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The influence of human papillomavirus type and HIV status on the lymphomononuclear cell profile in patients with cervical intraepithelial lesions of different severity

The influence of human papillomavirus type and HIV status on the lymphomononuclear cell profile... Background: Immunological alterations are implicated in the increased prevalence of high-grade squamous intraepithelial lesions (HG-SIL) and persistent human papillomavirus (HPV) infection. This study evaluated the expression of CD4, CD8, CD25 (IL-2Rα) and CD28 antigens from SIL biopsies, stratified by HIV status and HPV-type. Biopsies specimens from 82 (35 HIV ) women with a normal cervix, low-grade (LG-SIL) or high-grade lesions (HG-SIL) were studied. CD molecule expression was evaluated by immunohistochemistry and HPV detection/typing performed using PCR techniques. Results: CD4 stromal staining was increased in patients with HPV18. Women with HPV16 infection showed decreased: a) CD8 and CD25 stromal staining, b) CD25 staining in LG-SIL epithelium and in HG-SIL stroma. In HIV women samples, CD28 epithelial staining and CD8 stromal staining surrounding metaplastic epithelium were less intense and even absent, as compared to HIV women. Both epithelial and stromal CD8 staining was more intense in the HG- + - SIL/HIV group than in the HG-SIL/HIV group. Positive correlations were observed between CD4/ CD25, CD4/CD28 and CD25/CD28 in the stroma and CD25/CD28 in the epithelium. Conclusion: HIV status and HPV-type may influence the lymphomononuclear cell profile present in the spectrum of cervical lesions. The knowledge of the infiltrating cell profile in cervical tumours may help the development of specific anti-tumoural strategies. Introduction viral load, both of which have been associated with the Human Immunodeficiency Virus (HIV)-infected women HPV persistence and SIL severity [1,2]. have a high prevalence of persistent human papilllomavi- rus (HPV) co-infection and also of squamous intraepithe- HPV may evade immune surveillance by shifting Th cell lial lesions (SIL) [1]. The progression from HIV infection polarization, down-regulating the expression of Major status to acquired immune deficiency syndrome is Histocompatibility Complex (MHC) class I molecules marked by a decreasing CD4 count and increasing HIV and reducing the function of intraepithelial antigen-pre- Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 senting cells, which may cause a lack of Th1 polarization of HPV and cervical intraepithelial lesions. Median age from the beginning of HPV infection. This causes a shift to was 36 years (range 19 50) for HIV-negative women and the production of IL-4, IL-6 and/or the immunosuppres- 29 years (range 1947) for HIV-positive women. These sive cytokine IL-10 [3,4]. Similarly, the progression from women were enrolled at several Gynaecological Reference HIV infection to AIDS has been associated with the polar- Services in the State of São Paulo, Brazil, from 1996 to ization to the Th2 cytokine profile in cervicovaginal secre- 2001 and the study protocol was approved by the institu- tion[5] which might contribute to the persistence of HPV tional Ethics Committee on human experimentation. infection [6]. Colposcopies were performed for routine in all women In the general population, tumour infiltrating lym- and when indicated, the same physician performed colpo- phocytes in cervical cancer have been associated with a scopically directed cervical biopsies or conization loop depressed function of cytotoxic T cells [7,8]; a down-regu- excision. Cervical fragments were fixed with formalin and lation of CD25 (α chain of the IL-2 receptor-IL-2Rα) acti- then slides were stained with haematoxylin-eosin (Sigma, vated cells [9] and a decreased proportion of CD4 T cells St. Louis, MO, USA) and histologically evaluated by two with a reversed CD4/CD8 ratio [10]. In HIV-infected experienced histopathologists, who also provided scoring women, little information is available regarding the infil- representing the sum of intensity and distribution of trating T cell phenotypes; however, deregulation of staining in fields, in a double-blinded protocol. Thin 5 peripheral blood CD4/CD8 T cell function is progres- μm sections were cut, placed on organosilane-pretreated sively impaired [11] with hyporesponsiveness of T CD8 slides and submitted to immunohistochemical assays. An cells to activation by the co-stimulatory CD28 and CD40L additional 10 μm section was used for DNA extraction molecules [12]. To date it is not clear whether these and HPV typing. defects in HIV-infected women also occur in the cervicov- aginal milieu and whether these might be implicated in Cervical biopsies were selected from HIV-negative or HIV- the increased prevalence of HG-SIL. positive women and they were stratified into 3 groups: normal (no evidence of SIL or HPV DNA), low-grade SIL The interaction of the T-cell receptor with the MHC mole- (cervical intraepithelial neoplasia I) and high-grade SIL cule is considered to be the first signal for T cell activation. (cervical intraepithelial neoplasia II-III and in situ carci- CD4 and CD8 antigens, observed on the surface of T cells, noma). are co-receptors that bind to non-polymorphic regions of MHC class II and class I molecules, respectively and trans- Immunohistochemistry duce signals that initiate T cell activation. The second sig- Serial sections (34 μm) obtained from paraffin-embedded nal for T cells stimulation is provided by the interaction of blocks, cut and mounted on APTS (3-aminopropyltri- co-stimulator molecules, including CD28 antigen on T ethoxy-silane, Sigma, Saint Louis, USA)-pre-treated glass cells and CD80/CD86 on the surface of activated antigen slides were dried by the method of Alves et al. [14]. To pre- presenting cells. Activation of T cells by antigens and co- vent cross-reaction with endogenous peroxidase, the stimulators stimulate the production and secretion of IL- slides were treated with 3% H O for 20 min and then 2 2 2, which in turn induces the production of the IL-2 recep- incubated overnight at room temperature with the follow- tor (IL-2R). The IL-2R is formed by three non-covalently ing primary antibodies at the following dilutions: CD4 associated protein called a (IL-2Ra), b (IL-2b) and g (IL- (mouse monoclonal, clone 1F6, IgG1 NovoCastra, New- 2g) chains. The activation of T cells by antigens, co-stimu- Castle upon Tyne, UK) 1:80; CD8 (mouse monoclonal, lators and IL-2 leads the expression of IL-2a (CD25) [13]. clone 4B1L, IgG2b NovoCastra), 1:200; CD25 (mouse To determine the possible associations between CD4, monoclonal, clone 4C9, IgG2b NovoCastra) 1:300; CD8, CD25 