Manganese-Doped Silica-Based Nanoparticles Promote the Efficacy of Antigen-Specific Immunotherapy.

Prophylactic human papillomavirus (HPV) vaccines are commercially available for prevention of infection with cancerogenic HPV genotypes but are not able to combat pre-existing HPV-associated disease. In this study, we designed a nanomaterial-based therapeutic HPV vaccine, comprising manganese (Mn4+)-doped silica nanoparticles (Mn4+-SNPs) and the viral neoantigen peptide GF001 derived from the HPV16 E7 oncoprotein. We show in mice that Mn4+-SNPs act as self-adjuvants by activating the inflammatory signaling pathway via generation of reactive oxygen species, resulting in immune cell recruitment to the immunization site and dendritic cell maturation. Mn4+-SNPs further serve as Ag carriers by facilitating endo/lysosomal escape via depletion of protons in acidic endocytic compartments and subsequent Ag delivery to the cytosol for cross-presentation. The Mn4+-SNPs+GF001 nanovaccine induced strong E7-specific CD8+ T cell responses, leading to remission of established murine HPV16 E7-expressing solid TC-1 tumors and E7-expressing transgenic skin grafts. This vaccine construct offers a simple and general strategy for therapeutic HPV and potentially other cancer vaccines.

CD8+ lineage dendritic cells determine adaptive immune responses to inflammasome activation upon sterile skin injury.

The molecular links between sterile inflammation and induction of adaptive immunity have not been fully identified. Here, we examine how damage-associated molecular patterns (DAMPs), as opposed to pathogen-associated molecules (PAMPs), regulate the immune response to non-self-antigens presented at the site of a physical injury. Heat applied briefly to the skin invokes sterile inflammation, characterized by local cell death and caspase-1 activation without demonstrably disrupting skin integrity. Co-delivery of ovalbumin (OVA) with heat injury induces OVA-specific CD8+ T-cell responses, and this is dependent on caspase-1 activation and MyD88 signalling. Using Id2flox/flox-CD11cCre+ mice, we demonstrate that CD8+ lineage DCs are required to induce OVA-specific CD8+ T-cell responses following heat injury. Consistent with this observation, intradermal administration of CD8+ lineage DCs but not CD11b+ lineage DCs restores priming of CD8+ T-cell responses in Casp-1-/- mice. Thus, we conclude that a sterile injury induces CD8+ T-cell immune responses to local antigen through caspase-1 activation and requires CD8+ lineage DCs, a finding of significance for immunotherapy and for the pathogenesis of autoimmunity.

Batf3 selectively determines acquisition of CD8+ dendritic cell phenotype and function.

Batf3 is a transcription factor that impacts the development of CD103+ tissue-resident dendritic cells (DCs). However, whether Batf3 is absolutely required for the development of CD8+ DCs remains controversial. Id2 is required for CD8+ DC development. Here we show that bone marrow chimeric mice with a deletion of Id2 in the CD11c compartment lose the ability to reject a skin graft expressing a non-self protein antigen or mount a delayed hypersensitivity response. In contrast, Batf3-/- mice remained competent for skin graft rejection and delayed hypersensitivity, and retained a CD8+ DC population with markers characteristic of the CD11b+ DC lineage, including CD11b, CD4 and CD172α, as well as the key regulator transcription factor IRF4, but lacked IRF8 expression. CD8+ DCs in Batf3-/- mice took up and cleaved protein antigen and larger particles but were unable to phagocytose dying cells, a characteristic feature to the CD8+ DC lineage. These data clarify a requirement for CD8+ lineage DCs to induce effectors of neo-antigen-driven skin graft rejection, and improve our understanding of DC subtype commitment by demonstrating that in the absence of Batf3 CD8+ DCs can change their fate and become CD11b+ DCs.

Characterization of the skin microbiome in normal and cutaneous squamous cell carcinoma affected cats and dogs.

