After obtaining informed consent, peripheral blood was isolated from healthy volunteers (n = 10) and from patients with melanoma (n = 21). Patients were staged and classified according to the American Joint Committee on Cancer Melanoma Staging and Classification criteria [60] . B cells were isolated by negative selection using RosetteSepH B cell enrichment cocktail (Stem Cell Technologies, Vancouver, Canada) according to the manufacturer's instructions. B cell purity was assessed by flow cytometry by staining for mature B cells (CD22), T cells (CD3), monocytes (CD14) and plasmacytoid dendritic cells (BDCA3) using fluorescently-labeled monoclonal antibodies, all from BD Biosciences, Oxford, UK ( Figure S1 ). Flow cytometry experiments were conducted with either the FACSAria or FACSCanto (BD Biosciences) and flow cytometric data were analyzed using Flow Jo (Tree Star, Ashland, OR).