VOLUME 1 NUMBER 5 | SEPTEMBER 2008 |www.nature.com/mi
nature publishing group
See REVIEW page 372
Infection of the cervix by human papillomavirus (HPV) is a
common and usually clinically innocuous event. HPV encodes
discrete viral proteins that are recognized by the local immune
system, with the result that the vast majority of HPV infections
are asymptomatic, resolve spontaneously, and result in protective
immunity. In rare instances, however, the virus causes dysplastic
changes of the cervix known as cervical intraepithelial neopla-
sia (CIN), which is graded as CIN1, -2, or -3. CIN lesions are
clinically heterogeneous and can spontaneously regress, persist,
or in rare instances, progress to invasive cancer. Rates of CIN
regression vary with grade, occurring in approximately 50 – 90 %
of women with CIN1, 40 % with CIN2, and 30 % with CIN3. 1 – 3
Hence, the immune system can eradicate HPV even in the face
of established neoplasia, although the likelihood of clearance
declines as the lesions progress along the neoplastic spectrum.
The fact that more than 95 % of cervical cancers contain HPV
genomes 4 indicates that in certain cases the immune response
is unable to eradicate HPV.
The role of the inflammatory response in HPV infection and
disease is multifaceted. On the one hand, clearance of HPV
infection involves a cytotoxic immune response. For example,
spontaneous regression of HPV-induced genital warts is accom-
panied by infiltration of lesions with T cells and macrophages. 5
Treatment-induced regression using an immune-response modi-
fier, 5 % imiquimod, is accompanied by upregulation of inter-
feron (IFN)- ? , - ? , and - ? , as well as tumor necrosis factor- ? . 6
On the other hand, tumor-associated inflammation has been
implicated in promoting carcinogenesis, presumably through
secretion by inflammatory cells of growth-promoting cytokines,
chemokines, angiogenic factors, and proteases. 7 – 12 In addition
to playing a permissive role, the inflammatory milieu in tumors
can actively suppress specific antitumor responses. Polarized
macrophages in the tumor microenvironment contribute locally
to immunosuppression. 9 – 11 A subset of immature or semimature
myeloid dendritic cells (DCs) in the tumor microenvironment
can produce immunosuppressive cytokines such as interleukin
(IL)-10 and transforming growth factor (TGF)- ? . 13 – 15 Immature
DCs produce indoleamine 2,3-dioxygenase (IDO), an enzyme
that catabolizes tryptophan and thereby suppresses T cell-
dependent immune response. 16 – 18 In addition, arginine metabo-
lism by immature DCs contributes to T-cell inhibition by local
depletion of arginine, generation of nitric oxide and reactive
oxygen species, and other mechanisms. 19 Regulatory T cells pro-
duce T-cell anergy and permit cancer progression through local
production of cytokines such as IL-10 and TGF- ? . 20,21 T cells are
Evolving immunosuppressive microenvironment
during human cervical carcinogenesis
A Kobayashi 1 , V Weinberg 2 , T Darragh 3 and K Smith-McCune 1
Chronic infection with human papillomavirus (HPV) can result in cervical cancer. To understand how HPV escapes
immune eradication, we examined biophenotypes of immune cells in human normal cervix, cervical intraepithelial
neoplasia (CIN), and cancer. Expression and cellular localization of Forkhead box protein-3 (FOXP3), indolamine
2,3-dioxygenase (IDO), interleukin (IL)-10, and interferon (IFN)- ? were examined by immunofluorescence and
immunohistochemistry. Mean cell densities of stromal FOXP3 + cells, IDO + cells, IL-10 + cells, CD1a + cells, and
macrophages significantly increased from normal cervix to cancer, whereas densities of IFN- ? + and MMP-9 + cells
increased from normal cervix to CIN but decreased in cancer. Flow cytometry confirmed significant elevation of
cervical T cells expressing IFN- ? and transforming growth factor- ? in CIN compared with normal cervix. Upon activation, a
significantly increased proportion of cervical T cells expressed IFN- ? in CIN than normal. A unique subset of morphologically
immature stromal dendritic cells expressing IL-10 and IDO was more numerous in cancer than in normal cervix and CIN.
The potentially suppressive immune milieu in the cervix may be permissive of HPV-associated cervical carcinogenesis.
