functional attributes of mucosal immunity in cervical ... · vaccine indicate that protection...

[CANCER RESEARCH 64, 6766–6774, September 15, 2004]

Functional Attributes of Mucosal Immunity in Cervical Intraepithelial Neoplasiaand Effects of HIV Infection

Akiko Kobayashi,1,4 Ruth M. Greenblatt,2,5 Kathryn Anastos,6 Howard Minkoff,7 Leslie S. Massad,8 Mary Young,9

Alexandra M. Levine,10 Teresa M. Darragh,3 Vivian Weinberg,4 and Karen K. Smith-McCune1,4

1The Cancer Research Institute and Department of Obstetrics, Gynecology, and Reproductive Sciences, 2the Department of Medicine, 3the Department of Pathology, and 4theComprehensive Cancer Center at University of California at San Francisco, San Francisco, California; 5the Connie Wofsy Study Consortium of Northern California, University ofCalifornia, San Francisco; 6New York City/Bronx Consortium, Bronx-Lebanon Hospital/Montefiore Medical Center, Bronx, New York; 7Maimonides Medical Center, Brooklyn,New York; 8Southern Illinois University School of Medicine, Springfield, Illinois; 9Washington DC Metropolitan Consortium, Washington DC; 10University of Southern CaliforniaConsortium, Los Angeles, California

ABSTRACT

The role of mucosal immunity in human papillomavirus (HPV)-relatedcervical diseases is poorly understood. To characterize the local immunemicroenvironment in cervical intraepithelial neoplasia (CIN) 2/3 anddetermine the effects of HIV infection, we compared samples from threegroups: normal cervix, CIN 2/3 from immunocompetent women (HIV�

CIN 2/3), and CIN 2/3 from HIV seropositive women (HIV� CIN 2/3).CIN 2/3 lesions contained increased numbers of immune cells from boththe acquired and innate arms of the immune response in stroma [CD4�and CD8� T cells, macrophages, mast cells, B cells, neutrophils, andnatural killer (NK) cells] and dysplastic epithelium (CD4� T cells, macro-phages, and NK cells). Immune cells in CIN 2/3 expressed activationmarkers, as measured by interleukin-2 receptor (IL-2R) and transcriptionfactor T bet. Interferon-� production was significantly up-regulated inCIN lesions and was expressed by CD4� and CD8� T cells and NK cells,indicating the activation of immune cells. Abundant presence of trans-forming growth factor-�� CD25� cells in the infiltrates associated withCIN lesions, and of immature CD1a� dendritic cells expressing IL-10 andtransforming growth factor-�, indicate that CIN is associated with aninflux of immune cells that produce a mixture of proinflammatory andregulatory cytokines. In HIV� CIN, immune cell densities (CD4� T cells,macrophages, neutrophils, and NK cells) and expression of interferon-�were significantly decreased compared with HIV� CIN. Regulatory cyto-kines were also down-regulated in this group. Therefore, both pro- andanti-inflammatory responses present in CIN 2/3 lesions are suppressed inHIV-seropositive women.

INTRODUCTION

Cervical cancer is preceded by well-defined changes in the epithe-lium known as CIN, changes that are associated with genital infectionwith human papillomavirus (HPV). The majority of genital HPVinfections are clinically undetectable and do not result in CIN or

cancer. CIN 1 reflects active HPV replication and is rarely precan-cerous, most often resolving spontaneously. In contrast, CIN 2 and 3represent potential cancer precursors; �12% of CIN 3 lesions willprogress to cancer if left untreated (1, 2). More than 90% of CIN 2/3and cervical cancers are associated with a few high-risk HPV types,most commonly HPV 16 (3–5).

Host immune response to HPV appears to be critical in determiningthe outcome of infection, because HPV infection is detected morefrequently, the incidence of CIN is higher, and the risk of CINrecurrence after treatment is higher among immunocompromisedwomen (6–9). Recent and promising results with an HPV 16-specificvaccine indicate that protection against HPV infection is possible;vaccinated women mounted a systemic humoral response to HPV 16and were resistant to infection with this HPV type for at least 1 year(10). Cytotoxic immune responses are likely to be important in de-termining the sequelae of HPV infection once it is established.Women who clear HPV infection are more likely to have detectablelevels of interferon-� (IFN-�) in cervical cytobrush samples thanwomen whose infection persists (11). In addition, the correlationbetween presence of HPV protein E6-specific CTLs and the absenceof CIN in women with HPV16 infection again indicates that CTLresponses may be protective (12). Overall, however, the detection andmeasurement of potentially protective immune response to HPV has beendifficult, raising the possibility that HPV does not generate a strongimmune response and may in fact induce immunotolerance (13, 14).

The main purpose of this study was to investigate the functionalattributes of the local immune microenvironment in normal cervix, inCIN 2/3 lesions from immunocompetent women, and in CIN 2/3 fromHIV� women. The cytokine profiles in these cohorts indicated thatCIN lesions were characterized by both proinflammatory, potentiallycytotoxic responses as well as anti-inflammatory potentially immu-nosuppressive responses. We also found significant differences in thefunctional properties of the immune response in HIV� CIN 2/3, mostnotably failure to generate CTLs despite the abundance of CD8� Tcells. In addition, the production of regulatory cytokines [i.e., trans-forming growth factor (TGF)-� and IL-10] was reduced in HIV� CIN2/3. These findings have important ramifications for understanding ofthe nature of the immune response in women with CIN 2/3 and theeffects of vaccines and therapies that target local immune responses.

