Glucocorticoid-induced tumor necrosis factor receptor expression in patients with cervical human papillomavirus infection

Introduction: The progression of human papillomavirus (HPV) infection in the anogenital tract has been associated with the involvement of cells with regulatory properties. Evidence has shown that glucocorticoid-induced tumor necrosis factor receptor (GITR) is an important surface molecule for the characterization of these cells and proposes that GITR ligand may constitute a rational treatment for many cancer types. We aimed to detect the presence of GITR and CD25 in cervical stroma cells with and without pathological changes or HPV infection to better understand the immune response in the infected tissue microenvironment. Methods: We subjected 49 paraffi n-embedded cervical tissue samples to HPV DNA detection and histopathological analysis, and subsequently immunohistochemistry to detect GITR and CD25 in lymphocytes. Results: We observed that 76.9% of all samples with high GITR expression were HPV-positive regardless of histopathological fi ndings. High GITR expression (77.8%) was predominant in samples with ≥1,000 RLU/PCB. Of the HPV-positive samples negative for intraepithelial lesion and malignancy, 62.5% had high GITR expression. High GITR expression was observed in both carcinoma and high-grade squamous intraepithelial lesion (HSIL) samples (p = 0.16). CD25 was present in great quantities in all samples. Conclusions: The predominance of high GITR expression in samples with high viral load that were classifi ed as HSIL and carcinoma suggests that GITR+ cells can exhibit regulatory properties and may contribute to the progression of HPV-induced cervical neoplasia, emphasizing the importance of GITR as a potential target for immune therapy of cervical cancer and as a disease evolution biomarker.

Human papillomavirus (HPV) infects the basal and parabasal cells of squamous epithelium in the female anogenital tract, and HPV types 16, 18, 31, 33, and 45 in particular are believed to put patients at high risk for the development of high-grade cervical intraepithelial neoplasia (CIN) and cervical carcinoma 1 .
Infection progression has been associated with several factors, including the persistence of HPV, the presence of high-risk oncogenic HPV types, high viral load, integration of viral DNA, and E6 and E7 viral oncoprotein activity 1 .Evidence shows that regulatory T cells (T reg ) are also involved in the progression to cervical neoplasia in HPV-infected patients [2][3][4][5] .HPV-specifi c CD4 + regulatory cells isolated from lymph node biopsies of patients with cervical carcinoma were found to suppress proliferation and cytokine (interferon-γ, interleukin [IL]-2) production by responder T cells 5 .
T reg cells play a crucial role in modulating the elimination of pathogens and tumor antigens and perform their function through immunosuppressive cytokine production and immunosuppression induction mediated by cell-to-cell contact, having the ability to suppress the activation, proliferation, and effector function of different cell types contributing to the immune response 6,7 .T reg cells are subdivided into several subpopulations, one being the natural T reg cells (CD4 + CD25 + T reg ), which numerically represent the largest group of cells with suppressor activity 8 .
Studies show that T reg cells are activated with greater sensitivity than naïve effector T cells after antigenic stimulation, which has been attributed mainly to their semi-activated state that is thought to be due to the increased expression of CD25 (α-chain of the IL-2 receptor), glucocorticoid-induced tumor necrosis factor receptor (GITR) markers, and others [9][10][11] .
The detection of T reg cells has been challenging owing to the lack of exclusive surface molecules for these cells.Studies have shown that the presence of transcription factor

DISCUSSION
FOXP3 (forkhead box p3) is highly specific and that its transduction into naïve T cells increases the molecular expression associated with T reg cells, such as that of CD25 and GITR 12,13 .Evidence shows that another characteristic surface molecule of cells with regulatory properties, T reg cells in particular, is the GITR 14 -a tumor necrosis factor receptor superfamily member -which is predominantly expressed in CD25 + CD4 + T reg cells and plays an important role in the regulation of mucosal immune responses [15][16][17][18][19] .Recent studies have demonstrated that in vivo GITR ligation using an agonist anti-GITR monoclonal antibody, DTA-1, can augment anti-tumor T-cell responses by modulating T reg cells, which makes targeting GITR a potential immunotherapeutic approach to cancer treatment [20][21][22] .
Given the fi ndings that indicate the involvement of cells with regulatory properties, especially T reg cells, in the progression of cervical malignant lesions 3,4,23,24 , this study aimed to both CD25 and GITR markers in lymphocytes of cervical stroma to better understand the immune response in the microenvironment of HPV infection, which may shed light on novel therapeutic interventions against intraepithelial neoplasia and cervical cancer of viral etiology, and perhaps also make GITR a possible candidate biomarker for disease evolution.

