An infection, human papillomavirus (HPV), is the major causal factor in the development of cervical cancer and its precursor lesions . Studies of circulating, peripheral blood mononuclear cells have suggested that the T helper type 1 (Th1) immune response, involving the production of cytokines that promote cell-mediated immunity, is important in the regression of HPV infection and cervical lesions [2–5]. Furthermore, in women who have compromised immunity as a result of HIV, low circulating levels of CD4 T cells are associated with high rates of HPV infection and cervical neoplasia [6–9]. To our knowledge, however, no studies in HIV-seropositive women or other immune compromised populations have investigated the functional status of local cellular immunity in epithelium or skin, presumably the most important immunological factor, to measure its relationship with HPV infection or the development of cervical epithelial lesions.
Cutaneous anergy testing evaluates the ability to mount a delayed type-hypersensitivity (DTH) response to previously encountered antigens (e.g. mumps) and is an in-vivo measure of cell-mediated immunity at a local skin surface that reflects the number and functional capacity of the several different types of immune cells that comprise the Th1 immune response [10,11]. In the past, anergy testing was used in screening for tuberculosis. Although anergy testing is no longer recommended in routine clinical use, largely because of its limited reproducibility and other factors , it does provide information about the capacity of HIV-infected patients to mount an effective local immune response [13–25]. We have previously found that anergy status before the initiation of HAART independently predicts post-HAART morbidity and death [26,27], even after controlling for CD4 T-cell count, HIV-RNA level, and other factors.
Anergy testing in HIV-seropositive women might, therefore, measure aspects of local cellular immune status in the skin and epithelium not reflected by circulating CD4 T-cell counts and plasma HIV-RNA levels that could be important to the control of infections, including HPV. To study this issue, we measured the independent associations of anergy with the detection of cervical HPV infection and cervical neoplasia in a large prospective cohort of HIV-seropositive and seronegative women.
Materials and methods
Subjects and data collection
A total of 2058 HIV-seropositive and 568 seronegative women were enrolled into the Women's Interagency HIV Study (WIHS) between October 1994 and November 1995, as previously described . At baseline, the HIV-seropositive women had a similar distribution of demographic and risk behaviors as female AIDS cases reported in national US registries, suggesting that enrollment achieved a representative sample . All participants provided written informed consent, and approval for the study was obtained from each local institutional review board. Study visits were scheduled every 6 months, during which the women completed a detailed questionnaire administered by study personnel and underwent physical and gynecological examinations. As previously described [6,29,30], a cervicovaginal lavage for HPV-DNA testing was collected followed by a Pap smear, using an Ayres spatula and cytological brush. All Pap smears were interpreted centrally using the 1991 Bethesda System criteria . T-cell subsets were determined by immunofluorescence using flow cytometry in laboratories participating in the AIDS Clinical Trials Quality Assurance Program. Plasma HIV-RNA levels were measured using a nucleic acid sequence-based amplification technique (Organon Teknika, Durham, North Carolina, USA) with a lower threshold of detection of 4000 copies/ml.
Anergy testing was performed annually through March 2000 using the Mantoux technique with 0.1 cc Candida albicans antigen (Candin; Allermed Laboratories, Inc., San Diego, California, USA), tetanus toxoid (1: 5 dilution of fluid toxoid; Connaught Pharmaceuticals, now Aventis Pasteur, Swiftwater, Pennsylvania, USA), and mumps skin test antigen (Connaught Pharmaceuticals). Each antigen was separately injected intradermally into the volar surface of the forearm, and the reaction to each antigen was measured and recorded separately as the size of the induration in millimeters (mm). If no induration was present, 0 mm was recorded. Test administration and reading were standardized using a training video from the US Centers for Disease Control and Prevention. Anergy results were included in the analysis only if a measurement was recorded for all three antigens (C. albicans, tetanus toxoid, mumps) and the test was evaluated 2–3 days after administration as per protocol.