MATERIALS AND METHODS

Clinical Specimens. Specimens for the normal cervix group were obtainedas paraffin-embedded blocks from the Department of Pathology Archives atUniversity of California, San Francisco (UCSF) from women who underwenthysterectomies for benign uterine disease with no cervical abnormalities andwith no evidence of immuncompromise as stated on the pathology report(n � 21, mean age 51 range 39–81). Specimens and data for the immuno-competent (HIV�) CIN 2/3 group were obtained as paraffin-embedded sec-tions of cone and loop excisions from two sources: the UCSF Department ofPathology Archives and the Women’s Interagency HIV Study (WIHS; n � 19,mean age 33, range 19–84). The samples obtained from WIHS (n � 5) were

Received 4/5/04; revised 6/29/04; accepted 7/12/04.Grant support: Supported by grants from the UCSF AIDS Innovative Grants Program

and California AIDS Research Center (to K. Smith-McCune), the Terry Fox Fund (to K.Smith-McCune), the Integrated Training Program in Reproductive Endocrinology (T32HD07263 to A. Kobayashi), and the National Institute of Allergy and Infectious Diseases,with supplemental funding from the National Cancer Institute, the National Institute ofChild Health & Human Development, the National Institute on Drug Abuse, the NationalInstitute of Dental Research, the Agency for Health Care, Policy and Research, and theCenters for Disease Control and Prevention, U01-AI-35004, U01-AI-31834, U01-AI-34994, AI-34989, U01-HD-32632 (National Institute of Child Health and Human Devel-opment), U01-AI-34993, U01-AI-42590 (for WIHS). Additional funding from the UCSFCancer Center Support Grant CA82103 (to V. Weinberg), and the General ClinicalResearch Centers at UCSF and the San Francisco General Hospital through the NationalCenter for Research Resources Grants M01-RR-00079 and M01-RR-0083 (for the Gen-eral Clinical Research Centers) and National Institute of Allergy and Infectious DiseasesGrant UO1-AI-34989 (for WIHS). We thank Dr. L. H. Glimcher from Harvard School ofPublic Health, Boston, MA, for providing us the T bet clone.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Karen Smith-McCune, Cancer Research Institute and Depart-ment of Obstetrics, Gynecology and Reproductive Sciences, 2340 Sutter Street, RoomS331, UCSF, San Francisco, CA 94143-0128 (Zip for courier 94115). Phone: 415-476-7882; Fax: 415-502-3179. E-mail: [email protected].

©2004 American Association for Cancer Research.

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from women who were HIV seronegative whereas HIV serostatus was notavailable for most of the samples from the UCSF Department of PathologyArchives (n � 14). However, the women contributing samples to this groupwere clinically immunocompetent.

Specimens and data for the HIV� CIN 2/3 cohort in this study wereobtained in collaboration with WIHS, which is a longitudinal multi-site cohortstudy of women with or at risk for HIV infection (n � 28, mean age 32, range21–46). The mean CD4 count was 381 (range 1–1277). All samples obtainedfrom the WIHS cohort were selected from women who had HPV 16 detectedin cervicovaginal lavage specimens. WIHS sites included consortia based inBronx and Brooklyn New York, Chicago, Southern California, WashingtonDC, and the San Francisco Bay area. Detailed methods for this study werepublished previously (15). All study procedures and consent materials werereviewed and approved by human subject protection committees at each of thecollaborating institutions. Verification of HIV infections and HPV testing wasdescribed previously (15, 16). Loop and cone biopsies were performed forstandard indications, and tissues archived as paraffin blocks after pathologicalevaluation were accessed for this study. The histologic diagnosis of CIN 2/3was determined at the originating institution and confirmed after receipt atUCSF. Because of limited availability, we did not include a cohort of normalcervical samples from HIV� women. For each assay, sample selection wasnon-biased and was based on availability of appropriate specimens fromconsecutively accrued women.

