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Loss of New Chemokine CXCL14 in Tumor Tissue Is Associated
with Low Infiltration by Dendritic Cells (DC), while Restoration
of Human CXCL14 Expression in Tumor Cells Causes Attraction
of DC Both In Vitro and In Vivo
1
Galina V. Shurin,* Robert Ferris,
†§
Irina L. Tourkova,* Lori Perez,* Anna Lokshin,*
Levent Balkir,* Bobby Collins,
¶
Gurkamal S. Chatta,
‡
and Michael R. Shurin
2
*
†
Breast and kidney-expressed chemokine (BRAK) CXCL14 is a new CXC chemokine with unknown function and receptor selec-
tivity. The majority of head and neck squamous cell carcinoma (HNSCC) and some cervical squamous cell carcinoma do not
express CXCL14 mRNA, as opposed to constitutive expression by normal oral squamous epithelium. In this study, we demonstrate
that the loss of CXCL14 in HNSCC cells and at HNSCC primary tumor sites was correlated with low or no attraction of dendritic
cell (DC) in vitro, and decreased infiltration of HNSCC mass by DC at the tumor site in vivo. Next, we found that recombinant
human CXCL14 and CXCL14-positive HNSCC cell lines induced DC attraction in vitro, whereas CXCL14-negative HNSCC cells
did not chemoattract DC. Transduction of CXCL14-negative HNSCC cell lines with the human CXCL14 gene resulted in stim-
ulation of DC attraction in vitro and increased tumor infiltration by DC in vivo in chimeric animal models. Furthermore,
evaluating the biologic effect of CXCL14 on DC, we demonstrated that the addition of recombinant human CXCL14 to DC
cultures resulted in up-regulation of the expression of DC maturation markers, as well as enhanced proliferation of allogeneic T
cells in MLR. Activation of DC with recombinant human CXCL14 was accompanied by up-regulation of NF-
B activity. These
data suggest that CXCL14 is a potent chemoattractant and activator of DC and might be involved in DC homing in vivo. The
Journal of Immunology, 2005, 174: 5490 –5498.
The destructive disease head and neck squamous cell car-
cinoma (HNSCC)
3
annually afflicts 40,000 new persons
in the United States (1), and 3,000,000 new cases develop
worldwide annually (2, 3). Despite improvements in therapy and
diagnosis, the overall survival rate of generally 50% for persons
diagnosed with HNSCC has remained practically unchanged over
the last two decades (1). For this reason, new therapeutic strategies
need to be developed to treat HNSCC and the evaluation of alter-
native treatment strategies for patients with this malignancy is
highly justified. Immunotherapy has a long history, but is only
rarely considered as the treatment of choice. However, it seems
that increasing efficacy of immunotherapy will make it one of the
possible therapeutic options.
Specific active immunotherapy is based on the principle that
malignant cells contain immunogenic determinants against which
an antitumor immune response can be induced. Dendritic cells
(DC) that acquire Ags from tumor cells are able to induce and
regulate specific antitumor immunity. Several clinical trials have
been initiated to evaluate the efficacy of DC-based immunothera-
pies in cancer, including stimulation of endogenous DC (4 – 6).
However, it is still unclear why endogenous DC do not mediate
efficient antitumor immunity in cancer patients. Whereas success-
ful immunotherapy requires a functional immune system, a defect
in the immune response may contribute to tumor growth. Such
defects include active suppression of immune cells including DC
by the tumor causing disturbed longevity and cell dysfunction (7,
8). For instance, it has been shown that many tumor cell lines,
including melanoma and colon adenocarcinoma can effectively
chemoattract DC in vitro, modulate their phenotype, and eventu-
ally, severely damage DC mobility (9). From this point of view,
recent reports about loss of certain chemokines in several tumors,
including HNSCC, initially sound surprising (10 –12). However, it
is conceivable to hypothesize a new mechanism of tumor escape:
loss of certain chemokines by tumor cells results in a low attraction
of DC, decreased number of tumor-infiltrating DC and thus inhib-
ited ability of the immune cells to recognize tumor and initiate
specific antitumor immune responses. In fact, analysis of pheno-
type and distribution of immunocompetent cells in oral leukopla-
kia with different levels of dysplasia revealed that the levels of
immune effector cells varied according to the degree of dysplasia
(13). Examining distribution of S100
⫹
DC in the tumor tissues and
regional lymph nodes of 60 patients with HNSCC, Deng et al. (14)
reported that the S100
⫹
DC density in tumor tissues was correlated
with the tumor histologic grade, and the density of S100
⫹
DC was
significantly higher in regional lymph nodes without tumor than in
those with metastases. A similar conclusion was reported after
Departments of *Pathology,
†
Immunology,
‡
Medicine, and
§
Otolaryngology, Univer-
sity of Pittsburgh Medical Center, Pittsburgh, PA 15213; and
¶
Department of Oral
Medicine and Pathology, University of Pittsburgh School of Dental Medicine, Pitts-
burgh, PA 15261
Received for publication December 3, 2004. Accepted for publication February
8, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grant 2RO1
CA084270 (to M.R.S.) and University of Pittsburgh Cancer Institute Pilot Research
grant (to G.V.S.).
2
Address correspondence and reprint requests to Dr. Michael R. Shurin, Clinical
Immunopathology, Room 5725 Children’s Hospital of Pittsburgh-Medical Towers,
University of Pittsburgh Medical Center, 200 Lothrop Street, Pittsburgh, PA 15213.
E-mail address: shurinmr@upmc.edu
3
Abbreviations used in this paper: HNSCC, head and neck squamous cell carcinoma;
SCC, squamous cell carcinoma; DC, dendritic cell.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
evaluation of 36 cases of primary HNSCC of the lip mucosa or
vermillion border for the correlation between tumor-associated DC
density and tumor grade, mitotic rate, diameter, ulceration, depth
of invasion, muscle invasion, and metastasis (15). Goldman et al.
