CD47 blocking on RBCs enhances the induction of TT-specific CD4+T-cell proliferation. (A) RBC phagocytosis by neutrophils was measured by incubating neutrophils ± DiD-labeled plus RBCs or RBC-ops, ± F(ab′)2 CD47 for 45 minutes. Subsequently, RBC were lysed and the percentage of RBC-phagocytosing neutrophils was measured by flow cytometry. Comparing the RBC-ops condition with the RBC-ops- F(ab′)2 CD47 condition, RBC phagocytosis increases from 45% to 73% (mean ± SEM; n = 5). (B) The percentage of proliferation measured for T cells incubated with neutrophils plus RBCs or RBC-ops, ± F(ab′)2 CD47 and ± TT. T cells served as a negative control and T cells plus PBMCs as positive control (mean ± SEM; n = 8). (C) Surface expression of HLA-DR, CD40, and CD80 on nonphagocytosing and phagocytosing neutrophils (mean ± SEM; n = 4). (D) Western blot analysis for HLA-DR expression performed on neutrophils ± RBCs or RBC-ops and ± F(ab′)2 CD47. Fluorescence intensity of the bands was quantified using Odyssey Imaging system and normalized for GAPDH expression. (E) Western blot analysis for CD40 expression performed on neutrophils ± RBCs or RBC-ops and ± F(ab′)2 CD47. Fluorescence intensity of the bands was quantified using Odyssey Imaging system and normalized for actin expression. (D-E) May-Grünwald Giemsa stain; original magnification ×500. Asterisks above the straight-line bars represent significant differences compared with T = 0 control neutrophils; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (**P < .01; *P < .05).