Human cutaneous squamous cell carcinomas (SCCs) and actinic keratoses (AK) display microbial dysbiosis with an enrichment of staphylococcal species, which have been implicated in AK and SCC progression. SCCs are common in both felines and canines and are often diagnosed at late stages leading to high disease morbidity and mortality rates. Although recent studies support the involvement of the skin microbiome in AK and SCC progression in humans, there is no knowledge of this in companion animals. Here, we provide microbiome data for SCC in cats and dogs using culture-independent molecular profiling and show a significant decrease in microbial alpha diversity on SCC lesions compared to normal skin (P ≤ 0.05). Similar to human skin cancer, SCC samples had an elevated abundance of staphylococci relative to normal skin-50% (6/12) had >50% staphylococci, as did 16% (4/25) of perilesional samples. Analysis of Staphylococcus at the species level revealed an enrichment of the pathogenic species Staphylococcus felis in cat SCC samples, a higher prevalence of Staphylococcus pseudintermedius in dogs, and a higher abundance of Staphylococcus aureus compared to normal skin in both companion animals. Additionally, a comparison of previously published human SCC and perilesional samples against the present pet samples revealed that Staphylococcus was the most prevalent genera across human and companion animals for both sample types. Similarities between the microbial profile of human and cat/dog SCC lesions should facilitate future skin cancer research. IMPORTANCE: The progression of precancerous actinic keratosis lesions (AK) to cutaneous squamous cell carcinoma (SCC) is poorly understood in humans and companion animals, despite causing a significant burden of disease. Recent studies have revealed that the microbiota may play a significant role in disease progression. Staphylococcus aureus has been found in high abundance on AK and SCC lesions, where it secretes DNA-damaging toxins, which could potentiate tumorigenesis. Currently, a suitable animal model to investigate this relationship is lacking. Thus, we examined the microbiome of cutaneous SCC in pets, revealing similarities to humans, with increased staphylococci and reduced commensals on SCC lesions and peri-lesional skin compared to normal skin. Two genera that were in abundance in SCC samples have also been found in human oral SCC lesions. These findings suggest the potential suitability of pets as a model for studying microbiome-related skin cancer progression.

Targeting Staphylococcus aureus dominated skin dysbiosis in actinic keratosis to prevent the onset of cutaneous squamous cell carcinoma: Outlook for future therapies?

Cutaneous squamous cell carcinoma (cSCC) and its premalignant precursor, actinic keratosis (AK), present a global health burden that is continuously increasing despite extensive efforts to promote sun safety. Chronic UV exposure is a recognized risk factor for the development of AK and cSCC. However, increasing evidence suggests that AK and cSCC is also associated with skin microbiome dysbiosis and, in particular, an overabundance of the bacterium Staphylococcus aureus (S. aureus). Studies have shown that S. aureus-derived toxins can contribute to DNA damage and lead to chronic upregulation of proinflammatory cytokines that may affect carcinogenesis. Eradication of S. aureus from AK lesions and restoration of a healthy microbiome may therefore represent a therapeutic opportunity to alter disease progression. Whilst antibiotics can reduce the S. aureus load, antibiotic resistant S. aureus pose an increasing global public health threat. The use of specific topically delivered probiotics has been used experimentally in other skin conditions to restore eubiosis, and could therefore also present a non-invasive treatment approach to decrease S. aureus colonization and restore a healthy skin microbiome on AK lesions. This article reviews mechanisms by which S. aureus may contribute to cutaneous carcinogenesis, and discusses hypotheses and theories that explore the therapeutic potential of specific bacterial species which compete with S. aureus in an attempt to restore microbial eubiosis in skin.

HPV status represents dominant trait driving delineation of survival-associated gene co-expression networks in head and neck cancer.

Integration of high-dimensional tumor gene expression data with clinicopathological data can increase our understanding of disease diversity, enable retrospective patient stratification, and identify new potential biomarkers and therapeutic targets. Using a systems biology approach, we provide a holistic overview of gene co-expression networks in head and neck squamous cell carcinomas (HNSCC). Weighted gene co-expression network analysis of HNSCC RNA sequencing data from 519 patients from The Cancer Genome Atlas (TCGA) was used to determine correlates of 5-year survival, using regression tree-based optimal threshold calculations. Survival-associated gene sets were transformed to gene set scores that were assessed for correlation with clinicopathological data. We identified 8 gene co-expression modules for HNSCC tumors, each of which contained co-expressed genes associated significantly with 5-year survival. Survival-associated co-expression gene signatures correlated dominantly with tumor HPV and p16 status. Network analysis identified that survival was associated with signaling networks of infection, immunity, epithelial-mesenchymal transition (EMT), hypoxia, glycolysis, focal adhesion, extracellular matrix, MYC signaling, autophagy and transcriptional regulation. EMT-associated gene signatures were expressed dominantly in fibroblasts, and cancer-associated fibroblasts were inversely correlated with immune activity. Interestingly, a high Immune Suppression Score based on expression of 21 genes associated with immune inhibition and including immune checkpoints, cytokines and regulatory T cell factors, was also associated with increased survival probability, and was significantly higher in HPV+ HNSCC. Networks associated with HNSCC survival were further associated with survival in cervical cancer, melanoma and lung cancer. This study defines 5129 genes associated with HNSCC survival, organized into co-expressed networks, their correlation with clinicopathological data, and with gene expression data from other malignant diseases, and provides a source for the discovery of biomarkers and novel therapies for HNSCC.