(16) have determined that survival and recurrence rates for patients
with squamous cell carcinoma (SCC) of the tongue correlate with
the degree of DC infiltration of the primary tumor or adjacent
tongue tissue. Patients who had greater numbers of CD1a
⫹
DC
adjacent to tumor had better survival and decreased recurrence
rates. These suggest that the distribution of DC subsets in HNSCC
may reflect the degree of tumor immunity induced in the host-
bearing HNSCC. Altogether, these suggest a functional role of DC
in the immune response to HNSCC. Localized absence of DC
might impair mucosal immunologic protection, allow microbial
colonization, and enhance carcinogenesis. However, the mecha-
nisms and chemokines responsible for DC homing and accumula-
tion in HNSCC are unknown.
The various members of chemokine are subdivided into four
families known as either the CXC, C-C, C, and CX3C, or the
␣
,

,
␥
, and
␦
subfamilies, respectively (17). Approximately 50 human
chemokines and 20 receptors are currently known. This large num-
ber reflects the highly complex traffic pattern of blood leukocytes,
including granulocytes, monocytes, lymphocytes, and DC. Accu-
mulating evidence indicates critical regulatory roles for chemo-
kines during the development of metastatic tumors by stimulating
angiogenesis and tumor growth. In addition, by regulating immu-
nity, chemokines critically regulate antitumor immune responses
and chronic inflammation such as that associated with various neo-
plasias (18 –20).
Breast and kidney-expressed chemokine (BRAK) CXCL14 is a
new CXC chemokine with unknown function and receptor selec-
tivity (11, 12, 21). CXCL14 transcripts are highest in human kid-
ney, small intestine, and liver tissues and expressed constitutively
by a variety of epithelia including the basal keratinocytes and der-
mal fibroblasts of skin (21). Importantly, Hromas et al. (12) re-
ported that CXCL14 mRNA was expressed ubiquitously in normal
tissues, but absent in a variety of in vitro established tumor cell
lines. Moreover, using differential display and in situ mRNA hy-
bridization, Frederick et al. (11) have recently reported that squa-
mous epithelium constitutively express CXCL14, whereas expres-
sion in tumors was heterogeneous, with the majority of HNSCC
and some cervical SCC showing loss of CXCL14 mRNA. This
study demonstrates for the first time up-regulation of CXCL14
mRNA in the inflammatory sites in the tumor microenvironment
and lost expression from certain cancers in vivo. The loss of ex-
pression in tumors and the presence of CXCL14 in nonmalignant
tissues suggest that this chemokine may play a role in host-tumor
interactions. It is also possible that down-regulation of the
CXCL14 gene expression in tumor cells might be beneficial for
tumor growth. However, the role of CXCL14 in the regulation of
migration of DC in cancer and their biologic significance has not
yet been investigated.
In the present work, we have established new in vitro and in
vivo models to address the chemotactic interaction among human
DC, CXCL14, and HNSCC tumor cells. We have demonstrated
that HNSCC tissues are low in tumor infiltrating DC although DC
are present in oral dysplasia lesions. Decreased infiltration of
HNSCC by DC was correlated with no or low expression of
CXCL14 protein at the tumor site. However, intense CXCL14
staining was observed in oral dysplasia (premalignant) lesions.
Furthermore, we showed that CXCL14 is a potent DC chemoat-
tractant in vitro and in vivo and DC are recruited to genetically
modified CXCL14-expressing HNSCC cells. In addition to being
potent DC chemoattractant, CXCL14 also increased functional ac-
tivity of DC, which was associated with increased activity of
NF-
B.
Materials and Methods
Tumor cell lines and tissues
Human SCC-15, prostate adenocarcinoma LNCaP, and melanoma FemX
cell lines were obtained from American Type Culture Collection. The
HNSCC PCI-13, PCI-16, and PCI-4B cell lines were prepared from
HNSCC tumors (22). Conditioned medium from normal human lymph
node cell suspensions served as a positive control. Tumor cells were main-
tained in RPMI 1640 medium supplemented with 10% heat-inactivated
FCS, 100 U/ml penicillin, 100
g/ml streptomycin, 0.2 mM L-glutamine,
1 mM sodium pyruvate, and 0.1 mM HEPES (Invitrogen Life
Technologies).
The immunohistochemical studies were performed on a variety of for-
malin-fixed and paraffin-embedded tissue sections, including normal oral
tissue (7 blocks), oral epithelial hyperplasia (8 blocks), and oral SCC (8
blocks).
Mice
Male C.B-17 SCID (T
⫺/⫺
,B
⫺/⫺
) mice, 6- to 8-wk-old were obtained from
Taconic Farms. Animals were maintained in pathogen-free facility under
the controlled temperature, humidity, and a 12-h light to dark cycle.
Immunohistochemistry
Monoclonal Abs recognizing CD83, CD1a (Immunotech), CD11c
(DAKO), and S-100 (Sigma-Aldrich) were used for the detection of DC
infiltration in formalin-fixed and paraffin-embedded tumor tissue sections.
Five-micrometer sections were cut, mounted on positively precharged
slides (Superfrost Plus; Fisher Scientific), and allowed to dry overnight at
56°C to ensure optimal adhesion. The sections were deparaffinized and
rehydrated. After endogenous peroxidase quenching (0.3% H
2
O
2
in PBS
for 30 min), Ags were retrieved by boiling the sections in 1 mM EDTA/
NaOH solution, pH 8.0, in a microwave oven for three cycles (5 min each).
Appropriately diluted mouse anti-human Abs against CD83 (1/50), CD1a
(nondiluted), CD11c (1/100), and S-100 protein (1/1000) were applied to
each section. Immunohistochemistry was performed using an avidin-biotin
peroxidase technique. Staining was developed with peroxidase and amino-
9-ethylcarbazole or diaminobenzidine (Vector Laboratories).
Expression CXCL14 protein in tissue sections and tumor cell lines was
determined with anti-CXCL14 mAbs (1/100, overnight incubation; R&D
Systems). Staining with normal murine IgG2a was performed as a negative
control for CXCL14 stain. Immunohistochemical and immunocytochemi-
cal staining was performed using avidin-biotin peroxidase technique
described.
