Modulation of antigen presenting cell functions during chronic HPV infection.

High-risk human papillomaviruses (HR-HPV) infect basal keratinocytes, where in some individuals they evade host immune responses and persist. Persistent HR-HPV infection of the cervix causes precancerous neoplasia that can eventuate in cervical cancer. Dendritic cells (DCs) are efficient in priming/cross-priming antigen-specific T cells and generating antiviral and antitumor cytotoxic CD8+ T cells. However, HR-HPV have adopted various immunosuppressive strategies, with modulation of DC function crucial to escape from the host adaptive immune response. HPV E6 and E7 oncoproteins alter recruitment and localization of epidermal DCs, while soluble regulatory factors derived from HPV-induced hyperplastic epithelium change DC development and influence initiation of specific cellular immune responses. This review focuses on current evidence for HR-HPV manipulation of antigen presentation in dendritic cells and escape from host immunity

Immune-Inhibitory Gene Expression is Positively Correlated with Overall Immune Activity and Predicts Increased Survival Probability of Cervical and Head and Neck Cancer Patients

Background: Limited immunotherapy options are approved for the treatment of cervical cancer and only 10–25% of patients respond effectively to checkpoint inhibition monotherapy. To aid the development of novel therapeutic immune targets, we aimed to explore survival-associated immune biomarkers and co-expressed immune networks in cervical cancer. Methods: Using The Cancer Genome Atlas (TCGA) Cervical Squamous Cell Carcinoma (CESC) data (n = 304), we performed weighted gene co-expression network analysis (WGCNA), and determined which co-expressed immune-related genes and networks are associated with survival probability in CESC patients under conventional therapy. A “Pan-Immune Score” and “Immune Suppression Score” was generated based on expression of survival-associated co-expressed immune networks and immune suppressive genes, which were subsequently tested for association with survival probablity using the TCGA Head Neck Squamous Cell Carcinoma (HNSCC) data (n = 528), representing a second SCC cancer type. Results: In CESC, WGCNA identified a co-expression module enriched in immune response related genes, including 462 genes where high expression was associated with increased survival probability, and enriched for genes associated with T cell receptor, cytokine and chemokine signaling. However, a high level of expression of 43 of the genes in this module was associated with decreased survival probability but were not enriched in particular pathways. Separately, we identified 20 genes associated with immune suppression including inhibitory immune checkpoint and regulatory T cell-related genes, where high expression was associated with increased survival probability. Expression of these 20 immune suppressive genes (represented as “Immune Suppression Score”) was highly correlated with expression of overall survival-associated immune genes (represented as “Pan-Immune Score”). However, high expression of seven immune suppression genes, including TWEAK-R, CD73, IL1 family and TGFb family genes, was significantly associated with decreased survival probability. Both scores also significantly associated with survival probability in HNSCC, and correlated with the previously established “Immunophenoscore.” Conclusion: CESC and HNSCC tumors expressing genes predictive of T cell infiltrates (hot tumors) have a better prognosis, despite simultaneous expression of many immune inhibitory genes, than tumors lacking expression of genes associated with T cell infiltrates (cold tumors) whether or not these tumor express immune inhibitory genes.</p

HPV16 E7-driven epithelial hyperplasia promotes impaired antigen presentation and regulatory T cell development

Human papillomaviruses (HPV) infect keratinocytes and can lead to hyperproliferative dysplasia and malignant transformation if not cleared by the immune system. HPV has evolved an array of mechanisms to evade and manipulate the immune system to improve replication efficiency and promote persistent infection. We here demonstrate that hyperproliferative skin expressing the high-risk HPV16 E7 oncogene as a transgene drives immune-modulation of dendritic cells (DCs) resulting in reduced capacity to take up antigen and prime effector CD4 T cell responses. The phenotype of DCs in the E7-expressing hyperproliferative skin was not reversible by activation through intradermal immunization. Naïve CD4 T cells primed by E7-driven hyperproliferative skin acquired FoxP3 expression and an anergic phenotype. DC and T help modulation was dependent on E7-Rb interaction-driven epithelial hyperproliferation, rather than on expression of E7, as inhibition of binding of E7 to retinoblastoma protein, and of consequent epithelial hyperplasia was associated with normal skin DC phenotype, and Th1 effector responses to immunization were restored. We conclude that HPV-induced epithelial hyperplasia modulates epithelial DCs and inhibits Th1 immunity while polarizing T cell differentiation to a regulatory or anergic phenotype

A model of impaired Langerhans cell maturation associated with HPV induced epithelial hyperplasia.

Funder: National Health and Medical Research CouncilLangerhans cells (LC) are skin-resident antigen-presenting cells that regulate immune responses to epithelial microorganisms. Human papillomavirus (HPV) infection can promote malignant epithelial transformation. As LCs are considered important for controlling HPV infection, we compared the transcriptome of murine LCs from skin transformed by K14E7 oncoprotein and from healthy skin. We identified transcriptome heterogeneity at the single cell level amongst LCs in normal skin, associated with ontogeny, cell cycle, and maturation. We identified a balanced co-existence of immune-stimulatory and immune-inhibitory LC cell states in normal skin that was significantly disturbed in HPV16 E7-transformed skin. Hyperplastic skin was depleted of immune-stimulatory LCs and enriched for LCs with an immune-inhibitory gene signature, and LC-keratinocyte crosstalk was dysregulated. We identified reduced expression of interleukin (IL)-34, a critical molecule for LC homeostasis. Enrichment of an immune-inhibitory LC gene signature and reduced levels of epithelial IL-34 were also found in human HPV-associated cervical epithelial cancers

