remained at an intermediate level on DCs follow-
ing HCMV infection, it is unlikely that viral inhibition of DC
migration involves altered TNFR expression.
Inhibition of NF-?B DNA binding in HCMV-infected DCs.
Our results so far indicate that, while TNFR-p75 expression is
not inhibited by HCMV infection, the induction of CCR7
expression is prevented. This suggests that the signals that
induce CCR7 may be inhibited. TNF-? has been shown to
cause an increase in CCR7 at the level of mRNA (13, 32, 38),
suggesting that it regulates CCR7 transcriptionally. TNF sig-
nals through a protein complex that is associated with the TNF
receptors. These proteins include TNFR-associated death do-
main and TNFR-associated factors. They in turn link to at least
three distinct nuclear signals, including NF-?B, AP1, and JNK.
Of these three transcription factors, the best characterized is
the transcription factor NF-?B, which has been implicated as
an important transcription factor for DC (35) mediating CCR7
upregulation in lymphoid cells (18). To characterize a mecha-
nism for the inhibition of CCR7, we investigated whether
HCMV infection affects NF-?B binding to cellular DNA. Day
5 DCs were mock infected or infected with HCMV for 24 h
and were either left untreated or treated with LPS/TNF-?/
IFN-? for a further 24 h. Nuclear extracts from these cells were
prepared, and NF-?B DNA binding was measured using an
electrophoretic mobility shift assay. In this assay32P-radiola-
beled DNA containing a specific NF-?B binding site was incu-
bated with DC nuclear extracts. The results in Fig. 6 show that
a substantial increase in NF-?B is observed upon treatment
with LPS/TNF-?/IFN-? (compare lanes 1 and 3). HCMV in-
fection alone did not increase the level of NF-?B binding
(compare lanes 2 and 1). However, an inhibition of NF-?B
binding is seen in nuclear extracts from cells infected with
HCMV and then treated with LPS/TNF-?/IFN-? compared to
that of cells receiving treatment alone (lane 4 versus 3). These
data agree with observations in the U373MG astrocytoma cell
line where NF-?B DNA binding was not induced by HCMV,
and the infection resulted in a general loss of NF-?B binding
by 96 h postinfection (data not shown). The findings indicate
that HCMV can prevent NF-?B–DNA interaction in DCs and
that this inhibition is likely to contribute to the inability of
infected DCs to express CCR7.
This study describes, for the first time, an important effect of
HCMV infection on DC function, namely, the inhibition of DC
migration. Characterization of the molecular mechanism be-
hind this viral effect reveals that the inhibition of DC migration
is due to an inability of infected cells to express CCR7, the
gene of which is regulated by NF-?B via TNFR signaling. We
found that the induction of NF-?B DNA binding is inhibited in
infected DC. The possible physiological consequences of this
inhibition are also discussed.
DC migration depends upon the downregulation of inflam-
matory cytokine receptors, such as CCR5, and upregulation of
lymphoid chemokine receptors, such as CCR7. HCMV very
efficiently triggers the downregulation of CCR5 but does not
induce the expression of CCR7 in infected DCs. Moreover,
even following stimulation with LPS/TNF-?/IFN-?, a normally
potent stimulus for CCR7 induction, infected DCs remain neg-
ative for CCR7.
DCs survey the periphery and deliver Ag to the draining
lymph nodes to generate an immune response. Their impor-
tance as key regulators of the immune system makes their
manipulation a good target for altering immune responsive-
ness. This report, our previous data (25) and work with murine
cytomegalovirus (1) demonstrate that infection of DC with
cytomegalovirus is an important element of the virus-host in-
teraction. Despite viral alteration of DC mobility and function,
frequencies of T cells specific for HCMV Ags are among the
highest in PBMC of healthy HCMV-seropositive subjects. This
FIG. 4. Coculture of HCMV-infected fibroblasts with DC upregu-
lates CCR7 expression. Immature DCs were either left untreated for
24 h (a), treated with LPS/TNF-?/IFN-? for a further 24 h (b), infected
with 5 MOI of HCMV AD169 (c) or TB40/E (d) for 24 h, or mixed at
a 1:1 ratio with HFFs which were either uninfected (e) or infected with
5 MOI of TB40/E for 24 h (f), and the cells were cultured for a further
24 h together. Fluorescence-activated cell sorter analysis was carried
out following three-color staining for surface class II and CCR7 and
intracellular HCMV pp52 Ag expression. DCs were gated upon for-
ward-scatter and FL3 (class II expression) parameters. The numbers
represent the MFI of CCR7 expression in HCMV pp52 Ag-negative
(left panels) or -positive (right panels) DCs. Coculture of DCs with
HCMV-infected fibroblasts (f) resulted in a marked upregulation of
CCR7 on uninfected DCs. The results are representative of two ex-
VOL. 78, 2004HCMV INFECTION INHIBITS DC MIGRATION 3051
suggests the presence of other mechanisms by which HCMV-
specific T-cell responses are generated. One possibility would
be that the effect of HCMV infection differs on different sub-
sets of DCs and that HCMV-mediated inhibition of migration
of monocyte-derived DCs (MDDCs) is not representative.
