CD103 defines intraepithelial CD8+ PD1+ tumour-infiltrating lymphocytes of prognostic significance in endometrial adenocarcinoma

IntroductionIntraepithelial CD8+ tumour-infiltrating T-lymphocytes (TIL) are associated with a prolonged survival in endometrial cancer (EC). By contrast, stromal infiltration of CD8+ TIL does not confer prognostic benefit. A single marker to discriminate these populations would therefore be of interest for rapid assessment of the tumour immune contexture, ex vivo analysis of intraepithelial and stromal T-cells on a functional level and/or adoptive T-cell transfer. Here we determined whether CD103, the αE subunit of the αEβ7integrin, can be used to specifically discriminate the epithelial and stromal CD8+ TIL populations in EC.MethodsCD103+ TIL were quantified in a cohort of 305 EC patients by immunohistochemistry. Localization of CD103+ cells and co-expression of CD103 with CD3, CD8, CD16 and FoxP3 were assessed by immunofluorescence. Further phenotyping of CD103+ cells was performed by flow cytometry on primary endometrial tumour digests.ResultsCD8+CD103+ cells were preferentially located in endometrial tumour epithelium, whereas CD8+CD103− cells were located in stroma. CD103+ lymphocytes were predominantly CD3+CD8+ T-cells and expressed PD1. The presence of a high CD103+ cell infiltration was associated with an improved prognosis in patients with endometrial adenocarcinoma (p = 0.035). Moreover, this beneficial effect was particularly evident in high-risk adenocarcinoma patients (p = 0.031).ConclusionsBecause of the restricted expression on intraepithelial CD8+ T-cells, CD103 may be a suitable biomarker for rapid assessment of immune infiltration of epithelial cancers. Furthermore, this intraepithelial tumour-reactive subset might be an interesting T-cell subset for adoptive T-cell transfer and/or target for checkpoint inhibition therapy.     

High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer

In a prospective study, we have examined the tumor-specific immune response in a group of 59 patients with human papillomavirus (HPV) 16-positive (HPV16(+))-induced or HPV18(+)-induced cervical cancer. Local antitumor immunity was analyzed by the enumeration of tumor-infiltrating dendritic cells and CD4+, CD8+, and regulatory T cells as well as by calculation of the ratio of CD8+/CD4+ T cells and CD8+/regulatory T cells. Systemic tumor-specific immunity was assessed by determination of the HPV E6- and/or E7-specific T-cell response in the blood of these patients. Finally, these variables were evaluated with respect to known histopathologic prognostic variables, including the absence (LN-) or presence (LN+) of lymph node metastases. Stratification according to the lymph node status of patients revealed a significantly stronger CD8+ T-cell tumor infiltration, a higher CD8+/CD4+ T-cell ratio, and higher CD8+/regulatory T-cell ratio in the group of patients in which the tumor failed to metastasize to the tumor-draining lymph node. Subdivision according to the presence (IR+) or absence (IR-) of circulating HPV-specific T cells disclosed that the highest number of tumor-infiltrating CD8+ T cells was found in the group of LN- patients displaying a concomitant systemic tumor-specific immune response (LN-IR+). CD8+ T-cell infiltration in LN-IR- patients was comparable with that of LN+ patients. In cervical cancer, the absence of lymph node metastases is strongly associated with a better prognosis. Our data indicate that, especially in a subgroup of LN- patients, a strong and effective interaction between immune system and tumor exists. This subgroup of cervical cancer patients may have the best prognosis.     

