Recognition of ADP-ribosylation factor 4-like by HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes from patients with brain tumors

Although specific immunotherapy is one candidate treatment of brain tumor, the molecular basis of T-cell-mediated recognition of brain tumors has not yet been elucidated. In this study, we tried to identify brain tumor antigens using HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs). As an HLA-A2-restricted OK-CTL line contained CTLs capable of responding to HLA-A2+ malignant glioma cells, this cell line was used for identification of brain tumor antigens. After screening a cDNA library from brain tumor cells, this CTL line was found to produce interferon (IFN)-gamma when cultured with COS-7 cells, which were cotransfected with both a cDNA clone (clone 1) and HLA-A0207 cDNA. Data base searches indicated that the clone 1 was 98% identical to that of the human ADP-ribosylation factor 4-like (ARF4L). Two peptides, ARF4L 15-24 and ARF4L 69-77, possessed the ability to induce HLA-A2-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells of patients with brain tumors. Although ARF4L seemed to be ubiquitously expressed at the mRNA level, ARF4L-reactive CTLs failed to exhibit cytotoxicity against normal lymphoid blasts. These results indicate that these two ARF4L peptides could be targets for immunotherapy of HLA-A2+ patients with brain tumors.     

Genetic susceptibility of cervical cancer

Epidemiological and laboratory-based studies have identified infection with one of 15 high-risk human papillo-mavirus (HPV) types as a necessary but not sufficient cause of cervical cancer. The prevalence of genital HPV in-fections is high in young women, but most of the infections regress without interventions. Host genetic variations in genes involved in immune response pathways may be related to HPV clearance, and HPV E6/E7 oncoproteins interacting or downstream genes, both coding and non-coding, may contribute to the outcome of high risk HPV infection and cervical cancer. Of specific interest for this review has been the selection of genetic variants in genes involved in the above-referred pathways with a summary of their applications in association studies. Because the supportive and opposing data have been reported in different populations, well-designed international collabora-tive studies need to be conducted to define the consistency of the associations, paving the way to better define the patients at high risk of developing cervical cancer.     

Induction of human papillomavirus type 16-specific immunologic responses in a normal and an human papillomavirus-infected populations

Human papillomavirus (HPV) infection, especially with the oncogenic genotypes, is the most important risk factor for developing cervical cancer. We focused on generating HPV16 E7-specific cytotoxic CD8(+) T lymphocytes and evaluating HPV16 E7-specific immune responses in HPV16-infected and uninfected populations. Peripheral blood mononuclear cells (PBMCs) were first collected from an uninfected group with an human lymphocyte antigen (HLA) A2 haplotype (four volunteers). Mature monocyte-derived dendritic cells (DCs) were generated from the PBMCs and pulsed with one of two HLA-A2-restricted E7 peptides, aa 11-20 [YMLDLQPETT] and aa 86-93 [TLGIVCPI], as antigen presenting cells. The autologous naive or cultured PBMCs were then cultured with peptide-pulsed DCs to detect the HPV16 E7-specific immune responses by a variety of techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT) assay and cytotoxic T lymphocyte assay. Interferon-gamma (IFN-gamma) from E7-specific cytotoxic CD8(+) T lymphocytes stimulated with the respective peptide was detected by ELISA. Using ELISPOT analysis, a marked increase in the number of IFN-gamma-secreting CD8(+) E7-specific lymphocytes was observed following peptide stimulation. Cultured CD8(+) T lymphocytes were highly cytotoxic against the CaSki cells. PBMCs were then collected from an HPV16-infected population of the HLA-A2 haplotype, including four persons of HPV16 infection only, four with cervical intraepithelial neoplasia (CIN) lesions, and four cervical cancer patients. We then compared the immunologic responses to E7 between HPV16-infected and uninfected populations by ELISA and ELISPOT assay. The E7-specific immunologic responses of the HPV16-infected populations were significantly higher than those of the uninfected population. In addition, persons with an HPV16 infection only or those with CIN lesions generated higher E7-specific immunologic responses than cervical cancer patients. Our results demonstrate methods for evaluating E7-specific immunologic responses and reflect the biological responses of HPV16-infected people during different periods of cervical disease.     