However, a recent study on CD34?progenitor cell-derived
Langerhans cells (16) revealed that in HCMV TB40/E (100
MOI)-infected DCs which have been matured with granulo-
downregulation of class I, class II, and costimulatory molecules
is similar to that observed in MDDCs. Furthermore, our pre-
liminary results for blood DCs, isolated from the peripheral
blood of healthy donors with the Miltenyi magnetic separation
kit, also indicate that in HCMV-infected blood DCs, class I
molecules become downregulated and CCR7 surface expres-
sion is not induced upon infection (M. Moutaftsi, unpublished
data). Another mechanism of efficient T-cell stimulation dur-
ing HCMV infection may be cross-presentation of HCMV Ags
by uninfected DCs as proposed earlier (2, 31) and observed for
other herpesvirus Ags (6). Thus, while infection of DCs makes
recognition of HCMV Ags by naive CD8?T cells on the
surface of infected DCs unlikely, due to spatial segregation of
DCs and T cells, the cross-presentation mechanism would
make CD8?-T-cell priming, via the alternative Ag presentation
pathway, possible. In this work we studied secondary CD8?-
T-cell responses induced by infected DCs and found that mi-
grated DCs did not stimulate HCMV-specific memory CD8?T
cells. This was likely to be due to a very low proportion of DCs
carrying endogenous viral Ags, which alone would be sufficient
to prevent optimal T-cell activation. But, prevention of DC
maturation (25) and production of inhibitory cytokines such as
viral IL-10 (21) should also be considered as factors seriously
hampering T-cell priming. We also found that HCMV pp52
negative bystander DCs, which migrated toward CCL19, were
very efficient stimulators of HCMV-specific CD4?T cells. This
observation suggests that effective presentation of viral Ags,
probably derived from the inoculum (23) in the absence of viral
replication, is undisturbed, which may explain the high in vivo
frequencies of HCMV Ag-specific CD4?T cells (29).
The viral inhibition of the induction of CCR7 expression by
DCs is the principal element in the impaired DC migration.
Following infection with HCMV, DCs produce significant lev-
els of TNF-? (25) and express unchanged levels of both TNFR-
p75 and -p55, indicating that inhibition of CCR7 induction is
FIG. 5. TNFR-p75 expression following HCMV infection is not impaired on DCs. DCs were either mock infected (a and c) or infected with
HCMV for 24 h (b and d). DCs were then either left untreated (a and b) (immature DCs) or were treated with LPS/TNF-?/IFN-? for a further
24 h (c and d) (mature DCs). Surface TNFR-p75 or TNFR-p55 expression was tested by flow cytometry. The broken lines represent the binding
of an irrelevant antibody while the continuous lines represent the binding of TNFR-p75 (first row) or TNFR-p55 (second row) antibodies. The
numbers represent the percentage of positive cells after deducting the percentage of positive cells in the control groups. The results are
representative of three experiments.
FIG. 6. NF-?B activity in HCMV-infected DCs. DCs were infected
with HCMV TB40/E (lanes 2 and 4) or were mock-infected (lanes 1
and 3) for 48 h. Lanes 1 and 2 represent immature DC while the cells
in lanes 3 and 4 were treated with LPS/TNF-?/IFN-? for the last 24 h
before the NF-?B assay, and thus they represent mature DC. Nuclear
extracts were analyzed for consensus binding sites of NF-?B by incu-
bation with32P-labeled oligonucleotides containing the binding site of
NF-?B. The results are representative of three experiments.