Epithelium expressing the E7 oncoprotein of HPV16 attracts immune-modulatory dendritic cells to the skin and suppresses their antigen-processing capacity

Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs

Dysregulation of stemness pathways in HPV mediated cervical malignant transformation identifies potential oncotherapy targets

Human papillomavirus (HPV) infection is associated with a range of malignancies that affect anogenital and oropharyngeal sites. α-HPVs dominantly infect basal epithelial cells of mucosal tissues, where they dysregulate cell division and local immunity. The cervix is one of the mucosal sites most susceptible to HPV infections. It consists of anatomically diverse regions, and the majority of cervical intraepithelial neoplasia and cancers arise within the cervical squamo-columnar junction where undifferentiated basal progenitor cells with stem cell properties are found. The cancer stem cell theory particularly associates tumorigenesis, invasion, dissemination, and metastasis with cancer cells exhibiting stem cell properties. In this perspective, we discuss evidence of a cervical cancer stem cell niche and explore the association of stemness related genes with 5-year survival using a publicly available transcriptomic dataset of a cervical cancer cohort. We report that poor prognosis in this cohort correlates with overexpression of a subset of stemness pathway genes, a majority of which regulate the central Focal Adhesion pathway, and are also found to be enriched in the HPV infection pathway. These observations support therapeutic targeting of stemness genes overexpressed by mucosal cells infected with high-risk HPVs

Skin-resident DCs of K14.E7 mice are impaired in their ability to process antigen in vivo.

<p><b>(A)</b> Whole dermal and epidermal single cell suspensions of C57BL/6 and K14.E7 ear skin were incubated with 1ug/ml DQ-OVA for 1 hour and analyzed for photolytic degradation by flow cytometry. DC subsets were gated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152886#pone.0152886.t001" target="_blank">Table 1</a>. The green median fluorescent intensity of whole LCs, DQ-OVA+ CD103+ DCs and DQ-OVA+ CD11b+ DCs was compared. <b>(B)</b> C57BL/6 and K14.E7 mice were injected intradermally into the ear with 20μl of 1mg/ml labeled DQ-OVA. 24 hours later, single cell suspensions of treated and untreated ear dermis and epidermis were prepared and analyzed by flow cytometry for photolytic degradation of applied DQ-OVA, as recognized by fluorochrome unmasking. Histogram overlays of fluorescence in LCs, CD103⁺ dDCs and CD11b⁺ dDCs determined according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152886#pone.0152886.t001" target="_blank">Table 1</a> in one representative C57BL/6 (grey line) and K14.E7 (black line) mouse ear treated with DQ-OVA. The change in green median fluorescent intensity from untreated to DQ-OVA treated in dermal and epidermal LCs, CD103⁺ dDCs and CD11b⁺ dDCs from K14E7 (●) and C57BL/6 (■) mouse ears (n = 4) was compared. <b>(C)</b> C57BL/6 and K14.E7 mice were injected intravenously with OT-I CD8+ T cells and immunized intradermally into the ear pinnae with 20μl of 1mg/ml OVA. One week later, IFNγ secretion by OT-I T cells was analyzed by recalling ear-draining lymph node cells with SIINFEKL antigen in an ELISpot assay. Depicted are spots per 2x10⁵ cells. Each data point represents means of triplicates of one individual animal (n = 5).</p

Antigen presenting cells are increased and of altered phenotype in K14.E7 transgenic mouse epidermis.

<p>Single cell suspensions of C57BL/6 (C57) and K14.E7 epidermis were analyzed for different CD45⁺ (myeloid) antigen presenting cell subsets. <b>(A)</b> Gating strategy to identify LCs, CD11b⁺ and CD103⁺ DCs in C57BL/6 and K14.E7 epidermis. Plots were pre-gated on live CD45⁺ cell singlets. <b>(B)</b> Relative proportions of LCs, CD103+ and CD11b+ DC subsets from CD11c⁺ DCs in K14.E7 and C57BL/6 epidermis. <b>(C)</b> Relative (as % of CD45⁺ live cells) and absolute number of LCs in the epidermis of two K14.E7 and C57BL/6 mouse ears <b>(D)</b> Expression of cell surface markers MHCII, CD86, CD80, CD207 and CD11b on epidermal LCs from C57BL/6 and K14.E7 ear skin. Typical histograms (C57 = black, K14.E7 = white) and pooled median fluorescent intensity (MFI) data (n = 6, two independent determinations) are shown.</p

Skin-resident DCs of K14.E7 mice take up antigen and migrate to the draining lymph nodes.

<p>C57BL/6 and K14.E7 mice were painted with 100μl of 5mg/ml FITC dissolved in 1:1 acetone:dibutylphalate on shaved flanks and ears. 24 hours later, axillary and inguinal lymph nodes were analyzed by flow cytometry for presence of FITC⁺ DC subsets. <b>(A)</b> FITC uptake by DC subsets in lymph nodes pre-gated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152886#pone.0152886.t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152886#pone.0152886.s001" target="_blank">S1 Fig</a>. <b>(B)</b> MFI of FITC⁺ LCs in the ear epidermis pre-gated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152886#pone.0152886.g001" target="_blank">Fig 1A</a>. <b>(C)</b> MFI and total number of FITC⁺ DCs in lymph node. Shown is one of two independent experiments, n = 4.</p