Differential sensitivity of human papillomavirus type 16(+) and type 18(+) cervical carcinoma cells to CD95-mediated apoptosis

When cervical carcinoma cells were monitored for apoptotic signals, HPV18(+) lines were found to be highly sensitive to agonistic CD95 antibodies or recombinant CD95 ligands after co-exposure with CHX (CD95(S)). In contrast, HPV16(+) cervical carcinoma cells and HPV16-immortalized non-malignant human keratinocytes were CD95-resistant (CD95(R)) under equivalent conditions. Somatic cell hybridization between CD95(S) and CD95(R) cervical carcinoma cell lines revealed that CD95 sensitivity was a dominant trait, which could be correlated with abundant c-Myc and low Bcl-X(L) expression. Although CD95(R) cervical carcinoma cells expressed even higher levels of p53 and CD95 receptor at the surface, resistance could be attributed to the inability to form a functional DISC, necessary for successful transmission of the apoptogenic response. These data indicate that resistance to apoptotic stimuli represents an important immunological escape mechanism during virus-induced carcinogenesis.     

Naturally processed and HLA-B8-presented HPV16 E7 epitope recognized by T cells from patients with cervical cancer

Several major histocompatibility complex (MHC) alleles have been reported to present peptides derived from the HPV16 E7 oncoprotein to T cells. We describe an overrepresentation of the HLA-B8 allele (28.44%) in cervical cancer patients as compared to the MHC class I allele frequency in a local healthy control population (18.80%) and the identification of an HLA-B8-binding peptide TLHEYMLDL (HPV16 E7(7-15)), which is able to drive HPV16 E7-specific and MHC class I-restricted T-cell responses in peripheral blood lymphocytes from healthy individuals. TLHEYMLDL-specific T cells recognize the naturally processed and presented peptide on HPV16+ cervical cancer cells transfected with the HLA-B8 gene defined by IFN-gamma production. This peptide epitope is also recognized by freshly harvested tumor-infiltrating T cells or T cells from tumor-draining lymph nodes from patients with cervical cancer determined by flow cytometry as well as by tetramer in situ staining. HLA-B8-restricted HPV E7(7-15)-specific T cells reside predominantly in the CD8+ CD45RA+ CCR7+ precursor or in the differentiated CD8+ CD45RA+ CCR7- T-cell population.     

Methylation of viral and host genes and severity of cervical lesions associated with human papillomavirus type 16

Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16‐positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p < 0.0001). Area under the curve (AUC) was highest among the CIN2/3 vs. cancer ranging from 0.786 to 0.853 among the different sites. Site‐specific methylation levels strongly discriminated CIN2/3 from NILM/CIN1 and cancer from CIN2/3 (range of odds ratios [OR]: 3.69–12.76, range of lower 95% confidence bounds: 1.03–4.01). When methylation levels were mutually adjusted for each other EPB41L3 was the only independent predictor of CIN2/3 vs. NILM/CIN1 contrasts (OR = 9.94, 95%CI: 2.46–40.27). High methylation levels of viral and host genes are common among precancerous and cancer lesions and can serve as independent risk biomarkers. Methylation of host genes LMX1 and EPB41L3 and of the viral HPV16 L1 sites has the potential to distinguish among precancerous lesions and to distinguish the latter from invasive disease.     

The detection of circulating human papillomavirus‐specific T cells is associated with improved survival of patients with deeply infiltrating tumors

A detailed analyses of HPV‐specific immunity was performed in a large group of patients with HPV‐induced cervical cancer (CxCa) in relation to HLA‐types and prognostic factors. Patients were HLA‐typed and HPV16/18‐specific T‐cell immunity was assessed by proliferation assay and cytometric bead array using freshly isolated PBMC and by phenotypic analysis of HPV‐specific T cells. The results were analyzed in relation to known disease‐related HLA‐types (DR7, DR13, DR15/DQ06), invasion‐depth and size of tumor, lymph node (LN) status and disease free survival. In total 119 HLA‐typed patients with CxCa were analyzed. Patients expressing the HLA‐DR13 haplotype were underrepresented as compared to the Dutch population (p = 0.014), whereas HLA‐DR7 was overrepresented in patients with HPV16+ CxCa (p = 0.006). In 29 of 94 patients (31%) from whom blood could be tested, a proliferative response to HPV16/18 was detected, which was associated with increased numbers of HPV‐specific CD4+CD25+ (activated) T cells (p = 0.03) and HPV‐specific CD4+CD25+FoxP3‐positive T cells (p = 0.04). The presence of both FoxP3‐positive and negative HPV‐specific CD4+CD25+ T cells was significantly correlated (p = 0.01). Interestingly, the detection of HPV‐specific proliferation was associated with invasion depth (p = 0.020) but not with HLA type, tumor size nor LN status. Moreover, the detection of HPV‐specific immunity was associated with an improved disease free survival (p = 0.04) in patients with deeply infiltrating tumors. In conclusion, HPV‐specific proliferative T‐cell response, comprising higher percentages of HPV‐specific CD25+ and CD25+FoxP3‐positive CD4+T cells, are more frequently detected in patients with deep infiltrating CxCa tumors and associated with an improved survival.     