3052MOUTAFTSI ET AL.J. VIROL.
not dependent on these factors. LPS-induced CCR7 upregu-
lation is prevented in the presence of NF-?B inhibitors (7), and
the gene encoding CCR7 is described as a direct target of
NF-?B (18). Although HCMV infection leads to the activation
of NF-?B in fibroblasts (22, 37) and to its nuclear translocation
in monocytes (36), the effect may be cell type dependent, as in
HCMV-infected retinal pigment epithelial cells where NF-?B
activation was not observed (10). The effect of HCMV infec-
tion on NF-?B DNA binding in DCs has not been investigated
previously. Our findings show that HCMV fails to activate and
inhibits LPS/TNF-?/IFN-?-mediated induction of NF-?B
DNA binding in MDDCs. This effect also correlates with the
observed inhibitory effect of the virus on DC maturation. As
for the exact nature of viral product(s) responsible for the
inhibitory effect, although we know that the inhibition of DC
migration occurs as an immediate-early–early effect of HCMV
infection, further experiments are needed to reveal the fine
In summary, we have shown here a hitherto-undescribed
viral evasion mechanism, the prevention of DC migration by
HCMV. This effect of HCMV has the potential of avoiding or
delaying the activation of HCMV-specific primary T-cell re-
sponses by infected DCs in vivo. The interactions between
HCMV and DCs and the effects on the chemokine receptor
switch suggest a complex host-virus interplay. The findings also
emphasize the importance of alternative Ag presentation
mechanisms in the generation of HCMV-specific CD8?-T-cell
This work was supported by a University of Wales College of Med-
icine Ph.D. scholarship to M.M., by the Leukemia Research Fund
(P.B.), and by the Wellcome Trust (Z.T.).
We thank S. Man, M. Rowe, and A. Clayton for critical reading of
the manuscript and C. Sinzger for providing the TB40/E HCMV strain.
1. Andrews, D. M., C. E. Andoniou, F. Granucci, P. Ricciardi-Castagnoli, and
M. A. Degli-Esposti. 2001. Infection of dendritic cells by murine cytomega-
lovirus induces functional paralysis. Nat. Immunol. 2:1077–1084.
2. Arrode, G., C. Boccaccio, J. Lule, S. Allart, N. Moinard, J. P. Abastado, A.
Alam, and C. Davrinche. 2000. Incoming human cytomegalovirus pp65
(UL83) contained in apoptotic infected fibroblasts is cross-presented to
CD8?T cells by dendritic cells. J. Virol. 74:10018–10024.
3. Baekkevold, E. S., T. Yamanaka, R. T. Palframan, H. S. Carlsen, F. P.
Reinholt, U. H. von Andrian, P. Brandtzaeg, and G. Haraldsen. 2001. The
CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and
mediates T cell recruitment. J. Exp. Med. 193:1105–1112.
4. Baltathakis, I., O. Alcantara, and D. H. Boldt. 2001. Expression of different
NF-?B pathway genes in dendritic cells (DCs) or macrophages assessed by
gene expression profiling. J. Cell. Biochem. 83:281–290.
5. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of
immunity. Nature 392:245–252.
6. Blake, N., T. Haigh, G. Shaka’a, D. Croom-Carter, and A. Rickinson. 2000.
The importance of exogeneous antigen in priming the human CD8? T cell
response: lessons from the EBV nuclear antigen EBNA1. J. Immunol. 165:
7. Bouchon, A., C. Hernandez-Munain, M. Cella, and M. Colonna. 2001. A
DAP12-mediated pathway regulates expression of CC chemokine receptor 7
and maturation of human dendritic cells. J. Exp. Med. 194:1111–1122.
8. Brennan, P., and L. A. O’Neill. 1995. Effects of oxidants and antioxidants on
nuclear factor kappa B activation in three different cell lines: evidence
against a universal hypothesis involving oxygen radicals. Biochim. Biophys.
9. Caux, C., S. Ait-Yahia, K. Chemin, O. de Bouteiller, M. C. Dieu-Nosjean, B.
Homey, C. Massacrier, B. Vanbervliet, A. Zlotnik, and A. Vicari. 2000.
Dendritic cell biology and regulation of dendritic cell trafficking by chemo-
kines. Springer Semin. Immunopathol. 22:345–369.
