Robust, systemic tolerance after mixed chimerism induction with ABT-737 and CsA. B6 recipients were treated with an irradiation-free conditioning protocol including ABT-737 (50 mg/kg/day), CsA (10 mg/kg/day) from day −3 to day 12, MR1 (2 mg), and 25 × 106 BM cells from CBA donors. Six weeks after BMT, skin transplantation from CBA and BALB/c donors was performed. (A) FACS analysis 33 weeks after BMT revealed a significant percentage of donor-derived antigen-presenting cells (CD11c+) in the thymus, but not in T cells and in T-cell precursors (DN, double negative; DP, double positive). (B) Serum samples were collected 60 days after skin transplantation and analyzed by indirect FACS using CBA and BALB/c cells: a complete absence of CBA-reactive IgG and a normal seroconversion toward BALB/c were measured. MFI, mean fluorescence intensity. Statistical comparison between mixed chimeras (MC) and a group of naïve mice is shown. *P < .05; N = 5. (C) Thirty-three weeks after BMT, recipient mice were killed and their splenocytes stimulated in vitro with irradiated B6 (syn), CBA (BM donor), and BALB/c (3rd party) splenocytes in a classical MLR. T-cell proliferation analysis, measured by 3H-thymidin incorporation, revealed a complete lack of T-cell reactivity against CBA and a normal response toward BALB/c. CPM, counts per minute; ***P < .001; N = 5 per group. (D) A group of mice received a second skin graft 58 days after initial transplantation. All BALB/c grafts were rejected within 10 days, whereas CBA grafts were accepted for more than 40 days without signs of rejection. N = 7 per group. (E) Photograph of a representative example at day 100 after first transplantation.
