The incidence of viral sexually transmitted infections (STIs) has shown an upward trend worldwide.1 Condyloma acuminata (anogenital warts) are one of the commonest STIs caused by human papillomaviruses (HPVs),2 primarily by HPV-6 and HPV-11 (in 90% of cases)3-6 and occasionally by HPV-16 and HPV-18.7,8 Warts are generally papillomatous lesions occurring on the mucus membrane or skin of the external genitalia as well as in the perianal region. Patients are not generally satisfied with the standard treatment of warts because of high recurrence rate caused due to persistence of HPV in normal-presenting adjacent skin,9 adding to the difficulty of the physicians. For successful treatment outcome, a more comprehensive understanding of the host immune response and its interaction with HPV is necessary. Detailed understanding of the host immune mechanism will lead to better clinical management in the future. Few studies have reported suppression of local immune response mediated by dysfunctional natural killer (NK) cells, dysfunctional Langerhans cells, and resultant T-helper cells skewing toward Th2 phenotype at the local disease site.10-12 The Langerhans cells in HPV-infected lesions appeared in clumps with altered distribution and morphology13,14 and no surface expression of maturation marker CD83.15 It has been found that proportion of T-regulatory cell correlated with the size of the warts. Studies have also reported reduced interleukin (IL)-10 and transforming growth factor (TGF)-□1 expression and increased interferon (IFN)-□ and IL-2 expression in small-sized warts.16 In addition, the serum levels of IL-6 were increased and IFN-□ were decreased in HPV-infected patients.17 All these findings suggested an immune imbalance in patients with HPV-infected genital warts. The aim of this study was to understand how HPV antigen (6 and 11)-specific immune responses in peripheral blood of patients with venereal warts are related. Here we tried to assess T-helper cell response in vitro on stimulation with HPV-6 and -11 antigens both in patients and in controls, as well as IFN-□ and IL-4 response to detect skewing of T-helper cell to decreased cellular immunity. We demonstrated functional skewing of HPV antigen-specific T-helper cell response in these patients that may have future implications in treatment strategies to boost protective immune response.
MATERIALS AND METHODS
A total of 67 subjects (56 males, 11 females with age range from 18 to 64 years) with venereal warts for an average period of 5 months [minimum 1 month and maximum 15 months of duration, human immunodeficiency virus (HIV) negative without having any other immunosuppressive disease or having any history of treatment with immunosuppressive drugs in past or present or a history of skin warts] and a total of 26 age- and sex-matched healthy controls (HCs) with no clinical evidence or history of any viral warts were also recruited from the outpatient Department of Dermatology and Venereology at Dr Ram Manohar Lohia Hospital, New Delhi.
Sample Collection and Peripheral Blood Mononuclear Cell Isolation
After obtaining duly signed informed consent form, detailed history and findings were recorded as per the pro forma from both experimental and control subjects. Peripheral venous blood (8–10 mL) was collected in heparinized tubes, from the enrolled patients and controls. The patients under the study were provided standard treatment regimen of the disease. We isolated peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient (Lymphoprep, Oslo, Norway) and cultured in RPMI 1640 medium which contained 10% fetal bovine serum supplemented with antimycotic agents and antibiotics.
In vitro Stimulation of Peripheral Blood Mononuclear Cells
The PBMCs (2 × 106 cells/mL) from each experimental and control subject were cultured with recombinant HPV-6 and HPV-11 peptide (Genxbio) in separate wells for 24 hours at 37°C in 5% CO2 incubator [10 μg/mL brefeldin A/Golgi transport blocker (Sigma) was added to the culture]. Unstimulated PBMCs were used as negative control and fluorescent minus one control was also included. All the samples were further processed for cell surface and intracellular staining with titrated antibody dose.
Staining for Cell Surface Markers and Intracellular Cytokines
Cells were stained with fluorescent conjugated antibodies including □-CD3 PE-Cy5 (eBiosciences, CA, USA) followed by intracellular staining with □-IFN-□-fluorescein isothiocyanate and □-IL-4 PE (eBiosciences, CA, USA) by standard protocol. We used 2% paraformaldehyde for sample fixation prior to acquisition.
Flow Cytometry and Data Analysis
Stained cells were acquired on FC500 (Beckman Coulter) and Listmode raw cytometer data files were analyzed by the in-built CXP analysis STATA version 14.2. Lymphocytes were gated on forward and side scatter, and CD3 T cells were evaluated for IFN-□ or IL-4 positivity. We determined % frequency of CD3+IFN-□+ and CD3+IL-4+ T cells in comparison to unstimulated control.
In this study, we recruited 67 patients with anogenital warts. The number of warts at presentation varied from 1 to more than 50 in some patients. The size varied from 1 to 30 mm. The site of venereal warts in males was primarily penile shaft, glans penis, prepuce, perianal, gluteal, and buttocks, while labia majora and minora, vulva, perineal and perianal region were affected in females. Peptide-specific (HPV-6 and -11) Th1 and Th2 immune responses were determined in anogenital wart patients and HCs. After stimulation of PBMCs obtained from study subjects (SSs) and HCs with HPV-6 and HPV-11 peptide, cells were immunofluorescently stained with antibodies against T-cell surface marker (CD3) along with anti-IFN-□ (Th1 cytokine) and anti-IL-4 (Th2 cytokine). The mean percent frequency of HPV-6 peptide-stimulated T cells expressing IFN-□ was 0.26 ± 0.18 in wart patients and 0.53 ± 0.75 in HCs (Graph 1A). The statistically significant p-value between the two groups was 0.008. On the contrary, the mean percent frequency of HPV-6 peptide-stimulated T cells expressing IL-4 of 0.65 ± 0.55 in the wart patients was significantly higher (p = 0.0001) than those of HCs (0.18 ± 0.1) (Graph 1B). Furthermore, similar pattern of HPV antigen-specific T-cell immune response was observed after stimulation with HPV-11 peptide. As observed with HPV-6 peptide stimulation, the mean percent frequency of HPV-11 peptide-stimulated T cells expressing IFN-□ was decreased with a value of 0.27 (standard deviation [SD] ±0.23) in subjects with warts in contrast to 0.43 (SD ±0.5) in HCs (Graph 2A) with a significance of p = 0.04 (Graph 2A). Consistent significant increase in the mean percent frequency of HPV-11 peptide-stimulated T cells expressing IL-4 was observed (p = 0.001) in subjects with wart with mean value of 0.68 (SD ± 0.7) than with mean value of 0.2 (SD ±0.2) in HCs (Graph 2B).