1 Department of Obstetrics, Gynecology, Reproductive Science, University of California at San Francisco , San Francisco , California , USA . 2 The UCSF Comprehensive
Cancer Center Biostatistics Core, University of California at San Francisco , San Francisco , California , USA . 3 Department of Pathology, University of California at
San Francisco , San Francisco , California , USA . Correspondence: K Smith-McCune ( firstname.lastname@example.org )
Received 4 December 2007; accepted 23 April 2008; published online 2 July 2008. doi: 10.1038/mi.2008.33
MucosalImmunology | VOLUME 1 NUMBER 5 | SEPTEMBER 2008
also inhibited by interaction of the PD-1 receptor with negative
costimulatory ligands such as PD-L2 (B7-DC) expressed by DCs
and macrophages and PD-L1 (B7-H1) expressed on multiple cell
types including antigen-presenting cells. 22,23 Therefore, there
are several mechanisms by which stromal inflammatory cells
contribute to tumor escape from immune eradication.
The cervix is an ideal organ for studying the evolution of the
stromal microenvironment during carcinogenesis, for several
reasons. CIN lesions and cancers are routinely excised for ther-
apy, thereby providing a unique human experimental system
for assessing the stromal milieu across the carcinogenic spec-
trum. CIN and cancer samples express high levels of the HPV
oncogene E7, 24 – 26 hence cervical cancer represents a dramatic
example of failure of the immune response to eliminate an onco-
genic pathogen. We have previously shown increased numbers of
cells expressing pro-inflammatory (IFN- ? ) as well as tolerogenic
(IL-10 and TGF- ? ) factors in CIN2 and / or -3 (high-grade CIN
(HGCIN)) compared with normal cervix, suggesting the presence
of a dynamic immune equilibrium in precancerous lesions. 27 In
this report, we have characterized the biochemical evolution of
the local immune microenvironment in HPV-negative normal
cervix, HGCIN, and invasive squamous cancer with regard to the
phenotypes of stromal DCs, macrophages, and T cells.
The mean age of women with samples in the normal cervix
group was 58 years (range 43 – 74), in the HGCIN group was 29
years (range 17 – 40), and in the cancer group was 43 years (range
26 – 70). There was a significant overall difference in mean age
among the three groups ( P < 0.0001). Women in the HGCIN
group were significantly younger than women with a normal
cervix ( P = 0.0001) or cancer ( P = 0.005). In addition, women
with cancer were significantly younger than those with a normal
cervix ( P = 0.002).
The results of HPV typing showed no HPV detected in any
of the eight samples of normal cervix. Several HPV types were
detected in HGCIN samples (4 out of 14 samples with HPV16;
2 with HPV31; 1 each with HPV18, -33, -45, -52, or -58; 1 with
an untyped HPV from a probe mix for types -2, -13, -34, -42,
-57, -62, -64, -67, -72, and -82; and 2 with undetectable HPV).
In the cancer samples, HPV16 was the predominant type found
(8 out of 11 samples with HPV16, 1 with HPV18, and 2 with
Stromal IFN- ? + cells in normal cervix, HGCIN, and cervical
IFN- ? production indicates activation of local cell-mediated
immunity, commonly seen in response to viral infection or
cancer. We have previously reported that IFN- ? was expressed
in CD4 + and CD8 + T cells and to a lesser extent in natu-
ral killer cells in the cervix, and that the density of IFN- ? + cells
in the stroma of HGCIN samples was significantly increased com-
pared with normal cervix. 27 In the current analysis, stroma associ-
ated with cervical cancer demonstrated a significant reduction in
mean IFN- ? + cell density compared with HGCIN ( Figure 1a ).
The mean age of women contributing samples of HGCIN was
significantly lower than that of women contributing samples of
normal cervix and cancer. Therefore, we explored whether age
and IFN- ? + cell density were correlated within each patient
subset. No significant correlations existed between age and den-
sities of IFN- ? + cells in any of the three groups ( P = 1.00 for
normal, P = 0.41 for CIN2 / 3, and P = 0.55 for cancer). Therefore,
age is unlikely to contribute to the higher density of IFN- ? +
cells observed in HGCIN compared with normal or malignant
DCs in normal cervix, HGCIN, and cancer
The significant reduction in IFN- ? cell density from HGCIN to
cervical cancer suggested possible inhibition of IFN- ? response
during carcinogenesis. As DCs play a key role in directing local
immune responses toward either an effector response or toler-
ance, we analyzed DC biophenotypes in human cervical carcino-
genesis. Stromal CD1a + DCs were morphologically immature
(lacking pseudopodia) and expressed DC-SIGN ( Figure 2a ).
They also expressed human lymphocyte antigen class II
(HLA-II) ( Figure 2b ), indicating antigen-presenting capability.
Fluorescence-activated cell sorting (FACS) analysis of cells
extracted from fresh cervical tissues indicated a higher percent-
age of CD1a + CD123 + cells expressing DC-SIGN in HGCIN
samples than in normal cervix ( Figure 1b and Supplementary
Figure 1 ), suggesting that immature DCs are increased in
HGCIN. The majority of cervical CD1a + cells expressed CD123
regardless of disease status ( Supplementary Figure 1 ).