Transduction of HNSCC cell lines with the CXCL14 encoding
vector
Frederick et al. (11) have demonstrated expression of CXCL14 in PBMC
stimulated with LPS. We isolated total RNA from human PBMC activated
with Escherichia coli LPS (0.5
g/ml; Sigma-Aldrich) for 6 h using the
TriReagent (Molecular Research Center) and according to the supplier’s
instructions. Up to 2
g of total RNA was reverse-transcribed in a final
reaction volume of 25
l containing 2.5
M oligonucleotides, 1⫻reaction
buffer (Invitrogen Life Technologies), 0.5 mM each of dNTP (Invitrogen
Life Technologies), 10 mM DTT, 1
l of RNase inhibitor (Boehringer
Mannheim), and 200 U of Superscript II reverse transcriptase (Invitrogen
Life Technologies). For PCR, 4
l of cDNA was amplified in a final vol-
ume of 30
l containing 1⫻Taq buffer, 50
M each dNTP, and 2.5 U of
Taq polymerase enzyme (Invitrogen Life Technologies). Primers for hu-
man CXCL14 were: 5⬘-CAG GTC GAC ATG AGG CTC CTG GCG GCC
GCG and 3⬘-CGG GGA TCC CTA TTC TTC GTA GAC CCT GCG. PCR
amplification was performed at 94°C for 10 min, followed by 35 cycles at
94°C for 1 min, 64°C for 1 min, 72°C for 1 min, and a final extension at
72°C for 10 min. PCR products were resolved by agarose electrophoresis
and stained with ethidium bromide. To construct a eukaryotic expression
vector, the CXCL14 gene was cloned into pCR3.1 plasmid. The PCR prod-
ucts were purified using QIAEX II gel extraction kit (Qiagen) and cloned
into pCR3.1 mammalian expression vector using the Eukaryotic TA Ex-
pression kit (Invitrogen Life Technologies) according to the manufactur-
er’s instructions.
Human HNSCC primary cell line PCI-16 was transfected with the human
CXCL14 gene using Effectene Transfection Reagent (Qiagen) according to the
supplier’s instructions. Briefly, tumor cells were counted and plated at 70%
density in 10-cm petri dish 1 day before the transfection. On the next day, the
5491The Journal of Immunology
medium was removed, the cells were washed in HBSS (Invitrogen Life Tech-
nologies), and transfection mixture containing 2
g of DNA was added to the
tumor cells. The cells were incubated with a transfection mixture at 37°C for
6 h. After incubation, fresh medium (RPMI 1640, 10% FCS) was added and
tumor cells were incubated at 37°C for additional 48 h. Next, transfected tumor
cells were split and fresh medium containing 1 mg/ml geneticin (Invitrogen
Life Technologies) was added for selection of transfected cells. Culture me-
dium with geneticin was changed twice a week for 2–3 mo. Expression of
CXCL14 protein was confirmed by Western blot (recombinant human
CXCL14 served as a positive control; RDI) and immunocytochemistry.
RT-PCR
Analysis of mRNA expression of human chemokines in normal oral mu-
cosa and HNSCC tissues was performed using RT-PCR technique. RNA
was extracted from five normal mucosae and five oral SCC specimens,
transcribed into cDNA using 200 U of superscript reverse transcriptase,
and cDNA was amplified with 2.5 U of Taq polymerase using 1.5 pM of
the primers specific for MIP-3
␣
, MIP-3

, CXCL8, and GAPDH. RT-PCR
was conducted as described earlier (23).
Generation of human monocyte-derived DC
CD14-derived DC were generated as described earlier (24). Briefly, PBMC
were isolated from buffy coats by Ficoll gradient centrifugation. The
PBMC were further plated at 10
7
cells/well in 2 ml of AIM-V medium
(Invitrogen Life Technologies) in six-well plates. After 1-h incubation at
37°C in a humidified 5% CO
2
atmosphere, nonadherent cells were removed
and adherent monocytes were gently washed with warm AIM-V medium.
Adherent monocytes were cultured with recombinant human GM-CSF
(1000 U/ml; PeproTech) and IL-4 (1000 U/ml; PeproTech) in complete
RPMI 1640 medium for 7 days. Maturation of DC was stimulated by ad-
ditional supplementation with 20 ng/ml TNF-
␣
(PeproTech) on day 6.
Analysis of DC migration and chemotaxis in vitro and in vivo
Spontaneous and chemokine-induced migration of DC in vitro was as-
sessed in the Transwell system with DC placed in the upper chamber (10
6
cells/ml, 100
l) and chemokines added to the bottom chamber (600
l) in
a 4-h migration assay. Cell migration was measured in 48-well Transwell
plates (5-
m pores; Corning Costar). Recombinant human MIP-1
␣
(10 –20
ng/ml; PeproTech), synthetic chemotactic peptide N-fMLP (0.5–5
g/ml;
Sigma-Aldrich) and recombinant human CXCL14 (5–200 ng/ml; RDI)
were diluted in RPMI 1640 medium contained 1% FBS (assay medium),
and 600-
l aliquots were placed in the lower chamber of Transwell plates.
Assay medium was used to measure a spontaneous migration of DC. After
4-h incubation at 37°C, the Transwell inserts were removed and cells from
the lower chamber were collected. Cells transmigrated through the 5-
m
pore size membrane were acquired on FACScan (BD Biosciences) for 60 s.
Data are reported as mean number of transmigrated cells from triplicate
wells.
To test whether human tumor cell lines produce chemokines that attract
DC, cell-free conditioned media were collected from FemX, LNCaP, and
different HNSCC cell lines. Tumor cells were seeded at 1 ⫻10
6
in4mlof
assay medium. Twenty-four hours later, cell-free supernatant was collected
and centrifuged. Conditioned medium from normal human lymph node cell
suspensions served as a positive control. Tumor-conditioned or control
media were placed in the lower chamber of the Transwell plate and mi-
gration of DC was assessed as previously described.
