Regenerative Medicine Department, Operational and Undersea Medicine Directorate at the Naval Medical Research Center, Silver Spring, MD 20910-7500, USA.
Transpl Immunol. 2010 Oct;24(1):69-75. doi: 10.1016/j.trim.2010.10.003. Epub 2010 Oct 13.
Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.
利用高剂量供体骨髓细胞（BMC）进行供体特异性免疫耐受已在混合嵌合体耐受诱导方案中得到证实；然而，移植物抗宿主病（GVHD）的发展仍然存在风险。在本研究中，我们证明在同种异体移植后 7 天输注低数量的供体 Lin(-)骨髓细胞（Lin(-)BMC）有助于高水平的 macros 嵌合体诱导和移植物耐受。将 BALB/c 全厚皮肤同种异体移植物移植到 C57BL/6 小鼠上。在第 0、+2、+5、+7 和+14 天，用抗 CD4 和抗 CD8 mAb 处理小鼠，并用低剂量白消安处理第+5 天。在第+7 天，输注来自 BALB/c 供体小鼠的低剂量高度纯化的 Lin(-)BMC。使用流式细胞术评估嵌合率和克隆细胞缺失。通过供体和第三方皮肤移植和混合白细胞反应（MLR）测试供体特异性耐受性。低剂量免疫抑制联合 Lin(-)BMC 输注可导致稳定、混合、多谱系 macros 嵌合体和长期同种异体移植物存活（>300 天）。观察到混合供体-受者 macros 嵌合体。供体反应性 T 细胞被克隆删除，脾中观察到 CD4(+)CD25(+)Foxp3(+)调节性 T 细胞（Tregs）增加 130%。耐受的小鼠随后接受了第二个供体，但不能接受第三个（C3H）皮肤移植物，并且受体脾细胞不能与同种异体供体细胞反应，表明已获得供体特异性免疫耐受。我们得出结论，在没有细胞减少受体条件的情况下输注供体 Lin(-)BMC 可以通过建立多谱系 macros 嵌合体状态的机制诱导皮肤同种异体移植物的无限期存活，主要通过同种反应性 T 细胞的克隆删除和外周诱导的 CD4(+)Foxp3(+)Tregs。