Immunohistochemistry and Immunofluorescence. Immunohistochemis-try (IHC) was performed with primary antibodies against CD4� T cells withmonoclonal antibody to CD45R0 (clone OPD4), CD8� T cells with mono-clonal antibody to CD8 (clone C8/144B), B cells with CD20 (clone L26),tissue macrophages with CD68 (clone KP1), myeloid-derived dendritic cells(DCs) with CD1a (clone 010), and tissue mast cells with mast cell tryptase(clone AA1), purchased from DAKO (Carpenteria, CA). Monoclonal antibodyagainst CD15 (clone MY-1) for neutrophils, monoclonal antibody againstCD25 (clone IL-2R.1) for IL-2R-�, and monoclonal antibody to CD56 (clone123C3.D5) for natural killer (NK) cells were purchased from ID Labs (Lon-don, Ontario, Canada). Polyclonal anti-IFN-� antibody was purchased fromR&D Systems (Minneapolis, MN). Each primary antibody was diluted in PBSwith 1% BSA as follows: 1:500 (CD45R0), 1:200 (CD8), 1:1200 (CD20),1:500 (CD68), 1:100 (CD1a), 1:2000 (tryptase), 1:300 (CD15), 1:400 (CD25),1:25 (CD56), and 1:2400 (IFN-�). Routine IHC was performed after manu-facturer’s guidelines (Innogenex, San Ramon, CA). Before the inactivation ofendogenous peroxidase with 0.1% hydrogen peroxide, all tissue slides exceptfor IFN-�, CD25, and CD56 staining were digested with 0.025% trypsin for 10minutes at 37°C. IFN-� and CD56 antibodies were performed by boiling theslides in a 1.25 kW microwave for 2 minutes in 10 mmol/L Sorensen’s citricbuffer (pH 6) and cooling in citric buffer for 30 minutes. Antigen retrieval forprimary antibodies against IFN-� and CD56 was performed by boiling theslides in the same microwave for 10 minutes (5 minutes �2) in 1 mmol/LEDTA solution (pH 8), and then the slides were immediately transferred toPBS at room temperature (RT). Antibodies against CD4, CD8, CD20, CD68,and tryptase were incubated on tissue sections for 1 hour at RT. Antibodiesagainst CD15, CD25, CD56, CD1a, and IFN-� were incubated over night at4°C. A 1:3 dilution of hematoxylin gill 1 was used for counter staining. Thechromogen used to detect secondary antibodies was 3,3�-diaminobenzidine(Vector Laboratories, Burlingame, CA).

Double-staining immunofluorescence (IF) was performed to detect IL-10 orTGF-� producing cells in the cervical tissue. IL-10 antibody (PE-conjugatedrat IgG2a, clone: JES3–19F1, BD Bioscience, San Jose, CA) or TGF-� thatwas detected by antibody against TGF-�1 latency-associated peptide (goat IgGlatency-associated peptide antibody, R&D Systems) was combined with CD4,CD25, CD1a, CD68, mast cell tryptase, or S100 (anticow S-100 polyclonalantibody, DAKO). Secondary fluorescent antibodies were antimouse or anti-goat Alexa Fluor 488 or 633 (Molecular Probes, Eugene, OR). Each primaryantibody was diluted in PBS with 1% BSA as follows: 1:200 (IL-10-PE), 1:400(TGF-�), 1:5,000 (S100), and all fluorescent secondary antibodies were at1:200 except antimouse Alexa Fluor 488 (1:1,200). Routine IF was performedas described for IHC except that trypsin digestion was omitted. Before over-night incubation at 4°C with each primary antibody, slides were blocked with1� Power Block (BioGenex, San Ramon, CA), and each correspondingsecondary antibody was incubated for 1 hour at RT after the primary antibodyincubation. Slides then were incubated with 1:200 4�,6-diamidino-2-phenylin-

dole (D9542, Sigma-Aldrich, St. Louis, MO) for 5 minutes at RT and mountedwith Prolong Anti-fade kit (Molecular Probes). Between each step, slides werewashed thoroughly in PBS with 0.05% Tween 20. All steps involving fluo-rescent dyes were processed in the dark.

Quantification of Immune Cell Numbers. The numbers of immunophe-notyped cells in cervical tissues were determined using image analysis soft-ware. In each photographic image of a microscopic field (40� objective),stromal and epithelial areas were separately measured in mm2 (Openlab 2.2.4,Improvision, Lexington, Massachusetts), and the stained cells were counted byhand after visual inspection. Blood vessels and endocervical glands in thestroma that were larger than 500 �m2 were subtracted from the area. Thenumber of cells was then divided by area to determine cell density (cell no. permm2). Intraobserver variability of cell counts was �10% as determined bytriplicate counts of 10 fields. For counts of stromal cells, 10 microscopic fieldswithin 600 �m of the epithelial basement membrane were randomly selectedfrom each slide. Stromal areas containing organized aggregates describedpreviously (17) were excluded from this analysis. The average densities from10 fields from each sample were used for statistical comparisons. Ten micro-scopic fields for epithelial cell counts were not always available becausedysplastic epithelium was partially denuded in some samples. Cell densitiesfrom five 40� objective microscopic fields with CIN 2/3 were averaged foreach sample for statistical comparison. To avoid counting pseudopodia whencounting macrophages, we focused on stained areas larger than 200 pixels.Tryptase, secreted by mast cells into surrounding stroma, was often diffuselypresent surrounding mast cells. Therefore, to be counted as a mast cell, thestained area must have included a nucleus.

For quantification of cells stained by IFN-� antibody, the stromal andepithelial areas are combined to obtain generalized density in the cervicaltissue. Five 40� microscopic fields that contained both stroma and epitheliumwere randomly selected from each slide, and the cell counts per mm2 from fivefields were averaged and compared statistically.

Because IL-10–positive cells were widely distributed in the stroma, a slidescanner (Aperio Technologies, Vista, CA) was used to determine the numberof IL-10–positive cells. Five stromal areas not limited to 600 �m from thebasement membrane were randomly selected from each sample of normalcervix (n � 4), HIV� CIN 2/3 (n � 4), and HIV� CIN 2/3 (n � 9), and thedensity of IL-10–positive cells was measured as stated above.