Environmental pathways affecting gene expression (E.PAGE) as an R package to predict gene-environment associations.

The purpose of this study is to manually and semi-automatically curate a database and develop an R package that will act as a comprehensive resource to understand how biological processes are dysregulated due to interactions with environmental factors. The initial database search run on the Gene Expression Omnibus and the Molecular Signature Database retrieved a total of 90,018 articles. After title and abstract screening against pre-set criteria, a total of 237 datasets were selected and 522 gene modules were manually annotated. We then curated a database containing four environmental factors, cigarette smoking, diet, infections and toxic chemicals, along with a total of 25,789 genes that had an association with one or more of gene modules. The database and statistical analysis package was then tested with the differentially expressed genes obtained from the published literature related to type 1 diabetes, rheumatoid arthritis, small cell lung cancer, COVID-19, cobalt exposure and smoking. On testing, we uncovered statistically enriched biological processes, which revealed pathways associated with environmental factors and the genes. The curated database and enrichment tool are available as R packages at https://github.com/AhmedMehdiLab/E.PATH and https://github.com/AhmedMehdiLab/E.PAGE respectively.

Therapeutic DNA Vaccine Against HPV16-Associated Cancer.

Human papillomavirus (HPV) is a contagious cause of anogenital and oropharyngeal cancers developing from persistently infected and subsequently transformed basal keratinocytes of mucosal epithelium. DNA-based immunotherapy offers great potential for the treatment of persisting HPV infections and associated cancers. Preclinical testing of therapeutic DNA-based HPV-targeted immunotherapy requires robust animal models which mimic HPV-associated cancer disease in humans. Here we describe a detailed protocol of intradermal delivery of a therapeutic DNA vaccine and a grafting model of neoantigen expressing skin to evaluate vaccine efficacy against HPV16 mediated hyperproliferative epithelium in mice.

Antigen Nonspecific Induction of Distinct Regulatory T Cell States in Oncogene-Driven Hyperproliferative Skin.

Regulatory T cells (Tregs) are recruited to nonlymphoid tissues in chronic disease, including cancer, and the tissue environment is held to shape the Treg phenotype diversity. Using single-cell RNA sequencing, we examined the transcriptomic and TCR profile of Tregs recruited to hyperproliferative HPV16 E7-expressing transgenic and control nontransgenic murine skin grafts. Tregs were more abundant in E7 transgenic skin grafts than control grafts, without evidence of E7 specificity. E7 transgenic grafts attracted both Klrg1 + Tregs and Il1r2 + Tregs, which were phenotypically distinct but shared a core gene signature with previously described tumor-infiltrating Tregs. Pseudotime trajectory analysis of Tregs of defined TCR clonotypes predicted phenotypic plasticity within the skin and between the skin and draining lymph nodes. Thus, oncogene-induced hyperproliferative skin expressing a single defined non-self-antigen can attract and induce non-Ag-specific Tregs that acquire distinct regulatory phenotypes characterized by specific effector gene signatures.

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.

Regulatory T Cells but Not IL-10 Impair Cell-Mediated Immunity in Human Papillomavirus E7+ Hyperplastic Epithelium.

High-risk human papillomavirus infection can induce cervical and other intraepithelial neoplasia and invasive cancers. A transgenic mouse expressing keratin 14 promotor-driven HPV16 E7 oncoprotein exhibits epithelial hyperplasia and mimics many features of human papillomavirus-related intraepithelial precancers. We have previously demonstrated that HPV16 E7-mediated epithelial hyperplasia suppresses T helper type 1 responses to intradermally delivered antigen and directs differentiation of CD4+ T cells towards a Foxp3+ regulatory phenotype (Treg). Here we establish that Foxp3+ Treg expansion from a transferred naive T-cell population is driven directly by the hyperplastic skin and is independent of pre-existing immune-modulated lymphocytes. However, depletion of endogenous CD25+ Tregs before priming of adoptively transferred T cells significantly improves antigen-specific CD8+ T-cell responses but not T helper type 1 responses. Deletion of IL-10 had no effect on Treg expansion, epidermal dendritic cell alteration, and suppression of induced T helper type 1 immunity in HPV16 E7-driven hyperplastic mice. Thus, HPV16 E7-mediated epithelial hyperplasia promotes expansion of peripheral Tregs in response to intradermal immunization that suppress antigen-specific CD8+ T-cell responses independently of IL-10, but depletion of these Tregs is not sufficient to restore T helper type 1 immunity.