Regarding cytokine production by cervical DCs, we have
previously shown that a subset of CD1a + cells in the stroma
of HGCIN lesions expressed IL-10. 27 Our data indicate that
immunosuppressive enzyme IDO was expressed by both stromal
CD1a + cells ( Figure 2b ) and IL-10 + cells (data not shown).
In normal cervix, stromal CD1a + IDO + cells were widely
distributed in the stroma not closely approximated to the epi-
thelium, a distribution pattern similar to CD1a + IL-10 + cells
previously reported. 27 In HGCIN and cervical cancer samples,
stromal IDO + cells also localized within lymphocytic infiltrates
adjacent to neoplastic epithelium (data not shown). The IDO
staining pattern within these cells was cytoplasmic and granular,
consistent with that of a secretory protein.
We quantified the densities of stromal CD1a + cells in
samples of normal cervix, HGCIN, and cervical cancer, and
compared the mean cell densities among the three groups
( Figure 1c ). There was a significant overall difference in CD1a +
cell densities among the three groups ( P = 0.0009) and a signifi-
cant increase in CD1a + cell density in cervical cancer samples
compared with normal cervix and HGCIN.
These stromal immature DCs expressing immunosuppressive
factors described above are distinct from CD1a + Langerhans
cells residing within the epithelium. CD1a + cells within the
tumor, but not within the stroma, colocalized with S100, a
marker for Langerhans cells (data not shown). The majority of
CD1a + S100 + DCs within cancer had extended pseudopodia
and did not express HLA-II, IDO, or DC-SIGN. The intensity
of CD1a staining by immunohistochemistry (IHC) was greater
VOLUME 1 NUMBER 5 | SEPTEMBER 2008 |www.nature.com/mi
on cells within tumors than in stroma, and this was confirmed
by measuring the mean fluorescence intensity of CD1a staining
within tumor versus stroma from one representative sample. The
mean fluorescence intensities of nine CD1a + cells within the
tumor (range 117 – 203, median = 165) were significantly higher
than the mean fluorescence intensities of 13 CD1a + cells in the
stroma (range 44 – 188, median = 84, P = 0.002).
Stromal IL-10 + cells and IDO + cells in normal cervix,
HGCIN, and cancer
We examined stromal alterations in immune-modulating factors
during carcinogenesis by quantifying the densities of IL-10 +
cells and IDO + cells in normal cervix, HGCIN, and cancer.
Among the three groups, there was a significant overall differ-
ence among the mean cell densities for both stromal IL-10 +
( P = 0.008) and IDO + ( P = 0.002) cells. Pair-wise comparisons
revealed significant increases in mean IL-10 + cell density in
cervical cancer samples compared with normal cervix sam-
ples and in mean IDO + cell density in cervical cancer samples
compared with normal cervix or HGCIN samples ( Figure 1d
and e ). Significant trends in increasing density of IDO + and
IL-10 + cells were observed in HGCIN lesions compared with
normal cervix (Cuzick trend test: P = 0.01 for each test).
Macrophages in normal cervix, HGCIN, and cancer
Using CD68 as a marker for macrophages, we quantified the
densities of stromal macrophages in samples of normal cervix,
HGCIN, and cervical cancer, and compared the mean cell density
Figure 1 Quantitative comparisons of stromal cell phenotypes in normal cervix, HGCIN, and cancer. ( a , c – f ) Comparisons of mean cell densities
(no. of cells per mm 2 ) (MCD) of immunohistochemically stained cells; MCD of each sample is represented by a dot, and the mean of MCDs for each
group is represented by a horizontal bar. In f , MCD of one cancer sample was 479 per mm 2 and is marked with a black star (not in scale). Statistically
significant differences in MCDs are indicated. For each assay, inclusion for analysis was based on availability of appropriate specimens. ( b ) FACS
analysis of CD1a + cells in the cervix in normal and HGCIN samples; y axis is the percentage of CD1a + cells that are CD123 + and DC-SIGN hi . Each
dot represents one sample, and the mean for each group is represented by a horizontal bar. No statistical comparisons were performed due to the
small sample size. HGCIN, high-grade CIN.
MucosalImmunology | VOLUME 1 NUMBER 5 | SEPTEMBER 2008
among the three groups ( Figure 2f ). There was a significant increase
in macrophage density in HGCIN and cancer compared with normal
cervix. Stromal macrophages in HGCIN and cancer expressed IDO
and matrix metallopeptidase (MMP)-9 ( Figure 2d and e ). Although
MMP-9 + cells were virtually absent in normal cervical stroma, there
was a significant increase in the mean cell density of MMP-9 + cells
in the stroma of HGCIN, and the density significantly decreased in
cancer compared with HGCIN ( Figure 2g ).
