Lymphoma Gal-1 expression correlates with CD20 mAb resistance. (A-C) Gal-1 expression inversely correlates with lymphoma sensitivity to CD20 immunotherapy. Scatter plots compare normalized (A) and quantitative (B) Lgals1 transcript expression and Gal-1 secretion (C) relative to CD20 mAb sensitivity for each lymphoma analyzed. (D) Addition of Gal-1 blocks CD20 mAb-dependent phagocytosis of lymphoma cells. Peritoneal macrophages were cocultured with labeled BL3750 lymphoma cells as in Figure 2A, with or without CD20 mAb or rGal-1 added to the cultures as indicated. Each dot represents the results of individual experiments, with bars indicating means. (E) Representative GFP and cell surface CD20 expression by BL3750^Ctrl or BL3750^Gal-1 cells (open histograms) vs BL3750 cells (top panels, shaded histograms) or isotype-matched control mAb staining (middle panels, shaded histograms). Values represent mean (± SEM) Gal-1 secretion by BL3750, BL3750^Ctrl, and BL3750^Gal-1 cells (bottom panel, pooled results from 5 experiments). (F) Gal-1 blocks CD20 mAb-dependent phagocytosis of lymphoma. Peritoneal macrophages were cocultured for 24 hours with labeled BL3750^Ctrl or BL3750^Gal-1 lymphomas previously incubated with control (top panels) or CD20 mAb (middle panels), with representative contour plots showing F4/80 vs labeled BL3750 staining and mean (± SEM) frequencies of phagocytosed lymphomas cells shown. Values represent mean (± SEM) frequencies of BL3750^Ctrl (circles) or BL3750^Gal-1 (squares) previously incubated with control (open shapes), CD20 (closed shapes), or CD19 mAb (gray shapes) and cultured for 3, 6, and 24 hours with macrophages from 3 to 5 experiments (bottom panel, n = 7-20 mice per group).