Human papillomavirus DNA testing
HPV testing was conducted using an L1 consensus primer MY09/MY11/HMB01 polymerase chain reaction (PCR) assay, as previously described [6,29,30]. Amplification of a 268 base pair cellular β-globin DNA fragment was included in each assay as an internal control. After amplification, the presence of HPV-DNA sequences was assessed using filters individually hybridized with biotinylated oligonucleotide probes for specific HPV types, as well as a general probe mixture able to detect most anogenital HPV DNA. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73 were considered oncogenic, and HPV types 6, 11, 26, 32, 40, 53, 54, 55, 61, 66, 69, 70, 71, 81, 82, 83, 84, 85, and 92 were considered non-oncogenic. This classification is consistent with one suggested by the International Agency for Research on Cancer based on a meta-analysis , except that we did not include HPV types 26, 53, and 82 as oncogenic, in keeping with a subsequent International Agency for Research on Cancer expert panel . We also did not include HPV type 66 as one of the oncogenic HPV types because of the paucity of data supporting its classification as an oncogenic type . In any event, whether or not HPV type 66 was considered oncogenic or non-oncogenic was highly unlikely to affect our findings (e.g. at baseline HPV type 66 was found in only 3.8% of HIV-seropositive subjects). Specimens that were positive by the general probe mixture, but were not positive for a specific HPV type, were categorized as being non-oncogenic HPV positive.
We restricted a priori the analysis to women who had their first available anergy test result before 1 July 1996 (n = 1301), which is when HAART came into widespread use in the WIHS cohort. This restriction was made because of the bias inherent in which patients do and do not initiate HAART, as well as when they start, termed “selection by indication”; i.e., patients started on HAART by their physicians are on average less healthy than those not started on HAART, even within a given CD4 T-cell count/HIV-RNA stratum . Furthermore, the impact of HAART usage on anergy test results and their proper interpretation is uncertain. The definition of HAART was based on the DHHS/Kaiser Panel guidelines . Women who had had a hysterectomy (n = 146) were excluded at baseline, or censored at the visit before hysterectomy if it occurred during the investigation. Overall, 1029 HIV-seropositive and 272 HIV-seronegative women contributed at least one anergy test result to the analysis.
Anergy was defined as an induration of less than 2 mm for all three antigens. If any one of the antigens induced induration that measured 2–4 mm, but none measured 5 mm or more, the results were considered moderately reactive, whereas patients were defined as non-anergic if there was induration of 5 mm or more for any one of the antigens. Although a cutoff of 2 mm has often been used clinically , the Centers for Disease Control and Prevention suggest that a cutoff of 5 mm or more be used if a clinician does elect to use anergy testing in screening for tuberculosis . Non-anergic women were the comparison group in all analyses. The CD4 T-cell count was categorized into three conventional clinical strata (i.e. > 500, 200–500, and < 200 CD4 cells/μl), and the plasma HIV-RNA level into four categories (i.e. < 4000, 4000–20 000, 20 001–100 000, and > 100 000 copies/ml) that were found to be associated with HPV infection in previous studies [6,29,30]. Women were categorized as having a non-oncogenic infection if only non-oncogenic HPV types were detected or HPV DNA was detected but could not be typed (not representing a known oncogenic type). Cervical specimens in which β-globin could not be amplified were considered inadequate, and were excluded from analysis.
The association between anergy and the prevalent detection of HPV DNA or squamous intraepithelial lesions (SIL) was measured using multivariate logistic regression models that incorporated generalized estimating equations (GEE) with an exchangeable correlation structure to summarize results across all HPV types and across all visits before 1 July 1996 (see above), and to adjust the estimates of standard error for repeated observations involving the same women over time . The GEE approach used made few assumptions regarding the nature of the intra-individual (i.e. subject-specific) correlations. For the analysis of SIL, women with borderline cytological results (called atypical squamous cells of undetermined significance) at a particular visit were excluded from analysis for that particular visit.