Antigen-specific immunotherapy of cervical and ovarian cancer

Summary: We contrast the efforts to treat ovarian cancer and cervical cancer through vaccination because of their different pathobiology. A plethora of approaches have been developed for therapeutic vaccination against cancer, many of which target defined tumor-associated antigens (TAAs). Persistent infection with oncogenic human papillomavirus (HPV) types causes cervical cancer. Furthermore, cervical cancer patients frequently mount both humoral and T-cell immune responses to the HPV E6 and E7 oncoproteins, whose expression is required for the transformed phenotype. Numerous vaccine studies target these viral TAAs, including recent trials that may enhance clearance of pre-malignant disease. By contrast, little is known about the etiology of epithelial ovarian cancer. Although it is clear that p53 mutation or loss is a critical early event in the development of epithelial ovarian cancer, no precursor lesion has been described for the most common serous histotype, and even the location of its origin is debated. These issues have complicated the selection of appropriate ovarian TAAs and the design of vaccines. Here we focus on mesothelin as a promising ovarian TAA, because it is overexpressed and immunogenic at high frequency in patients, is displayed on the cell surface, and potentially contributes to ovarian cancer biology.     

过继转输的肿瘤特异性T杀伤细胞在小鼠体内维持杀伤功能的研究

体外试验从免疫小鼠脾细胞中诱导出对ＦＢＬ－３瘤细胞具有特异性杀伤功能的Ｔ杀伤细胞，转输到同系正常小鼠体内后发现，同时给予肿瘤抗原刺激和７ｄｒＩＬ－２作用，可使受鼠脾细胞获得杀伤ＦＢＬ－３细胞的活性，其杀伤功能与转输的Ｔ杀伤细胞有关，若转输后连续２１ｄ只给予ｒＩＬ－２不能维持体内建立的杀瘤活性，而间断给予肿瘤抗原再刺激，并联合应用短程ｒＩＬ－２，以１４ｄ为１周期，则可长期维持体内的肿瘤特异性杀伤功能     

Are 20 human papillomavirus types causing cervical cancer?

In 2012, the International Agency for Research on Cancer concluded that there was consistent and sufficient epidemiological, experimental and mechanistic evidence of carcinogenicity to humans for 12 HPV types (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58 and HPV59) for cervical cancer. Therefore, these types were considered as 1A carcinogens. They all belong to the family of the α‐Papillomaviridae, in particular to the species α5 (HPV51), α6 (HPV56), α7 (HPV18, HPV39, HPV45, HPV59) and α9 (HPV16, HPV31, HPV33, HPV35, HPV52, HPV58). Less evidence is available for a thirteenth type (HPV68, α7), which is classified as a 2A carcinogen (probably carcinogenic). Moreover, seven other phylogenetically related types (HPV26, HPV53, HPV66, HPV67, HPV68, HPV70 and HPV73) were identified as single HPV infections in certain rare cases of cervical cancer and were considered possibly carcinogenic (2B carcinogens). Recently, Halec et al [7] demonstrated that the molecular signature of HPV‐induced carcinogenesis (presence of type‐specific spliced E6*| mRNA; increased expression of p16; and decreased expression of cyclin D1, p53 and Rb) was similar in cervical cancers containing single infections with one of the eight afore‐mentioned 2A or 2B carcinogens to those in cancers with single infections with group 1 carcinogens. Ninety six percent of cervical cancers are attributable to one of the 13 most common HPV types (groups 1 and 2A). Including the additional seven HPV types (group 2B) added 2.6%, to reach a total of 98.7% of all HPV‐positive cervical cancers. From recently updated meta‐analyses, it was shown that HPV68, HPV26, HPV66, HPV67, HPV73 and HPV82 were significantly more common in cancer cases than in women with normal cervical cytology, suggesting that for these HPV types, an upgrading of the carcinogen classification could be considered. However, there is no need to include them in HPV screening tests or vaccines, given their rarity in cervical cancers. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