The number of samples contributing data for analyses and P values areindicated in Table 1 unless otherwise stated in the text and reflects the numbersof available appropriate specimens, which in some cases are less than thenumbers stated above attributable to depletion of the sample reservoir overtime.

For the intensity measurement of IL-10 and TGF-� staining, a confocalmicroscope (Carl Zeiss, Inc, Thornwood, NY) was used. For IL-10 intensity,five randomly selected stained cells from each sample of HIV� CIN 2/3(n � 4) and of HIV� CIN 2/3 (n � 4) were analyzed. For TGF-� intensity, fiverandomly selected stained cells from each sample of HIV� CIN 2/3 (n � 3)and HIV� CIN 2/3 (n � 2) were analyzed. The z-stack images from each cellwere projected for maximum value after background thresholding and a meanintensity score of each projection as calculated by Zeiss LSM software wasrecorded.

In situ Hybridization. The T bet clone, kindly provided by Dr. L. Glim-cher, was inserted into pcDNA3 vector (Invitrogen, Carlsbad, CA). PlasmidDNA used to produce antisense and sense probes was linearized with therestriction enzymes NotI and BamHI. One microgram of linearized plasmidwas used as a template for in vitro transcription and labeling using a digoxi-genin RNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN).Paraffin-embedded cervical tissue sections were deparaffinized, rehydrated,and digested in 50 �g/ml proteinase K (Roche Diagnostics, Indianapolis, IN)for 30 minutes at 42°C. After denaturation of probes at 95°C for 5 minutes, thetissue sections were hybridized with the RNA antisense or sense probe (3 ng/�lin 2� standard saline citrate, 1� Denhardt’s solution, 10% dextran sulfate,0.02% SDS, and 50% formamide) at 62°C for 16 hours. Subsequently theslides were vigorously washed twice in 2� standard saline citrate with 1mmol/L EDTA, in 0.1 standard saline citrate and 1 mmol/L EDTA at 62°C for2 hours), and twice in 0.5� standard saline citrate. Slides were incubated withanti-digoxigenin antibody (1:800 dilution) for 1 hour at RT and color devel-opment was carried out using nitroblue tetrazolium and 5-bromo-4-chloro-3-inodolyl phosphate solution at RT in the dark.

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MUCOSAL IMMUNITY IN CERVICAL INTRAEPITHELIAL NEOPLASIA



Statistical Analysis. ANOVA methods were carried out for comparativeanalyses of the mean values of quantified immune cells among the cohorts.Pair-wise tests of means were performed using linear contrast statements forplanned a priori comparison. The Spearman rank correlation method was usedto identify any correlation between age and the quantified immune cell den-sities and between peripheral and cervical CD4 counts. No adjustment formultiple comparisons was performed in these analyses.

RESULTS

A Distinctive Immune Microenvironment Is Present in CIN 2/3.To investigate the nature of the mucosal immune response in CIN 2/3lesions, we performed IHC on cervical samples from women withnormal cervix or with CIN 2/3, half of whom were immunosuppressedsecondary to HIV infection. The sources of samples for each of thesegroups are described in Materials and Methods. Staining patternsrevealed that infiltrates in the stroma were localized to areas subtend-ing the CIN lesions, suggesting localized responses to events withinthe epithelium. Examples of IHC for CD8� T cells and macrophagesare shown in Fig. 1A.

Cell densities were quantitated using image analysis as described inMaterials and Methods, and statistical comparisons of the mean celldensities for each group were performed. Cells involved in the ac-quired immune response (CD4� and CD8� T cells and B cells) weresignificantly increased in the stroma among HIV� CIN 2/3 samplescompared with normal cervix. In addition, the numbers of CD4� T

cells in the epithelium were significantly elevated in HIV� CIN 2/3compared with normal cervical epithelium (Table 1).

The cells in the innate immune response (macrophages, NK cells,and neutrophils) in CIN 2/3 were also studied. Neutrophils wereessentially restricted to the stroma and were significantly increased inHIV� CIN 2/3 samples compared with normal cervix (Table 1).Densities of macrophages and NK cells in the stroma and the epithe-lium were both elevated significantly in CIN 2/3 compared withnormal cervix (Table 1).

When HIV� CIN 2/3 samples were compared with normal cervix,significant increases were found in CD4� and CD8� T cells andmast cells in the stroma, and macrophages in the epithelium of CIN2/3 samples (Table 1). The vast majority of CD8� cells also expressCD3 (17)11, indicating that the majority of CD8� cells are indeed Tcells rather than NK cells.

Because hysterectomies are usually performed in older women, themean age of the group contributing samples of normal tissue (51years) was significantly greater than the HIV� and HIV� CIN 2/3groups (31 and 32 years, respectively). Therefore we exploredwhether age and cell densities were correlated among women withnormal cervical tissue. Strong correlations existed between age andnumbers of epithelial CD4� T cells (r � 0.83, P � 0.002), stromal Bcells (r � 0.92, P � 0.0002), and stromal macrophages (r � 0.72,P � 0.03) with older age having higher cell counts. Given the youngermean ages of the CIN 2/3 groups, age is unlikely to contribute to thehigher densities of these cell types observed in CIN 2/3. There was nocorrelation between age and epithelial cell counts of CD8� T cells, Bcells, macrophages or mast cells.