We used Cox models to evaluate the relationship between the first available anergy result before 1 July 1996, and the incident detection of HPV infection. For this analysis, follow-up time was allowed to go beyond 1 July 1996, although women who did subsequently use HAART were censored at the time of HAART initiation (an additional analysis, described below, was restricted to the period before 1 July 1996). If a subject did not have an anergy test result from their initial study visit, the first available anergy result was used and that visit was set as their baseline. The mid-interval (i.e. the midpoint calendar date between two consecutive visits) was defined as the time of each incident event, and non-anergic women were used as the reference group. As in the GEE analysis of prevalent HPV detection, the detection of each HPV type was modelled separately and the results were summarized across all the types. This was accomplished in the Cox models by using the Wei Lin Weissfeld marginal model approach to adjust the results for possible correlations between multiple incident infections. The major assumption of this approach is that anergy affects all HPV types fairly equally. Although recent data suggests that the relationship of immune status with HPV infection may vary by HPV type, this is a matter of degree, and immunodeficiency is associated with an increased risk of HPV infection for essentially all types evaluated . Our current results represent an appropriate weighted average of the overall effects of anergy on the risk of HPV infection, and we did not have sufficient data to analyse the HPV-DNA results on an HPV type-specific basis. Patients who had missing HPV data (or cytology) for two visits in a row for any reason were censored at the time of their last visit with complete data and, as mentioned, subjects who had a hysterectomy during follow-up were censored at the visit before that procedure. Factors that changed over time were modelled as time-dependent covariates.
As an alternative approach to the analysis of incident HPV detection, we used GEE to measure the relationship of anergy status at each visit with the detection of a new (previously undetected) HPV type at the following visit; restricted to visits before 1 July 1996 when HAART use began to become common in our cohort. We could not, however, prospectively study HPV persistence, because there were too few endpoints. That is, HPV persistence is optimally assessed on a type-specific basis using only incident infections so that the time of onset can be estimated, and too few events of new HPV detection followed by resolution occurred before 1 July 1996 to analyse in this study. Similarly, there were too few incident SIL to assess risk factors for persistent lesions. The GEE models for these endpoints did not converge (i.e. the software could not calculate an odds ratio because there were insufficient data).
Patient characteristics at the time of their first anergy test result (the ‘baseline visit’) are shown in Table 1. Anergic women were older than non-anergic women, had greater immunosuppression (as reflected by the CD4 T-cell count and HIV-RNA level), and were less likely to have had a recent male sex partner (Table 1). Moderately reactive women were also more likely than non-anergic women to be immunosuppressed, and both moderately reactive and anergic women were more likely than non-anergic HIV-seropositive women to be using some form of antiretroviral therapy (i.e. monotherapy or combination therapy). The cross-sectional prevalence of oncogenic HPV detection at baseline showed a significant gradient (P trend < 0.0001), with the highest prevalence among anergic women, the lowest among non-anergic women, and an intermediate prevalence of oncogenic HPV among moderately reactive women.
In univariate GEE analysis (summarizing cross-sectional data across all visits), oncogenic HPV infection was significantly associated with anergy [odds ratio (OR) 1.97; 95% confidence interval (CI) 1.54–2.52; Table 2]. This association was of only borderline statistical significance [adjusted odds ratio (AOR) 1.22; 95% CI 0.92–1.63] after adjustment for all covariates, including combined CD4/HIV-RNA strata (13 separate strata); HIV treatment (none, monotherapy, combination therapy); age (< 25, 25–29, 30–34, 35–39, 40–44, ≥ 45 years); race/ethnicity (white, black, Hispanic, other); lifetime number of male sex partners reported at baseline (0–4, 5–9, 10–49, ≥ 50); number of male sex partners in past 6 months (0, 1, 2, ≥ 3); and self-report of cervical treatment (yes/no). We termed this our ‘complete’ model. Conversely, anergy had a highly significant association (P < 0.007) with prevalent SIL even in our complete model (AOR, 1.70; 95% CI 1.16–2.48).
Moderate reactivity, as expected, showed an intermediate level of association with SIL and oncogenic HPV detection, which for SIL (but not for oncogenic HPV) approximated statistical significance in multivariate models (Table 2). Neither moderate reactivity nor anergy, though, showed any association with the prevalent detection of non-oncogenic HPV (Table 2).
The incident detection of oncogenic HPV and its relationship with anergy was assessed in multivariate Cox models (Table 3). Similar to the findings in the GEE analysis of HPV prevalence, there was a highly significant univariate association between anergy and the incident detection of oncogenic HPV [hazard ratio (HR) 1.79; 95% CI 1.48–2.17] and whereas control for other factors, especially the CD4 T-cell count, attenuated this statistical association, it remained of borderline significance in the complete model [adjusted hazard ratio (AHR) 1.24; 95% CI 0.99–1.56]. To examine further this relationship we used GEE to evaluate the relationship of anergy status at a given visit with the detection of a new oncogenic HPV type at the following visit (Table 4). This association was highly significant in univariate analysis (OR 2.36; 95% CI 1.51–3.67), and was of borderline significance in the complete model (AOR 1.50; 95% CI 0.92–2.45).
