Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies.

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Prevention and treatment of papillomavirus-related cancers through immunization.

Cervical and other anogenital cancers are initiated by infection with one of a small group of human papillomaviruses (HPV). Virus-like particle-based vaccines have recently been developed to prevent infection with two cancer-associated HPV genotypes (HPV16, HPV18) and have been ∼95% effective at preventing HPV-associated disease caused by these genotypes in virus-naive subjects. Although immunization induces virus-neutralizing antibody sufficient to prevent infection, persistence of antibody as measured by current assays does not appear necessary to maintain protection over time. Investigators have not identified a reliable surrogate immunological marker of protection against disease following immunization. The prophylactic vaccines are not therapeutic for existing infection. Trials of HPV-specific immunotherapy have shown some efficacy for existing disease, although animal modeling suggests that a combination of immunization and local enhancement of innate immunity may be necessary for optimal therapeutic outcome. HPV prophylactic vaccines are the first vaccines designed to prevent a human cancer and are the practical outcome of a global collaborative effort between basic and applied scientists, clinicians, and industry.

No Vacillation on HPV Vaccination.

Evidence of the safety and protective benefits of human papillomavirus virus (HPV) vaccines as an anti-cancer measure is overwhelming. However, vaccine uptake varies widely across countries and falls short of levels needed to achieve population immunity. We highlight policy measures that would help ensure greater worldwide coverage and save lives.

Single-dose HPV vaccine immunity: is there a role for non-neutralizing antibodies?

A single dose of human papillomavirus (HPV) vaccine against HPV infection (prerequisite for cervical cancer) appears to be as efficacious as two or three doses, despite inducing lower antibody titers. Neutralizing antibodies are thought to be the primary mediator of protection, but the threshold for protection is unknown. Antibody functions beyond neutralization have not been explored for HPV vaccines. Here, we discuss the immune mechanisms of HPV vaccines, with a focus on non-neutralizing antibody effector functions. In the context of single-dose HPV vaccination where antibody is limiting, we propose that non-neutralizing antibody functions may contribute to preventing HPV infection. Understanding the immunological basis of protection for single-dose HPV vaccination will provide a rationale for implementing single-dose HPV vaccine regimens.

Scavenging of soluble and immobilized CCL21 by ACKR4 regulates peripheral dendritic cell emigration.

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130-4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623-630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341-3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.

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.

Analysis of human leukocyte antigen associations in human papillomavirus-positive and -negative head and neck cancer: Comparison with cervical cancer.

BACKGROUND: Although the majority of human papillomavirus (HPV) infections are cleared by the immune system, a small percentage of them progress to develop HPV-driven cancers. Cervical cancer studies highlight that HPV persistence and cancer risk are associated with genetic factors, especially at the human leukocyte antigen (HLA) genes. This study was conducted to investigate such associations in head and neck cancer (HNC). METHODS: In all, 192 patients with HNC and 384 controls were genotyped with the Infinium Global Screening Array (Illumina, Inc). HLA variants were imputed with SNP2HLA, and an association analysis was performed by logistic regression. RESULTS: HPV-positive HNCs were significantly associated with single-nucleotide polymorphisms (SNPs) at DRB1_32660090 (P = 1.728 × 10-6 ) and DRB1_32660116 (P = 1.728 × 10-6 ) and with the amino acid variant DRB1_11_32660115 (P = 1.728 × 10-6 ). None of these associations were observed in the HPV-negative cohort, and this suggested their specificity to convey risk for HPV-associated HNCs. In general, associations observed for HPV-negative HNC were relatively weak, and variants in the HLA-DPA1 region were the strongest among them (P = 4.531 × 10-4 ). Several lead signals reported by previous HNC genome-wide association studies, including SNPs rs3135001 (P = .012), rs1049055 (P = .012), and rs34518860 (P = .029) and allele HLA-DQB1*06 (P = .009), were replicated in the current study. However, these associations were limited to the HPV-positive HNC group. Several cervical cancer-associated HLA variants, including SNPs rs9272143 (P = .002) and rs9271858 (P = .002) and alleles HLA-B-1501 (P = .009) and HLA-B-15 (P = .015), were also exclusively associated with HPV-positive HNC. CONCLUSIONS: HPV-positive HNC risk is associated with distinct HLA variants, and some of them are shared by both cervical cancer and HPV-positive HNC. Human papillomavirus (HPV)-positive head and neck cancer (HNC) risk is associated with distinct human leukocyte antigen variants, and some of them are shared by both cervical cancer and HPV-positive HNC. LAY SUMMARY: Cervical cancer studies highlight that human papillomavirus (HPV)-driven cancer risk is linked with human leukocyte antigen (HLA) polymorphism. Hence, the current study was designed to investigate the HLA associations in HPV-positive and HPV-negative head and neck cancer (HNC) and compare these associations with cervical cancer. Several lead signals reported by previous HNC and cervical genome-wide association studies were replicated in the current study. However, these associations were limited to the HPV-positive HNC group, and this suggests that HPV-positive HNC risk is associated with distinct HLA variants, and some of them are shared by both cervical cancer and HPV-positive HNC.