Up‐regulation of interleukin‐17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer

BACKGROUND:

Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)‐17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line.

METHODS:

IL‐17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL‐17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6‐transfected H1299 human NSCLC cells.

RESULTS:

Immunohistochemical data showed that 48.1% of lung tumors had IL‐17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL‐17 with IL‐6 (P < .001) and IL‐17 with Mcl‐1 (P < .001) expression were also observed. Cell growth rate was increased, and IL‐17/Mcl‐1 expression levels were elevated in HPV 16 E6‐transfected H1299 cells. The transfected E6 oncoproteins can significantly up‐regulate expression levels of IL‐17 and antiapoptotic protein Mcl‐1.

CONCLUSIONS:

The study suggests that HPV infection‐induced IL‐17 levels can stimulate Mcl‐1 expression through the PI3K pathway and promote lung tumor cell progression through a p53‐ and IL‐6‐independent pathway. Cancer 2010. © 2010 American Cancer Society.     

Invader human papillomavirus (HPV) type 16 and 18 assays as adjuncts to HPV screening of cervical papanicolaou smears with atypical squamous cells of undetermined significance

BACKGROUND:

High‐risk (HR) human papillomavirus (HPV) testing is standard practice for triaging women who have Papanicolaou (Pap) smears with atypical squamous cells of undetermined significance (ASC‐US), however, only 5% to 17% of these women have underlying cervical intraepithelial neoplasia 2 (CIN‐2)/CIN‐3. Recent reports have demonstrated that the presence of either HPV type 16 (HPV‐16) or HPV‐18 confers an elevated risk for CIN‐2/CIN‐3. The current study was designed to determine the prevalence of HPV‐16 and HPV‐18 in ASC‐US Pap smears and to determine whether further typing would enhance the risk stratification of patients for CIN‐2/CIN‐3.

METHODS:

One hundred seventy‐eight Pap smears with ASC‐US were screened retrospectively for HR HPV by using the proprietary Invader screening assay followed by typing for HPV‐16 and HPV‐18 by using Invader type‐specific probes on 100 of the samples. Clinical follow‐up results were correlated with HPV types.

RESULTS:

Fifty‐one percent of the ASC‐US samples were positive for HR HPV, the majority of which (70%) harbored non‐HPV‐16/HPV‐18 HR HPV types; 27% were associated with HPV‐16, whereas only 3% contained HPV‐18. The screening assay indicated that 46% of women who had Pap smears with ASC‐US were in need of further HPV‐16/HPV‐18 typing. Testing for HPV‐16 stratified women with ASC‐US into 3 groups: 1) 14% of women were positive for HPV‐16 and had a high risk (54%) of CIN‐2/CIN‐3 on follow‐up biopsy, 2) 35% of women were positive for non‐HPV‐16 HPV types and had an intermediate risk (9%), and 3) 51% of women were negative for HPV and had a negligible risk for CIN‐2/CIN‐3.

CONCLUSIONS:

The combined application of a proprietary screening assay and a type‐specific HPV‐16 assay demonstrated global potential for the development of tailored management protocols for women who have Pap smears with ASC‐US. Cancer 2009. © 2009 American Cancer Society.     

Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia

It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV-16 E7 specific immune responses in patients with different grades of cervical intra-epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T-cell responses were measured by IFN-gamma ELISPOT analysis, after stimulation with recombinant HPV-16 E7 protein. As expected, HPV-16 E7 specific IFN-gamma T cell responses were more frequent in HPV-16 DNA positive patients compared with that in HPV-16 DNA negative patients (39/50 vs. 16/36, (p=0.006, chi2 test). After invasive procedures, a small number of HPV-16 DNA positive CIN patients, but a considerable proportion of HPV-16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV-16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4+ and CD8+ T cells showed E7 specific IFN-gamma production upon in-vitro stimulation. Our study shows that invasive procedures may enhance HPV-specific cell-mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV-specific immune responses is used as intermediate end-point.     