The incident detection of SIL and its relationship with anergy was also assessed in Cox models (Table 3). The relationship was significant in univariate analysis (HR 1.38; 95% CI 1.02–1.88), but there was no association in the complete model, and moderate reactivity had only a non-significant positive association with incident SIL in the complete model (AHR 1.30; 95% CI 0.85–2.00). Incident SIL is less common than the incident detection of HPV, though, and the smaller number of endpoints probably accounts for the wide confidence intervals in the fully adjusted models.
In this large observational cohort study of HIV-seropositive and seronegative women, we found that anergic women were more likely to have prevalent SIL and incident detection of oncogenic HPV infection than non-anergic women, after adjustment for combined CD4 T-cell/HIV-RNA strata and other factors. These results suggest that anergy status provides information regarding immune control of HPV and SIL in the cervical epithelium not accounted for by CD4 T-cell and HIV-RNA levels in circulation.
Anergy testing evaluates the ability of the immune system to mount a DTH reaction, a cell-mediated immune response, to antigens previously encountered through natural exposure or vaccination . In brief, at the time of initial antigen exposure, memory T cells specific for that antigen are generated. Then during anergy testing, antigen-presenting cells process the injected antigen, and present the relevant epitopes to CD4 T cells through binding of the processed antigen to human leukocyte antigen (HLA) class II molecules expressed on the antigen-presenting cell surfaces. These complexes are recognized by the memory T cells (usually CD4 Th1 T cells, but CD8 T cells can also participate), which activates the T cells to produce inflammatory cytokines (IFN-γ, TNF-β), and results in the accumulation of fibrin, phagocytes, and plasma at the injection site, which is recognized on the skin surface as induration. The absence of induration 48–72 h after injection with ubiquitously encountered antigens (i.e. anergy) indicates deficient local cellular immune function in the skin.
Although the association of anergy with HPV infection and the development of SIL observed in this study was undoubtedly affected by the well-documented problems associated with anergy testing [10,12], including the poor reproducibility of anergy test results, this would most likely have caused random misclassification and, therefore, bias towards the null . The odds ratios and hazard ratios we have reported are thus probably conservative, although we can not fully rule out the possibility that some misclassification occurred in a non-random fashion (e.g. greater difficulty, for some reason, in the interpretation of anergy results in those with worse immune function). Another limitation is that we were unable to evaluate each HPV type separately. The relationship between immune status and HPV infection may differ depending on the HPV type , and it would be of interest in future studies to measure the association between anergy and HPV infection on a type-specific basis. Immunodeficiency is, however, associated with an increased risk of HPV infection for most types, and our results represent an appropriate weighted average of the overall effects of anergy on the risk of HPV infection.
Few previous studies examined the relationship between the results of antigen skin testing and HPV or anogenital lesions. A small study of men with recurrent penile condylomata acuminata (n = 30) found that these patients had smaller reactions to skin test antigens than controls, and that cases with disease duration of more than one year were significantly more likely to be anergic than the controls . In a small study of skin responses to HPV 16 E7 (a viral oncoprotein), responses were observed in eight out of 11 cervical neoplasia regressors compared with two out of 30 with persistent/progressive cervical neoplasia, and none out of seven cervical cancer patients .