Appearance of warts associated with suppression of cell-mediated immunity (decreased Th1 and increased Th2 response) has been reported in HPV-6 and/or -11-infected patients.18,19 To the best of our knowledge, till date, no study has been initiated or done on the role of HPV antigen-specific immune response in subjects with anogenital warts (SSs) due to highly localized nature, lack of significant systemic manifestations of the infection, and social taboo. Here, we have demonstrated HPV antigen-specific functional skewing of protective (Th1) vs suppressive (Th2) immune response in peripheral blood from subjects with warts (SSs). Earlier studies had demonstrated virus-specific antibodies in the sera of genital wart patients.20 However, Almeida et al21 had demonstrated that sera from genital wart patients specifically reacted with virus from genital warts only and not with that from skin warts unlike sera from skin wart patients which reacted with both types of viruses. The seroconversion of 27.6 to 75% following skin test, specifically in patients testing positive (36.9–86.6%), supported presence of low levels of antibodies specific to wart virus in many cases. Several studies had attempted to understand the role of host T-cell immune response against HPVs, specifically cell-mediated immunity. The supporting role of cell-mediated immunity in controlling HPV infection is evident in many reviews of literature, which has documented that prevalence of HPV infection and associated disease is increased in immunosuppressed populations. Another study showed HPV infection in all immunosuppressed women presenting with neoplasia of lower genital tract.22 In the 1990s, as the diagnosis of HPV infection by molecular diagnosis was developed, there were many reports confirming the rise in the incidence of infection with HPV23 and other comorbidities, which included warts24 and cervical squamous intraepithelial lesions25 in patients who were immunosuppressed. The association of reduced cell-mediated immunity and consequent increase in HPV infection and associated disease in HIV-infected individuals has been demonstrated in various studies wherein these patients have shown increased prevalence26-31 and longer period of persistence of HPV infection,26,32-35 which also provides a strong evidence to support the role of cell-mediated immunity. One of the major limitations in studies of cell-mediated immune responses to HPV infection due to its highly localized nature of tissue tropism is squamous epithelial sites. Therefore, it is challenging to demonstrate HPV-specific cell-mediated immune responses in peripheral blood of wart patients. Studies had reported the evidence of the role of IFN-□, TGF-□, and tumor necrosis factor in inhibition of HPV-infected cell growth, and persistence of viral infection and progression of disease could be due to escape from these mechanisms. As the epithelial cells have shown to be the producers of these cytokines except for IFN-□, some authors have suggested the autocrine role of cytokines in growth regulation of keratinocytes infected with HPV.36-38 This points to the fact that a large number of cell types possibly are contributing to the growth-inhibitory function; these cytokines are secreted by many cells like T cells, macrophages, NK cells, and others. Cell-mediated immunity to HPV had been studied by in vitro tests,39,40 but there is much controversy regarding the significance of these tests,41 and, in general, patients with anogenital warts were excluded from these studies.39 Moreover, there is scarcity of data on HPV-6 and -11 peptide-specific cell-mediated immune responses in peripheral blood of genital wart patients. In our study, we investigated the HPV-6 and -11 antigen-specific T cell-mediated immune responses in PBMCs in patients with warts. Our observation of incidence of reduced frequency of HPV-specific IFN-□ T-cell responders in peripheral blood is consistent with the earlier studies conducted in localized warts. In contrast, increased frequency of HPV antigen-specific IL-4-producing T cells observed in peripheral blood of wart patients suggested that genital warts induced a systemic immune suppression in the host. Cell-mediated immunity plays a major role in the cure of skin warts.42 Our results are consistent with other studies and strengthen the evidences pointing to the role of HPV-specific host T-cell immune response in warts.43
Our study demonstrated strong association of anogenital warts with Th2 skewed peripheral T-cell response. Our findings warrant future investigations to design specifically targeted, effective immune-prophylactic and immune-therapeutic strategies against HPV-induced lesions of the genital tract, which might lead to early clearance and prevention of relapse.
The study findings may be helpful in designing future immunomodulation strategies for venereal wart patients, specifically for management of recurrence after standard treatment. Such approach might pave way for potential adjunct therapy with immunomodulators to augment host immune response.
The authors would like to acknowledge the valuable contributions of Professor PK Sharma, Head, Department of Dermatology and Venereology, all residents especially Dr Neha, from the Department of Dermatology, Dr RML Hospital, New Delhi, Mr NP Singh, Pankaj, Arvind, Lab Technicians, Abhinav Saurabh, Prabin, Divya, Girija, Manju, SRF, Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences. Special thanks to Dr. V Sreenivas, Professor, Department of Biostatistics, AIIMS, New Delhi for help with the statistical analysis.