The clinical utility of the local inflammatory response in colorectal cancer

BackgroundThe host immune response is important in the prevention of tumour progression in solid organ cancers. The aim was to evaluate the clinical utility of the local inflammatory response in patients with colorectal cancer.MethodsThree hundred and sixty-five patients with primary operable colorectal cancer were included. The local inflammatory response was assessed using three different methods; (1) individual T-cell subtypes (CD3, CD8, CD45R0, FOXP3), (2) an immunohistochemistry-based immune score (Galon Immune Score) and (3) a histopathological assessment (Klintrup–Makinen grade). Relationships with tumour and host characteristics were established and the prognostic value of each method compared.ResultsA strong infiltration of tumour infiltrating lymphoctyes (TIL’s) was associated with improved cancer-specific survival. When individual T-cell subtypes were considered, CD3-positive cells were the strongest predictor of survival at the invasive margin (CD3⁺ IM) while CD8-positive cells were the strongest predictor in the cancer cell nests (CD8⁺ CCN). Infiltration of T-cells was related to early tumour stage, expanding growth pattern and lower levels of venous invasion but was not influenced by host characteristics or degree of systemic inflammation. In summary, CD3⁺ IM, CD8⁺ CCN, The Galon Immune Score and the Klintrup–Makinen grade all exhibited similar survival relationships in both node-positive and node-negative colorectal cancer.ConclusionA coordinated adaptive immune response is an important factor in predicting outcome in patients with primary operable colorectal cancer. By comparing different methodologies we have provided a foundation on which to develop a standardised approach for assessing the local inflammatory response in these patients.     

Radiochemotherapy induces a favourable tumour infiltrating inflammatory cell profile in head and neck cancer

Head and neck squamous cell cancers (HNSSC) generate an immune-suppressive micro-environment by a specific pattern of tumour infiltrating inflammatory cells. The aim of our study was to evaluate the impact of radiochemotherapy on the numbers and composition of inflammatory cells and its influence on outcome. Fifty-eight patients suffering from oral cavity cancer were studied, whose therapy consisted of concurrent radiochemotherapy followed by surgery. Numbers and ratios of tumour infiltrating inflammatory cells were compared prior to and after radiochemotherapy. Intraepithelial and stromal location of tumour infiltrating inflammatory cells was analysed separately. Infiltration of CD3(+), CD4(+), CD25(+), FoxP3(+), CD8(+), Granzyme B(+), CD20(+) and CD68(+) cells predominated in the peritumoural stromal compartment, whereas CD1a(+) dendritic cells were found more frequently in the intraepithelial compartment. Neoadjuvant treatment was associated with a general decrease of tumour infiltrating inflammatory cells in both compartments. The CD8(+) and Granzyme B(+) cytotoxic cells decreased only slightly after RCT. In contrast, the decrease of FoxP3(+) regulatory T cells was more pronounced and the cytotoxic T-cell/FoxP3(+) ratio increased 2- to 3-fold in both compartments, respectively. Patients with high cytotoxic cell numbers, high dendritic cell numbers and a high ratio of cytotoxic cells to regulatory T cells had a better disease free survival. Concurrent radiochemotherapy of oral squamous cell carcinoma was shown to drive the composition of inflammatory cells in a direction which is supposed to be prognostically favourable.     

Stromal infiltration of CD8 T cells is associated with improved clinical outcome in HPV-positive oropharyngeal squamous carcinoma

Background:

Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) have a better prognosis than those with HPV-negative tumours. There is interest in de-escalating their treatment but strategies are needed for risk stratification to identify subsets with a poor prognosis. This study investigated tumour-infiltrating lymphocytes (TILs) in relation to HPV tumour status and patient survival.

Methods:

Biopsies from 218 patients diagnosed with OPSCC between 2002 and 2011, who underwent chemo/radiotherapy were analysed for HPV by PCR, in-situ hybridisation and p16 immunohistochemistry (IHC). One hundred and thirty-nine samples with concordant HPV detection were analysed for CD3, CD4, CD8 and FoxP3 expression in tumour and stromal regions using multiplexIHC and multispectral image analysis. Labelling of smooth muscle actin (SMA) identified activated stroma.

Results:

Human papillomavirus-positive compared with HPV-negative OPSCC had higher infiltration in both tumour and stromal areas of CD4 and CD8 T cells but not FoxP3 T regulatory cells. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). There was significantly higher SMA expression in HPV-positive compared with -negative tumours, which did not correlate with survival.

Conclusions:

Studies of TILs for risk stratification in OPSCC should assess stromal infiltration.     