and CD28 antigens expression in several CD28 (goat policlonal, clone N20, SC 1625 Santa Cruz stages of cervical cancer development, we assessed the Biotechnology, Santa Cruz, CA, USA), 1:300. Sections of expression of these molecules in cervical biopsies strati- human tonsils were used as positive controls, and a nega- fied according to the severity of the lesion and HPV type tive control to detect background staining was performed detected (HPV16 and 18). by omitting the primary antibody. Isotype specificity for tonsil sections was confirmed by comparison of staining with irrelevant antibodies of the same isotype as the pri- Materials and methods Sample selection mary antibody. A control incubation to detect back- The study was conducted retrospectively on 82 (47 HIV- ground staining was performed omitting the primary negative and 35 HIV-positive), non-pregnant, non-lactat- antibody. ing, premenopausal women, which were recruited sequentially according to their arrival at the outpatient After incubation with the primary antibodies, immu- clinic. They were colposcopically and cytologically noperoxidase staining was performed using a universal screened, selected for cause, i.e., the presence or absence biotinylated secondary antibody mixed with a preformed Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 avidin and biotinylated horseradish peroxidase macro- from HPV16 (HPV16E7.667 and HPV16E7.774 primers) molecular complex (Novostain super ABC kit-NovoCas- and another one from HPV18 (HPV18E7.696 and tra), diluted 1:200 for CD4, CD8, CD25 and a different HPV18E7.799 primers) [20]. All DNAs were amplified biotinylated secondary antibody (Vector, BA 5000, anti- with these 4 sets of primers together with a set of primers goat IgG Burlingame, CA, USA) diluted 1:200 for CD28. for a housekeeping gene (globin) [21] as an internal con- Slides were alternately washed 3 times with PBS and TBST trol of amplification. Amplified DNA was applied to a (Tris-buffered saline; 0.05 M Tris, pH 7.4, containing 10% polyacrylamide gel, electrophoresed at 200 volts for 0.3% Tween 20) and diaminobenzidine (0.5 mg/mL) 1.45 h and stained with AgNO3 by the method of Sangui- (Sigma) was used as chromogen. Slides were then sequen- netti et al [22]. It is important to note that the absence of tially counter-stained with haematoxylin without acid for HPV16 or HPV18 did not exclude the presence of HPV 30 sec, washed in tap water, stained blue with ammonia- types other than 16 or 18, which could be identified if spe- cal water for 20 sec and exhaustively rinsed with tap water. cific primers for them were used. Only unambiguous Finally, slides were dehydrated and mounted for light amplifications were considered as positive. Lack of ampli- microscopy evaluation at 400× magnification using an fication with these primers or ambiguous amplifications eyepiece graticule connected to an objective lens, as after several repetitions were assigned as other HPV types. detailed elsewhere [15]. Statistical Analysis A minimum of 10 fields (total area equivalent to 0.75 According to the distribution of the variables and the mm ) was assessed per case. The expression of CD anti- number of groups compared, statistical analysis was per- gens was evaluated using a previously described semi- formed using the unpaired t, Mann-Whitney and Kruskall- quantitative method [16]. A total score representing the Wallis tests. The Spearman test (r) was used to calculate sum of intensity and distribution of staining in fields pre- correlations. P values were two-sided and the level of sig- senting dysplastic epithelium and in the respective subep- nificance was set at ≤ 0.05. All data were analysed using ithelial stroma was assigned to each case. The intensity of the Instat Mac 2.01 software (GraphPad software, CA, cellular staining in epithelium and stroma was scored as: USA). (0) no staining, (1) weak; (2) moderate; and (3) intense. Staining in the epithelium was scored as follows: (0), Results patchy basal; (1), diffuse basal; (2), diffuse full-thickness; Patient data (3), patchy and diffuse full-thickness positivity. Staining Among the 47 HIV-negative women, 4 exhibited no cervi- distribution in stroma was categorised as: (1), patchy sub- cal lesions and 43 presented SIL (13 LG-SIL and 30 HG- epithelial; (2) diffuse full-extent; (3) patchy and diffuse SIL). Among 35 women presenting HIV-infection, 5 full-extent positivity. Only sections showing epithelial exhibited no cervical lesions and 30 women presented SIL and stromal tissue were considered for the analysis. (21 LG-SIL and 9 HG-SIL). According to CD4 counts, 9 HIV-positive women presented CD4 counts > 500 cells/μL HPV identification and typing and 26 of them CD4 counts < 500 cells/μL. The median HPV DNA obtained from paraffin blocks [17] was ampli- peripheral CD4 T-cell count of the group presenting with fied by PCR using 12.5 pmoles of dNTP, 25 pmoles of both SIL and HIV infection was 360 cells/μL. Patient data each primer, 1.5 U Taq DNA polymerase (Gibco, USA), 5 stratified by lesion severity (LG-SIL, HG-SIL), HIV status μL of 10× enzyme buffer, 20 μg of genomic DNA (50 ng) and HPV type are shown in Table 1. and distilled deionised H O to complete a total volume of 50 μL. The mixture was processed in a thermocycler appa- CD4 and CD8 staining ratus (MJ Research, MA, USA) under the following cycling CD4 and CD8 staining was observed in cells presenting conditions: 1 cycle at 95°C for 5 min, 30 cycles at 95°C the morphology of lymphomononuclear cells. CD4 stain- for 30 sec, at 55°C for 30 sec and at 72°C for 1 min and ing was observed throughout the squamous epithelium, finally 1 cycle at 72°C for 10 min and then at 4°C indefi- and remarkably more intense in the stroma under the nitely. transformation zone and surrounding endocervical glands, especially near inflammatory areas. CD4 stromal Since formalin may degrade DNA, producing DNA frag- staining was increased, particularly in specimens present- ments of different length, several pairs of primers were ing HPV18, when compared with those without HPV18 used. Primers GP5+ and GP6+ [18], which amplify small infection. However, in the HIV-positive/HPV18 group, DNA fragments and primers MY09 and MY11 [19], which CD4 stromal staining showed to be decreased, when com- amplify longer DNA fragments, were used for generic HPV pared with those in the HIV-negative/HPV18 group (P = amplification. Since HPV16 and HPV18 are the types 0.02) (Fig 1). Few koilocytotic cells presented CD8 stain- most frequently associated with cervical neoplasia, 2 sets ing. A significant decreasing of CD8 stromal staining was of specific primers were used to detect the E7 gene, one observed when specimens with