Trafficking of DC in vivo was assessed in immunodeficient SCID mice
(Taconic Farms) bearing human CXCL14-positive or CXCL14-negative
PCI-16 HNSCC. Mice were injected s.c. with 10
7
CXCL14-positive or
CXCL14-negative HNSCC cells and 2 ⫻10
6
human DC labeled with
fluorescent dye 5-sulfofluorescein diacetate/succinimidyl ester (SFDA/SE,
2.5
M; Molecular Probes) were injected i.v. 1 wk after tumor cells ad-
ministration. Tumors were harvested 48 h later and tissue sections were
analyzed by confocal microscopy and immunohistochemistry with
anti-CD1a Abs.
Flow cytometry
Expression of DC specific markers was determined as described earlier
(25) by flow cytometry on a FACSCalibur (BD Biosciences) using the
following Abs: CD14-FITC, HLA-DR-PE (BD Biosciences), CD1a-PE,
CD40-PE, CD80-PE, CD83-PE, (Immunotech/Coulter), and CD86-FITC
(BD Pharmingen). The analyses were done using the CellQuest software
(BD Biosciences).
MLR assay
MLR assays were performed to evaluate the effect of CXCL14 on the
ability of human DC to stimulate proliferation of allogeneic T cells. Con-
trol and CXCL14-treated (200 ng/ml) DC were added in triplicates in
graded doses (10
2
–10
6
cells per well) to T cells (1 ⫻10
5
per well) in
round-bottom 96-well plates. Proliferation of T cells was measured 72 h
later by incorporation of [
3
H]thymidine (1
Ci/well; DuPont-NEN) added
for the last 16 h. Cells were harvested onto GF/C glass fiber filter paper
(Whatman) and isotope incorporation was assessed by 1450 MicroBeta
TRILUX liquid scintillation counter (Wallac). The counts were expressed
as cpm ⫾SEM.
NF-
B activity assay
Monocyte-derived DC were treated with CXCL14 200 ng/ml for 0 –30
min. TNF-
␣
(50 ng/ml, 15 min) served as a well-known activator of NF-
B
in DC. Nuclear extract from Jurkat cells was used as an internal control.
The effect of CXCL14 on NF-
B activation in DC was determined using
a method developed by Active Motif. This method was developed in an
ELISA format and uses binding of the active form of NF-
B to immobi-
lized oligonucleotides corresponding to NF-
B nuclear consensus site 5⬘-
GGGACTTTCC-3⬘. The assay was performed according to the manufac-
turer’s specifications.
We additionally quantitated NF-
B in DC by an activity assay recently
developed by Marligen Biosciences using a Luminex technology. The as-
say is based on a specific binding of transcription factors to cognate se-
quences on labeled probes. Nuclear extracts were incubated with a mixture
of PE-conjugate oligonucleotides containing appropriate cognate DNA
binding sequences. This mixture was then incubated with a digestion re-
agent. In the presence of active transcription factors, label remains asso-
ciated with the probes, whereas it is removed in the absence of transcription
factor binding. Finally, the oligonucleotides were captured onto distinctly
colored agarose microspheres that allow each of the reactions to be indi-
vidually scored, and the quantitative signal generated by the label was
detected with a Bio-Plex (Bio-Rad) reader. The amount of label remaining
correlates with the amount of active transcription factor derived from the
nuclear extract. This format allows better sensitivity and dynamic range
than does EMSA. Furthermore, quantitative results allow comparisons
among treatments. The assays were performed according to manufacturer’s
protocol.
Statistical analysis
For a single comparison of two groups, the Student ttest was used after
evaluation for normality. If data distribution was not normal, a Mann-
Whitney rank sum test was performed. For the comparison of multiple
groups, one- or two-way ANOVA was applied. For all statistical analysis,
the level of significance was set at a probability of 0.05 to be considered
significant. All experiments were repeated at least two or three times. Data
are represented as the mean ⫾SEM.
Results
Immunohistochemical analysis of human HNSCC tissues for
infiltration by DC and expression of CXCL14
First, we confirmed and expanded the published data concerning
the reduction of DC numbers in the tumor mass when compared
with nonmalignant tissues (14, 15). We analyzed a variety of par-
affin specimens of HNSCC and oral dysplastic lesions for the pres-
ence of CD1a, S-100, CD83, and CD11c DC by immunohisto-
chemistry. The biopsy specimens were from different patients
diagnosed with mild to moderate dysplasia from oral mucosal
sites, including buccal mucosa, lateral tongue, and floor of the
mouth. For comparison, specimens were also obtained from pa-
tients diagnosed with invasive HNSCC from similar oral mucosal
sites. The results of the analysis of multiple oral epithelial hyper-
plasia, and oral SCC specimens suggest that CD83- and CD11c-
positive DC were essentially absent in HNSCC tissues, and the
numbers of CD1a- and S-100-positive DC were markedly lower in
the tumor tissues than in oral dysplasia lesions (Fig. 1A). These
data allowed us to hypothesize that DC migration into the HNSCC
tissues might be inhibited compared with their migration to the
hyperplastic or premalignant lesions. It is likely that chemokines
5492 REGULATION OF DC ATTRACTION BY CXCL14 CHEMOKINE
are, at least in part, responsible for differential homing of DC in
normal and malignant tissues.
To test whether decreased infiltration of HNSCC by DC might
be associated with a low expression of chemokines, we have mea-
sured expression of DC attracting chemokines in HNSCC and nor-
mal mucosa tissues by RT-PCR. Our data revealed similar mRNA
expression of MIP-3
␣
(CCL20) and MIP-3

(CCL19) in HNSCC
and normal mucosa (Fig. 2). These chemokines interact with
CCR6 and CCR7, expressed on immature and mature DC, respec-
tively. Given that expression of new chemokine CXCL14 mRNA
was reported to be lost in different tumors (11, 12), we also ex-
amined CXCL14 protein in different human tissues by immuno-
histochemistry. Because expression of CXCL14 in tissues has been
previously determined only by in situ hybridization (11, 12), we
have developed a new immunohistochemical procedure to analyze
CXCL14 protein in paraffin-embedded tissues. Fig. 1Bdemon-
strates that both normal oral mucosa tissues (n⫽7) and oral dys-
plasia specimens (n⫽8) were strongly positive for CXCL14,
whereas HNSCC tissues (n⫽8) were low or negative for
CXCL14 staining. Thus, these data suggest that CXCL14 protein
is lost in human HNSCC, which led us to the hypotheses that low
infiltration of HNSCC by DC might be associated with the lost
expression of certain chemokines (i.e., CXCL14) and whether
CXCL14 is chemoattractive for DC.
