title: In vitro VLA-4 blockade results in an impaired NK cell-mediated immune surveillance against melanoma.
abstract: Natalizumab (NTZ) is a monoclonal antibody targeting the α4β1 integrin (CD49d/CD29), very late antigen-4 (VLA-4), which is approved for treatment of relapsing-remitting multiple sclerosis (RR-MS). A possible association between NTZ treatment and a higher risk of melanoma is under debate. Natural Killer (NK) cells, which express VLA-4, represent an innate barrier limiting spreading of melanoma under steady state conditions. Indeed, because of their expression of activating receptors, they are very efficient in recognizing and killing melanoma cells without the need of a previous priming. For this reason, we aimed at assessing whether NK-cell functions might be impaired by sustained exposure to NTZ. To investigate this possibility we isolated NK cells from healthy donors and tested their cytotoxic and migratory functions against primary melanoma cells derived from subcutaneous and lymph node metastases. Flow cytometry analysis demonstrated expression of CD49d on both freshly isolated NK cells and activated NK cells. Moreover, VLA-4 and its receptor, vascular cell adhesion protein-1 (VCAM-1) were similarly expressed on freshly isolated NK cells. However, upon a short exposure to NTZ, expression of VLA-4 on NK cells decreased. Analysis of NK receptor expression upon exposure of NK cells from three healthy donors to NTZ indicated that DNAM-1 and NKp46 are apparently decreased, while NKG2A is increased. The degranulation of NK cells towards melanoma cells, which express both VLA-4 and VCAM-1, was not affected when NTZ was added to the co-culture or when both NK cells and melanoma cells were each pre-exposed to NTZ for over 12h. In contrast, degranulation was significantly inhibited after 48h of pre-incubation indicating that NTZ can influence NK-cell degranulation towards melanoma cells only after a prolonged exposure. Using a migration chamber assay, we observed that the migration of NK cells towards melanoma cells was dependent upon the concentration of melanoma cells in the lower chamber, and that it was significantly reduced in presence of NTZ. Our results show that upon exposure to NTZ both cytolytic activity and migration toward melanoma cells were affected, suggesting that binding of NTZ to NK cells affects pathways involved in these NK-cell functions. We analyzed the expression of CD49d on NK cells from MS patients treated with NTZ and observed that it decreases with time of treatment. These data suggest that blockade of VLA-4 on NK-cell surface alters some key functions involved in the immune surveillance toward melanoma by NK cells and may provide a mechanistic explanation for the reported occurrence of melanoma in MS patients treated with NTZ.
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Gandoglia
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Ivaldi
authors.affiliation: Laboratory of Clinical and Experimental Immunology, Giannina Gaslini Institute, Genoa, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Carrega
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy.
authors.forename: Eric
authors.initials: E
authors.lastname: Armentani
authors.affiliation: Laboratory of Immunology and Biotherapy, Department of Human Pathology, University of Messina, Messina, Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Ferlazzo
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy; IRCCS Azienda Ospedaliera Universitaria San Martino - IST, Genoa, Italy.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Mancardi
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy.
authors.forename: Nicole
authors.initials: N
authors.lastname: Kerlero de Rosbo
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy; IRCCS Azienda Ospedaliera Universitaria San Martino - IST, Genoa, Italy. Electronic address: auccelli@neurologia.unige.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Uccelli
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy; IRCCS Azienda Ospedaliera Universitaria San Martino - IST, Genoa, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Laroni
mesh_terms: D000328:Adult; D015550:Cell Degranulation / Q000187:drug effects / Q000276:immunology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology; D003430:Cross-Sectional Studies; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D006801:Humans; D007157:Immunologic Surveillance / Q000187:drug effects*; D039041:Integrin alpha4beta1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D008297:Male; D008545:Melanoma / Q000235:genetics / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D008875:Middle Aged; D000069442:Natalizumab / Q000494:pharmacology; D055607:Receptors, Natural Killer Cell / Q000378:metabolism; D055815:Young Adult
chemical_list: D039041:Integrin alpha4beta1; D000069442:Natalizumab; D055607:Receptors, Natural Killer Cell
keywords: Melanoma; Multiple sclerosis; NK cells; Natalizumab; VLA-4