转染自体胃癌细胞总RNA的树突状细胞诱导个体化免疫治疗的体外实验

目的探讨转染自体胃癌细胞总RNA的树突状细胞(DC)体外介导抗胃癌的免疫效应。方法制备短期培养的原代胃癌细胞。用rhGM-CSF、rhIL-4和TNF-α体外诱导胃癌患者外周血单个核细胞(PBMC)中DC的发育和成熟,并转染自体肿瘤细胞总RNA,激活自体T细胞产生CTL,用CCK-8试剂盒检测CTL的杀伤活性。应用流式细胞术及混合淋巴细胞培养技术检测DC的免疫功能状态。用ELISA法测定IL-12和INF-γ的水平。结果转染自体肿瘤细胞总RNA的成熟DC,不仅可高表达MHC-I、II类分子及CD80、CD83和CD86协同刺激分子,并可获得高效刺激自体或异体T细胞增殖的能力。转染RNA的成熟DC,分泌IL-12的水平及其刺激产生的CTL培养上清液中INF-γ的水平显著高于单纯成熟DC及未成熟DC;且CTL对自体胃癌细胞的杀伤率显著高于异体组。结论转染自体胃癌细胞总RNA的成熟DC能够体外诱导产生对自体肿瘤细胞具有高度抗原特异性杀伤活性的CTL。     

The role of antigen-specific and non-specific immunotherapy in the treatment of cancer

Immunotherapy in the treatment of cancer is increasing, particularly with the recent FDA approval of sipuleucel-T and ipilimumab. The efficacy of anti-tumor immunotherapies has been modest compared to their theoretical and pre-clinical promise. This review evaluates the promise and pitfalls of immunotherapy and highlight some of the obstacles to improving anti-tumor immunotherapy: the need for technical refinement of therapies, the need for an increased understanding of how best to combine therapies with traditional cytotoxic therapies, the inability of patients to mount an effective immune response either due to disease burden or tumor induced immune suppression, the significant toxicities associated with many immunotherapies, and the lack of strongly immunogenic antigens required by many therapies. Further, antigen-non-specific immunotherapies, including cytokines such as interleukins and interferons, immuno-stimulatory agents such as CpG oligonucleotides, or BCG, antibodies targeted against receptors such as the agonistic CD40 or inhibitory CTLA-4 antibodies, and enzyme inhibitors such as those targeting cyclo-oxygenase or indolamine-2,3-dioxygenase are discussed. In addition, potential mechanisms of these therapies such as direct anti-tumor effects, reversal of immune suppression, activation of innate immunity, and antigen-non-specific T-cell activation are reviewed. We also appraise the potential of these antigen-non-specific therapies to overcome some of the previously described pitfalls of immunotherapy. Lastly, we discuss a recent series of studies from our laboratory demonstrating the importance of antigen-non-specific ‘bystander activation’ of memory T-lymphocytes by immunomodulatory therapies such as interleukin-2 and the antigen-non-specific anti-tumor effects of these cells.     

Detection of human papillomavirus in urine and cervical swabs from patients with invasive cervical cancer

Despite the high prevalence of both human papillomavirus (HPV) infections and cervical cancer among Zimbabwean women, the ability to test for HPV infection of the uterine cervix is limited by a lack of an easy sample collection method that does not require gynecological examination. The presence of HPVs in urine and cervical swab samples collected from 43 women who presented with invasive cervical cancer was investigated. HPV detection was done by means of degenerate primers in a nested polymerase chain reaction (PCR). Typing of HPVs was done using restriction fragment length polymorphism (RFLP) analysis. HPV was identified and typed in 98% (42/43) of cervical swabs and 72% (31/43) of paired urine samples. HPV type 16 was the most common (25/42, 59%), followed by types: 33 (13/42, 31%), 18 (6/42, 14%), and 31 (1/42, 2%). Type-specific concordance between cervical and urine samples was high (22/28, 79%). Therefore, the HPV types identified in urine samples in most cases represent the same HPV type infecting the cervical epithelium. The results suggest that urine may be a practical sample for testing of HPV urogenital infection. Further research is required before the detection of HPV in urine can be applied in the routine cervical screening programs.     