Chemoattractive properties of CXCL14 and tumor cell lines
toward DC
To determine whether DC could be attracted by a CXCL14, we
compared its chemotactic activity toward DC with the known DC
chemokines. Analysis of DC migration revealed that CXCL14 and
two control DC chemokines fMLP, a prototypic bacterial chemo-
tactic stimulus (26) and MIP-1
␣
(27), all dose-dependently che-
moattracted human DC (Fig. 3A). For example, in the presence of
20 ng/ml MIP-1
␣
migration of immature DC reached 6360 ⫾650
cells/min vs 3620 ⫾380 cells/min spontaneously transmigrated in
control wells ( p⬍0.05). A comparable chemoattraction of DC
(5960 ⫾568 cells/min, p⬍0.05) was also detected in the presence
of 200 ng/ml (20 nM) CXCL14 (Fig. 3A). Thus, CXCL14 is a
potent DC chemokine with a chemoattractive activity in the nano-
grams per milliliter range.
Next question was whether CXCL14 chemoattracts both imma-
ture and mature DC. Fig. 3Bdemonstrates that only immature, but
not mature DC, are chemoattracted by CXCL14. These data are in
agreement with Shellenberger et al. (28) and the general concept
that immature DC are attracted to nonlymphoid tissues where a
number of potent DC chemokines, including CXCL14, may be
ubiquitously expressed.
In the next set of experiments, we tested whether human tumor
cell lines, including different HNSCC, prostate adenocarcinoma,
and melanoma, might produce chemokines that could attract hu-
man DC in vitro. Cell-free conditioned media were collected from
FemX melanoma, LNCaP prostate adenocarcinoma, and HNSCC
cell lines SCC-15, PCI-13, PCI-16, PCI-38, and PCI-4B as de-
scribed in Materials and Methods. Conditioned medium from nor-
mal human lymph node cell suspensions served as a positive con-
trol. Fig. 3Cdemonstrates that conditioned media from FemX
melanoma cells (5620 ⫾483 vs 3630 ⫾250 cells in control, p⬍
0.005), normal lymph node cells (5880 ⫾602 cells, p⬍0.005),
and PCI-4B (6340 ⫾436 cells, p⬍0.005), but not from LNCaP
(3920 ⫾286 cells) and HNSCC lines PCI-13 (3720 ⫾405), PCI16
(3320 ⫾241), and SCC-15 (3390 ⫾301) ( p⬎0.1), displayed
chemoattractive activity toward human DC. Selective attraction of
DC by several tumor cell lines raises the question whether it might
correlate with the expression of CXCL14.
The next series of experiments focused on evaluating the ex-
pression of CXCL14 protein in different human tumor cell lines.
Tumor cells were cultured on microscopic slides for 48 –72 h, and
CXCL14 protein was detected by the immunocytochemical pro-
cedure. Human PBMC-derived monocytes stimulated with 0.5
g/ml LPS for 6 h served as a positive control for the expression
of CXCL14. Nonstimulated PBMC were used as a negative con-
trol. We found that all tested tumor cells, with the exception of
PCI4B and FemX, were CXCL14-negative (Fig. 3D). Thus,
HNSCC cell line SCC-15 and primary HNSCC cell lines PCI-13,
PCI-16, and PCI-38 as well as prostate adenocarcinoma cell line
LNCaP express no CXCL14 protein. Interestingly, CXCL14-neg-
ative HNSCC cell lines did not attract DC in a chemotaxis assay,
FIGURE 1. Immunohistochemical evaluation of tumor-infiltrating DC
(A) and CXCL14 expression (B) in HNSCC, oral dysplasia, and normal
oral tissue specimens. Five-micrometer sections were dried overnight, de-
waxed, rehydrated, followed by Ag retrieval with 1 mM EDTA/NAOH
solution (see Materials and Methods). A, For evaluating tumor-infiltrating
DC, the following DC-specific Abs were used: CD83, CD1a, CD11c, and
S-100. Secondary Abs were biotinylated with goat anti-mouse. B, For de-
tection of CXCL14 expression in HNSCC tissues anti-CXCL14 Abs or
control murine IgG2a were applied to the tissues overnight. Biotinylated
horse anti-mouse secondary Abs were added for 30 min. Staining was
developed with amino-9-ethylcarbazole and counterstained with hematox-
ylin. Positive staining is red-brown. The representative immunohistochem-
ical data from the analysis of 10 –12 specimens are shown.
FIGURE 2. Analysis of chemokine mRNA expression in normal oral
mucosa and HNSCC tissues by RT-PCR. mRNA was extracted from nor-
mal mucosa and HNSCC specimens, transcribed into cDNA using 200 U
of superscript reverse transcriptase, and cDNA was amplified with 2.5 U
Taq polymerase using 1.5 pM of the primers specific for MIP-3
␣
, MIP-3

,
CXCL8, and GAPDH, as described in Materials and Methods. The results
of a representative experiment are shown (n⫽3).
5493The Journal of Immunology
whereas CXCL14-expressing cell lines PCI-4B and FemX dem-
onstrated significant chemoattractive potential for DC in the same
assay (Fig. 3D).
Migration of DC toward CXCL14-transduced tumors in vitro
and in vivo
Our results indirectly support the hypothesis that DC are chemoat-
tracted toward CXCL14-producing cells and do not migrate to-
ward at least certain types of tumors that have lost expression of
this chemokine. To test this possibility in direct in vitro and in vivo
experiments, we have generated a vector encoding human
CXCL14, which was used for a stable transduction of human
CXCL14-negative HNSCC cell lines. The HNSCC cell line
PCI-16 was transfected with the human CXCL14 gene and, after
selection, expression of CXCL14 protein was confirmed by West-
ern blot (Fig. 4A). These data suggest that CXCL14-negative tu-
mor cells could be efficiently engineered to produce high levels of
CXCL14 protein. Functional activity of synthesized CXCL14 pro-
tein in tumor cells was next tested in in vitro and in vivo
experiments.