HPV16 infection alone Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 Table 1: Data stratified by histopathological diagnosis, HIV status and HPV types. HPV- HPV16 HPV18 HPV16/18 HPVX n(%) n(%) n(%) n(%) n(%) Normal LG-SIL HG-SIL LG-SIL HG-SIL LG-SIL HG-SIL LG-SIL HG-SIL LG-SIL HG-SIL HIV-negative (n = 47) 4 (8.5) 1(2.1) 3(6.4) 5(10.7) 10(21.3) 2(4.3) 6(12.8) 2(4.3) 6(12.8) 3(6.4) 5(10.7) HIV-positive (n = 35) 5 (14.3) - - 4(11.4) 1(2.9) - 2(5.7) 1(2.9) 1(2.9) 16(45.7) 5(14.3) Total (n = 82) 9(11.0) 1(1.2) 3(3.7) 9(11.0) 11(13.4) 2(2.4) 8(9.8) 3(3.7) 7(8.6) 19(23.2) 10(12.2) HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; HPV X = Unidentified HPV types; LG-SIL = Low-grade Squamous Intraepithelial Lesion; HG-SIL = High-grade Squamous Intraepithelial Lesion. were compared with specimens without HPV16. How- CD25 (IL-2Rα) and CD28 staining ever, CD8 stromal staining seemed to be increased in In most specimens, CD25 staining was observed in basal specimens with both HIV and HPV16 infections, when and immature epithelium, independent of HIV status (Fig compared with specimens from patients HIV-negative/ 4). However, CD25 epithelial staining varied according to HPV16 (P = 0.004) (Fig 2). CD8 stromal staining was both HPV status and lesion severity, being more frequent more frequently localised surrounding the metaplastic in the HPV16-positive/HG-SIL group than in the HPV16- epithelium and more intensively in specimens from HIV- negative/LG-SIL group (P = 0.03) (Fig 5). CD25 stromal positive group, compared to the HIV-negative group (P = staining was primarily observed in lymphomononuclear 0.001). In the HIV-positive/SIL group, clusters of lympho- cells. More intense CD25 stromal staining was observed mononuclear cells were observed around vessels and adja- in HPV16-negative/HG-SIL group than in the HPV16-neg- cent to the basement membrane. HIV-positive/SIL group ative/LG-SIL group (P = 0.002) (Fig 6). Both epithelial exhibited a more intense CD8 stromal staining than and stromal CD25 staining (respectively, P = 0.02 and observed in the HIV-negative/SIL group (P = 0.0005) 0.005) were associated with HPV type infection (HPV16, (Fig 3). HPV16/18 and other HPV types), when compared to HPV-negative group. No significant difference was found A (n=8) B (n=35) C (n=18) D (n=3) E (n=22) F (n=15) G (n=13) H (n=3) Mean (± SD) CD4 stromal staining H Figure 1 Increased immunohistochemica PV18 infection (Kruskal-Wallis = 0.02) l expression of CD4 stromal staining of cervical specimens (mean ± SD) in the presence of Increased immunohistochemical expression of CD4 stromal staining of cervical specimens (mean ± SD) in the presence of HPV18 infection (Kruskal-Wallis = 0.02). A = HPV-negative; B = HPV18-negative; C = HPV18-positive; D = both HIV and HPV are negative; E = both HIV and HPV18 are negative; F = HIV-negative, HPV18-positive; G = HIV-positive, HPV18-negative; H = both HIV and HPV18 are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus. Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 A (n=11) B (n=33) C (n=26) D (n=3) E (n=14) F (n=22) G (n=19) H (n=5) Mean (± SD) CD8 stromal staining Reduced immu H Figure 2 PV16 infection (one way A nohistochemical NOVA test = 0.004) expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of Reduced immunohistochemical expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of HPV16 infection (one way ANOVA test = 0.004). A = HPV-negative; B = HPV16-negative; C = HPV16- positive; D = both HIV and HPV are negative; E = both HIV and HPV16 are negative; F = HIV-negative, HPV16-positive; G = HIV-positive, HPV16-negative; H = both HIV and HPV16 are positive. HPV = Human papillomavirus; HIV = Human Immunode- ficiency Virus. A (n= 40) B (n= 30) C ( n= 7) D (n= 33) E (n= 30) F (n= 9) G (n= 6) M ean ( ± S D ) C D 8 s tr o m a l s taining Increasin H Figure 3 IV-infection (one way ANOVA test = 0.0005) g immunohistochemical expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of Increasing immunohistochemical expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of HIV-infection (one way ANOVA test = 0.0005). A = HIV-negative; B = HIV-positive; C = both HIV and SIL are negative; D = HIV-negative, SIL-positive; E = HIV-positive, SIL-negative; F = both HIV and SIL are positive; G = all HIV, SIL and HPV16 are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; SIL = Squamous Intraepithelial Lesion. Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 CD25 staining Figure 4 in a basal and immature cervical squamous epithelium CD25 staining in a basal and immature cervical squamous epithelium. A (n=4) B (n=35) C (n=21) E (n=13) D (n=16) F (n=18) Mean (± SD) CD25 epithelial staining H Figure 5 Reduced immu PV16 infection (Kruskal-Wallis = 0.03) nohistochemical expression of CD25 epithelial staining of cervical specimens (mean ± SD) in the absence of Reduced immunohistochemical expression of CD25 epithelial staining of cervical specimens (mean ± SD) in the absence of HPV16 infection (Kruskal-Wallis = 0.03). A = all HIV, HPV and SIL are negative; B = HPV16 total; C = HPV16-negative, LG-SIL-positive; D = both HPV16 and LG-SIL are positive; E = HPV16-negative, HG-SIL-positive; F = both HPV16 and HG-SIL are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; SIL = Squamous Intraepithelial Lesion; LG-SIL = Low-grade Squamous Intraepithelial Lesion; HG-SIL = High-grade Squamous Intraepithelial Lesion. Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 A (n=4) B (n=21) C (n=17) D (n=16) E (n=13) F (n=18) Mean (± SD) CD25 stromal staining H Figure 6 Reduced immu PV16 infection (Kruskal-Wallis = 0.002) nohistochemical expression of CD25 stromal staining of cervical specimens (mean ± SD) in the absence of Reduced immunohistochemical expression of CD25 stromal staining of cervical specimens (mean ± SD) in the absence of HPV16 infection (Kruskal-Wallis = 0.002). A = all HIV, HPV and SIL are negative; B = HPV16 total; C = HPV16-negative, LG-SIL-positive; D = HPV16-negative, HG-SIL-positive; E = both HPV16 and LG-SIL are positive; F = both HPV16 and HG-SIL are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; SIL = Squamous Intraepithelial Lesion; LG-SIL = Low-grade Squamous Intraepithelial Lesion; HG-SIL = High-grade Squamous Intraepithelial Lesion. in epithelial or stromal CD25 staining in patients present- mens with SIL, stratified according to HIV status and HPV ing or not HG-SIL, regardless of HIV status. type. CD28 stromal staining was observed in lymphomononu- In the present study, specimens harbouring HPV18 clear cells, particularly under the epithelial lesion and showed a decreased CD4 stromal staining and HPV16 under the transformation zone, sometimes as patchy infection was associated with