Sickle mice treated with BMT after busulfan and costimulation blockade developed stable WBC mixed chimerism in the peripheral blood and the hematopoietic organs.

(A) Mean WBC chimerism in the peripheral blood. Sickle mice (H2-K^b) recipients were treated with 20 mg/kg busulfan (intraperitoneally) on day −1, with 500 μg CTLA4-Ig and anti-CD40L intraperitoneally on days 0, 2, 4, and 6, and underwent transplantation with allogeneic BALB/c (H2-K^d) TDBM (2 × 10⁷ cells intravenously on day 0). The data in this figure represent the mean percent engraftment for 7 engrafted mice that were fully analyzed for more than 150 days (▪). Three mice became transiently engrafted but rejected their grafts on or before 60 days. These mice completely reverted to the host WBC and RBC phenotype by 3 to 4 months after transplantation (data not shown). Control animals received 1 of 3 treatments: TDBM alone (n = 5, ○), TDBM plus busulfan (no costimulation blockade, n = 5, ▵), or TDBM plus costimulation blockade but no busulfan (n = 9, □) Animals in each of the control groups showed less than 2.5% peripheral WBC chimerism. (B) Lineage-specific chimerism over time. The percent of each lineage (granulocytes [GR1], macrophages [CD11b], B cells [B220], and T cells [CD3]) that was donor type (H2-K^d) was identified by flow cytometry over time. Values shown are the mean for each time point from 7 engrafted animals ± SEM. ▪ indicates B220; ●, GR1; ▴, CD11b; ⧫, CD3. (C) Representative flow cytometric analysis. At left is a histogram showing H2-K^d staining of a chimeric animal with both host (H2-K^d-negative) and donor (H2-K^d-positive) WBCs. At right is a dot plot showing a representative quadrant analysis of CD3 cells versus H2-K^d. (D) WBC chimerism in the bone marrow (▪), spleen (■), and thymus (░). Engrafted sickle cell mice (n = 4) showed 45% ± 15% chimerism (H2-K^d-positive cells by flow cytometry) in the splenic compartment, 50% ± 10% chimerism in the bone marrow, and 25% ± 15% chimerism in the thymus.