Next we demonstrated that human HNSCC tumor cells trans-
duced with the CXCL14 gene attract significantly higher levels of
human DC both in vitro and in vivo. First, cell-free supernatants
from CXCL14-negative wild type PCI-16 cultures and PCI-16
cells transduced with CXCL14 were collected and tested for the
attraction of human monocyte-derived DC in the chemotaxis as-
say. Fig. 4Bshows that wild type PCI-16 cells did not attract DC
(2100 ⫾77 vs 2500 ⫾105 cells transmigrated in control wells),
whereas CXCL14-expressing PCI-16 cells were chemoattractive
for DC (6400 ⫾135 transmigrated cells, p⬍0.05). Together with
the results demonstrating no attraction of DC toward control neo-
transduced tumor cells, this suggests that CXCL14-transduced tu-
mor cells release biologically active CXCL14 protein. Second, we
evaluated trafficking of human DC labeled with a fluorescent dye
in SCID mice in vivo. Fluorescent-labeled human DC were i.v.
injected in immunodeficient SCID mice (n⫽5) bearing both wild
type (or control neo-transduced) and CXCL14-transduced PCI-16
cells for 7 days. Two days later, tumors were harvested and fluo-
rescent cells were examined on 6-
m sections immediately by
confocal microscopy. The results revealed that infiltration of
CXCL14-expressing tumors by labeled DC was significantly
higher than in wild type or neo-transduced tumors in all tested
mice (Fig. 4C). Similar data were obtained when nonlabeled hu-
man DC were i.v. transferred in SCID mice (n⫽5) bearing PCI-
16/wild type (or neo-transduced) and PCI-16/CXCL14 and infil-
tration of tumors by injected DC was assessed 48 h later by
immunohistochemistry. Fig. 4Ddemonstrates that the levels of
accumulation of CD1a
⫹
human DC in CXCL14-expressing tu-
mors were markedly higher than the number of DC in control
tumors. Thus, these results suggest that the recovery of CXCL14
expression in HNSCC cells is associated with increased attraction
of DC both in vitro and in vivo.
Regulation of DC function by CXCL14
We next tested whether CXCL14, in addition to being a DC che-
moattractant, may also increase functional activity of DC. We first
examined whether CXCL14 alters phenotype characteristics of
DC. Fig. 5 shows that the addition of 200 ng/ml CXCL14 to DC
markedly up-regulated expression of CD83, HLA-DR, CD86, and
CD80 molecules when compare with CXCL14-untreated DC. For
example, the percentage of CD83
⫹
cells increased from 8.4 ⫾
0.9% in control DC cultures to 38.0 ⫾2.3% in DC cultures treated
with CXCL14 ( p⬍0.01). The same pattern was observed for the
expression of CD86 and CD80 molecules on DC (Fig. 5). Inter-
estingly, not only the percentage of DC expressing the specific
markers was up-regulated after addition of CXCL14, but also the
FIGURE 3. Analysis of migration of human DC toward different chemokines and tumor cell lines in vitro. DC were generated from CD14
⫹
monocytes,
and DC chemotaxis was assessed in the 5-
m pore size Transwell system in 4-h migration assay. The numbers of transmigrated DC were determined by
a 60 s FACScan analysis of triplicate samples. A, Comparative analysis of DC migration toward three chemokines revealed a chemoattractive potential of
CXCL14. The results of a representative experiment are shown as mean ⫾SEM. Three independent experiments have shown similar results. B, Immature
(GM-CSF ⫹IL-4, Day 6), but not mature (GM-CSF ⫹IL-4 ⫹TNF-
␣
, Day 8) DC migrate toward CXCL14. The representative results are shown as
mean ⫾SEM (n⫽4). ⴱ,p⬍0.05, one-way ANOVA and ttest. C, Differential chemoattraction of human DC to different human tumor cell lines. The
results from three independent experiments are shown and presented as the mean ⫾SEM. ⴱ,p⬍0.05, Student ttest. D, Immunohistochemical evaluation
of CXCL14 expression in tumor cell lines was done as described in Materials and Methods. Note the correlation between CXCL14 expression in tumor
cell lines (D) and their chemoattractive potential toward DC in a migration assay (C).
5494 REGULATION OF DC ATTRACTION BY CXCL14 CHEMOKINE
levels of CD83, CD86, CD80, and HLA-DR expression on DC
were significantly up-regulated (Fig. 5). For instance, the mean
fluorescence intensity values for CD83 and HLA-DR markers
were increased from 4.4 ⫾0.5 in control DC to 15.9 ⫾1.4 ( p⬍
0.01) on DC treated with CXCL14 and from 123.6 ⫾5.8 to
287.7 ⫾9.9 ( p⬍0.01), respectively (Fig. 5). Thus, it is conceiv-
able that CXCL14 stimulates maturation of DC.
Further confirmation of the biologic activity of CXCL14 was
obtained in the MLR assay using DC generated from different
donors with or without the addition of 200 ng/ml CXCL14 (Fig.
6A). Significantly higher induction of allogeneic T cell prolifera-
tion by CXCL14-treated DC ( p⬍0.01), as compared with un-
treated DC, was observed. For instance, at DC to T cell ratio 1:30,
uptake of [
3
H]thymidine increased from 17252 ⫾897 cpm in con-
trol to 30385 ⫾689 cpm ( p⬍0.01) in group treated with
CXCL14.
To explore the molecular mechanisms of CXCL14-mediated ac-
tivation of DC, monocyte-derived DC were treated with CXCL14
(200 ng/ml) and TNF-
␣
(50 ng/ml). The levels of p65 in nuclear
extracts were determined using NF-
B Transcription Factor Assay
kit. TNF-
␣
served as a well-known activator of NF-
BinDC.