Correction: Defining the genetic susceptibility to cervical neoplasia-A genome-wide association study.

[This corrects the article DOI: 10.1371/journal.pgen.1006866.].

Oral HPV16 DNA as a screening tool to detect early oropharyngeal squamous cell carcinoma.

Given that oropharyngeal squamous cell carcinoma (OPSCC) have now surpassed cervical cancer as the most common human papillomavirus (HPV)-driven cancer, there is an interest in developing non-invasive predictive biomarkers to early detect HPV-driven OPSCC. In total, 665 cancer-free individuals were recruited from Queensland, Australia. Oral HPV16 DNA positivity in those individuals was determined by our in-house developed sensitive PCR method. Individuals with (n = 9) or without (n = 12) oral HPV16 infections at baseline were followed for a median duration of 24 mo. Individuals with persistent oral HPV16 infection (≥ 30 mo) were invited for clinical examination of their oral cavity and oropharynx by an otolaryngologist. Oral HPV16 DNA was detected in 12 out of 650 cancer-free individuals (1.8%; 95% confidence interval [CI]: 1.0-3.2). Of the 3 individuals with persistent oral HPV16 infection, the first individual showed no clinical evidence of pathology. The second individual was diagnosed with a 2 mm invasive squamous cell carcinoma (T1N0M0) positive for both p16INK4a expression and HPV16 DNA. The third individual was found to have a mildly dysplastic lesion in the tonsillar region that was negative for p16INK4a expression and HPV16 DNA and she continues to have HPV16 DNA in her saliva. Taken together, our data support the value of using an oral HPV16 DNA assay as a potential screening tool for the detection of microscopic HPV-driven OPSCC. Larger multicenter studies across various geographic regions recruiting populations at a higher risk of developing HPV-driven OPSCC are warranted to extend and confirm the results of the current investigation.

Antibody-Free Multiplex Measurement of 23 Human Cytokines in Primary Cell Culture Secretome Using Targeted Mass Spectrometry.

Cytokines are commonly measured by immunoassays; however, these have limited multiplexing capacity, are costly, and can exhibit cross-reactivity. Multiple reaction monitoring (MRM) mass spectrometry is a robust method to quantify analytes with high specificity and multiplexing ability, hence we aimed to investigate its suitability as an alternative cost-effective method for cytokine measurement. Human keratinocyte conditioned media spiked with recombinant cytokines was used as an experimental system to evaluate sensitivity, linearity, and reproducibility of an MRM assay targeting 79 peptides representing 23 human cytokines. Our MRM method was able to identify 21 cytokines by two or more unique peptides and two cytokines by a single unique peptide. In a serum-free matrix, the median LOD and LOQ for cytokine peptides was 130 and 433 pg/mL, respectively. The presence of serum increased median LOD and LOQ by about 2.3-fold. The assay shows excellent replicate consistency with 8% intra- and 12% interday coefficient of variations. We found high pH reversed-phase fractionation a useful tool to increase assay sensitivity with the drawback of increasing its variability by approximately 10%. Overall, our results suggest utility of a multiplex cytokine MRM for routine measurement of secreted cytokines in cellular experiments under low serum conditions. Additional enrichment steps will be required in high complexity matrices such as serum.

Defining the genetic susceptibility to cervical neoplasia-A genome-wide association study.

