Induction of donor-specific tolerance with splenocytes-for clinical application to organ transplantation

同种异体嵌合体在移植受体中已经建立，宿主中的供体细胞可能在促进耐受的诱导中发挥重要作用。在本研究中，我们研究了通常在身体器官捐献手术中获取的异基因脾细胞(SPLC)混合物对骨髓细胞耐受诱导的影响。1)注射3x10^6骨髓细胞不能建立有效的混合嵌合体。单次注射10×10~(-6)SPLCs可完全排斥供体细胞。然而，在一组非常低剂量的3x10^6骨髓细胞和10×10^6 SPLC的混合物中，小鼠成功地诱导了嵌合体。这些嵌合小鼠在体内表现出供者特异性的耐受性；皮肤移植。富含T细胞的SPLCs与少量的BMCs混合后可诱导有效的嵌合体，而B细胞富集群的SPLCs与BMCs混合后不能形成有效的嵌合体。不含巨噬细胞的SPLC在诱导耐受方面与单纯SPLC一样有效。即使是低剂量的骨髓细胞，如果与强大的SPLC相结合，也可以建立一种同种异体嵌合体和供体特异性耐受的状态。SPLC中的T细胞可能在支持骨髓细胞诱导供者特异性免疫耐受中发挥重要作用。2)研究了供者脾细胞与外周血中干细胞混合的效果。给予粒细胞刺激因子，检测外周血干细胞。低剂量的干细胞与脾细胞混合物建立了有效的嵌合体。这一结果表明了嵌合体诱导的临床应用的可能性，因为从PBL中安全地获取干细胞而不是骨髓细胞。3)GVHD仍然是一个非常令人担忧的问题。T细胞可能在移植物抗宿主病中起主要作用，但它们也在嵌合体的建立中起作用。有必要建立一种在不消除T细胞的情况下预防GVHD的方法。

Allogeneic chimerism has been established in the graft-accepting recipients and the donor cells in the host may act in a major role to facilitate the induction of tolerance. In the present study, we investigated effects of allogeneic splenocytes (SPLCs) mixture, which we usually procure at cadaveric organ donor surgery, to the bone marrow cells on the tolerance induction.1) Injection of 3x10^6 BMCs did not establish effective mixed chimerism. Donor cells were completely rejected by single 10x10^6 SPLCs injection. In a group of a mixture of very low dose of 3x10^6 BMCs and 10x10^6 SPLCs, however, mice were successfully induced chimerism. These chimeric mice showed donor-specific tolerance in vivo; skin grafting. T-cell enriched SPLCs could induce effective chimerism when mixed with a small amount of BMCs, but a mixture of B-cell enrich population of SPLCs and BMCs failed to establish effective chimerism. SPLCs without macrophages were as effective as naive SPLCs to induce tolerance. Even low dose of BMCs can establish a state of allogeneic chimerism and donor specific tolerance if combined with vigorous SPLCs. T-cells in SPLCs may play a important role to support BMCs for induction of donor-specific tolerance.2) Efficacy of a donor splenocytes mixed with stem cells in the peripheral blood were also investigated. Granulocyte stimulating factor was given and stem cell from peripheral blood was examined. Effective chimerism was established with a low dose of stem cell with a mixture of splenocytes. This results indicates the possibility of clinical usage of a chimerism induction because of safe procurement of stem cell in PBL instead of bone marrow cells.3) GVHD remains as a great concern. T-cells may play a major role for GVHD, however, they do for establishment of chimerism. It is necessary to establish a method to prevent GVHD without eliminating T-cells.