Recent developments in immunotherapy of cancers caused by human papillomaviruses

A subset of oncogenic human papillomaviruses (HPVs) is the main cause of genital cancers, most importantly cervical cancer and an increasing number of head and neck cancers. Despite the availability of prophylactic vaccines against the most prevalent oncogenic HPV types, HPV‐induced malignancies are still a major health and economic burden. Besides conventional treatment with surgery, chemotherapy and radiation, immunotherapy is emerging as an efficient adjuvant option. Here, we review relevant studies and ongoing clinical trials using immune checkpoint inhibitors, therapeutic vaccines, gene editing approaches and adoptive T cell therapies, with special focus on engineered TCR T cells, which are showing encouraging results and could lead to significant improvement in the treatment of HPV+‐infected cancer patients.     

Increase of human papillomavirus-16 E7-specific T helper type 1 response in peripheral blood of cervical cancer patients after radiotherapy

It has been suggested that tumour cell lysis by gamma-radiation induces a tumoral antigen release eliciting an immune response. It is not clear how a specific immune response in cervical cancer patients is developed after radiotherapy. This study is an attempt to investigate the role of the human papillomavirus type 16 (HPV-16) E7-specific T helper response before and after radiotherapy. Lymphocytes were isolated from 32 cervical cancer patients before and after radiotherapy and from 16 healthy women. They were stimulated for 12 hr with autologous HPV-16 E7-pulsed monocyte-derived dendritic cells or directly with HPV-16 E7 synthetic peptides: E7(51-70), E7(65-84) and E7(79-98). The cells were stained for CD4, CD69, intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) cytokines and analysed by flow cytometry. A specific CD4(+) CD69(+) IFN-gamma(+) immune response against HPV-16 E7(79-98) peptide was observed in 10 of 14 patients (71.4%) after treatment, compared with 4 of 14 (28.5%) before radiotherapy (P = 0.039); however, this response was not associated with a successful clinical response. Before treatment, 5 of 31 patients showed a HPV-16 E7(79-98)-specific T helper type 2 (Th2) response. Interestingly, this response was significantly associated with a decrease in disease-free survival (P = 0.027). These results suggest that a Th2-type cellular response could be useful as a predictor of recurrence and poor prognosis. An increase of the HPV-specific immune response was observed after radiotherapy; however, it is not enough to control completely the disease after treatment. Our results support that the E7-specific T-cell IFN-gamma response in cervical cancer patients, rather than reflecting the host's capability of controlling tumour growth, might be an indicator for disease severity.     

IL-21 synergizes with IL-7 to augment expansion and anti-tumor function of cytotoxic T cells

IL-21, a recently identified member of the common gamma-chain (gammac) receptor cytokine family, has been shown to be an important regulator of both innate and adaptive immune responses. In this study, we investigated whether IL-21 could synergize with another gammac cytokine, IL-7, to induce enhanced proliferation and effector function of tumor antigen-specific CD8(+) T cells. Our results showed that IL-21 could significantly augment the IL-7-induced expansion of cytotoxic T cells, possibly by preventing the cytokine-induced down-regulation of the IL-7Ralpha (CD127) on antigen-stimulated T cells. IL-21 also greatly enhanced the production of T(h)1 and inflammatory cytokines by activated T cells, including IFN-gamma, IL-2, tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, IL-1beta and IL-6. Finally, the addition of IL-21 to IL-7-cultured CTLs resulted in a considerably higher level of cytolytic activity, as measured by specific killing of tumor cells or antigen-pulsed target cells. These results suggest that IL-21 may play a cooperative role with IL-7 in modulating primary CD8(+) T-cell responses and may have important implications for immunotherapy of cancer.     