Figure 2 Phenotypic characterization of cervical stromal DCs, T cells, and macrophages, and quantitative comparisons of macrophages in normal
cervix, HGCIN, and cancer. ( a ) Fluorescent double staining of stromal DCs in cervical cancer; CD1a in green, DC-SIGN in red, and DAPI in blue.
Bar = 20 ? m. ( b ) Fluorescent triple staining of stromal DCs in cervical cancer; CD1a in green, HLA-II in red, and IDO in blue. Bar = 20 ? m.
( c ) Fluorescent double staining of stromal lymphocytes in cervical cancer: CD25 in red, FOXP3 in green, and DAPI in blue. Bar = 10 ? m.
( d and e ) Fluorescent double staining of stromal macrophages; CD68 in red, IDO ( d ) or MMP-9 ( e ) in green, DAPI in blue. Bar = 20 ? m. ( a – e ) Right
lower images show colocalization of fluorescent staining. ( f and g ) Comparisons of MCDs of immunohistochemically stained cells, macrophages
( f ), and MMP-9 + cells ( g ) in normal cervix, HGCIN, and cancer. MCD for each sample is represented by a dot, and the mean of MCDs for each
group is represented by a horizontal bar. Statistically significant differences in MCDs are indicated. Inclusion for analysis was based on availability of
appropriate specimens. DAPI, 4 ’ -6-Diamidino-2-phenylindole; DCs, dendritic cells; HGCIN, high-grade CIN; HLA-II, human lymphocyte antigen class II;
IDO, indolamine 2,3-dioxygenase; MMP-9, matrix metallopeptidase-9.
VOLUME 1 NUMBER 5 | SEPTEMBER 2008 |www.nature.com/mi
IDO expression by other cell types in HGCIN and cancer
In tumor-associated stroma, a portion of CD56 + CD3- cells
express IDO (data not shown), supporting a previous report that
natural killer cells express IDO. 28 Although T cells and B cells
have been reported to express IDO, 28 neither stromal T cells
(CD4 + , CD8 + , and CD3 + ) nor B cells (CD20 + ) colocalized
with IDO in the cervix (data not shown).
IDO staining within the tumor did not colocalize with CD8
or CD3, markers for tumor-infiltrating lymphocytes. However,
fluorescent double staining of IDO demonstrated colocalization
with cytokeratin 7, a marker for epithelial cancer, indicating that
the cancer cells themselves are also a source of IDO (data not
shown). The IDO staining pattern in cancer cells was different
from that in DCs, natural killer cells, and macrophages, in that
it was not confined to the cytoplasm but included nucleis of the
tumor cells, consistent with a previous report of IDO staining
within cervical cancer. 29
Putative regulatory T cells in normal cervix, HGCIN, and
We have previously reported the presence of CD4 + CD25 +
putative regulatory T cells in stroma associated with HGCIN
lesions. 27 We further characterized the phenotype of CD4 + T
cells in cervical cancer and HGCIN by examining expression
of Forkhead box protein-3 (FOXP3), a transcription factor, the
expression of which is generally restricted to regulatory T cells
and which is involved in T cell-dependent immune suppres-
sion. IHC showed that most FOXP3 + cells were distributed
in the stroma of cervical tissues within lymphocytic infiltrates
approximated to the neoplastic epithelium, and the vast majority
of FOXP3 + cells colocalized with CD3 + (data not shown) and
CD25 + cells ( Figure 2c ). FOXP3 staining localized to nuclei,
consistent with its function as a transcription factor. Some
FOXP3 + T cells were also observed within cervical cancer (data
not shown). It has been shown that some FOXP3 + T cells can
express IFN- ? in response to antigenic stimulation. 30 Triple-
color staining demonstrated that FOXP3 + CD25 + T cells in the
cervix did not express IFN- ? ( Supplementary Figure 2 ).
Comparing the densities of stromal FOXP3 + cells in
normal cervix, HGCIN, and cancer, there was a significant overall
difference ( P = 0.01), and pair-wise comparisons revealed a
significant elevation of FOXP3 + cell density in cervical cancer
samples compared with normal cervix or HGCIN ( Figure 1f ).
A significant trend in increasing density of FOXP3 + cells was
observed in HGCIN lesions compared with normal cervix
(Cuzick trend test: P = 0.01).
Intracellular cytokine flow cytometric analysis of cervical
To assess functional capability of T cells, we used flow cytom-
etry to measure cytokine production in T cells isolated from
fresh cervical tissues with or without activation by phorbol
12-myristate 13-acetate and ionomycin. The percentages
of viable cervical T cells expressing IFN- ? or TGF- ? were
significantly higher in HGCIN than in normal cervix ( Table 1 ;
Supplementary Figure 3 ). The percentages of IFN- ? + T cells
increased significantly upon activation in both normal cervix
and HGCIN ( P = 0.02 and 0.01, respectively). The increase in
percentages of TGF- ? + T cells upon activation was significant
in normal cervix ( P = 0.02) but not in HGCIN ( P = 0.14). Upon
activation, a significantly increased proportion of cervical
T cells expressed IFN- ? in HGCIN than normal.