In HIV-seropositive patients clinical staging is accomplished, largely, by measuring the CD4 T-cell count and plasma HIV-RNA levels. These biomarkers only partly characterize the immune status of HIV-seropositive patients. The functional capacity of CD4 T cells that are present (e.g. the ability to recognize antigen HLA complexes and secrete cytokines) also matters, as do the levels and functions of other immune cells [41,42]. We and others have, for example, shown that the HIV viral load has an inverse association with the density of cervical Langerhans cells (professional antigen-presenting cells) , and also that anergy status predicts morbidity and death independently of the CD4 T-cell count in HIV-seropositive subjects [14–16,27]. To the best of our knowledge, this is the first study in HIV-seropositive women (or any immune compromised populations) to show that anergy is also associated with an increased risk of oncogenic HPV infection and the development of cervical epithelial lesions. The current data, though, do not allow us to identify the specific components of DTH and cellular immunity that are deficient or the nature of these deficiencies that account for the relationship of anergy with HPV and SIL. Furthermore, although the relationship between DTH responses in the skin and control of HPV infection in the cervix raises the question of whether there might be important biological linkages between immune function at the two sites, analogous to the concept (based primarily on mouse models) of there being a common mucosal immune system, we did not directly measure local immune responses in the cervical epithelium. The incident detection of oncogenic HPV infection and the detection of cervical neoplasia were themselves our only measures of immune control at the level of the cervix (representing only indirect evidence). Collaboration between clinical and laboratory investigators may be useful in correlating functional deficiencies, in particular DTH-related cells with HPV infection and HPV-related cervical disease, and in more directly correlating cutaneous DTH responses with cellular immunity in the cervix.
Whereas knowledge of the underlying mechanisms is incomplete, and we are limited by the lack of a direct measure of cervical immune function, we would point out that the current study provides empiric evidence that cutaneous anergy testing may be a biomarker of the immune system's ability to control HPV infection in the cervical epithelium in HIV-seropositive women. The current findings could even have implications for HPV infection and SIL in HIV-seronegative women. HPV prevalence has been reported to be increased in elderly women in some populations, possibly as a result of the immune senescence associated with aging [44,45], and even in younger women the capacity to respond to the antigens of infectious agents at epithelial surfaces may vary. It is possible, therefore, that cutaneous anergy testing could play some role clinically in identifying women at increased risk of oncogenic HPV infection and cervical disease. Anergy testing, however, would first need to be vastly improved (e.g. through the use of HPV antigens), as the risks of HPV infection and disease associated with anergy test results in this study were relatively modest. We note that despite the availability of an effective HPV vaccine , an entire generation of women will remain unvaccinated, and methods for identifying those at high risk of cervical disease remain an important public health goal.
In conclusion, the findings of this study suggest that cutaneous anergy testing may measure aspects of local cellular immune function in epithelial tissues that are important to the control of HPV and the development of SIL, and that in HIV-seropositive women are not fully accounted for by the circulating CD4 T-cell count and HIV-RNA level. Further research is, therefore, warranted to provide a better understanding of the functional deficiencies in DTH-related immune cells that correlate with HPV infection, and the relationship of this with cellular immunity in the cervix. In addition to its scientific interest, such an effort could have potential clinical implications.
The authors wish to thank Melissa Fazzari, Ji Yon Bang, and Dr Ruth Greenblatt for their contributions to this study, and Victor Kamensky for his programming support.
Sponsorship: HPV-DNA testing was funded through R01-CA-085178. All specimens and other data in this study were collected by the Women's Interagency HIV Study (WIHS) Collaborative Study Group with centers (principal investigators) at New York City/Bronx Consortium (Kathryn Anastos, MD); Brooklyn, NY (Howard Minkoff, MD); Washington, DC Metropolitan Consortium (Mary Young, MD); The Connie Wofsy Study Consortium of Northern California (Ruth Greenblatt, MD); Los Angeles County/Southern California Consortium (Alexandra Levine, MD); Chicago Consortium (Mardge Cohen, MD); Data Coordinating Center (Stephen Gange, PhD). The WIHS is funded by the National Institute of Allergy and Infectious Diseases with supplemental funding from the National Cancer Institute and the National Institute on Drug Abuse (U01-AI-35004, U01-AI-31834, U01-AI-34994, U01-AI-34989, U01-AI-34993, and U01-AI-42590). Funding is also provided by the National Institute of Child Health and Human Development (U01-HD-32632) and the National Center for Research Resources (M01-RR-00071, M01-RR-00079, M01-RR-00083).
This study was presented in part at the 22nd International Papillomavirus Conference, May 2005, Vancouver, Canada [abstract M-02].
Conflicts of interest: None of the authors have a commercial or other association that might pose a conflict of interest.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
anergy; CD4 T-lymphocyte count; cellular immunity; HIV; human papillomavirus