Immune cell infiltration of primary and metastatic lesions: mechanisms and clinical impact

The infiltration of tumors and their metastases by hematopoietic cells can contribute both positively and negatively to tumor growth, invasion, and patient outcomes. These differing outcomes are associated with both tumor heterogeneity and the diversity of leukocytes infiltrating neoplastic lesions. Tumors infiltration by histiocytes (macrophages and dendritic cells (DCs)) is associated with poor clinical outcomes, although infiltration by a subset of DCs is related to improved outcomes. T-cell infiltration of tumors and metastases are surrogates for positive outcomes, although subset analysis suggests that not all infiltrating T-cells have this potential. Overall, tumor infiltration by CD8(+) T-cells is associated with a positive outcome, while the frequency of infiltrating CD4(+) cells may be a negative predictor. In addition to tumor infiltration by macrophages and T-cells, recent studies have shown that myeloid-derived suppressor cells (MDSCs), also infiltrate tumors, inhibiting T-cell and DC number and function and facilitate tumor growth, angiogenesis, and metastasis. In summary, hematopoietic cell infiltration of tumors can regulate tumor progression and provide a useful diagnostic surrogate. Further, strategies focused on the manipulation of cellular infiltration via cellular, gene and molecular immunotherapies have the potential to provide a novel target for adjuvant therapy.     

CD4(+)CD25hi regulatory T-cell frequency correlates with persistence of human papillomavirus type 16 and T helper cell responses in patients with cervical intraepithelial neoplasia

CD4(+)CD25(hi)CTLA4(+)FoxP3(+) regulatory T cells (Treg) have been shown to maintain immune tolerance against self antigens and increased circulating frequencies have been reported in various types of cancers. Circulating invariant natural killer T-cells (iNKT) are reduced in cancer patients and low iNKT frequency is related to poor prognosis. It is not yet clear whether high Treg numbers and low iNKT cell numbers pose an increased risk for the progression of premalignant lesions or whether Treg and iNKT cell numbers are influenced by dysplasia. We therefore studied prospectively the relation between iNKT cell and Treg frequencies and the natural course of human papillomavirus type 16 (HPV16) induced pre-malignant cervical dysplasia in 82 patients who participated in a nonintervention cohort study of women with abnormal cytology. Treg frequencies were significantly increased in women who had persistent HPV16 infection. Within the HPV16 persistence group there was no difference in Treg frequencies among patients who developed a CIN3 lesion and patients who did not progress to CIN3. Furthermore, Treg frequencies were increased in patients who had detectable HPV16 E7 specific IL-2 producing T-helper cells, which suggests a causal role of HPV infection in Treg development in parallel with HPV16 specific T helper cells. No evidence was found for a role for iNKT cells in persistence of HPV16 and progression of HPV16 induced CIN lesions. However, HPV-persistence-associated Tregs may explain the inefficacy of concomitant persistence associated immunity and may contribute to subsequent progression to neoplasia.     

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.     

Adenovirus vector‐based prime‐boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV‐infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus‐specific antigens for vaccine‐induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV‐specific CD8⁺ T cells producing IFN‐γ and TNF‐α to the cervicovaginal tract. Importantly, the cervicovaginal CD8⁺ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue‐resident memory CD8⁺ T cells. This prime‐boost strategy targeting heterologous locations also induced circulating HPV‐specific CD8⁺ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.     

Human papillomavirus genotype attribution for HPVs 6, 11, 16, 18, 31, 33, 45, 52 and 58 in female anogenital lesions