When cervical and peripheral blood samples were compared
from the same patient, significantly higher proportions of
T cells in cervical tissue than in peripheral blood expressed
IFN- ? ( P = 0.01 for both normal and HGCIN) and TGF- ?
( P = 0.03 for both normal and HGCIN) (data not shown).
We examined cytokine expression in cervical T cells from
three samples containing histologically confirmed low-grade
CIN. In unactivated samples, percentages of cytokine-express-
ing viable T cells were low for both IFN- ? (2.3, 2.2, and 1.4 % )
and TGF- ? (4.5, 1.7, and 1.3 % ), resembling mean values seen in
T cells from normal cervix ( Table 1 ).
Evolution of DC and macrophage phenotypes during
The tumor microenvironment is known to contain immature
myeloid cells recruited to the primary tumor site. 31 In the pres-
ence of IL-10 and / or TGF- ? , DCs undergo incomplete differen-
tiation and thereby direct the differentiation of CD4 + T cells into
regulatory T cells. 15,32 – 34 The stroma of HGCIN lesions is known
to contain a novel subset of stromal DCs expressing immunosup-
pressive factors. 27,35 The data reported herein demonstrate that
stromal DCs in the cervix evolve during carcinogenesis, with
progressively increased numbers of cells expressing DC-SIGN
and the immunosuppressive factors IL-10 and IDO. This unique
subset of stromal CD1a + DCs is distinct from Langerhans cells
by virtue of being S100-negative, expressing HLA-II, and express-
ing lower levels of CD1a. In Figure 3 , we propose a model of
cervical carcinogenesis in which these novel immature stromal
DCs direct the differentiation of regulatory T cells and thereby
determine the polarity of the local immune response.
Our findings that the number of stromal macrophages is
increased in HGCIN and cancer confirm those of others, 36 – 39
Table 1 Comparison of percentages of viable CD3+ cells
expressing intracellular cytokines in normal cervix and
Cytokine Normal %
( ± s.d.) N =8
( ± s.d.) N =9
2.3 ( ± 1.5) 8.6 ( ± 9.5) 0.03
4.0 ( ± 2.9) 10.2 ( ± 8.0) 0.03
Abbreviations: IFN- ? , interferon- ? ; HGCIN, high-grade CIN; PMA, phorbol
12-myristate 13-acetate; TGF- ? , transforming growth factor- ? .
a Treated with PMA and ionomycin as described in Methods.
16.0 ( ± 12) 30.4 ( ± 12.7) 0.02
5.9 ( ± 3.5) 12.3 ( ± 7.9) (0.08)
MucosalImmunology | VOLUME 1 NUMBER 5 | SEPTEMBER 2008
but we further demonstrate that they have a polarized or
alternatively activated phenotype, by virtue of expression of IDO
and MMP-9. Their phenotype evolves across the carcinogenic
spectrum, given the increase in IDO + but dramatic reduction
in MMP-9 + cells in cancer compared with HGCIN lesions. In
a murine model of cervical carcinogenesis, MMP-9 produced
by infiltrating macrophages has been implicated in angiogen-
esis and tumor development; genetic or pharmacologic abla-
tion of MMP-9 resulted in diminished bioavailability of vascular
endothelial growth factor, reduced microvessel count, dimin-
ished progression of CIN lesions to invasive cancer, and smaller
tumor volume. 40 In analogy with data from the transgenic mouse
model, increased numbers of MMP-9 + macrophages in human
HGCIN may facilitate the pronounced angiogenesis associated
with these lesions. 41
Evolution of T-cell phenotypes during cervical
Previous studies have demonstrated that patients with cervi-
cal cancer exhibit systemic immune tolerance to HPV antigens.
For example, vaccination of cervical cancer patients with HPV
antigens failed to generate a cytotoxic T-cell response. 42 The
findings that T cells in HGCIN lesions expressed increased levels
of regulatory cytokines 35,43,44 and that FOXP3 + T-cell density
increased significantly across the disease spectrum to cancer 29
support a model in which there is a progressive evolution of
T-cell phenotypes along the disease spectrum. These data
indicate that mechanisms for immune tolerance occur before
invasive cancer develops, consistent with the model in Figure 3 .