HIV Infection Results in Alterations of the Immune Microen-vironment in CIN 2/3. The mean densities of CD4� T cells in thestroma and epithelium and the macrophage density in the epitheliumin HIV� CIN 2/3 samples were significantly lower than HIV� CIN2/3 samples. In addition, significant elevations of stromal B cells andmacrophages seen in HIV� CIN 2/3 were not observed in HIV� CIN2/3. Densities of neutrophils and NK cells were also attenuated inHIV� CIN 2/3 compared with HIV� CIN 2/3 (Table 1). There was noapparent correlation between cervical CD4� T cell densities (instroma, epithelium, or both combined) and peripheral CD4 counts (atthe visit proximate to the surgical procedure or nadir).

IFN-� Production of Lymphocytes Infiltrating CIN 2/3 Lesions.To determine whether the cells in the CIN 2/3 lesions were committedto a Th1 pathway, we examined expression of cytokines and cytokinereceptors in the clinical samples. Expression of the IL-2R-�, which isnecessary for lymphocyte proliferation and differentiation involved inthe Th1 response, was abundantly expressed in the infiltrates associ-ated with CIN 2/3, and no differences were apparent between HIV�

and HIV� women (data not shown). IFN-�–positive cells were abun-dantly distributed in the stroma near dysplastic epithelium from HIV�

women but were dramatically decreased in samples from HIV�

women with CIN 2/3 (Fig. 1B), even in CD8� cell aggregates (Fig.1C). Quantification of IFN-�–positive cells (Fig. 1D) indicated thatthe mean density of IFN-�–positive cells in the HIV� CIN 2/3 groupwas significantly lower than what we observed in HIV� CIN 2/3 andwas similar to the density in normal cervix. Hence, despite abundantnumbers of T cells in CIN 2/3 from HIV� women, IFN-� productionin these cells is significantly impaired.

IFN-� expression in HIV� CIN 2/3 was localized to both CD4�(Fig. 2A) and CD8� (Fig. 2B) T cells by double staining IF, indicatingthe presence of Th1 responses in CIN 2/3 lesions of immunocompe-tent women. In addition, NK cells also expressed IFN-� (data not

11 A. Kobayashi and K. K. Smith-McCune, unpublished data.

Table 1 Summary of cell densities among the three clinical groups.

Normal cervix HIVneg CIN HIVpos CIN

CD4� T cellsEpithelium N � 11 N � 14 N � 17

62 � 51*,‡ 167 � 161‡,¶ 85 � 85¶Stroma N � 10 N � 14 N � 20

215 � 157§,¶¶ 1083 � 689�,¶¶ 665 � 488§,�CD8� T cells

Epithelium N � 11 N � 13 N � 17408 � 560 434 � 321 585 � 445

Stroma N � 10 N � 13 N � 20330 � 264¶,‡‡ 850 � 577¶ 1045 � 704‡‡

B cellsEpithelium N � 11 N � 13 N � 17

4 � 9 10 � 21 12 � 27Stroma N � 10 N � 14 N � 20

26 � 38† 206 � 242† 125 � 123Macrophages

Epithelium N � 11 N � 14 N � 1764 � 101¶,§§ 483 � 325‡,§§ 268 � 252‡,¶

Stroma N � 9 N � 14 N � 20277 � 204¶ 550 � 273¶ 503 � 361

Mast cellsEpithelium N � 10 N � 12 N � 16

27 � 57 43 � 55 36 � 39Stroma N � 9 N � 12 N � 20

85 � 47†† 151 � 54 209 � 121††Neutrophils

Stroma N � 10 N � 11 N � 1448 � 83** 351 � 250§,** 177 � 174§

NK cellsEpithelium N � 8 N � 11 N � 13

27 � 37‡ 103 � 100‡,‡‡ 17 � 32‡‡Stroma N � 8 N � 11 N � 13

8 � 13§§ 116 � 102§§,¶¶ 13 � 16¶¶

* Cell densities are expressed by cell number/mm2 � SD.† P � 0.01.‡ P � 0.02.§ P � 0.03.¶ P � 0.04.� P � 0.03.** P � 0.001.†† P � 0.002.‡‡ P � 0.003.§§ P � 0.001.¶¶ P � 0.001.

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shown), although given that their cumulative density (219 cells/mm2)is much lower than CD8� (1284 cells/mm2) or CD4� (1250 cells/mm2) T cells (Table 1), the predominant source of IFN-� is likely tobe from T cells.