ObjectiveHuman papillomavirus (HPV) vaccines can potentially control cervical cancer and help to reduce other HPV-related cancers. We aimed to estimate the relative contribution (RC) of the nine types (HPVs 16/18/31/33/45/52/58/6/11) included in the recently approved 9-valent HPV vaccine in female anogenital cancers and precancerous lesions (cervix, vulva, vagina and anus).MethodsEstimations were based on an international study designed and coordinated at the Catalan Institute of Oncology (Barcelona-Spain), including information on 10,575 invasive cervical cancer (ICC), 1709 vulvar, 408 vaginal and 329 female anal cancer cases and 587 Vulvar Intraepitelial Neoplasia grade 2/3 (VIN2/3), 189 Vaginal Intraepitelial Neoplasia grade 2/3 (VaIN2/3) and 29 Anal Intraepitelial Neoplasia grade 2/3 (AIN2/3) lesions.Consecutive histologically confirmed paraffin-embedded cases were obtained from hospital pathology archives from 48 countries worldwide. HPV DNA-detection and typing was performed by SPF₁₀-DEIA-LiPA₂₅ system and RC was expressed as the proportion of type-specific cases among HPV positive samples. Multiple infections were added to single infections using a proportional weighting attribution.ResultsHPV DNA prevalence was 84.9%, 28.6%, 74.3% and 90.0% for ICC, vulvar, vaginal and anal cancers, respectively, and 86.7%, 95.8% and 100% for VIN2/3, VaIN2/3 and AIN2/3, respectively. RC of the combined nine HPV types was 89.5% (95% confidence interval (CI): 88.8–90.1)-ICC, 87.1% (83.8–89.9)-vulvar, 85.5% (81.0–89.2)-vaginal, 95.9% (93.0–97.9)-female anal cancer, 94.1% (91.7–96.0)-VIN2/3, 78.7% (71.7–84.2)-VaIN2/3 and 86.2% (68.3–96.1)-AIN2/3. HPV16 was the most frequent type in all lesions. Variations in the RC of HPVs 31/33/45/52/58 by cancer site were observed, ranging from 7.8% (5.0–11.4)-female anal cancer to 20.5% (16.1–25.4)-vaginal cancer.ConclusionsThe addition of HPVs 31/33/45/52/58 to HPV types included in current vaccines (HPV16/18) could prevent almost 90% of HPV positive female anogenital lesions worldwide. Taking into account that most HPV-related cancers are ICC ones, the 9-valent HPV vaccine could potentially avoid almost 88% of all female anogenital cancers.     

Midgut carcinoid patients display increased numbers of regulatory T cells in peripheral blood with infiltration into tumor tissue

INTRODUCTION: Our aim was to investigate the immune status of midgut carcinoid patients. Cancer patients generally display suppressed Th1-type immunity that disables mounting of an efficient anti-tumor response. However, little is known about patients with neuroendocrine midgut carcinoids. MATERIAL AND METHODS: Circulating regulatory T cells were determined in patient blood by staining for CD4, CD25 and FoxP3 in flow cytometric analysis. T cell proliferation was measured by Alamar Blue in response to polyclonal activation and the regulatory phenotype of patient CD25+ cells was validated by allogeneic stimulation of CFSE labelled responders. Cytokine levels in patient peripheral blood were measured by ELISA and CBA. Tumor infiltrating T cells were analyzed by immunohistochemistry and immunofluorescence. RESULTS: The results demonstrate that midgut carcinoid patients exhibit increased frequencies of circulating Tregs and patient T cells have a decreased proliferative capacity compared to healthy donors. Systemic Th1-promoting cytokines are reduced. Midgut carcinoid tumors display CD4+ and CD8+ T cell infiltration, always in the presence of regulatory CD4+FoxP3+ cells. DISCUSSION: Midgut carcinoid patients display elevated T regulatory cell numbers and T cell dysfunction. Therapeutic strategies to overcome tumor-induced Th1 immunosuppression are required in combination with anti-tumor vaccinations.     

Prognostic value of intra‐tumoral CD8+/FoxP3+ lymphocyte ratio in patients with resected colorectal cancer liver metastasis

Background and Objectives

Patients with isolated colorectal‐cancer‐liver‐metastases (CRCLM) frequently undergo metastatectomy. Tumor‐infiltrating‐lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor‐infiltrating CD8⁺ cytotoxic T‐cells and FoxP3⁺ regulatory T‐cells at the metastatic site of CRCLM patients.