Samples
Forty-nine patient cervical samples embedded in paraffi n and selected on a non-probabilistic form by convenience sampling from 2000 to 2002 in the Cancer Prevention Center of Campo Grande, Mato Grosso do Sul, Brazil, were used.These samples previously underwent a Hybrid Capture II reaction (Digene, Gaithersburg, MD, USA) to quantify the viral load for group B -high oncogenic risk types that were classifi ed into scores from 0 to 3: 0 (HPV-negative samples); 1 (1 to < 100U of light released for probe; relative light units/positive control to group B (RLU/PCB); 2 (100 to <1,000 RLU/PCB); and 3 (≥1,000 RLU/PCB).On the basis of histopathological analysis, the samples were classifi ed as lowgrade squamous intraepithelial lesions (LSIL) (CIN I); high-grade squamous intraepithelial lesions; (HSIL) (CIN II, III); carcinoma, and negative for intraepithelial lesion and malignancy (NILM).
The detection system was a Universal LSAB + Kit/HRP (Dako ® , Carpinteria, CA, USA), and diaminobenzidine (Dako ® ) was used as a chromogen.Counterstaining was performed in hematoxylin, and the slides were observed under common optical microscopy with 10× and 40× objective lenses.Samples showing brown staining on characteristic cells were considered positive.Human tonsil tissue was used as the external control of the reaction over which the primary antibody (positive control) and phosphate buffer pH 7.4 containing 1% albumin (negative control) was applied.

Quantitative analysis
According to the presence of immunomarked cells, the histological sections were classifi ed in low (small quantities) and high (large quantities) scores.The slides were analyzed by 2 independent observers, previously cal ibrated (κ = 0.98), and the fi nal result of discordant cases was obtained by common analysis to produce a consensus.

Statistical analysis
Analysis of the frequencies among the histopathological fi ndings and viral load according to GITR expression intensity were compared using Fisher's exact test.

Ethical considerations
This study was approved by the Research Ethics Committee of the Universidade Federal de Mato Grosso do Sul, protocol number 975, July 31, 2007.
A frequency analysis of the histopathological findings according to GITR expression intensity is shown in Table 2. High GITR expression was predominant in the carcinoma and HSIL samples (p = 0.16) (Figure 1).
All samples showed intense staining for CD25 regardless of the result of viral load or histopathological fi ndings (Figure 2).
In the present study, we observed that among the high GITR expression samples 76.9% were HPV-positive and 23.1% were HPV-negative.The expression of this marker was predominant in samples with high viral load as well as high-grade lesions and carcinoma.
A number of surface and secreted molecules have been associated with T reg , and GITR has been recognized as CD4 + T reg markers in mice and humans 22,26,27 .In this context, it is of interest that GITR + T reg cells might be involved in the failure of the immune system to control the development of HPV-induced cancer.Studies have demonstrated increased frequencies and suppressive activity of T reg cells in patients with high-grade lesions and cervical cancer.In addition, compared to colorectal cancer, skin melanoma, and bronchial carcinoma, HPV-derived CIN lesions and cervical carcinomas have higher numbers of T reg cells 23,24 .
One study that investigated the infl uence of tumor-infi ltrating T reg cells on tumor-specifi c T cell responses found that T reg cells in patients with liver cancer upregulated GITR expression compared with T reg cells in tumor-free liver tissue and blood 28 .Another study identifi ed increased numbers of T reg GITR + cells in tumorpositive lymph nodes compared with tumor-negative nodes in the same patient 29 .Both studies propose that GITR ligand could be a promising treatment for cancer and that GITR and GITR ligand are good candidates for disease evolution biomarkers.
Studies investigating the natural history of HPV infection have shown that viral clearance may vary from 4-16 months according to the virus' oncogenicity [30][31][32] .However, it has been observed that persistent infection with a higher likelihood of progression to high-grade lesions and invasive carcinoma can occur in the face of an ineffective immune response.In this context and considering that HPV infection is restricted to epithelial cells, the importance of the local immune response is highlighted, making the components present in the microenvironment crucial for lesion development or regression [33][34][35][36] .The role of GITR has been unclear until now, emphasizing the importance of the present study to clarify the immune response in the cervical microenvironment.
The presence of high GITR and CD25 expression levels found in HPV-derived CIN lesions and cervical carcinomas indicates that these cells may play an important role in the downregulation of immune responses 37 .A strong association between these markers and T reg cells was demonstrated in a study that found GITR expression in only those cells that also expressed CD4 and CD25, and most of them co-expressed FOXP3 38 .The association of GITR and CD25 with negatively regulated T helper-activated lymphocytes has been demonstrated in experiment with C57BL/6 GITR +/+ mice (wild type), which showed decreased IL-2 expression compared to C57BL/6 GITR -/-39 .
The relevance of cells expressing the studied markers in immune response suppression is emphasized by studies that evaluate in vitro regulatory activity through cytokine expression by CD4 + T cells, CD4 + CD25 + GITR + cells, and CD4 + CD25 - GITR + cells.These studies showed that the fi rst produced cytokines that activated the immune response and the last 2 increased immunosuppressive cytokine levels 15,17 .

