Figure 1. B cell depletion prevents tolerance.

(A) C57BL/6 mice i.v. injected with anti-mCD20 mAb (100μg/mouse) or isotype control IgG. After 3 days, CD19⁺B220⁺ cells in the spleen, LN and thymus analyzed by flow cytometry. In bone marrow, IgM⁺IgD⁺ cells analyzed by gating on CD19⁺ cells. Based on FSC-A vs. SSC-A profile, all the cells gated on the lymphocytic population. (B) C57BL/6 mice i.v. injected with anti-mCD20 mAb (100μg/mouse). CD19⁺B220⁺ cells as a percentage of total lymphocytes in spleen, LN and thymus analyzed by flow cytometry at different time points after gating on lymphocytic population. (C) C57BL/6 recipient mice given tolerogen (DST, 1 X 10⁷ cells/mouse d−7 relative to transplantation; and anti-CD40L mAb 250 μg/mouse d−7, −4, 0 and +4) and received BALB/c cardiac allografts or C57BL/6 syngeneic grafts on d0. Recipients given anti-mCD20 mAb (100μg/mouse) d+1. Graft survival monitored (left). After 5 days, grafts harvested, sections made and stained with hematoxylin and eosin (H & E), and parenchymal rejection scores calculated (right). H & E staining of graft (bottom). (D) C57BL/6 recipients given tolerogen and BALB/c allografts as described above, and various doses of anti-mCD20 mAb on d +1. Graft survival plotted (left). After 5 days, grafts harvested, sections stained with H & E, and parenchymal rejection scores calculated (right). (E) Human CD20 transgenic (H-2^d) mice given tolerogen and C57BL/6 (H-2^b) cardiac allografts. Anti-human CD20 mAb (100μg/mouse) given i.v. on d+1. Graft survival (left), Masson’s trichrome staining and parenchymal rejection score calculated (middle), and H & E staining on d+5 (right).