Methods

TILs were isolated from fresh metastatic tissues of 47 patients with CRCLM. Archived paraffin‐embedded tissue, from the same patients, was retrieved. CD8⁺ and FoxP3⁺ cells, both in the intra‐tumoral and the peri‐tumoral compartments, were measured by immunohistochemistry on full tissue sections. Proportions of cytotoxic T‐cells (CD8⁺) and regulatory T‐cells (CD4⁺CD25⁺FoxP3⁺), within CD45⁺TILs, were measured by flow‐cytometry.

Results

By immunohistochemistry, individual densities of intra‐tumoral or peri‐tumoral CD8⁺ and FoxP3⁺ cells were not prognostic of survival. However, the intra‐tumoral, but not the peri‐tumoral, CD8⁺/FoxP3⁺ ratio was an independent predictor of survival (HR 0.43, 95%CI 0.19‐0.95, P = 0.032). By flow cytometry, the intra‐tumoral CD8⁺/regulatory T‐cell ratio was also an independent predictor of survival (HR 0.45, 95%CI 0.20‐0.99, P = 0.044).

Conclusions

The ratio of cytotoxic (CD8⁺) to regulatory (FoxP3⁺) T‐cells, in the intra‐tumoral compartment, but not in the peri‐tumoral compartment, can predict survival after resection of CRCLM.     

Eradication of established tumors by vaccination with Venezuelan equine encephalitis virus replicon particles delivering human papillomavirus 16 E7 RNA.

The etiological role of human papillomaviruses (HPV) in cervical and other cancers suggests that therapeutic vaccines directed against requisite viral antigens may eradicate tumors or their precursors. A Venezuelan equine encephalitis (VEE) alphavirus vector delivering the HPV16 E7 RNA was evaluated for antitumor efficacy using a murine E7+ tumor model. Vaccination with VEE replicon particles expressing E7 (E7-VRP) induced class I-restricted CD8+ T-cell responses as determined by IFN-gamma enzyme-linked immunospot (ELISPOT), tetramer, and cytotoxicity assays. E7-VRP vaccination prevented tumor development in all of the mice and effectively eliminated 7-day established tumors in 67% of tumor-bearing mice. The induction of protective T-cell responses was dependent on CD8+, but not CD4+ T cells. Long-lasting T-cell memory responses developed in E7-VRP-vaccinated mice as determined by complete protection from tumor challenge 3 months after the final vaccination. These promising results highlight the potent CD8+ T-cell-mediated antitumor effects elicited by VEE replicon-based vectors and support their further development toward clinical testing against cervical intraepithelial neoplasia or carcinoma.     

Interrater agreement of anal cytology

BACKGROUND:

The majority of anal cancers are caused by persistent infections with carcinogenic human papillomaviruses (HPV). Similar to cervical carcinogenesis, the progression from HPV infection to anal cancer occurs through precancerous lesions that can be treated to prevent invasion. In analogy to cervical cytology, anal cytology has been proposed as a screening tool for anal cancer precursors in high‐risk populations.

METHODS:

The authors analyzed the interobserver reproducibility of anal cytology in a population of 363 human immunodeficiency virus (HIV)‐infected men who have sex with men (MSM). Liquid‐based cytology (LBC) specimens were collected in the anal dysplasia clinic before the performance of high‐resolution anoscopy on all patients. Papanicolaou‐stained LBC slides were evaluated by 2 cytopathologists, each of whom was blinded to the clinical outcome and the other pathologist's results, using the revised Bethesda terminology.

RESULTS:

Overall agreement between the 2 observers was 66% (kappa, 0.54; linear‐weighted kappa, 0.69). Using dichotomizing cytology results (atypical squamous cells of undetermined significance [ASC‐US] or worse vs less than ASC‐US), the agreement increased to 86% (kappa, 0.69). An increasing likelihood of testing positive for markers associated with HPV‐related transformation, p16/Ki‐67, and HPV oncogene messenger RNA was observed, with increasing severity of cytology results noted both for individual cytologists and for consensus cytology interpretation (P value for trend [p_trend] < .0001 for all).

CONCLUSIONS:

Moderate to good agreement was observed between 2 cytopathologists evaluating anal cytology samples collected from HIV‐positive MSM. A higher severity of anal cytology was associated with biomarkers of anal precancerous lesions. Anal cytology may be used for anal cancer screening in high‐risk populations, and biomarkers of HPV‐related transformation can serve as quality control for anal cytology. Cancer (Cancer Cytopathol) 2013. Published 2012 by the American Cancer Society.     

Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine.

PURPOSE: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specific T-cell response in cervical cancer patients. EXPERIMENTAL DESIGN: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specific T-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFN gamma-ELISPOT, and cytokine production and phenotyped by the T-cell markers CD4, CD8, CD25, and Foxp3. RESULTS: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively. These responses were broad, involved both CD4(+) and CD8(+) T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4(+)CD25(+)Foxp3(-) type 1 cytokine IFN gamma-producing T cells but also included the expansion of T cells with a CD4(+)CD25(+)Foxp3(+) phenotype. CONCLUSIONS: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4(+) and CD8(+) T cells to a broad array of epitopes in all patients. The expansion of CD4(+) and CD8(+) tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-induced T cells display a CD4(+)CD25(+)Foxp3(+) phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of T cells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.     

Hyperthermia and irradiation of head and neck squamous cancer cells causes migratory profile changes of tumour infiltrating lymphocytes

Purpose: CD4⁺CD25⁺FoxP3⁺ regulatory T-cells (Treg) are responsible for immunoevasion mechanisms induced by cancer. Specific chemokines such as CCL22 are presumed to mediate active Treg trafficking into the tumour site. In this context, the effects of irradiation and hyperthermia of tumour cells on Treg migration and the CCL22 concentration in the tumour cell supernatants after treatment were studied. Moreover, the relationship between CCL22 concentration and Treg cell migration was also examined.

Materials and methods: Treg and CD4⁺CD25^? T-cells were isolated from human peripheral blood. Supernatants were obtained from primary cell cultures derived from head and neck carcinoma patients. Tumour cell cultures were treated with a dose of 2 Gy and hyperthermia (41.5°C) or with hyperthermia or irradiation alone. Cancer cell culture supernatants were then used for a transmigration assay.

Results: Treg and CD4⁺CD25^? T-cells showed an increased transmigration towards supernatants of hyperthermia-treated tumour cells. After combined application of hyperthermia and irradiation, Treg migration was similar to control levels, but CD4⁺CD25^? migration was still enhanced. Irradiation caused a significantly decreased Treg influx, whereas the CD4⁺CD25^? T-cell migration was not altered after the same treatment. Changes of Treg chemotaxis could be attributed to a treatment-associated escalation of the CCL22 in the tumour cell supernatants.

Conclusion: The combination of irradiation and hyperthermia is able to modify transmigration of tumour infiltrating lymphocytes beneficially and individually. In this in vitro system hyperthermia alone negatively impacts the immune response by selectively recruiting Treg, whereas hyperthermia with the addition of irradiation negates this effect.     

Characterization of HPV subtypes not covered by the nine-valent vaccine in patients with CIN 2-3 and cervical squamous cell carcinoma

Background: As the second most common female malignant tumor, cervical cancer is also one of the most preventable and avoidable cancers. The World Health Organization has launched a global plan to accelerate the elimination of cervical cancer. Therefore, in the era of postvaccine, the role of HPV subtypes in cervical precancerous lesions and cervical cancer that are not covered by vaccine should be further discussed. The purpose of this study was to explore the role of HPV subtypes not covered by the nine-valent vaccine in high-grade cervical precancerous lesions and cervical cancer. Materials and methods: A retrospective analysis was performed on the clinical data of 5220 patients with an HPV infection who were diagnosed and treated in the Department of Gynecology of Shanghai General Hospital between October 2016 and February 2020. In addition, the clinical characteristics of the biopsy results of 470 cases of cervical intraepithelial neoplasia (CIN) 2-3 and 205 cases of cervical squamous cell carcinoma were analyzed. Results: Among patients with HPV subtype infection not covered by the nine-valent vaccine, univariate analysis showed that compared with patients with CIN 2-3, age ≥ 50, not using condom and TCT reported as ASC-H were risk factors for cervical squamous cell carcinoma (P < 0.05). The detection rates of HPV subtype not covered by the nine-valent vaccine in CIN 2-3 and cervical squamous cell carcinoma patients were 7.23% and 6.34%, respectively. Conclusion: In patients with CIN 2-3 and cervical squamous cell carcinoma, the infection rates of HPV subtype not covered by the nine-valent vaccine were 7.23% and 6.34%, respectively. With the increasing popularity of the vaccine, the infection rates of the corresponding HPV subtype decreased; however, HPV subtype infection not covered by the nine-valent vaccine should not be ignored.     