A transcription factor, T bet, is required in the expression of IFN-�in the Th1 response (18). We analyzed T bet expression by in situhybridization to determine whether the low expression of IFN-� in

HIV� samples was attributable to the absence of this necessarytranscription factor (Fig. 2C). T bet RNA was detected in six of eightHIV� CIN 2/3 samples and in eight of nine HIV� CIN 2/3 samples.Hybridization with sense probe showed no staining (data not shown).All 6 T bet-positive samples from HIV� CIN 2/3 also expressedIFN-�. However, in HIV� CIN 2/3, three of eight T bet-positivesamples failed to produce any IFN-�, indicating that despite the

Fig. 1. Immune cell distributions and down-regulated expression of IFN-� in HIV� CIN 2/3. A,examples of immune cell distribution; top two im-ages are CD8� cells and bottom two are CD68�cells. Images on the left are normal cervix and onthe right are CIN 2/3 (40� objective). B, IFN-�staining by IHC is abundant in HIV� CIN 2/3 (left)but markedly decreased in HIV� CIN 2/3 (right;40� objective). C, abundant CD8 staining in HIV�

CIN 2/3 (top) but IFN-� is essentially absent (bot-tom; 20� objective). D, comparison of IFN-��cell density among the three study groups. Dashedline indicates basement membrane. E, epithelium;S, stroma.

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expression of a key transcription factor, IFN-� was not produced inthis group.

Regulatory Cytokines in CIN 2/3. Recent studies have shown thatIFN-� production in the cytotoxic (Th1) response is down-regulatedby TGF-� and IL-10, secreted by regulatory cells including Tr1 andTh3 (reviewed in ref. 19). We therefore tested whether regulatorycytokines might also be involved in the local immune microenviron-ment in CIN 2/3. In normal cervix, TGF-�� CD1a� cells werescattered throughout the stroma in a pattern similar to that of IL-10staining cells described below. In CIN 2/3 lesions, TGF-� stainingwas detected in CD25� cells (Fig. 3A), macrophages, CD1a� cells,and CD4� T cells and was not detected in cervical keratinocytes (datanot shown). Compared with normal cervix, there was an increaseddensity of stromal TGF-�� cells in the infiltrates adjacent to the CIN2/3 lesions, particularly in HIV� samples, although less intensivelystained TGF-�� cells were also detected in the HIV� group (Fig. 3B).Although the overall TGF-�� cell density comparison did not showa marked difference as a result of HIV infection, the apparent differ-ence in staining intensity was further investigated by immunofluores-cence (Fig. 3C). The mean fluorescent intensity score of TGF-��cells for HIV� CIN 2/3 was 62.5 (�13.4) while mean intensity scorefor HIV� CIN 2/3 was 36.8 (�8; Table 2). Although statisticalcomparisons are not applicable because of the small number of patient

samples in each group, the data indicate that the overall expression ofTGF-� is reduced in HIV� samples.

IL-10 Expressing Immature Dendritic Cells in CIN 2/3. Wealso investigated the expression of another regulatory cytokine,IL-10, attributable to its role in the regulation of Th1 responses(reviewed in ref. 19). Whereas all other immune cells were con-centrated in regions proximate to the dysplastic epithelium, IL-10 –positive cells had a distinctive distribution throughout thecervical stroma (Fig. 4A and B). IL-10 staining did not co-localizewith cervical keratinocytes, CD4� cells, CD25� cells, macro-phages, or mast cells (data not shown) but did co-localize withCD1a� cells (Fig. 4D). CD1a is used as a marker for myeloid-derived DCs. Epithelial tissues contain specialized antigen present-ing cells known as Langerhans cells; however, cervical CD1a�DCs did not co-localize with S100, a Langerhans cell marker.S100-positive cells were distributed primarily in the dysplasticepithelium and had a dendritic morphology (data not shown),whereas CD1a� cells were detected exclusively in the stroma. Asshown in Fig. 4B-D, CD1a�, IL-10�, S100� cells lack matureDC morphology. Triple color IF showed IL-10 and TGF-� co-localized with CD1a in cervical stroma, indicating that a portion ofCD1a� DCs produces both regulatory cytokines (Fig. 4D). Theseimmature cervical DCs demonstrated differences in the number

Fig. 2. IFN-� is expressed in CD4� and CD8�T cells in HIV� CIN 2/3 and T bet is expressed inboth HIV� and HIV� CIN 2/3. A, fluorescent dou-ble-staining in HIV� CIN 2/3; CD4 (left) is greenand IFN-� (middle) is red; co-localization (right) ofCD4 with IFN-� is indicated by yellow (40� objec-tive). B, double staining of CD8 (left) in green,IFN-� (middle) in red, and co-localization (right) ofCD8 with IFN-� is in yellow (40� objective). C, Tbet expression is detectable by ISH in HIV� CIN2/3 (20� objective, insert 40� objective).

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and intensity of IL-10 expression depending on HIV status. Thedensity of IL-10� cells were increased in HIV� CIN 2/3 (69cells/mm2 � 40) compared with normal cervical samples (23cells/mm2 � 22) or HIV� CIN 2/3 (15 cells/mm2 � 15). Inaddition, the mean intensity of IL-10 staining in positively stained

cells from HIV� CIN 2/3 (44.7 � 9.9) was markedly higher thanin HIV� CIN 2/3 (29.4 � 3.3; Fig. 4C; Table 2). Althoughstatistical comparison was not applied because of small samplesizes, the data indicate a difference in the levels of IL-10 expres-sion as an alteration in DC function in HIV infection.