an increased CD8 stromal agglomerates, resembling germinative centres. CD28 epi- staining, in HIV-infected women. Since CD8 cells are thelial staining was less frequently observed in HIV-nega- recruited preferentially to cervical lesions with progres- tive patients than in HIV-positive patients (P < 0.0001), sion to invasion [26], and in regressing CIN1 lesions, irrespective of the HPV type infection or the SIL severity CD4+ cells predominated within the stroma with highest (Data not shown). CD4/CD8 ratio compared with progressive CIN1 [27-29], these findings may explain the progression or regression Positive correlations were observed between CD4 and of the HPV infection towards carcinogenesis, particullarly CD25 in stroma (r = 0.32, p = 0.01), CD4 and CD28 in among HIV-infected women. stroma (r = 0.33, p = 0.03) and between CD25 and CD28 both in epithelium (r = 0.31, p = 0.03) and in stroma (r = HPV16 is considered to be the major etiologic agent of 0.40, p = 0.004). squamous cervical carcinoma and keratinocytes (non- professional antigen presenting cells) are reported to directly present viral antigens to cytotoxic T lymphocytes Discussion A cellular infiltration composed essentially of CD4 and by cross priming [1], whereas HPV18 is more frequently macrophages is frequently observed in condyloma under associated with adenocarcinoma. The mechanisms of spontaneous regression [23], and a decrease in the viral peptide presentation are less studied than those number of Langerhans cells in the transformation zone reported for squamous carcinoma. Increased HPV16 E7- has been associated with a reduced in vitro T cell prolifer- specific T helper cell responses are associated with persist- ation and IL-2 production [24]. With the progression to ent HPV16 infection, HG-SIL and invasive cervical carci- HG-SIL, the number of immature Langerhans cells noma [30]. It is accepted that HPV infects keratinocytes increase with a consequent deficient function [24,25]. In and interferes with the local cytokine expression [31-33], this study, we evaluated the intensity and distribution of but it is still unknown how HPV together with HIV influ- CD4, CD8, CD25 and CD28 molecules in cervical speci- ences local T cell function. Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 In this study, CD8 staining increased in HIV infected tory T cells [13], the role of these cells in SIL development group and further increased with concomitant SIL. In should be further evaluated performing longitudinal stud- /SIL addition, both the epithelium and stroma of the HIV ies double staining CD4 and CD25. group presented an increased expression of CD8 com- pared to the HIV /SIL group. These findings indicate a In conclusion, our results indicate that HPV type and HIV continuous stimulation by persistent HPV infection, in status may influence lymphomononuclear cell marker agreement with other authors [27,28]. Because of the staining in cervical lesions. A particular immune response proximity of the columnar epithelium to the stroma and may be triggered depending on the balance of these varia- the lack of keratinocytes in the endocervix, the findings of bles, culminating in the progression to cancer. Knowledge this study suggest that stromal lymphomononuclear CD4 of the lymphomonuclear cell profile and the tumour cells (T CD4 and macrophages) may cope better than CD8 immunogen may help understanding HPV evasion mech- in HPV18 infection. In other words, macrophages may be anisms and enable new proposals of anti-tumoral treat- the major APC in stroma and CD4 cells (T lymphocytes ment, especially therapeutic vaccines against cervical and macrophages) may be involved in immunity against lesions. HPV18. Competing interests Several studies have shown that peripheral blood lym- The authors declare that they have no competing interests. phocytes produce IL-2[34,35] and generate cytotoxic activity [36] after specific HPV16 E7 protein stimulation. Authors' contributions De Gruijl et al. [37] reported an increased IL-2 production MAG Gonçalves designed and coordinated the work, the by peripheral blood CD4 T cells, associated with persist- results analyses and manuscript writing. EA Donadi par- ent HPV infection and progression of a premalignant ticipated in the design of the study and in the immunolog- lesion and a higher production of Th2 cytokines related to ical evaluation and data interpretation, and EG Soares was cervical malignancy. In our study, HPV16 associated with responsible for slides histopathological interprestion. All decreased stromal CD8 and CD25 expression and authors read and approved the final manuscript. increased CD25 staining was observed in both epithelium and stroma with HG-SIL. The expression of IL-2R on Acknowledgements We wish to thank Ana Maria Anselmi Dorigan for excellent technical assist- keratinocytes and the IL-2 secretion has been reported in ance. different phases of cancer cell development [38,39]. Bind- ing of IL-2 to IL-2R on tumour cells may down-regulate Financial support: FAPESP (01/02908-2 MA Gonçalves). surface expression of IL-2R, the intercellular adhesion molecule-I (ICAM-I) and the MHC class I antigens and References may inhibit the in vitro growth of tumour cells by arresting 1. Cardillo M, Hagan R, Abadi J, Abadi MA: CD4 T cell count, viral these cells in the G0/G1 cell cycle [40]. Although intrale- load and squamous intraepithelial lesions in women infected with the human immunodeficiency virus. 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Bell MC, Schmidt-Grimminger D, Turbat-Herrera E, Tucker A, Har- scientist can read your work free of charge kins L, Prentice N, Crowley-Nowick PA: HIV+ patients have increased lymphocyte infiltrates in CIN lesions. Gynecol Oncol "BioMed Central will be the most significant development for 2000, 76:315-9. disseminating the results of biomedical researc h in our lifetime." 28. Ahmed SM, Al H, Reid WM, Johnson MA, Poulter LW: The cellular Sir Paul Nurse, Cancer Research UK response associated with cervical intraepithelial neoplasia in HIV+ and HIV- subjects. Scand J Immunol 2002, 56:204-211. Your research papers will be: 29. Patel S, Chiplunkar S: Host immune responses to cervical can- available free of charge to the entire biomedical community cer. Curr Opin Obtet Gynecol 2009, 21:54-9. 30. Bontkes HJ, De Gruijl TD, Muysenberg AJC van den, Verheijen RH, peer reviewed and published immediately upon acceptance Stukart MJ, Meijer CJ, Scheper RJ, Stacey SN, Duggan-Keen MF, Stern cited in PubMed and archived on PubMed Central PL, Man S, Borysiewicz LK, Walboomers JM: Human papillomavi- rus type 16 E6/E7-specific cytotoxic T lymphocytes in yours — you keep the copyright women with cervical neoplasia. Int J Cancer 2000, 88:92-8. BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