Natural killer cells infiltrating human nonsmall-cell lung cancer are enriched in CD56 bright CD16(-) cells and display an impaired capability to kill tumor cells

BACKGROUND: Despite natural killer (NK) cells being originally identified and named because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltrating malignant tumors, especially in humans. METHODS: NK cells infiltrating human nonsmall cell lung cancers (NSCLC) were analyzed with the aim of identifying their potential protective role in an antitumor immune response. Both relevant molecule expression and functions of NK cells infiltrating NSCLC were analyzed in comparison with autologous NK cells isolated from either peritumoral normal lung tissues or peripheral blood. RESULTS: The CD56 bright CD16(-) NK cell subset was consistently observed as being highly enriched in tumor infiltrate and displayed activation markers, including NKp44, CD69, and HLA-DR. Remarkably, the cytolytic potential of NK cells isolated from cancer tissues was lower than that of NK cells from peripheral blood or normal lung tissue, whereas no difference was observed regarding their capability of producing cytokines. With regard to their localization within tumor, NK cells were found in tumor stroma, whereas they were not in direct contact with cancer cells. CONCLUSIONS: For the first time NK cells infiltrating NSCLC have been characterized and it is shown that they are mainly capable of producing relevant cytokines rather than exerting direct cancer cell killing.     

Human T cell responses to HPV 16 E2 generated with monocyte-derived dendritic cells.

Persistent infection with human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical cancer. The E2 protein is required early in viral infection and therefore may serve as a useful immune target for a vaccine aimed at prevention or therapy of premalignant lesions. Dendritic cells (DC) prepared from monocytes and pulsed with bacterially produced HPV 16 E2 C-terminus protein were used to stimulate autologous T cells over several rounds of stimulation. T cells were tested for gamma-interferon release by ELISPOT and for cytotoxic activity by (51)chromium release assays. To generate E2-expressing target cells for cytotoxicity assays, we constructed a recombinant vaccinia virus encoding HPV 16 E2, which was used to infect autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL). The results show that DC pulsed with E2 C-terminus protein induce gamma-interferon-releasing T cells as demonstrated by ELISPOT. Furthermore, we demonstrate E2-specific lysis of vaccinia-E2 infected autologous LCL by CD8+ cytotoxic T lymphocytes (CTL). E2-specific CTL did not lyse untreated autologous LCL or LCL infected with wild-type vaccinia and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. In addition, T cells stimulated with DC in the absence of E2 failed to demonstrate lysis of vaccinia-E2-labeled targets. Phenotypically, CTL populations were CD3+/CD8+. These results will facilitate the study of naturally occurring T-cell responses to HPV E2 in patients with cervical intraepithelial neoplasia and the development of immunotherapeutic strategies designed to treat this and other HPV-associated diseases.     

Activation of cyclin D1 and D2 promoters by human T-cell leukemia virus type I tax protein is associated with IL-2-independent growth of T cells

Our aim was to examine the involvement of G(1) cell-cycle regulators in cell growth dysregulation induced by HTLV-I. Compared to uninfected cells, higher expression levels of cyclin D1 and D2 mRNA were detected in HTLV-I-infected T-cell lines, which were at least in part mediated by the viral transforming protein Tax since Tax activated both cyclin D1 and D2 promoters in the human T-cell line Jurkat. A Tax mutant that did not activate NF-kappaB failed to activate cyclin D1 and D2 promoters. Inhibitors of NF-kappaB (dominant negative IkappaBs mutants) suppressed Tax-dependent activation of cyclin D1 and D2 promoters, indicating that Tax-induced activation was mediated by NF-kappaB. Wild-type and mutant Tax capable of activating NF-kappaB, but not Tax mutant incapable of activating NF-kappaB, converted cell growth of a T-cell line from being IL-2-dependent to being IL-2-independent; and this conversion was associated with IL-2-independent induction of cyclins D1 and D2. Our data suggest that induction of cyclins D1 and D2 by Tax is involved in IL-2-independent cell-cycle progression as well as IL-2-independent transformation of primary human T cells by HTLV-I. High expression levels of cyclin D1 and D2 mRNAs were also detected in some patients with ATL. Our findings link HTLV-I infection to changes in cellular D-type cyclin gene expression, transformation of T cells and subsequent development of T-cell leukemia.