Aldehyde dehydrogenase in regulatory T-cell development,immunity and cancer

The role of aldehyde dehydrogenase (ALDH) in carcinogenesis and resistance to cancer therapies is well known. Mounting evidence also suggests a potentially important role for ALDH in the induction and function of regulatory T (Treg) cells. Treg cells are important cells of the immune system involved in promoting immune tolerance and preventing aberrant immune responses to beneficial or non-harmful antigens. However, Treg cells also impair tumor immunity, leading to the progression of various carcinomas. ALDH expression and the subsequent production of retinoic acid by numerous cells, including dendritic cells, macrophages, eosinophils and epithelial cells, seems important in Treg induction and function in multiple organ systems. This is particularly evident in the gastrointestinal tract, pulmonary tract and skin, which are exposed to a myriad of environmental antigens and represent interfaces between the human body and the outside world. Expression of ALDH in Treg cells themselves may also be involved in the proliferation of these cells and resistance to certain cytotoxic therapies. Hence, inhibition of ALDH expression may be useful to treat cancer. Besides the direct effect of ALDH inhibition on carcinogenesis and resistance to cancer therapies, inhibition of ALDH could potentially augment the immune response to tumor antigens by inhibiting Treg induction, function and ability to promote immune tolerance to tumor cells in multiple cancer types.     

Clinical course and immune response of a renal cell carcinoma patient to adoptive transfer of autologous cytotoxic T lymphocytes

The cytotoxic T lymphocyte (CTL) is a promising candidate for an effector cell in adoptive immunotherapy for renal cell carcinoma (RCC). Here we report the clinical course and in vivo immune responses of a RCC patient with bulky retroperitoneal lymph node (RPLN) metastases who received adoptive autologous CTL therapy. A 56-year-old woman diagnosed with RCC with multiple RPLN metastases underwent unilateral nephrectomy. Autologous RCC cells were primary-cultured from surgical specimens. Before addition of peripheral blood mononuclear cells (PBMC) for CTL induction, subconfluent RCC cells were irradiated with 50 Gy. The PBMCs were then cultured on RCC cells in the induction medium supplemented with four kinds of interleukins. The induced CTLs showed the potent killing activity against autologous RCC cells in a typical MHC-class I-restricted manner. The patient received three courses of CTL therapy with a total of 10.2 x 10(9) cells, and the RPLN mass decreased markedly in size after the second course. Eosinophilia and enhanced CTL inducibility from peripheral blood were observed after CTL administrations. The patient was progression free without further treatment; however, she developed rapidly progressive glomerulonephritis more than 1 year after the last treatment. The patient died of newly developed metastases 27 months after the start of CTL therapy. At autopsy, viable RCC cells were found in multiple metastatic sites. However, only diffuse fibrous tissue was observed in the responding RPLN mass. Apparent histological divergence was observed between primary and metastatic sites.     

Distribution of human papillomavirus genotypes in cervical intraepithelial neoplasia and invasive cervical cancer in Canada

Infection with high‐risk human papillomavirus (HPV) causes cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC). The distribution of HPV types in cervical diseases has been previously described in small studies for Canadian women. The prevalence of 36 HPV genotypes in 873 women with CIN and 252 women with ICC was assessed on cervical exfoliated cells analyzed with the Linear Array (Roche Molecular System). HPV16 was the most common genotype in CIN and ICC. The seven most frequent genotypes in order of decreasing frequency were HPV16, 51, 52, 31, 39, 18, and 56 in women with CIN1, HPV16, 52, 31, 18, 51, 39, and 33 in women with CIN2, HPV16, 31, 18, 52, 39, 33, and 58 in women with CIN3, and HPV16, 18, 45, 33, 31, 39, and 53 in women with ICC. HPV18 was detected more frequently in adenocarcinoma than squamous cell carcinoma (P = 0.013). Adjustment for multiple type infections resulted in a lower percentage attribution in CIN of HPV types other than 16 or 18. The proportion of samples containing at least one oncogenic type was greater in CIN2 (98.4%) or CIN3 (100%) than in CIN1 (80.1%; P < 0.001 for each comparison). Multiple type infections were demonstrated in 51 (20.2%) of 252 ICC in contrast to 146 (61.3%) of 238 women with CIN3 (P < 0.001). Adjusting for multiple HPV types, HPV16 accounted for 52.1% and HPV18 for 18.1% of ICCs, for a total of 70.2%. Current HPV vaccines should protect against HPV types responsible for 70% of ICCs in Canadian women. J. Med. Virol. 83:1034–1041, 2011. © 2011 Wiley‐Liss, Inc.     