The influence of human papillomavirus type and HIV status on the lymphomononuclear cell profile in patients with cervical intraepithelial lesions of different severity

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Springer Journals
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Copyright © 2009 by Goncalves et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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1750-9378
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10.1186/1750-9378-4-11
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19689792
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Abstract

Background: Immunological alterations are implicated in the increased prevalence of high-grade squamous intraepithelial lesions (HG-SIL) and persistent human papillomavirus (HPV) infection. This study evaluated the expression of CD4, CD8, CD25 (IL-2Rα) and CD28 antigens from SIL biopsies, stratified by HIV status and HPV-type. Biopsies specimens from 82 (35 HIV ) women with a normal cervix, low-grade (LG-SIL) or high-grade lesions (HG-SIL) were studied. CD molecule expression was evaluated by immunohistochemistry and HPV detection/typing performed using PCR techniques. Results: CD4 stromal staining was increased in patients with HPV18. Women with HPV16 infection showed decreased: a) CD8 and CD25 stromal staining, b) CD25 staining in LG-SIL epithelium and in HG-SIL stroma. In HIV women samples, CD28 epithelial staining and CD8 stromal staining surrounding metaplastic epithelium were less intense and even absent, as compared to HIV women. Both epithelial and stromal CD8 staining was more intense in the HG- + - SIL/HIV group than in the HG-SIL/HIV group. Positive correlations were observed between CD4/ CD25, CD4/CD28 and CD25/CD28 in the stroma and CD25/CD28 in the epithelium. Conclusion: HIV status and HPV-type may influence the lymphomononuclear cell profile present in the spectrum of cervical lesions. The knowledge of the infiltrating cell profile in cervical tumours may help the development of specific anti-tumoural strategies. Introduction viral load, both of which have been associated with the Human Immunodeficiency Virus (HIV)-infected women HPV persistence and SIL severity [1,2]. have a high prevalence of persistent human papilllomavi- rus (HPV) co-infection and also of squamous intraepithe- HPV may evade immune surveillance by shifting Th cell lial lesions (SIL) [1]. The progression from HIV infection polarization, down-regulating the expression of Major status to acquired immune deficiency syndrome is Histocompatibility Complex (MHC) class I molecules marked by a decreasing CD4 count and increasing HIV and reducing the function of intraepithelial antigen-pre- Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 senting cells, which may cause a lack of Th1 polarization of HPV and cervical intraepithelial lesions. Median age from the beginning of HPV infection. This causes a shift to was 36 years (range 19 50) for HIV-negative women and the production of IL-4, IL-6 and/or the immunosuppres- 29 years (range 1947) for HIV-positive women. These sive cytokine IL-10 [3,4]. Similarly, the progression from women were enrolled at several Gynaecological Reference HIV infection to AIDS has been associated with the polar- Services in the State of São Paulo, Brazil, from 1996 to ization to the Th2 cytokine profile in cervicovaginal secre- 2001 and the study protocol was approved by the institu- tion[5] which might contribute to the persistence of HPV tional Ethics Committee on human experimentation. infection [6]. Colposcopies were performed for routine in all women In the general population, tumour infiltrating lym- and when indicated, the same physician performed colpo- phocytes in cervical cancer have been associated with a scopically directed cervical biopsies or conization loop depressed function of cytotoxic T cells [7,8]; a down-regu- excision. Cervical fragments were fixed with formalin and lation of CD25 (α chain of the IL-2 receptor-IL-2Rα) acti- then slides were stained with haematoxylin-eosin (Sigma, vated cells [9] and a decreased proportion of CD4 T cells St. Louis, MO, USA) and histologically evaluated by two with a reversed CD4/CD8 ratio [10]. In HIV-infected experienced histopathologists, who also provided scoring women, little information is available regarding the infil- representing the sum of intensity and distribution of trating T cell phenotypes; however, deregulation of staining in fields, in a double-blinded protocol. Thin 5 peripheral blood CD4/CD8 T cell function is progres- μm sections were cut, placed on organosilane-pretreated sively impaired [11] with hyporesponsiveness of T CD8 slides and submitted to immunohistochemical assays. An cells to activation by the co-stimulatory CD28 and CD40L additional 10 μm section was used for DNA extraction molecules [12]. To date it is not clear whether these and HPV typing. defects in HIV-infected women also occur in the cervicov- aginal milieu and whether these might be implicated in Cervical biopsies were selected from HIV-negative or HIV- the increased prevalence of HG-SIL. positive women and they were stratified into 3 groups: normal (no evidence of SIL or HPV DNA), low-grade SIL The interaction of the T-cell receptor with the MHC mole- (cervical intraepithelial neoplasia I) and high-grade SIL cule is considered to be the first signal for T cell activation. (cervical intraepithelial neoplasia II-III and in situ carci- CD4 and CD8 antigens, observed on the surface of T cells, noma). are co-receptors that bind to non-polymorphic regions of MHC class II and class I molecules, respectively and trans- Immunohistochemistry duce signals that initiate T cell activation. The second sig- Serial sections (34 μm) obtained from paraffin-embedded nal for T cells stimulation is provided by the interaction of blocks, cut and mounted on APTS (3-aminopropyltri- co-stimulator molecules, including CD28 antigen on T ethoxy-silane, Sigma, Saint Louis, USA)-pre-treated glass cells and CD80/CD86 on the surface of activated antigen slides were dried by the method of Alves et al. [14]. To pre- presenting cells. Activation of T cells by antigens and co- vent cross-reaction with endogenous peroxidase, the stimulators stimulate the production and secretion of IL- slides were treated with 3% H O for 20 min and then 2 2 2, which in turn induces the production of the IL-2 recep- incubated overnight at room temperature with the follow- tor (IL-2R). The IL-2R is formed by three non-covalently ing primary antibodies at the following dilutions: CD4 associated protein called