10. Cinatl, J., S. Margraf, J. U. Vogel, M. Scholz, and H. W. Doerr. 2001. Human
cytomegalovirus circumvents NF-kappa B dependence in retinal pigment
epithelial cells. J. Immunol. 167:1900–1908.
11. Cumberbatch, M., and I. Kimber. 1995. Tumour necrosis factor-alpha is
required for accumulation of dendritic cells in draining lymph nodes and for
optimal contact sensitization. Immunology 84:31–35.
12. Cyster, J. G. 2000. Leukocyte migration: scent of the T zone. Curr. Biol.
13. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia,
F. Briere, A. Zlotnik, S. Lebecque, and C. Caux. 1998. Selective recruitment
of immature and mature dendritic cells by distinct chemokines expressed in
different anatomic sites. J. Exp. Med. 188:373–386.
14. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf,
and M. Lipp. 1999. CCR7 coordinates the primary immune response by
establishing functional microenvironments in secondary lymphoid organs.
15. Geissmann, F., M. C. Dieu-Nosjean, C. Dezutter, J. Valladeau, S. Kayal, M.
Leborgne, N. Brousse, S. Saeland, and J. Davoust. 2002. Accumulation of
immature Langerhans cells in human lymph nodes draining chronically in-
flamed skin. J. Exp. Med. 196:417–430.
16. Hertel, L., V. G. Lacaille, H. Strobl, E. D. Mellins, and E. S. Mocarski. 2003.
Susceptibility of immature and mature Langerhans cell-type dendritic cells to
infection and immunomodulation by human cytomegalovirus. J. Virol. 77:
17. Hirao, M., N. Onai, K. Hiroishi, S. C. Watkins, K. Matsushima, P. D.
Robbins, M. T. Lotze, and H. Tahara. 2000. CC chemokine receptor-7 on
dendritic cells is induced after interaction with apoptotic tumor cells: critical
role in migration from the tumor site to draining lymph nodes. Cancer Res.
18. Hopken, U. E., H. D. Foss, D. Meyer, M. Hinz, K. Leder, H. Stein, and M.
Lipp. 2002. Up-regulation of the chemokine receptor CCR7 in classical but
not in lymphocyte-predominant Hodgkin disease correlates with distinct
dissemination of neoplastic cells in lymphoid organs. Blood 99:1109–1116.
19. Jeannin, P., G. Magistrelli, N. Herbault, L. Goetsch, S. Godefroy, P. Char-
bonnier, A. Gonzalez, and Y. Delneste. 2003. Outer membrane protein A
renders dendritic cells and macrophages responsive to CCL21 and triggers
dendritic cell migration to secondary lymphoid organs. Eur. J. Immunol.
20. Kellermann, S. A., S. Hudak, E. R. Oldham, Y. J. Liu, and L. M. McEvoy.
1999. The CC chemokine receptor-7 ligands 6Ckine and macrophage inflam-
matory protein-3 beta are potent chemoattractants for in vitro- and in vivo-
derived dendritic cells. J. Immunol. 162:3859–3864.
21. Kotenko, S. V., S. Saccani, L. S. Izotova, O. V. Mirochnitchenko, and S.
Pestka. 2000. Human cytomegalovirus harbors its own unique IL-10 ho-
molog (cmvIL-10). Proc. Natl. Acad. Sci. USA 97:1695–1700.
22. Kowalik, T. F., B. Wing, J. S. Haskill, J. C. Azizkhan, A. S. Baldwin, Jr., and
E.-S. Huang. 1993. Multiple mechanisms are implicated in the regulation of
NF-?B activity during human cytomegalovirus infection. Proc. Natl. Acad.
Sci. USA 90:1107–1111.
23. Le-Roy, E., M. Baron, W. Faigle, D. Clement, D. M. Lewinsohn, D. N.
Streblow, J. A. Nelson, S. Amigorena, and J. L. Davignon. 2002. Infection of
APC by human cytomegalovirus controlled through recognition of endoge-
nous nuclear immediate early protein 1 by specific CD4?T lymphocytes.
J. Immunol. 169:1293–1301.
24. Lutz, M. B., and G. Schuler. 2002. Immature, semi-mature and fully mature
dendritic cells: which signals induce tolerance or immunity? Trends Immu-
25. Moutaftsi, M., A. M. Mehl, L. K. Borysiewicz, and Z. Tabi. 2002. Human
cytomegalovirus inhibits maturation and impairs function of monocyte-de-
rived dendritic cells. Blood 99:2913–2921.