Acquisition of murine splenic myeloid cells for protein and gene expression profiling by advanced flow cytometry and CITE-seq.

Here, we outline detailed protocols to isolate and profile murine splenic dendritic cells (DCs) through advanced flow cytometry of the myeloid compartment and single-cell transcriptomic profiling with integrated cell surface protein expression through CITE-seq. This protocol provides a general transferrable road map for different tissues and species. For complete details on the use and execution of this protocol, please refer to Lukowski et al. (2021).

Absence of Batf3 reveals a new dimension of cell state heterogeneity within conventional dendritic cells.

Conventional dendritic cells (cDCs) are traditionally subdivided into cDC1 and cDC2 lineages. Batf3 is a cDC1-required transcription factor, and we observed that Batf3-/- mice harbor a population of cDC1-like cells co-expressing cDC2-associated surface molecules. Using single-cell RNA sequencing with integrated cell surface protein expression (CITE-seq), we found that Batf3-/- mitotic immature cDC1-like cells showed reduced expression of cDC1 features and increased levels of cDC2 features. In wild type, we also observed a proportion of mature cDC1 cells expressing surface features characteristic to cDC2 and found that overall cDC cell state heterogeneity was mainly driven by developmental stage, proliferation, and maturity. We detected population diversity within Sirpa+ cDC2 cells, including a Cd33+ cell state expressing high levels of Sox4 and lineage-mixed features characteristic to cDC1, cDC2, pDCs, and monocytes. In conclusion, these data suggest that multiple cDC cell states can co-express lineage-overlapping features, revealing a level of previously unappreciated cDC plasticity.

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

Langerhans 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.

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.

HPV16 E7-Driven Epithelial Hyperplasia Promotes Impaired Antigen Presentation and Regulatory T-Cell Development.

Human papillomaviruses infect keratinocytes and can lead to hyperproliferative dysplasia and malignant transformation if not cleared by the immune system. Human papillomavirus 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 human papillomavirus 16 E7 oncogene as a transgene drives immunomodulation 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-retinoblastoma protein interaction-driven epithelial hyperproliferation, rather than on expression of E7. Inhibition of binding of E7 to retinoblastoma protein, and of consequent epithelial hyperplasia, was associated with normal skin DC phenotype, and T helper type 1 effector responses to immunization were restored. We conclude that human papillomavirus-induced epithelial hyperplasia modulates epithelial DCs and inhibits T helper type 1 immunity while polarizing T-cell differentiation to a regulatory or anergic phenotype.

Immune responses to a HSV-2 polynucleotide immunotherapy COR-1 in HSV-2 positive subjects: A randomized double blinded phase I/IIa trial.

BACKGROUND: Genital herpes simplex infection affects more than 500 million people worldwide. We have previously shown that COR-1, a therapeutic HSV-2 polynucleotide vaccine candidate, is safe and well tolerated in healthy subjects. OBJECTIVE: Here, we present a single center double-blind placebo-controlled, randomized phase I/IIa trial of COR-1 in HSV-2 positive subjects in which we assessed safety and tolerability as primary endpoints, and immunogenicity and therapeutic efficacy as exploratory endpoints. METHODS: Forty-four HSV-2+ subjects confirmed by positive serology or pathology, and positive qPCR during baseline shedding, with a recurrent genital HSV-2 history of at least 12 months including three to nine reported lesions in 12 months prior to screening, aged 18 to 50 years females and males with given written informed consent, were randomized into two groups. Three immunizations at 4-week intervals and one booster immunization at 6 months, each of 1 mg COR-1 DNA or placebo, were administered intradermally as two injections of 500 µg each to either one forearm or both forearms. RESULTS: No serious adverse events, life-threatening events or deaths occurred throughout the study. As expected, HSV-2 infected subjects displayed gD2-specific antibody titers prior to immunization. COR-1 was associated with a reduction in viral shedding after booster administration compared with baseline. CONCLUSIONS: This study confirms the previously demonstrated safety of COR-1 in humans and indicates a potential for use of COR-1 as a therapy to reduce viral shedding in HSV-2 infected subjects.

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.

DNA Vaccine Encoding HPV16 Oncogenes E6 and E7 Induces Potent Cell-mediated and Humoral Immunity Which Protects in Tumor Challenge and Drives E7-expressing Skin Graft Rejection.

We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinical studies evaluating the efficacy of this vaccine in patients with HPV16 premalignancies are planned.