a (IL-2Ra), b (IL-2b) and g (IL- (mouse monoclonal, clone 1F6, IgG1 NovoCastra, New- 2g) chains. The activation of T cells by antigens, co-stimu- Castle upon Tyne, UK) 1:80; CD8 (mouse monoclonal, lators and IL-2 leads the expression of IL-2a (CD25) [13]. clone 4B1L, IgG2b NovoCastra), 1:200; CD25 (mouse To determine the possible associations between CD4, monoclonal, clone 4C9, IgG2b NovoCastra) 1:300; CD8, CD25 and CD28 antigens expression in several CD28 (goat policlonal, clone N20, SC 1625 Santa Cruz stages of cervical cancer development, we assessed the Biotechnology, Santa Cruz, CA, USA), 1:300. Sections of expression of these molecules in cervical biopsies strati- human tonsils were used as positive controls, and a nega- fied according to the severity of the lesion and HPV type tive control to detect background staining was performed detected (HPV16 and 18). by omitting the primary antibody. Isotype specificity for tonsil sections was confirmed by comparison of staining with irrelevant antibodies of the same isotype as the pri- Materials and methods Sample selection mary antibody. A control incubation to detect back- The study was conducted retrospectively on 82 (47 HIV- ground staining was performed omitting the primary negative and 35 HIV-positive), non-pregnant, non-lactat- antibody. ing, premenopausal women, which were recruited sequentially according to their arrival at the outpatient After incubation with the primary antibodies, immu- clinic. They were colposcopically and cytologically noperoxidase staining was performed using a universal screened, selected for cause, i.e., the presence or absence biotinylated secondary antibody mixed with a preformed Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 avidin and biotinylated horseradish peroxidase macro- from HPV16 (HPV16E7.667 and HPV16E7.774 primers) molecular complex (Novostain super ABC kit-NovoCas- and another one from HPV18 (HPV18E7.696 and tra), diluted 1:200 for CD4, CD8, CD25 and a different HPV18E7.799 primers) [20]. All DNAs were amplified biotinylated secondary antibody (Vector, BA 5000, anti- with these 4 sets of primers together with a set of primers goat IgG Burlingame, CA, USA) diluted 1:200 for CD28. for a housekeeping gene (globin) [21] as an internal con- Slides were alternately washed 3 times with PBS and TBST trol of amplification. Amplified DNA was applied to a (Tris-buffered saline; 0.05 M Tris, pH 7.4, containing 10% polyacrylamide gel, electrophoresed at 200 volts for 0.3% Tween 20) and diaminobenzidine (0.5 mg/mL) 1.45 h and stained with AgNO3 by the method of Sangui- (Sigma) was used as chromogen. Slides were then sequen- netti et al [22]. It is important to note that the absence of tially counter-stained with haematoxylin without acid for HPV16 or HPV18 did not exclude the presence of HPV 30 sec, washed in tap water, stained blue with ammonia- types other than 16 or 18, which could be identified if spe- cal water for 20 sec and exhaustively rinsed with tap water. cific primers for them were used. Only unambiguous Finally, slides were dehydrated and mounted for light amplifications were considered as positive. Lack of ampli- microscopy evaluation at 400× magnification using an fication with these primers or ambiguous amplifications eyepiece graticule connected to an objective lens, as after several repetitions were assigned as other HPV types. detailed elsewhere [15]. Statistical Analysis A minimum of 10 fields (total area equivalent to 0.75 According to the distribution of the variables and the mm ) was assessed per case. The expression of CD anti- number of groups compared, statistical analysis was per- gens was evaluated using a previously described semi- formed using the unpaired t, Mann-Whitney and Kruskall- quantitative method [16]. A total score representing the Wallis tests. The Spearman test (r) was used to calculate sum of intensity and distribution of staining in fields pre- correlations. P values were two-sided and the level of sig- senting dysplastic epithelium and in the respective subep- nificance was set at ≤ 0.05. All data were analysed using ithelial stroma was assigned to each case. The intensity of the Instat Mac 2.01 software (GraphPad software, CA, cellular staining in epithelium and stroma was scored as: USA). (0) no staining, (1) weak; (2) moderate; and (3) intense. Staining in the epithelium was scored as follows: (0), Results patchy basal; (1), diffuse basal; (2), diffuse full-thickness; Patient data (3), patchy and diffuse full-thickness positivity. Staining Among the 47 HIV-negative women, 4 exhibited no cervi- distribution in stroma was categorised as: (1), patchy sub- cal lesions and 43 presented SIL (13 LG-SIL and 30 HG- epithelial; (2) diffuse full-extent; (3) patchy and diffuse SIL). Among 35 women presenting HIV-infection, 5 full-extent positivity. Only sections showing epithelial exhibited no cervical lesions and 30 women presented SIL and stromal tissue were considered for the analysis. (21 LG-SIL and 9 HG-SIL). According to CD4 counts, 9 HIV-positive women presented CD4 counts > 500 cells/μL HPV identification and typing and 26 of them CD4 counts < 500 cells/μL. The median HPV DNA obtained from paraffin blocks [17] was ampli- peripheral CD4 T-cell count of the group presenting with fied by PCR using 12.5 pmoles of dNTP, 25 pmoles of both SIL and HIV infection was 360 cells/μL. Patient data each primer, 1.5 U Taq DNA polymerase (Gibco, USA), 5 stratified by lesion severity (LG-SIL, HG-SIL), HIV status μL of 10× enzyme buffer, 20 μg of genomic DNA (50 ng) and HPV type are shown in Table 1. and distilled deionised H O to complete a total volume of 50 μL. The mixture was processed in a thermocycler appa- CD4 and CD8 staining ratus (MJ Research, MA, USA) under the following cycling CD4 and CD8 staining was observed in cells presenting conditions: 1 cycle at 95°C for 5 min, 30 cycles at 95°C the morphology of lymphomononuclear cells. CD4 stain- for 30 sec, at 55°C for 30 sec and at 72°C for 1 min and ing was observed throughout the squamous epithelium, finally 1 cycle at 72°C for 10 min and then at 4°C indefi- and remarkably more intense in the stroma under the nitely. transformation zone and surrounding endocervical glands, especially near inflammatory areas. CD4 stromal Since formalin may degrade DNA, producing DNA frag- staining was increased, particularly in specimens present- ments of different length, several pairs of primers were ing HPV18, when compared with those without HPV18 used. Primers GP5+ and GP6+ [18], which amplify small infection. However, in the HIV-positive/HPV18 group, DNA fragments and primers MY09 and MY11 [19], which CD4 stromal staining showed to be decreased, when com- amplify