Padovani CTJ et al -GITR expression in HPV infection
It is unclear whether increased frequencies of regulatory cells are a cause or consequence of high viral load and chronic infection 2,40,41 .The predominant expression of GITR in samples with high viral load and classifi ed as HSIL and carcinoma in this study suggest that GITR + cells can exhibit regulatory properties.The lack of a correlation between GITR and viral load or GITR and histopathological fi ndings can be explained by the small sample size.Additional studies are required to confi rm these observations.
Further longitudinal studies are required to assess the true association between HPV persistence and immunoregulatory cell involvement in lesion progression and the development of neoplasia.Studies have demonstrated increased frequencies and suppressive activity of T reg cells in HPV-infected patients with cervical cancer and its precursor lesions (CIN) and suggest that T reg cells may be a potential marker of cervical disease persistence.One longitudinal analysis of T reg cell frequencies showed a modest decline 1 year after curative surgery or chemoradiation 3,4 .
Finally, on the basis of the fi nding that GITR confi gures a surface molecule characteristic of cells with a regulatory profi le, our results suggest that GITR + cells may play a role in the development of a favorable microenvironment for the progression of HPV-induced cervical neoplasia that omits proper activation of the immune response for antigen elimination.Additional studies have been made by the same group including the characterization of FOXP3 + /CD25 + , CD4 + /transforming growth factor-β + and IL-10 -secreting cells in HPV-infected samples by using IHC to help elucidate the role of T reg cells in cervical intraepithelial neoplasia (CIN) and cervical cancer (manuscript in preparation).
The authors declare that there is no confl ict of interest.

FIGURE 1 -FIGURE 2 -
FIGURE 1 -GITR expression in HPV positive cervical lesions.a: positive control human tonsil stained with anti-human GITR antibody; b: negative control human tonsil -omitting primary antibody, showed no staining; c: CIN II and d: NILM, with GITR expressing cells in small quantities; e: CIN III and f: CIN I, with GITR expressing cells in large quantities.All fi gures are presented in the same magnifi cation (400X).Black arrows indicate positive-staining cells with anti-human GITR antibody.GITR: glucocorticoid-induced tumor necrosis factor receptor; HPV: human papillomavirus; CIN: cervical intraepithelial neoplasia.
ao Desenvolvimento do Ensino, REFERENCES 1.International Agency for Research Cancer (IARC).Working group on the evaluation of carcinogenic risks to humans.IARC monographs on A B C Ciência e Tecnologia de Mato Grosso do Sul (FUNDECT/ MS) and Conselho Nacional de Desenvolvimento Científi co e Tecnológico (CNPq).

TABLE 1 -
Distribution of histopathological fi ndings according to viral load and GITR expression (n=49).

TABLE 2 -
Frequence of histopathological fi ndings according to the intensity of GITR expression.