O20 Advanced melanoma is associated with distinct circulating B-cell profiles enriched with immunoregulatory and autoreactive features
Authors: Willsmore Zena , Crescioli Silvia , Booth Lucy , Laddach Roman , Yorke Rozalyn , Harris Robert  , Chauhan Jitesh , Chenoweth Alicia  , Adams Rebecca  , Osborn Gabriel , Di Meo Ashley  , Prassas Ioannis , Geh Jenny L C , Patel Akshay J   , Middleton Gary W , MacKenzie Ross Alastair D   , Healey Ciaran , Diamandis Eleftherios P , Sinha Khushboo , Wu Yin , Whittaker Sean J , Perucha Esperanza , Papa Sophie , Lacy Katie E   , Karagiannis Sophia N 
Published: July 2023

Abstract
There is increasing evidence that B cells play a significant role in the immunobiology of melanoma; however, reports of the pro- vs. antitumour roles of B cells are inconsistent. We hypothesized that advanced melanoma favours regulatory B-cell phenotypes and class switching to less effective antibody isotypes. We conducted an in-depth characterization of the circulating humoral compartment in patients with advanced stage III/IV melanoma (n = 46), compared to healthy volunteers (n = 16). Phenotyping of circulating B cells was performed using B-cell-directed mass cytometry (CyTOF); serum immunoglobulin antibody isotyping was conducted using multiplex immunoassay; and immuno-mass spectrometry was employed to screen serum IgGs for autoantibodies against &gt; 13 000 candidate human proteins. The melanoma B-cell compartment was enriched in naïve CD21lo B cells, plasmablasts and double-negative B cells, compared to samples from healthy controls. These populations shared significant characteristics with phenotypes enriched in classical autoimmune diseases, most notably systemic lupus erythematosus (SLE). An enriched plasmablast population showed the highest median scaled expression of regulatory B-cell markers interleukin (IL)-10 and CD95 (Fas) of all B-cell populations, suggesting a potential immunosuppressive role. Consistent with an IL-10-enriched, T helper 2-biased environment, serum antibody isotyping revealed enrichment of IgG4, a less effective antibody isotype that may promote tumour propagation, in patients with melanoma, compared to healthy controls. In support of an autoreactive immune response, immuno-mass spectrometry screening of serum IgGs revealed altered autoreactivity in sera from patients with melanoma vs. with healthy control sera. Some autoantibodies were found exclusively in patients with melanoma, and these were reactive against human proteins recognized in carcinogenesis and proteins localized to the skin. Correlations between autoreactivity to tubulin cytoskeletal components and tumour burden was also identified. These findings show parallels with SLE, where one of the principal defects is an increased presence of apoptotic cell components and a breakdown of tolerance to cell-associated autoantigens. This may also suggest that localized immune reactivity in the tumour microenvironment may be reflected in an altered regulatory and autoreactive circulating humoral compartment. Our data identify distinct immature and regulatory B-cell signatures in patients with advanced melanoma that share characteristics with those reported in autoimmune diseases. Tumour-associated immune suppression may skew class switching to less effective antibody isotypes and toward regulatory B-cell phenotypes likely to favour tumour progression.