We demonstrated a high prevalence of melanoma patientderived antibodies produced by circulating B cells in cancer patients that recognize melanoma cell lines (Figure 2 ). We observed that B cell culture supernatants from different patients displayed differential binding to each cell line, which reflects specificity and reactivity of different antibodies to a range of antigens expressed at different levels in a number of melanoma cell lines; these may also reflect binding to some antigens lowly expressed on human melanocytes ( Figure 4 and Table 1 ). Melanoma patients had a high percentage of melanoma-reactive antibody-producing B cell cultures, significantly higher than those from healthy volunteer-derived B cell cultures (Figure 2) , with 28% of melanoma patient-derived B cell cultures recognizing melanoma cells, compared to 2% of cultures from healthy volunteers (Figure 4) . Limiting dilution analyses of reactivity against melanoma cells versus normal melanocytes provided further evidence in support of the presence and frequency of tumor-reactive B cells in Figure 6 . Selection of a patient derived B antibody that recognizes melanoma cells but not melanocytes. A selected tumor-reactive culture from a stage III patient (Patient 6) which secreted antibodies that bound to tumor cell lines and compared to melanocytes by ELISA ( Figure 5 , C) was sub-cloned, and a monoclonal antibody 6_2G3 was selected and evaluated on live cells by flow cytometry (solid black line histograms) for reactivity to fibroblasts, melanocytes, and 6 melanoma cell lines. IgG isotype controls are shown in shaded grey histograms. doi:10.1371/journal.pone.0019330.g006 Figure 7 . A tumor-specific antibody derived from a patient with melanoma is able to induce tumor cell cytotoxicity. Two monoclonal antibodies were evaluated in vitro using a live cell imaging assay: 6_2G3 clone bound to A-375 cells compared to melanocytes; and 6_2D10, a clone also from Patient 6 did not, and served as a negative control antibody for these experiments. (A) Cell viability of A-375 melanoma cells incubated with patient blood (Figure 4) . For one stage II patient evaluated, the data indicate that metastatic melanoma cells are recognized by a higher proportion of B cells (estimated on average , 1 in 2,000 mature B cells) compared to primary melanoma cells or melanocytes, although in a cohort of 10 patients we measured reactivity of B cell cultures to both metastatic and primary melanoma cell lines ( Figure 2 ). Taking into consideration the expected variability in immune responses among patients, and the array of tumor antigens these patients may be exposed to, the observations that B cells from two patients with stage II melanoma yielded comparable reactivity to metastatic melanoma cells (estimated 1 in 1,790 for Patient 15, and 1 in 2,430 B cells for Patient 21) indicate the presence and support the prevalence of a circulating melanoma-reactive B cell compartment.