longer DNA fragments, were used for generic HPV pared with those in the HIV-negative/HPV18 group (P = amplification. Since HPV16 and HPV18 are the types 0.02) (Fig 1). Few koilocytotic cells presented CD8 stain- most frequently associated with cervical neoplasia, 2 sets ing. A significant decreasing of CD8 stromal staining was of specific primers were used to detect the E7 gene, one observed when specimens with HPV16 infection alone Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 Table 1: Data stratified by histopathological diagnosis, HIV status and HPV types. HPV- HPV16 HPV18 HPV16/18 HPVX n(%) n(%) n(%) n(%) n(%) Normal LG-SIL HG-SIL LG-SIL HG-SIL LG-SIL HG-SIL LG-SIL HG-SIL LG-SIL HG-SIL HIV-negative (n = 47) 4 (8.5) 1(2.1) 3(6.4) 5(10.7) 10(21.3) 2(4.3) 6(12.8) 2(4.3) 6(12.8) 3(6.4) 5(10.7) HIV-positive (n = 35) 5 (14.3) - - 4(11.4) 1(2.9) - 2(5.7) 1(2.9) 1(2.9) 16(45.7) 5(14.3) Total (n = 82) 9(11.0) 1(1.2) 3(3.7) 9(11.0) 11(13.4) 2(2.4) 8(9.8) 3(3.7) 7(8.6) 19(23.2) 10(12.2) HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; HPV X = Unidentified HPV types; LG-SIL = Low-grade Squamous Intraepithelial Lesion; HG-SIL = High-grade Squamous Intraepithelial Lesion. were compared with specimens without HPV16. How- CD25 (IL-2Rα) and CD28 staining ever, CD8 stromal staining seemed to be increased in In most specimens, CD25 staining was observed in basal specimens with both HIV and HPV16 infections, when and immature epithelium, independent of HIV status (Fig compared with specimens from patients HIV-negative/ 4). However, CD25 epithelial staining varied according to HPV16 (P = 0.004) (Fig 2). CD8 stromal staining was both HPV status and lesion severity, being more frequent more frequently localised surrounding the metaplastic in the HPV16-positive/HG-SIL group than in the HPV16- epithelium and more intensively in specimens from HIV- negative/LG-SIL group (P = 0.03) (Fig 5). CD25 stromal positive group, compared to the HIV-negative group (P = staining was primarily observed in lymphomononuclear 0.001). In the HIV-positive/SIL group, clusters of lympho- cells. More intense CD25 stromal staining was observed mononuclear cells were observed around vessels and adja- in HPV16-negative/HG-SIL group than in the HPV16-neg- cent to the basement membrane. HIV-positive/SIL group ative/LG-SIL group (P = 0.002) (Fig 6). Both epithelial exhibited a more intense CD8 stromal staining than and stromal CD25 staining (respectively, P = 0.02 and observed in the HIV-negative/SIL group (P = 0.0005) 0.005) were associated with HPV type infection (HPV16, (Fig 3). HPV16/18 and other HPV types), when compared to HPV-negative group. No significant difference was found A (n=8) B (n=35) C (n=18) D (n=3) E (n=22) F (n=15) G (n=13) H (n=3) Mean (± SD) CD4 stromal staining H Figure 1 Increased immunohistochemica PV18 infection (Kruskal-Wallis = 0.02) l expression of CD4 stromal staining of cervical specimens (mean ± SD) in the presence of Increased immunohistochemical expression of CD4 stromal staining of cervical specimens (mean ± SD) in the presence of HPV18 infection (Kruskal-Wallis = 0.02). A = HPV-negative; B = HPV18-negative; C = HPV18-positive; D = both HIV and HPV are negative; E = both HIV and HPV18 are negative; F = HIV-negative, HPV18-positive; G = HIV-positive, HPV18-negative; H = both HIV and HPV18 are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus. Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 A (n=11) B (n=33) C (n=26) D (n=3) E (n=14) F (n=22) G (n=19) H (n=5) Mean (± SD) CD8 stromal staining Reduced immu H Figure 2 PV16 infection (one way A nohistochemical NOVA test = 0.004) expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of Reduced immunohistochemical expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of HPV16 infection (one way ANOVA test = 0.004). A = HPV-negative; B = HPV16-negative; C = HPV16- positive; D = both HIV and HPV are negative; E = both HIV and HPV16 are negative; F = HIV-negative, HPV16-positive; G = HIV-positive, HPV16-negative; H = both HIV and HPV16 are positive. HPV = Human papillomavirus; HIV = Human Immunode- ficiency Virus. A (n= 40) B (n= 30) C ( n= 7) D (n= 33) E (n= 30) F (n= 9) G (n= 6) M ean ( ± S D ) C D 8 s tr o m a l s taining Increasin H Figure 3 IV-infection (one way ANOVA test = 0.0005) g immunohistochemical expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of Increasing immunohistochemical expression of CD8 stromal staining of cervical specimens (mean ± SD) in the presence of HIV-infection (one way ANOVA test = 0.0005). A = HIV-negative; B = HIV-positive; C = both HIV and SIL are negative; D = HIV-negative, SIL-positive; E = HIV-positive, SIL-negative; F = both HIV and SIL are positive; G = all HIV, SIL and HPV16 are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; SIL = Squamous Intraepithelial Lesion. Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 CD25 staining Figure 4 in a basal and immature cervical squamous epithelium CD25 staining in a basal and immature cervical squamous epithelium. A (n=4) B (n=35) C (n=21) E (n=13) D (n=16) F (n=18) Mean (± SD) CD25 epithelial staining H Figure 5 Reduced immu PV16 infection (Kruskal-Wallis = 0.03) nohistochemical expression of CD25 epithelial staining of cervical specimens (mean ± SD) in the absence of Reduced immunohistochemical expression of CD25 epithelial staining of cervical specimens (mean ± SD) in the absence of HPV16 infection (Kruskal-Wallis = 0.03). A = all HIV, HPV and SIL are negative; B = HPV16 total; C = HPV16-negative, LG-SIL-positive; D = both HPV16 and LG-SIL are positive; E = HPV16-negative, HG-SIL-positive; F = both HPV16 and HG-SIL are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; SIL = Squamous Intraepithelial Lesion; LG-SIL = Low-grade Squamous Intraepithelial Lesion; HG-SIL = High-grade Squamous Intraepithelial Lesion. Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 A (n=4) B (n=21) C (n=17) D (n=16) E (n=13) F (n=18) Mean (± SD) CD25 stromal staining H Figure 6 Reduced immu PV16 infection (Kruskal-Wallis = 0.002) nohistochemical expression of CD25 stromal staining of cervical specimens (mean ± SD) in the absence of Reduced immunohistochemical expression of CD25 stromal staining of cervical specimens (mean ± SD) in the absence of HPV16 infection (Kruskal-Wallis = 0.002). A = all HIV, HPV and SIL are negative; B = HPV16 total; C = HPV16-negative, LG-SIL-positive; D = HPV16-negative, HG-SIL-positive; E = both HPV16 and LG-SIL are positive; F = both HPV16 and HG-SIL are positive. HPV = Human papillomavirus; HIV = Human Immunodeficiency Virus; SIL = Squamous Intraepithelial Lesion; LG-SIL = Low-grade Squamous Intraepithelial Lesion; HG-SIL = High-grade Squamous Intraepithelial Lesion. in epithelial or stromal CD25 staining in patients present- mens with SIL, stratified according to HIV status and HPV ing or not HG-SIL, regardless of HIV status. type. CD28 stromal staining was observed in lymphomononu- In the present study, specimens harbouring HPV18 clear cells, particularly under the epithelial lesion and showed a decreased CD4 stromal staining and HPV16 under the transformation zone, sometimes as patchy infection was associated with an increased CD8 stromal agglomerates, resembling germinative centres. CD28 epi- staining, in HIV-infected women. Since CD8 cells are thelial staining was less frequently observed in HIV-nega- recruited preferentially to cervical lesions with progres- tive patients than in HIV-positive patients (P < 0.0001), sion to invasion [26], and in regressing CIN1 lesions, irrespective of the HPV type infection or the SIL severity CD4+ cells predominated within the stroma with highest (Data not shown). CD4/CD8 ratio compared with progressive CIN1 [27-29], these findings may explain the progression or regression Positive correlations were observed between CD4 and of the HPV infection towards carcinogenesis, particullarly CD25 in stroma (r = 0.32, p = 0.01), CD4 and CD28 in among HIV-infected women. stroma (r = 0.33, p = 0.03) and between CD25 and CD28 both in epithelium (r = 0.31, p = 0.03) and in stroma (r = HPV16 is considered to be the major etiologic agent of 0.40, p = 0.004). squamous cervical carcinoma and keratinocytes (non- professional antigen presenting cells) are reported to directly present viral antigens to cytotoxic T lymphocytes Discussion A cellular infiltration composed essentially of CD4 and by cross priming [1], whereas HPV18 is more frequently macrophages is frequently observed in condyloma under associated with adenocarcinoma. The mechanisms of spontaneous regression [23], and a decrease in the viral peptide presentation are less studied than those number of Langerhans cells in the transformation zone reported for squamous carcinoma. Increased HPV16 E7- has been associated with a reduced in vitro T cell prolifer- specific T helper cell responses are associated with persist- ation and IL-2 production [24]. With the progression to ent HPV16 infection, HG-SIL and invasive cervical carci- HG-SIL, the number of immature Langerhans cells noma [30]. It is accepted that HPV infects keratinocytes increase with a consequent deficient function [24,25]. In and interferes with the local cytokine expression [31-33], this study, we evaluated the intensity and distribution of but it is still unknown how HPV together with HIV influ- CD4, CD8, CD25 and CD28 molecules in cervical speci- ences local T cell function. Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:11 http://www.infectagentscancer.com/content/4/1/11 In this study, CD8 staining increased in HIV infected tory T cells [13], the role of these cells in SIL development group and further increased with concomitant SIL. In should be further evaluated performing longitudinal stud- /SIL addition, both the epithelium and stroma of the HIV ies double staining CD4 and CD25. group presented an increased expression of CD8 com- pared to the HIV /SIL group. These findings indicate a In conclusion, our results indicate that HPV type and HIV continuous stimulation by persistent HPV infection, in status may influence lymphomononuclear cell marker agreement with other authors [27,28]. Because of the staining in cervical lesions. A particular immune response proximity of the columnar epithelium to the stroma and may be triggered depending on the balance of these varia- the lack of keratinocytes in the endocervix, the findings of bles, culminating in the progression to cancer. Knowledge this study suggest that stromal lymphomononuclear CD4 of the lymphomonuclear cell profile and the tumour cells (T CD4 and macrophages) may cope better than CD8 immunogen may help understanding HPV evasion mech- in HPV18 infection. In other words, macrophages may be anisms and enable new proposals of anti-tumoral treat- the major APC in stroma and CD4 cells (T lymphocytes ment, especially therapeutic vaccines against cervical and macrophages) may be involved in immunity against lesions. HPV18. Competing interests Several studies have shown that peripheral blood lym- The authors declare that they have no competing interests. phocytes produce IL-2[34,35] and generate cytotoxic activity [36] after specific HPV16 E7 protein stimulation. Authors' contributions De Gruijl et al. [37] reported an increased IL-2 production MAG Gonçalves designed and coordinated the work, the by peripheral blood CD4 T cells, associated with persist- results analyses and manuscript writing. EA Donadi par- ent HPV infection and progression of a premalignant ticipated in the design of the study and in the immunolog- lesion and a higher production of Th2 cytokines related to ical evaluation and data interpretation, and EG Soares was cervical malignancy. In our study, HPV16 associated with responsible for slides histopathological interprestion. All decreased stromal CD8 and CD25 expression and authors read and approved the final manuscript. increased CD25 staining was observed in both epithelium and stroma with HG-SIL. The expression of IL-2R on Acknowledgements We wish to thank Ana Maria Anselmi Dorigan for excellent technical assist- keratinocytes and the IL-2 secretion has been reported in ance. different phases of cancer cell development [38,39]. Bind- ing of IL-2 to IL-2R on tumour cells may down-regulate Financial support: FAPESP (01/02908-2 MA Gonçalves). surface expression of IL-2R, the intercellular adhesion molecule-I (ICAM-I) and the MHC class I antigens and References may inhibit the in vitro growth of tumour cells by arresting 1. Cardillo M, Hagan R, Abadi J, Abadi MA: CD4 T cell count, viral these cells in the G0/G1 cell cycle [40]. Although intrale- load and squamous intraepithelial lesions in women infected with the human immunodeficiency virus. 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Bontkes HJ, De Gruijl TD, Muysenberg AJC van den, Verheijen RH, peer reviewed and published immediately upon acceptance Stukart MJ, Meijer CJ, Scheper RJ, Stacey SN, Duggan-Keen MF, Stern cited in PubMed and archived on PubMed Central PL, Man S, Borysiewicz LK, Walboomers JM: Human papillomavi- rus type 16 E6/E7-specific cytotoxic T lymphocytes in yours — you keep the copyright women with cervical neoplasia. Int J Cancer 2000, 88:92-8. BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes)

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Infectious Agents and Cancer – Springer Journals

Published: Aug 18, 2009

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