The data presented herein demonstrate for the first time that
IDO + cells, FOXP3 + T cells, and TGF- ? + T cells increased
across the disease spectrum in parallel with a sharp decline
in IFN- ? + cells in invasive cancer, suggesting that immu-
nosuppressive mechanisms may dominate in cervical can-
cer. Differences between peripheral blood and cervix in the
proportions of T cells expressing both IFN- ? and TGF- ?
reported herein underscore the importance of studying these
mechanisms at the mucosal site itself. In a cross-sectional
study of invasive cervical cancer samples, increased presence of
FOXP3 + cells in lymph nodes was associated with lymph node
metastasis, 29 indicating that immune suppression may extend
to sites beyond the tumor itself.
These data support a model of immune equilibrium proposed
by others to explain immunologic failure in the emergence of
cancer. 45,46 The finding that low-grade CIN lesions have low
numbers of cells expressing FOXP3, 29 IDO, 29 and TGF- ? (data
herein) indicates that immunosuppressive mechanisms emerge
in HGCIN lesions. We propose that HGCIN lesions are poised
at the tipping point between immune eradication and immune
escape, as shown in Figure 3, and that the ultimate clinical out-
come of HPV-associated disease may depend upon dynamic
elements within the stromal microenvironment. Given the
cross-sectional nature of our study design, we were unable to
test this hypothesis by making correlations with clinical out-
comes. In a recent prospective study, however, clearance of
HPV infection and low-grade CIN lesions was positively asso-
ciated with the presence of IFN- ? in the cervix, 47 supporting
our model. Appreciation of factors influencing the equilibrium
between immune eradication and escape may lead to therapeu-
tics to block immune tolerance and hence accelerate clearance
of HGCIN lesions.
Clinical specimens . All specimens of normal cervix, HGCIN, and
cervical cancer were obtained as paraffin-embedded blocks from the
archives of the Department of Pathology at University of California,
San Francisco (UCSF). Normal cervical specimens were obtained
from hysterectomies for benign uterine disease with no cervical abnor-
malities ( N = 8). HGCIN samples were obtained from loop and cone
biopsies performed for standard indications ( N = 14). Cervical cancer
samples were obtained from women undergoing radical hysterectomy
for invasive squamous cancer of the cervix ( N = 11). The histological
diagnosis of CIN2 or -3 or squamous cervical cancer was confirmed by
T. Darragh. For each assay, inclusion for analysis was based on avail-
ability of appropriate specimens.
HPV genotyping . All samples were tested for HPV genotyping using
the same paraffin-embedded tissue blocks used in IHC. HPV genotyp-
ing was performed on DNA extracted from a 20- ? m section by PCR
using MY09 / MY11 primer pairs followed by dot blot hybridization in
the laboratory of Joel Palefsky. 48 Sample adequacy was tested by PCR
amplification using specific primers for human ? -globin.
Immunohistochemistry and immunofluorescence . Immuno-
histochemistry was performed with primary anti-human antibod-
ies against myeloid-derived DCs with anti-CD1a mouse monoclonal
antibody (clone 010; DAKO, Carpenteria, CA); against IDO with
rabbit polyclonal (generous gift from J.M. McCune at UCSF); against
IFN- ? and IL-10 (goat polyclonal; R & D Systems, Minneapolis, MN);
Figure 3 A model for the central role of DCs in immune editing in HPV-
induced cervical disease and the location of HGCIN lesions at the
equilibrium between immune escale and immune eradication. Dashed
lines are based on our hypotheses. See Discussion for details. DCs,
dendritic cells; HGCIN, high-grade CIN; HPV, human papillomavirus.
VOLUME 1 NUMBER 5 | SEPTEMBER 2008 |www.nature.com/mi
against CD68 (mouse clone KP1; DAKO); against MMP-9 (rab-
bit polyclonal; Chemicon International, Temecula, CA); and against
FOXP3 (rabbit polyclonal; Abcam, Cambridge, MA). Each primary
antibody was used at the following dilutions: 1:100 (CD1a), 1:1500
(IDO), 1:25 (IFN- ? ), 1:20 (IL-10), 1:500 (CD68), 1:750 (MMP-9),
1:1500 (FOXP3, lot 61809), and 1:100 (FOXP3, lot 89864). Antigen
retrieval for IDO, IL-10, and FOXP3 was performed by digestion with
0.025 % trypsin for 10 min at 37 ° C, boiling the slides in a 1.25 kW
microwave for 2 min in 10 m M Sorensen ’ s citric buffer (pH 6.5)
and cooling in citric buffer for 30 min. Antigen retrieval for CD1a,
MMP-9, and CD25 was performed by boiling in citric buffer as described
above. For IFN- ? and CD68, antigen retrieval was carried out by
leaving slides in steamed 1 m M EDTA (pH 8) for 15 min. Before the
inactivation of endogenous peroxidase with 10 % hydrogen peroxide,
slides for IFN- ? staining were blocked for endogenous biotin according
to the manufacturer ’ s guidelines (DAKO). IHC for all other anti-
bodies was carried out as previously described. 27 For IFN- ? , slides
were incubated at 1:400 dilution of biotinylated anti-goat secondary
antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room
temperature (RT). Rabbit and goat IgG (Jackson ImmunoResearch)
and mouse IgG1 and IgG2a (DAKO) were used as negative isotype
Double- and triple-staining immunofluorescence (IF) was performed
to characterize CD1a + cells and IDO- or FOXP3-producing cells.