Cervical human papillomavirus among 19 753 women attending gynecological department of a major comprehensive hospital in north Anhui China 2013-2016: Implication for cervical cancer screening and prevention

Our study aimed to assess the prevalent, incident, and persistent infection, and clearance of HPV among 19 753 individual women attending the gynecological department at a major comprehensive hospital. HPV 16, 52, and 58 ranked top three types with the highest prevalence and incidence. The prevalence of high-risk (HR) HPV peaked among women aged 15 to 19 years, then sharply decreased with age, stabilized among women aged 25 to 44 years, and then surged again among women aged 45 years and older. HR HPV infection were more likely to be prevalent (15.9% vs 1.3%, P < 0.001), incident (17.3 vs 2.0 per 1000 person-months, P < 0.001), and persistent (33.0% vs 24.2%, P = 0.033), and less likely to clear (88 vs 115 per 1000 person-months, P = 0.040) compared to low-risk HPV types. The majority of women detected with HR HPV types did not retest within 12 months. Clinical guidelines on HPV DNA testing are needed and education and counseling about HPV infection and its implications for women detected with HPV at clinical settings are warranted.     

DNA疫苗与肿瘤免疫治疗

DNA疫苗因其能诱发有效及持久的免疫反应而被广泛应用于恶性肿瘤的免疫治疗。已构建出多种肿瘤DNA疫苗,包括TAAs全长DNA疫苗、TAAs表位DNA疫苗、肿瘤融合DNA疫苗。肿瘤DNA疫苗可以激发肿瘤特异性的细胞/体液免疫反应及延长实验动物的生存期。肿瘤DNA疫苗在动物试验中的有效性为其在临床治疗中的应用提供了理论依据。     

The p53 R72P polymorphism does not influence cervical cancer development in a Portuguese population: a study in exfoliated cervical cells

The interaction between the E6 protein of the high-risk human papillomaviruses (HPVs) with p53 seems to be crucial in cervical carcinogenesis. The presence of Arg/Arg genotype at codon 72 of TP53 gene was characterized as a risk factor in development of cervical cancer. However, the role of this polymorphism remains controversial and some authors suggested that the origin of DNA (blood or exfoliated cervical cells) might influence these results. This study analyzed the effect of the p53 codon 72 polymorphism (R72P) in exfoliated cervical cells of women from the northern region of Portugal using two methodologies: allele-specific polymerase chain reaction and real-time polymerase chain reaction. We studied 700 cervical exfoliated cells which showed: 334 cases from women without cervical cancer or cervical lesion (N), 114 low-grade squamous intraepithelial lesions (LSIL), 107 high-grade squamous intraepithelial lesions (HSIL), 20 invasive cervical cancers (ICC) and 125 atypical squamous cells of unknown significance (ASCUS). No statistically significant differences between cases and controls were found, regarding the influence of the R72P polymorphism with cytological classification, high risk-HPV infection and HPV16 presence (P = 0.336, P = 0.945, and P = 0.964, respectively). Also, the influence of this polymorphism in the median age of onset for LSIL, HSIL, and ICC was not statistically significant (P = 0.674, P = 0.810, and P = 0.928, respectively). Therefore, the hypothesis that women with Arg/Arg genotype have an increased risk of developing cervical cancer failed to be proven in this study. Moreover, our study reveals that results using exfoliated cervical cells are reliable as compared with studies on blood.