ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Downregulation of HLA-I by the molluscum contagiosum virus mc080 impacts NK-cell recognition and promotes CD8+ T-cell evasion.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.

Transpalpebral proton beam radiotherapy of choroidal melanoma.

BACKGROUND: Collateral damage to upper eyelid margin during proton beam radiotherapy (PBR) for choroidal melanoma may cause squamous metaplasia of the tarsal conjunctiva with keratinisation, corneal irritation, discomfort and, rarely, corneal perforation. We evaluated transpalpebral PBR as a means of avoiding collateral damage to the upper eyelid margin without increasing the risk of failure of local tumour control. METHODS: Retrospective study of consecutive patients who underwent PBR for choroidal melanoma between 1992 and 2007 at the Royal Liverpool University Hospital and the Douglas Cyclotron at Clatterbridge Cancer Centre, UK. RESULTS: Sixty-three patients were included in this study. Mean basal tumour diameter and tumour thickness were 11.8 mm and 3.6 mm, respectively. PBR mean beam range and modulation were 26.5 mm and 16.9 mm respectively. The eyelid margin was included in the radiation field in 15 (24%) eyes. The median follow-up was 2.5 years. Local tumour recurrence developed in 2 (3.2%) patients. In these two cases that developed tumour recurrence the transpalpebral treatment did not involve the eyelid margin. Six (9.5%) patients died of metastatic disease. No eyelid or ocular surface problems developed in any of the 48 patients who were treated without eyelid rim involvement, while 7 of the 15 patients with unavoidable irradiation of the eyelid rim developed some degree of madarosis. These seven patients all received more than 26.55 proton Gy to the eyelid margin. Symptoms, such as grittiness occurred in 12% of 48 patients without eyelid margin irradiation as compared with 53% of 15 patients whose lid margin was irradiated. CONCLUSIONS: Transpalpebral PBR of choroidal melanoma avoids eyelid and ocular surface complications without increasing failure of local tumour control.

Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation.

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αß and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1-6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12-US21; a genetic arrangement, which is suggestive of an 'accordion' expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.

Whole anterior segment proton beam radiotherapy for diffuse iris melanoma.

AIM: To report the results of whole anterior segment proton beam radiotherapy (PBR) for diffuse iris melanoma. METHODS: Between 2000 and 2011, 12 patients with iris melanoma received PBR to the entire iris and ciliary body. RESULTS: Patients had a mean age of 57 years and a median follow-up of 3.5 years (range 1-11.6 years). Tumour iris involvement was 1-4 h in five patients, 5-8 h in four and 9-12 h in three. Angle involvement was 6-8 h in five patients and 9-12 h in seven. The visual acuity (VA) before treatment was 6/5-6/6 in six patients, 6/8-6/9 in three and 6/18-6/38 in three. No tumour recurrence occurred during the follow-up period. Glaucoma treatment was required in 11 of 12 patients. The visual acuity at the last follow-up was 6/5-6/9 in five patients, 6/18-6/24 in three, 6/60-1/60 in two and no light perception in two. Four patients developed varying non-severe degrees of limbal stem cell deficiency, which was treatable with conservative measures. CONCLUSIONS: Whole anterior segment PBR is a useful alternative to enucleation for diffuse iris melanoma. Most patients will need treatment for glaucoma and some may require treatment for tear-film instability and/or stem cell failure.

Comparison of interventions to reduce sun exposure.

Exposure to ultraviolet (UV) light is the leading behavioral cause of skin cancer. This study evaluated the efficacy of 2 interventions to reduce UV exposure in college students prior to an opportunity for high-intensity exposure. Participants of 1 college campus were assigned randomly to 1 of 2 interventions prior to their spring holiday spent in a warm, sunny location: (1) a community-based informational campaign, or (2) a combination of the campaign and a cognitive-behavioral small group intervention. Participants of a second college campus served as a comparison group. The cognitive-behavioral group exhibited increased knowledge, more positive attitudes toward UV protection, greater advancement through stages of change, and greater protective clothing use relative to the comparison or community-education groups. The informational campaign had little apparent impact in this study.