Routine IF was performed as described for IHC. EDTA was used for
antigen retrieval for HLA-II antibody. Double-staining IF for characteri-
zation of IDO- or FOXP3-producing cells was performed as described
below. Primary antibodies against cytokeratin 7 (CK7, mouse IgG1,
clone OV-TL 12 / 30; DAKO, at 1:500), IL-10, CD68 (mouse IgG1, clone
KP1; DAKO, at 1:100), CD56 (mouse IgG1, clone 123C3.D5; ID Labs,
London, ON, Canada, at 1:10), CD4 (mouse IgG1, clone OPD4; DAKO,
at 1:100), CD8 (mouse IgG1, clone C8 / 144B; DAKO, at 1:400), CD20
(mouse IgG2a, clone L26; DAKO, at 1:300), CD3 (mouse IgG1, clone
F7.2.38; Gene Tex, San Antonio, TX, at 1:10), or CD25 (mouse IgG1,
clone IL2R.1; ID Labs, at 1:100) were combined with IDO or FOXP3
polyclonal rabbit antibody. For double staining of CD1a with DC-SIGN
(mouse IgG2b, clone120507; R & D Systems, at 1:20) and CD56 with CD3
(rabbit IgG; DAKO, at 1:100), trypsin digestion and EDTA were used for
antigen retrieval. For double staining of MMP-9 (Chemicon, at 1:600)
with CD68, citric buffer was used. All antibodies were incubated on slides
overnight at 4 ° C and incubated with corresponding secondary antibod-
ies, Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 555 anti-mouse IgG,
Alexa Fluor 546 anti-mouse IgG2b, and Alexa Fluor 488 anti-mouse IgG1
(all from Molecular Probes, Eugene, OR) at 1:1500, 1:1000, 1:800, and
1:200 dilutions respectively, for 1 h at RT.
For triple staining, antibodies against HLA-II (monoclonal mouse
IgG2a, clone 1QU9; Antigenix America, Huntington Sta., NY) at 1:10
and IDO rabbit antibody were combined and incubated overnight at
4 ° C and incubated with corresponding secondary antibodies (Alexa
Fluor 546-conjugated anti-mouse IgG2a and Alexa Fluor 633-conjugated
anti-rabbit, both at 1:200) for 1.5 h at RT. Slides were then incubated
overnight at 4 ° C with anti-CD1a antibody, and the following day, slides
were incubated with secondary anti-mouse IgG1 antibody conjugated
with Alexa Fluor 488 (1:1000). For triple staining of interferon- ? with
FOXP3 and CD25, slides were incubated with goat anti-INF- ? overnight
at 4 ° C. Alexa Fluor 633 anti-goat secondary antibody at 1:200 was added
for 1 h at RT. Following incubation with non-serum protein block, slides
were incubated overnight at 4 ° C with rabbit anti-FOXP3 and mouse
anti-CD25 antibodies combined. Corresponding secondary antibodies,
Alexa Fluor 488 anti-rabbit at 1:500 and Alexa Fluor 555 anti-mouse at
1:200 were combined and incubated for 1 h at RT. IF detection, confocal
microscopy, and mean fluorescence intensity measurements were per-
formed as previously described. 27
Quantification of immune cell numbers . The densities of IHC-stained
cells in cervical tissues were determined using image analysis software
as previously published using Openlab 4.0.3 (Improvision, Lexington,
MA). 27 For quantification of FOXP3 + cells, cells were counted as positive
only if the staining was nuclear. Cell densities from 5 to 10 fields were
averaged for each patient.
The number of samples included in the analysis of each assay is indi-
cated in Table 1 unless otherwise stated in the text. These counts reflect
the number of available specimens, which in some cases are less than
the total subset stated above due to depletion of the sample reservoir
FACS analysis of fresh cervical tissues . A protocol for fresh tissue
collection was approved by the Committee on Human Research at UCSF.