Expression of coinhibitory receptors on T cells in the microenvironment of usual vulvar intraepithelial neoplasia is related to proinflammatory effector T cells and an increased recurrence‐free survival

Human papillomavirus‐induced usual‐type vulvar intraepithelial neoplasia (uVIN) are infiltrated by immune cells but apparently not cleared. A potential explanation for this is an impaired T cell effector function by an immunesuppressive milieu, coinfiltrating regulatory T cells or the expression of coinhibitory molecules. Here, the role of these potential inhibitory mechanisms was evaluated by a detailed immunohistochemical analysis of T cell infiltration in the context of FoxP3, Tbet, indoleamine 2,3‐dioxygenase, programmed cell death 1, T cell immunoglobulin mucin 3 (TIM3), natural killer cell lectin‐like receptor A (NKG2A) and galectins‐1, ?3 and ?9. Paraffin‐embedded tissues of primary uVIN lesions (n = 43), recurrent uVIN lesions (n = 20), vulvar carcinoma (n = 21) and healthy vulvar tissue (n = 26) were studied. We show that the vulva constitutes an area intensely surveyed by CD8+, CD4+, Tbet+ and regulatory T cell populations, parts of which express the examined coinhibitory molecules. In uVIN especially, the number of regulatory T cells and TIM3+ T cells increased. The expression of the coinhibitory markers TIM3 and NKG2A probably reflected a higher degree of T cell activation as a dense infiltration with stromal CD8+TIM3+ T cells and CD3+NKG2A+ T cells was related to the absence of recurrences and/or a prolonged recurrence‐free survival. A dense coinfiltrate with regulatory T cells was negatively associated with the time to recurrence, most dominantly when the stromal CD8+TIM3+ infiltration was limited. This notion was sustained in vulvar carcinoma's where the numbers of regulatory T cells progressively increased to outnumber coinfiltrating CD8+TIM3+ T cells and CD3+NKG2A+ T cells.     

Regulatory (FOXP3+) T cells as target for immune therapy of cervical intraepithelial neoplasia and cervical cancer

Regulatory (FOXP3⁺) T cells (T_regs) comprise a subpopulation of CD4⁺ T cells that suppress autoreactive immune cells, thereby protecting organs and tissues from autoimmunity. T_regs have also been detected in human malignancies and their depletion or inactivation substantially improves cellular antitumor immunity in preclinical studies. Novel therapeutic strategies for cervical cancer and precancerous cervical intraepithelial neoplasia (CIN) focus on immune-modulatory and cancer vaccination approaches. In this context, the frequency of T_regs in cervical cancer and precancerous CIN could influence therapeutic strategies. We determined the frequency of infiltrating CD4⁺ and CD8⁺ T cells as well as FOXP3⁺ T_regs in high-grade CIN lesions (CIN III) and cervical carcinoma compared to colon carcinoma, skin melanoma, and bronchial carcinoma. We show that human papilloma virus-derived lesions have a significantly higher number of infiltrating lymphocytes and FOXP3⁺ T_regs compared to three other common tumor entities. In addition we explored the therapeutic effect of agonistic anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein antibodies that, by single systemic application, inactivate T_regs and induce strong intratumoral invasion of CD8⁺ T cells and complete tumor eradication in 70% of treated animals. The large number of T_regs in human papilloma virus-derived lesions suggests a pivotal role of T_regs for counteracting the host immune response. We therefore regard CIN and cervical cancer as prime targets for new immune-based non-invasive therapies. ( Cancer Sci 2009; 100: 1112–1117)