26. Mueller, S. N., C. M. Jones, C. M. Smith, W. R. Heath, and F. R. Carbone.
2002. Rapid cytotoxic T lymphocyte activation occurs in the draining lymph
nodes after cutaneous herpes simplex virus infection as a result of early
antigen presentation and not the presence of virus. J. Exp. Med. 195:651–
27. Riegler, S., H. Hebart, H. Einsele, P. Brossart, G. Jahn, and C. Sinzger.
2000. Monocyte-derived dendritic cells are permissive to the complete rep-
licative cycle of human cytomegalovirus. J. Gen. Virol. 81:393–399.
28. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble
antigen by cultured human dendritic cells is maintained by granulocyte/
macrophage colony-stimulating factor plus interleukin 4 and downregulated
by tumor necrosis factor alpha. J. Exp. Med. 179:1109–1118.
29. Sester, M., U. Sester, B. Gartner, B. Kubuschok, M. Girndt, A. Meyerhans,
and H. Kohler. 2002. Sustained high frequencies of specific CD4 T cells
restricted to a single persistent virus. J. Virol. 76:3748–3755.
30. Sozzani, S., P. Allavena, G. D’Amico, W. Luini, G. Bianchi, M. Kataura, T.
Imai, O. Yoshie, R. Bonecchi, and A. Mantovani. 1998. Differential regula-
tion of chemokine receptors during dendritic cell maturation: a model for
their trafficking properties. J. Immunol. 161:1083–1086.
31. Tabi, Z., M. Moutaftsi, and L. K. Borysiewicz. 2001. Human cytomegalovirus
pp65- and immediate early 1 antigen-specific HLA class I-restricted cytotoxic
T cell responses induced by cross-presentation of viral antigens. J. Immunol.
VOL. 78, 2004HCMV INFECTION INHIBITS DC MIGRATION 3053
32. Vecchi, A., L. Massimiliano, S. Ramponi, W. Luini, S. Bernasconi, R. Bonec- Download full-text
chi, P. Allavena, M. Parmentier, A. Mantovani, and S. Sozzani. 1999. Dif-
ferential responsiveness to constitutive vs. inducible chemokines of imma-
ture and mature mouse dendritic cells. J. Leukoc. Biol. 66:489–494.
33. Wang, B., H. Fujisawa, L. Zhuang, S. Kondo, G. M. Shivji, C. S. Kim, T. W.
Mak, and D. N. Sauder. 1997. Depressed Langerhans cell migration and
reduced contact hypersensitivity response in mice lacking TNF receptor p75.
J. Immunol. 159:6148–6155.
34. Wang, B., S. Kondo, G. M. Shivji, H. Fujisawa, T. W. Mak, and D. N. Sauder.
1996. Tumour necrosis factor receptor II (p75) signalling is required for the
migration of Langerhans’ cells. Immunology 88:284–288.
35. Yoshimura, S., J. Bondeson, F. M. Brennan, B. M. Foxwell, and M. Feld-
mann. 2001. Role of NFkappaB in antigen presentation and development of
regulatory T cells elucidated by treatment of dendritic cells with the protea-
some inhibitor PSI. Eur. J. Immunol. 31:1883–1893.
36. Yurochko, A. D., and E. S. Huang. 1999. Hum. cytomegalovirus binding to
human monocytes induces immunoregulatory gene expression. J. Immunol.
37. Yurochko, A. D., T. F. Kowalik, S. M. Huong, and E. S. Huang. 1995. Human
cytomegalovirus upregulates NF-?B activity by transactivating the NF-?B
p105/p50 and p65 promoters. J. Virol. 69:5391–5400.
38. Zhang, W., Z. Chen, F. Li, H. Kamencic, B. Juurlink, J. R. Gordon, and J.
Xiang. 2003. Tumour necrosis factor-alpha (TNF-alpha) transgene-express-
ing dendritic cells (DCs) undergo augmented cellular maturation and induce
more robust T-cell activation and antitumour immunity than DCs generated
in recombinant TNF-alpha. Immunology 108:177–188.
3054 MOUTAFTSI ET AL.J. VIROL.