A small percentage of women with cervical HPV infection progress to cervical neoplasia, and the risk factors determining progression are incompletely understood. We sought to define the genetic loci involved in cervical neoplasia and to assess its heritability using unbiased unrelated case/control statistical approaches. We demonstrated strong association of cervical neoplasia with risk and protective HLA haplotypes that are determined by the amino-acids carried at positions 13 and 71 in pocket 4 of HLA-DRB1 and position 156 in HLA-B. Furthermore, 36% (standard error 2.4%) of liability of HPV-associated cervical pre-cancer and cancer is determined by common genetic variants. Women in the highest 10% of genetic risk scores have approximately >7.1% risk, and those in the highest 5% have approximately >21.6% risk, of developing cervical neoplasia. Future studies should examine genetic risk prediction in assessing the risk of cervical neoplasia further, in combination with other screening methods.

HLA and KIR Associations of Cervical Neoplasia.

Background: Cervical cancer is the fourth most common cancer in women, and we recently reported human leukocyte antigen (HLA) alleles showing strong associations with cervical neoplasia risk and protection. HLA ligands are recognized by killer immunoglobulin-like receptors (KIRs) expressed on a range of immune cell subsets, governing their proinflammatory activity. We hypothesized that the inheritance of particular HLA-KIR combinations would increase cervical neoplasia risk. Methods: Here, we used HLA and KIR dosages imputed from single-nucleotide polymorphism genotype data from 2143 cervical neoplasia cases and 13858 healthy controls of European decent. Results: The following 4 novel HLA alleles were identified in association with cervical neoplasia, owing to their linkage disequilibrium with known cervical neoplasia-associated HLA-DRB1 alleles: HLA-DRB3*9901 (odds ratio [OR], 1.24; P = 2.49 × 10-9), HLA-DRB5*0101 (OR, 1.29; P = 2.26 × 10-8), HLA-DRB5*9901 (OR, 0.77; P = 1.90 × 10-9), and HLA-DRB3*0301 (OR, 0.63; P = 4.06 × 10-5). We also found that homozygosity of HLA-C1 group alleles is a protective factor for human papillomavirus type 16 (HPV16)-related cervical neoplasia (C1/C1; OR, 0.79; P = .005). This protective association was restricted to carriers of either KIR2DL2 (OR, 0.67; P = .00045) or KIR2DS2 (OR, 0.69; P = .0006). Conclusions: Our findings suggest that HLA-C1 group alleles play a role in protecting against HPV16-related cervical neoplasia, mainly through a KIR-mediated mechanism.

Role of Ultraviolet Radiation in Papillomavirus-Induced Disease.

Human papillomaviruses are causally associated with 5% of human cancers. The recent discovery of a papillomavirus (MmuPV1) that infects laboratory mice provides unique opportunities to study the life cycle and pathogenesis of papillomaviruses in the context of a genetically manipulatable host organism. To date, MmuPV1-induced disease has been found largely to be restricted to severely immunodeficient strains of mice. In this study, we report that ultraviolet radiation (UVR), specifically UVB spectra, causes wild-type strains of mice to become highly susceptible to MmuPV1-induced disease. MmuPV1-infected mice treated with UVB develop warts that progress to squamous cell carcinoma. Our studies further indicate that UVB induces systemic immunosuppression in mice that correlates with susceptibility to MmuPV1-associated disease. These findings provide new insight into how MmuPV1 can be used to study the life cycle of papillomaviruses and their role in carcinogenesis, the role of host immunity in controlling papillomavirus-associated pathogenesis, and a basis for understanding in part the role of UVR in promoting HPV infection in humans.

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.

Does the nature of residual immune function explain the differential risk of non-melanoma skin cancer development in immunosuppressed organ transplant recipients?

Patients receiving immunosuppression to prevent organ transplant rejection are at a greatly increased risk of developing nonmelanoma skin cancer. In recent years a correlation has been identified between the class of immunosuppressant that these patients receive and their subsequent cancer risk; in particular, patients switched from calcineurin inhibitors to mammalian target of rapamycin (mTOR) inhibitors not only displayed a dramatic reduction in new tumor formation but also in some cases a regression of their existing lesions. Studies of cancer models in mice and cell lines in the laboratory have attributed these discrepancies in cancer risk to the ability of immunosuppressants such as mTOR inhibitors to elicit direct anticancer effects, including suppressing angiogenesis and increasing autophagy-mediated DNA repair. Recent evidence from the immunological literature however, suggests a significant alternative contribution of mTOR inhibitors; namely the promotion of memory T-cell function. Recent advances in understanding memory T-cell establishment and the demonstration of their critical role in long-term immunity make it timely to review the available evidence as to whether the improved nonmelanoma skin cancer outcome shown by patients switched to mTOR inhibitor treatment regimens may be associated with the retainment of memory T-cell function.