Fig. 3. TGF-� co-localized to CD25� T cells and is down-regulated in CIN 2/3 samples from HIV� patients. A, fluorescentdouble staining shows co-localization of TGF-� (red) and CD25(green) in CIN 2/3 from an HIV� patient. Nuclei are stained bluewith 4�,6-diamidino-2-phenylindole, and the right bottom imageshows the combination of three colors, with yellow indicatingco-localization of TFG-� and CD25 (63� objective). B, TGF-�–positive cells are abundant by IHC in CIN 2/3 samples (left) but thestaining intensity is less in HIV� samples (right; 40� objective).An arrow in the HIV� example indicates a cell with strong TGF-�staining. C, flattened, projected z-stack images of TGF-�–positivecells by confocal microscopy, from HIV� (left) and from HIV�

CIN 2/3 lesions (right; nuclei in blue, TGF-� in green, 63�objective).

Table 2 Cellular IF intensity scores of regulatory cytokines

HIV� CIN2/3 samples Cell intensity score HIV� CIN2/3 samples Cell intensity score

IL-10 1 41, 53, 40, 42, 43, 56 A 34, 27, 27, 27, 26, 262 36, 38, 38, 41, 34 B 38, 34, 32, 31, 353 27, 26, 29, 31, 29 C 16, 16, 32, 37, 314 47, 39, 43, 46, 80 D 27, 17, 33, 33, 36

Mean intensity score 44.7 � 9.9 Mean intensity score 29.4 � 3.3TGF-� 1 69, 59, 60, 66, 69 E 36, 34, 46, 54, 31

2 66, 92, 61, 72, 82 F 32, 32, 40, 43, 304 55, 49, 45, 48, 45

Mean intensity score 62.5 � 13.4 Mean intensity score 36.8 � 8.0

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DISCUSSION

This report demonstrates that CIN 2/3 lesions are associated withmucosal immune responses characterized by activated CD4� andCD8� T cells as well as cells displaying the phenotype of an immu-nosuppressive regulatory T cell. HIV infection impairs the productionof IFN-� and regulatory cytokines in the lesions. Our data are the firstto provide absolute cell densities for B cells, T cells, macrophages,mast cells, neutrophils, NK cells, and IFN-�–expressing cells innormal cervix, and in CIN 2/3 lesions from both HIV� and HIV�

women. These findings are expressed as cell density (cell number/mm2), which has advantages over other less quantitative methodsbecause of its universal relevance, thereby allowing precise evaluationof interventions such as protective and therapeutic vaccines, andmaking possible comparisons across other cohorts or tissue samples.

Our results demonstrate that CIN 2/3 lesions are characterized bycomplex infiltrates of immune cells from both the acquired and theinnate arms of the immune response. The finding of increased num-bers of stromal CD8� T cells in CIN 2/3 compared with normalcervix confirms reports from other groups (20, 21). The presence ofabundant intraepithelial CD8� T cells in normal cervical epitheliumsuggests that the cervix, like the gut, is protected by a specific groupof resident intraepithelial lymphocytes (22).

Our results indicate that the local T cells are activated and produceboth IFN-� as well as regulatory cytokines in CIN 2/3. There areconflicting findings of IFN-� levels associated with CIN possiblyattributable to differences in type of sample [cervicovaginal washing(23) versus tissue levels reported here], or techniques used [relativemRNA expression in microdissected paraffin sections (24) versus the

Fig. 4. IL-10 is expressed in CD1a� cells inCIN 2/3 and is attenuated in HIV� samples. A,slide scanner image of an entire tissue section(HIV� sample) used for quantitative analysis ofIL-10–positive cells attributable to their uniquedistribution throughout the cervical stroma. B, �20objective image taken from panel A, brown indi-cates IL-10–positive cells. C, flattened, projectedz-stack images of IL-10–positive cells by confocalmicroscopy; nuclei in blue, IL-10 in red. Positivecells are from HIV� (top) and HIV� (bottom) CIN2/3 samples (63�). D, fluorescent triple staining ofIL-10, TGF-� with CD1a from HIV� sample(IL-10 in red, TGF-� in blue, CD1a in green, andthe right lower image shows combination of threecolors, 63� objective). Dashed line indicates base-ment membrane; E, epithelium; G, endocervicalgland; S, stroma.

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absolute cell density of positive IHC staining reported here]. Ourfindings are the first to localize IFN-� and other cytokines to specificcellular compartments and indicate the presence of a robust Th1response in CIN 2/3 lesions. This response is presumably elicited tocontain or eradicate local HPV infection and/or protect against devel-opment of HPV-induced neoplasia. The protective role of a localizedimmune response has been inferred from the correlation of a subset ofCD8� T cells called tumor-infiltrating lymphocytes with improvedoutcomes in several invasive cancers (25–27). In addition, infiltratinglymphocytes are correlated with regression of genital warts in bothhumans and animal models (28–31). In the setting of HIV infection,peripheral CD4 counts are inversely correlated with the presence ofCIN, indicating a protective role of CD4� T cells in this population(32). Because our study is a cross-sectional study, the functional roleof the lymphocytic infiltrates in CIN lesions cannot be ascertained.Recent insights into cancer biology have indicated that the role of theimmune response in cancer contributes both protective and permissivefactors (33). For example, inflammatory cells secrete matrix metallo-protease-9, which has been shown to be essential for neovasculariza-tion and lesion progression in a model of HPV-induced skin carcino-genesis (34). In human CIN, cells located within germinal centersexpress high levels of matrix metalloprotease-9 in the human CIN 2/3lesions.12