Nuclear extract from Jurkat cells was used as internal control. We
demonstrated that activation of DC with CXCL14 was accompa-
nied by a significant up-regulation of NF-
B activity in DC up to
200% ( p⬍0.01) (Fig. 6B). Next, these data were confirmed and
further explored by using Luminex-based technique for analyzing
NF-
B activation (Fig. 6C). The results also demonstrated that
CXCL14 is a strong inducer of NF-
B activation in human DC.
Interestingly, the kinetic analysis of transcription factor activity
revealed that NF-
B activation induced in DC by CXCL14 was
delayed compared with TNF-
␣
-induced activation reaching the
maximum at 30 min (Fig. 6C).
In summary, these data suggest that CXCL14, in addition to
being DC attractant, also increases functional activity of DC.
Discussion
We have demonstrated that expression of a new DC chemokine,
CXCL14, is frequently lost in HNSCC tissues, which was accom-
panied by a low infiltration of the tumor by DC. We speculate that
low levels of HNSCC infiltration by DC may be due to a low or
absent expression of CXCL14 in tumor cells. It is well known that
homing of leukocytes to the sites of hemopoiesis, Ag priming,
immune surveillance, and inflammation largely depends on the
presence of chemokines (29). The presence of DC, macrophages,
and lymphocytes in solid tumors is regulated by local production
of chemokines by tumor and stromal cells. In particular, CC che-
mokines are the major determinants of macrophage and lympho-
cyte infiltration in carcinomas of the breast and cervix, sarcomas,
and gliomas (30). CCL2 (MCP-1) has been implicated in mediat-
ing macrophage infiltration into breast (19) and ovarian cancers
(31), whereas CCL5 levels correlate with the extent of CD8 T cell
infiltrate in ovarian tumors (20). It is conceivable to speculate that
immature DC might be constitutively recruited to CXCL14-ex-
pressing tissues. This would allow DC and monocytes to leave the
circulation and enter these tissues in the absence of inflammation.
On the contrary, the loss of CXCL14 expression in malignant tis-
sues may explain a decreased rate of DC attraction and thus aug-
ments efficacy of tumor escape mechanisms.
CXCL14 was initially named BRAK because it was identified in
human breast and kidney derived cells (12). CXCL14 (KS1, Kec,
BMAC, NJAC, MIP-2
␥
) is a chemokine with an as yet unknown
function and receptor selectivity (11, 12, 21). The mature se-
quences of CXCL14 and its murine analog SK1 contain 77 amino
acids and are unique with regard to the short N-terminal end of
only two amino acids (Ser-Lys), preceding the first of four che-
mokine-typical Cys residues. The most closely related chemo-
kines, MIP-2
␣
and MIP-2

, share ⬃30% amino acid identity with
CXCL14. Kurth et al. (32) have recently provided evidence that
FIGURE 4. Transduction of CXCL14-negative human HNSCC cell line PCI-16 with the CXCL14 gene resulted in expression of high levels of CXCL14
protein. Primary HNSCC cells PCI-16 were transduced with the human CXCL14 gene as described in Materials and Methods. Immunocytochemical
analysis of CXCL14-positive cells was assessed by Western Blot (A) as described in Materials and Methods. Recombinant human CXCL14 protein served
as a positive control. B, CXCL14-transduced HNSCC cells secrete functionally active protein, which exhibits significant chemoattractive potential toward
human DC in vitro (B) and in vivo (Cand D). PCI-16 cells were transduced with the human CXCL14 gene and after selection with G148 supernatants
obtained from wild type (wt) and CXCL14-transduced PCI-16 tumor cells were tested for their ability to attract DC in Transwell-based cell migration assay
(B). Medium and CXCL14 served as negative and positive controls, respectively. ⴱ,p⬍0.05 vs medium (one-way ANOVA, n⫽5). Cand D, PCI-16
wild type or PCI-16/CXCL14 tumor cells (10
7
cells per mouse) were injected s.c. in SCID mice on day 1. Sulfofluorescein diacetate/succinimidyl ester
(SFDA/SE)-labeled or nonlabeled human DC (2 ⫻10
6
cells) were injected i.v. on day 7 and all tumors were harvested 48 h later. Tissue sections were
analyzed by confocal microscopy (C) or immunohistochemistry with anti-CD1a Abs (red staining) (D) as described in Materials and Methods. The results
from a representative experiment (n⫽3) are shown.
5495The Journal of Immunology
CXCL14 is not a chemoattractant for peripheral blood T cells, B
cells, and NK cells or neutrophils and is selectively chemotactic
for monocytes activated by the cyclic AMP-elevating agents PGE
2
and forskolin. The authors proposed that once monocytes enter
tissues in response to local inflammation, PGE
2
at the site renders
them responsive to the high levels of CXCL14 in these tissues,
attracting them to the subepithelial locations where they mature
into macrophages. In contrast, others have reported that CXCL14
regulates trafficking of B cells (21), is a potent chemoattractant for
neutrophils, and weak or inactive for DC, monocytes, NK cells,
and T and B lymphocytes (33). Thus, the data on the biologic role
of CXCL14 for chemoattraction of immune cells are controversial.
Our results demonstrate that human recombinant CXCL14 and
CXCL14-transduced HNSCC cell line PCI-4B are potent inducers
of DC migration in vitro and in vivo, whereas CXCL14-negative
HNSCC cell lines and prostate adenocarcinoma cell line LNCaP
do not attract DC in a chemotaxis assay. Several laboratories dem-
onstrated that CXCL14 mRNA is constitutively expressed in nor-
mal tissues, but absent in a number of tumors (11, 12, 34). The
majority of HNSCC and some cervical SCC show loss of CXCL14
mRNA. Analysis of the expression of 20,000 genes in human pros-
tate epithelial cells passaged to senescence revealed the CXCL14
gene among three genes whose expression was uniformly lost in
human prostate cancer cell lines and xenografts (34). The loss of
expression in tumors and the presence of CXCL14 in nonmalig-
nant tissues suggest that this chemokine may play a role in host-
tumor interactions. It is also possible that down-regulation of the
CXCL14 gene expression in tumor cells might be beneficial for
tumor growth. In agreement, our new data revealed that the growth
of CXCL14-transduced murine HNSCC cell line B7E3/6 in syn-
geneic BALB/c mice was significantly inhibited in comparison
with wild type tumors, which was associated with high infiltration
by DC and CD8
⫹
T cells (G. V. Shurin, R. Ferris, I. L. Tourkova,
L. Perez, G. S. Chatta, and M. R. Shurin, manuscript in
preparation).