HPV16-E7 expression in squamous epithelium creates a local immune suppressive environment via CCL2- and CCL5- mediated recruitment of mast cells.

Human Papillomavirus (HPV) 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions.

A pilot study to compare the detection of HPV-16 biomarkers in salivary oral rinses with tumour p16(INK4a) expression in head and neck squamous cell carcinoma patients.

BACKGROUND: Human papilloma virus-16 (HPV-16) infection is a major risk factor for a subset of head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal squamous cell carcinoma (OPSCC). Current techniques for assessing the HPV-16 status in HNSCC include the detection of HPV-16 DNA and p16(INK4a) expression in tumor tissues. When tumors originate from hidden anatomical sites, this method can be challenging. A non-invasive and cost-effective alternative to biopsy is therefore desirable for HPV-16 detection especially within a community setting to screen at-risk individuals. METHODS: The present study compared detection of HPV-16 DNA and RNA in salivary oral rinses with tumor p16(INK4a) status, in 82 HNSCC patients using end-point and quantitative polymerase chain reaction (PCR). RESULTS: Of 42 patients with p16(INK4a)-positive tumours, 39 (sensitivity = 92.9 %, PPV = 100 % and NPV = 93 %) had oral rinse samples with detectable HPV-16 DNA, using end-point and quantitative PCR. No HPV-16 DNA was detected in oral rinse samples from 40 patients with p16(INK4a) negative tumours, yielding a test specificity of 100 %. For patients with p16(INK4a) positive tumours, HPV-16 mRNA was detected using end-point reverse transcription PCR (RT-PCR) in 24/40 (sensitivity = 60 %, PPV = 100 % and NPV = 71 %), and using quantitative RT-PCR in 22/40 (sensitivity = 55 %, PPV = 100 % and NPV = 69 %). No HPV-16 mRNA was detected in oral rinse samples from the p16(INK4a)-negative patients, yielding a specificity of 100 %. CONCLUSIONS: We demonstrate that the detection of HPV-16 DNA in salivary oral rinse is indicative of HPV status in HNSCC patients and can potentially be used as a diagnostic tool in addition to the current methods.

Development and implementation of papillomavirus prophylactic vaccines.

Translation of basic scientific findings into practical patient outcomes is a significant exercise even when the goal is conceptually straightforward, as in the development of a vaccine for an infectious disease. Recognition of the association of cervical cancer with papillomavirus infection encouraged development of a vaccine to help with prevention of this very common cancer, causing over 250,000 deaths each year worldwide. To introduce a vaccine program, it was however necessary to develop a technology for making viral Ag, demonstrate that systemic immunization could provide mucosal surface protection in the genital tract, develop assays for vaccine potency, and understand enough about the epidemiology and natural history of the infection to plan effective intervention strategies. This process took ∼25 years. The major hurdle, now that effective vaccines are available, is to ensure their deployment in the countries where they are most needed. The development and deployment of human papillomavirus vaccines demonstrate the benefits of collaborative research activity across the globe, and between academia and industry, to translate scientific discoveries into public health benefits.

IL-17 suppresses immune effector functions in human papillomavirus-associated epithelial hyperplasia.

Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia that can progress to cancer and is thought to depend on immunosuppressive mechanisms that prevent viral clearance by the host. IL-17 is a cytokine with diverse functions in host defense and in the pathology of autoimmune disorders, chronic inflammatory diseases, and cancer. We analyzed biopsies from patients with HPV-associated cervical intraepithelial neoplasia grade 2/3 and murine skin displaying HPV16 E7 protein-induced epithelial hyperplasia, which closely models hyperplasia in chronic HPV lesions. Expression of IL-17 and IL-23, a major inducer of IL-17, was elevated in both human HPV-infected and murine E7-expressing lesions. Using a skin-grafting model, we demonstrated that IL-17 in HPV16 E7 transgenic skin grafts inhibited effective host immune responses against the graft. IL-17 was produced by CD3(+) T cells, predominantly CD4(+) T cells in human, and CD4(+) and γδ T cells in mouse hyperplastic lesions. IL-23 and IL-1ß, but not IL-18, induced IL-17 production in E7 transgenic skin. Together, these findings demonstrate an immunosuppressive role for IL-17 in HPV-associated epithelial hyperplasia and suggest that blocking IL-17 in persistent viral infection may promote antiviral immunity and prevent progression to cancer.