Another possible explanation for the presence of CIN 2/3 despitethe abundant expression of IFN-� could be attributable to a competingimmunosuppressive response. Recently, subsets of CD25� regulatoryT cells have been described to suppress Th1 response through secre-tion of IL-10 and TGF-� (35). Similar findings of T-cell activationand increase of IL-10 in CIN 2/3 have been reported by others usingdifferent techniques (36, 37). We have identified TGF-�–producingCD4� and CD25� cells in stroma of CIN 2/3 samples, suggestingthat regulatory T cells are recruited to CIN 2/3 lesions. Regulatory Tcells are produced in the context of persistent antigen exposurethrough a mechanism that triggers DCs to produce IL-10, which thendirects differentiation of naı̈ve CD4� T cells to regulatory T cells (38,39). HPV typically does not elicit strong local or systemic immuneresponses, through various mechanisms of immune evasion (reviewedin 13, 14, 40), including the relative paucity of DCs in the transfor-mation zone compared with the exocervix (41), the low expression ofCD3 � chain in T cells in women with CIN and cervical cancer (42,43) and direct effects of viral genes on suppression of the immuneresponse (reviewed in ref. 14). The presence of TGF�� CD25� cellsin CIN lesions described here suggests that putative T regulatory cellsare recruited to CIN lesions and adds a possible new mechanism bywhich HPV evades the immune response. In the context of a CIN 2/3lesion, the competing influences of cytotoxic and suppressive immuneresponses may govern the eventual clinical outcomes of regression orpersistence.

We have described a class of immature CD1a� DCs residentthroughout the cervical stroma expressing IL-10 and TGF-�. Imma-ture stromal DCs have been described previously in the cervix andfound to be less abundant in the transformation zone, the site of CINdevelopment, than in the exocervix (41). Submucosal DCs have alsobeen described in the vagina and were shown to be responsible fortrafficking of antigen to draining lymph nodes in an animal model ofherpes simplex virus infection (44). The presence of IL-10� CD1a�cells in the cervix is intriguing in light of recent descriptions of anovel class of DCs that differentiate in the presence of IL-10 and arecapable of inducing immunotolerance in mice (45) and humans (46).

The function of IL-10� TFG-�� immature DCs in cervical stroma isunder investigation.

Our study included an HIV� CIN 2/3 cohort to determine theeffects of immunosuppression on the local immune microenviron-ment. Samples from HIV� CIN 2/3 demonstrated a robust infiltrationof CD8� T cells, confirming the findings of others (37, 47). TheHIV� CIN 2/3 group had significantly lower numbers of stromal andepithelial CD4� T cells and NK cells, as well as epithelial macro-phages compared with HIV� women. It is worth noting, however, thatlocal CD4� T cell and macrophage numbers were significantly in-creased in HIV� CIN 2/3 compared with normal cervix, indicating acapability for effective extravasation and emigration of lymphocytesto the affected site despite systemic immunosuppression. Thereforethe attenuation of the local immune response in HIV� CIN 2/3 isrelative rather than absolute. Macrophages and DCs express CD4 andcoreceptors for HIV entry, and this might explain the decreasedimmune cell densities and consequent decrease of stromal IL-10�CD1a� regulatory cells in the HIV� CIN 2/3 group. We also foundthat the stromal density of mast cells was significantly increased inHIV� CIN 2/3 samples compared with normal cervix, a finding notpresent in HIV� CIN 2/3 samples, suggesting a shift in the mucosalimmune response from Th1 to Th2 in the HIV� group. In addition, weobserved a dramatic diminution of IFN-� production in CIN 2/3samples from HIV� women. Similar functional impairments in pe-ripheral T cells from HIV� patients have been described by others(48, 49) and are due in part to failure to signal through the STATpathway (50). The regulatory function of the local cervical immuneresponse is also affected in HIV� women, as seen in lower per-cellexpressions of IL-10 and TGF-�. Our finding of reduced IL-10production in HIV� samples is interesting in light of a recent reportthat HPV-specific CTLs were stimulated rather than inhibited byIL-10 (51), although these findings were generated using peripherallymphocytes, and hence their relevance to the cervical microenviron-ment is unknown.

In summary, our data indicate that both the innate and the cellularimmune responses are activated in CIN 2/3 lesions. In addition,regulatory T cells presumably contribute to an immunosuppressivemicroenvironment. Widespread stromal DCs expressing IL-10 andTGF-� may also contribute to immunotolerance against HPV andpossibly other cervical pathogens. Systemic HIV infection suppressesmultiple functional properties of the cervical immune responses, in-cluding IFN-� and regulatory cytokine production. These findingsprovide insights into the mechanisms governing both the protectiveand the potentially immunosuppressive effects of the mucosal im-mune response in HPV-induced cervical neoplasia and will contributeto further understanding of the effects of vaccines and therapiestargeting a local immune response.

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2004;64:6766-6774. Cancer Res Akiko Kobayashi, Ruth M. Greenblatt, Kathryn Anastos, et al. Intraepithelial Neoplasia and Effects of HIV InfectionFunctional Attributes of Mucosal Immunity in Cervical

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