Normal cervical tissues were collected from patients undergoing elec-
tive hysterectomies for indications unrelated to cervical dysplasia; the
final pathology report was checked to confirm that the cervix was histo-
logically normal. HGCIN cervical tissues were collected from patients
undergoing loop electrosurgical excision for treatment of CIN; samples
in which the final diagnosis did not show HGCIN were excluded from
the study. For CD1a + cell isolation, fresh tissues were aseptically minced
into small pieces ( < 1 mm 3 ) and were digested by incubation in colla-
genase type-2 (Worthington Biochemical, Lakewood, NJ) at 0.5 mg ml − 1
in Hanks buffer with D -glucose and antibiotics at 37 ° C for 30 min with
agitation. EDTA (1 m M, pH 7) was added for the last 5 min, and minced
tissues were disrupted by aspiration through a blunt needle. Cells were
collected by filtration through a 70 ? l filter, rinsed in phosphate-buffered
saline with 1 % bovine serum albumin, and incubated with an antibody
cocktail of CD1a PE (clone VIT6B; Caltag, Burlingame, CA) / DC-SIGN
FITC (clone DCN46; BD Bioscience, San Jose, CA) / CD123 APC (clone
AC145; Miltenyi Biotec, Auburn, CA) for FACS analysis.
For intracellular cytokine analysis, minced tissue pieces without diges-
tive enzymes were divided into two wells with 5 ml RPMI-1640 supple-
mented with 15 % Nu-Serum (BD Bioscience), penicillin (100 U ml − 1 ),
streptomycin (100 ? l ml − 1 ), 25 m M Hepes, and fungizone (2.5 ? l ml − 1 )
and incubated over night at 37 ° C. One sample was incubated in medium
containing Golgistop (BD) at 1:2500 dilution and the other with
10 ng ml − 1 phorbol 12-myristate 13-acetate and 300 ng ml − 1 ionomy-
cin in addition to Golgistop. The following morning, 1 m M EDTA was
added to each well, and tissues and medium were transferred into 50 ml
conical tubes through 70 ? l filter (Falcon / BD), centrifuged at 1000 r.p.m.
for 10 min, and washed in phosphate-buffered saline with 1 % bovine
serum albumin and 1 m M EDTA. Pelleted cells were incubated with
PerCP-conjugated anti-CD3 (clone SK7, BD), APC-anti-CD8 (clone
SK1, BD) and FITC-dead / live detection (Molecular Probes) on ice for
30 min in the dark and washed in phosphate-buffered saline with 1 %
bovine serum albumin and 1 m M EDTA. Intracellular cytokine stain-
ing with PE-anti-IFN- ? (clone 25723.11; BD) and PE-anti-TGF- ? (clone
TB21; IQProducts, DL Groningen, The Netherlands) was carried out
following the manufacturer ’ s guidelines (Cytofix / Cytoperm Plus Kit; BD
Permingen, San Diego, CA). For both CD1a and intracellular cytokine
analyses, stained cells were fixed in 4 % paraformaldehyde and stored at
4 ° C till flow cytometry (FACS Calibur; BD Immunocytometry Systems,
San Jose, CA). Cytometry data were analyzed with FlowJo software (Tree
Star, Ashland, OR).
Statistical analyses . For each antibody, the overall comparison among
the three patient groups of mean cell densities was performed using
analysis of variance methods recognizing the small sample sizes. When
the overall comparison was statistically significant, post hoc pair-wise
tests were performed using the Newman – Keuls method to determine
where differences occurred. The non-parametric Cuzick test for ordered
groups was performed to test for a trend in cell density with increasing
disease state. Due to the small number of patients in each subset, the data
were also analyzed using the non-parametric Kruskal – Wallis test (results
not shown). The comparison was defined as being significantly different
only when significance was confirmed. The non-parametric Spearman
rank correlation was calculated to evaluate the pair-wise association
between variables (e.g., age and IFN- ? ). The non-parametric Wilcoxon
test for matched pairs was used for the analysis of flow cytometry data
MucosalImmunology | VOLUME 1 NUMBER 5 | SEPTEMBER 2008
to compare the distributions of cervical T cells with or without activa-
tion for HGCIN and for normal cervix, and to compare the distribu-
tions of cervical T cells with peripheral blood T cells for HGCIN and for
normal cervix. To evaluate no activation and activation separately, the
Mann – Whitney test was used to compare the distributions of normal
cervical T cells and HGCIN T cells. Significance was defined as a prob-
ability value less than 0.05. No adjustment for multiple comparisons was
performed in these analyses. Probability values are stated in the text.
SUPPLEMENTARY MATERIAL is linked to the online version of the
paper at http://www.nature.com/mi
Supported by a Research Scholars grant from the American Cancer
Society (to K.S.M.) and NIH / NCI P30 CA 82103 Cancer Center support
grant to V.W. and to K.S.M. (PI Frank McCormick). We thank Jeff Mold and
M.J. McCune from UCSF School of Medicine for providing rabbit antibody
The authors declared no conflict of interest.
© 2008 Society for Mucosal Immunology
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