FIGURE 4.

CD45 regulation of galectin-1 susceptibility requires the intracellular phosphatase domain, and CD45 isoform usage determines the requirement for core 2 O-glycans. A, T cells lacking the intracellular domain of CD45 do not require C2GnT expression for galectin-1 susceptibility. Left, T200⁻ or Rev1.1 cells were transfected with cDNA encoding C2GnT or vector alone, and the presence of core 2 O-glycans determined by staining with 1B11-PE (open) or isotype control (filled). CD45 expression was determined with CD45-FITC (open) or isotype control (filled). Histograms are of representative clones. Right, T200-C2GnT, T200-vector, Rev-C2GnT, or Rev-vector cells were treated with galectin-1 or buffer control, and cell death was analyzed by staining with annexin-V-FITC and 7-AAD. T200⁻ and Rev1.1 cells were susceptible to galectin-1 death, and cells with or without core 2 O-glycans had comparable levels of cell death. Data are mean ± S.D. of at least duplicate samples, and representative of at least four experiments. B, expression of core 2 O-glycans does not enhance galectin-1 susceptibility in T cells expressing CD45RABC. Left, T200⁻ cells were transfected with CD45RABC and C2GnT (T200 RABC C2) or CD45RABC and vector control (T200 RABC vec), and core 2 O-glycan expression was determined as in A. Histograms are of representative clones. Right, cell death assays were performed as in A. Data are mean ± S.D. of triplicate samples. C, loss of CD45 phosphatase activity enhances T cell susceptibility to galectin-1. Left, T200⁻ cells transfected with CD45RABC (T200RABC) or the inactive phosphatase mutant CD45RABC-C817S (T200C817S) were stained for CD45 as above. Right, while cells expressing CD45RABC were sensitive to galectin-1, complete loss of CD45 phosphatase activity in the C817S mutant enhanced susceptibility to galectin-1. Cell death assays were performed as above. Data are mean ± S.E. of triplicate samples from three independent experiments. Significance was determined by Student's t test.