Importantly, a leukocyte and chemokine balance in tumors can
be manipulated. When murine tumors are engineered to overex-
press certain chemokines, the increased intratumoral infiltrate
stimulates antitumor responses. For instance, overexpression of
CCL19 (MIP-3

) mediated rejection of murine breast tumors in an
NK cell and CD4 T cell-dependent mechanism (35). CCL21
(6Ckine) reduced growth of colon adenocarcinoma in mice using
a similar pathway (36). Overproduction of CCL20 (MIP-3
␣
) might
activate tumor-specific CTLs by attracting DC (37), whereas over-
production of secondary lymphoid tissue chemokine by DC may
FIGURE 5. CXCL14 up-regulated DC maturation. Monocyte-derived
DC were coincubated with CXCL14 (200 ng/ml, 72 h) and surface expres-
sion of CD83, CD80, CD86, and HLA-DR was assessed by FACScan.
Both the percentage and the mean fluorescent intensity (%/MFI) are dem-
onstrated. The results of a representative experiment are shown (n⫽3).
FIGURE 6. CXCL14 stimulated APC function of human DC and up-
regulates activation of NF-
B. A, CXCL14 (200 ng/ml, daily day 3– 6)
significantly up-regulates Ag-presenting activity of human DC in vitro, as
was determined in an allogeneic MLR assay. Data are shown as mean ⫾
SEM. ⴱ,p⬍0.01, two-way ANOVA (n⫽3). B, Monocyte-derived DC
were treated with CXCL14 (200 ng/ml, 30 min) and p65 was assessed in
nuclear extracts as described in Material and Methods. TNF-
␣
(50 ng/ml,
15 min) served as well-known activator of NF-
B in DC. Nuclear extract
from Jurkat cells was used as an internal control. The levels of p65 in
nuclear extracts were determined using NF-
B Transcription Factor Assay
kit (Active Motif). Data are expressed as mean ⫾SEM from two inde-
pendent experiments. ⴱ,p⬍0.05 (ANOVA). C, NF-
B activity in human
DC was determined 0, 15, 30, and 60 min after stimulation with CXCL14
(200 ng/ml) or TNF-
␣
(50 ng/ml) using Luminex-based technique as de-
scribed in Materials and Methods. The results are shown as mean ⫾SEM
from two independent experiments. ⴱ,p⬍0.01 (ANOVA).
5496 REGULATION OF DC ATTRACTION BY CXCL14 CHEMOKINE
enhance T cell recruitment and immune priming to tumor-associ-
ated Ags (38). In fact, injection of recombinant secondary lym-
phoid tissue chemokine in the axillary lymph node region in mice
with bilateral multifocal pulmonary adenocarcinomas led to a
marked reduction in tumor burden with extensive lymphocytic and
DC infiltration of the tumors and enhanced survival (39). Together
with clinical evidence demonstrating that infiltration of tumor
mass by DC is associated with a better patient survival, these re-
sults suggest that regulated induction of DC migration into the
tumor site might induce efficient antitumor immune responses.
However, there are no data on whether CXC cytokines play a role
in attraction of immune cells to the tumor site and inducing anti-
tumor immunity. We have shown, that genetic modification of
CXCL14-negative PCI-16 HNSCC cell line with the CXCL14
gene results in stimulation of DC attraction in vitro and increased
infiltration of the tumor by DC in vivo. In fact, we have shown on
a murine HNSCC model that CXCL14-expressing tumors were
highly infiltrated by CD11c
⫹
DC suggesting their potential role in
developing antitumor immune response at the tumor site (G. V.
Shurin, R. Ferris, I. L. Tourkova, L. Perez, A. Lokshin, L. Balkir,
B. Collins, G. S. Chatta, and M. R. Shurin, manuscript in
preparation).
Next, we evaluated the effect of CXCL14 on DC function. It is
known that chemokines may regulate cellular adhesion, prolifera-
tion, and cell survival (10, 18, 40). Based on the current knowledge
of the life cycle of DC, it has been postulated that chemokines can
play an important role at several stages of DC development (18).
Basal chemokine production and expression at the surface of en-
dothelial cells can mediate DC precursor recruitment into periph-
eral tissues, which is important for the maintaining DC levels
within tissues. Once in the tissue, chemokines, such as MIP-1
␣
,
MIP-1

, MIP-3
␣
, MIP-5, MCP-3, MCP-4, RANTES, TECK, and
SDF-1 (41), may participate in differentiation of DC precursors
into immature DC that are programmed to pick up and process
Ag(s). Upon initiation of an inflammatory response, chemokines
that recruit immature DC may be up-regulated, resulting in DC
accumulation within the tissue. When DC have matured, they enter
tissue-draining lymphatic vessels and migrate to the T cell zones in
secondary lymphoid organs under the influence of chemokines
produced there, such as MIP-3

and 6Ckine. In the T cell zones,
DC can produce chemokines that stimulate DC-T cell interaction,
thereby enhancing the likelihood of clonal selection (18, 41). Our
data show that CXCL14 chemoattracts only immature, but not ma-
ture DC, which is in agreement with the concept that nonlymphoid
tissue chemokines should attract immature DC. Importantly, we
demonstrated that CXCL14 also activated DC through NF-
B-
mediated pathways and up-regulated expression of costimulatory
molecules on DC as well as enhanced the proliferation of alloge-
neic T cells in MLR. Thus our results support the hypothesis that
CXCL14 might be a novel DC chemokine regulating their homing
and activation in nonlymphoid tissues.
Disclosures
The authors have no financial conflict of interest.
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5498 REGULATION OF DC ATTRACTION BY CXCL14 CHEMOKINE
