Establishment of drug-induced tolerance for heart transplantation in large animals

大型动物心脏移植药物诱导耐受性的建立

• 批准号: 12470242
• 负责人: YASUI Hisataka
• 金额: $ 9.15万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470242/
• 关键词: cyclophosphamide; Chimerism; drug-induced tolerance; bone marrow cells; 骨髄細胞; 副作用軽減; 大動物; 皮膚移植; ドナー特異的免疫寛容; Busulfan; α-Gal; B細胞

Recently, we have described a drug (cyclophosphamide (CP) plus busulfan (BU))-induced skin allograft tolerance in mice that cagularly overcome fully H-2 mismatched barriers. Using this method, we have investigated whether or not this regimen can prolong survival of heart allografts and inhibit the development of post-transplant cardiac allograft vasculopathy (CAV).Method. The components of the method are intravenous administration of 1x108 allogeneic spleen cells on day 0, intraperitoneaction of 200 mg/kg CP and 30 mg/kg BU on day 2, and intravenous injection of T cell-depleted 1x107 allogeneic bone marrow cells in the same strain of mice on day 3. Heart grafting (HG) was performed on day 28. Chimerism in peripheral blood was followed by cytometric (FCM) analysis, and the transplantation tolerance was assessed by specific acceptance of heart grafting followed by set-skin grafting. The frequency of certain Vb families was determined by FCM to assess deletion of donor-reactive T cells. H … More i al analysis (elastica van Gieson) was performed at various timings after grafting. Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytopressions in the heart grafts were analyzed with RT-PCR.Results. In a fully MHC mismatched combination of B10.D2 (H-2d, IE+)→B10 (H-2b, IE-), stable multilineage mixed chimerism was observed permanently, and IE-reactive Vb11 + T cells were specifically reduced in the periphery in the recipient B10 mice. B10.D2 grafts were accepted permanently in a donor specific manner, and post-transplant CAV did not develop. Induction of the transplan tolerance was confirmed by specific acceptance of second set-skin grafting from donor strain. In the donor B10.D2 heart grafter Th1 (IL-2, IFN-γ) nor Th2 (IL-4, IL-10) cytokine was not accumulated.Conclusions. These results demonstrated that the drug-induced tolerance recently established by us can regularly induce a long-g heart allograft tolerance without development CAV or intragraft mRNA accumulation of Th1 or Th2.Recently, we have described a drug (cyclophosphamide (CP) plus busulfan (BU))-induced skin allograft tolerance in mice that can ularly overcome fully H-2 mismatched barriers. Using this method, we have investigated whether or not this regimen can prolong urvival of heart allografts and inhibit the development of post-transplant cardiac allograft vasculopathy (CAV).Methods. The components of the method are intravenous administration of 1x108 allogeneic spleen cells on day 0, intraperitoneaction of 200 mg/kg CP and 30 mg/kg BU on day 2, and intravenous injection of T cell-depleted 1x107 allogeneic bone marrow cells in the same strain of mice on day 3. Heart grafting (HG) was performed on day 28. Chimerism in peripheral blood was followed by cytometric (FCM) analysis, and the transplantation tolerance was assessed by specific acceptance of heart grafting followed by set-skin grafting. The frequency of certain Vb families was determined by FCM to assess deletion of donor-reactive T cells. Hi al analysis (elastica van Gieson) was performed at various timings after grafting. Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytopressions in the heart grafts were analyzed with RT-PCR.Results. In a fully MHC mismatched combination of B10.D2 (H-2d, IE+)→B10 (H-2b, IE-), stable multilineage mixed chimerism was observed permanently, and IE-reactive Vb11+ T cells were specifically reduced in the periphery in the recipient B10 mice. B10.D2 grafts were accepted permanently in a donor specific manner, and post-transplant CAV did not develop. Induction of the transplan tolerance was confirmed by specific acceptance of second set-skin grafting from donor strain. In the donor B10.D2 heart grafter Th1 (IL-2, IFN-γ) nor Th2 (IL-4, IL-10) cytokine was not accumulated.Conclusions. These results demonstrated that the drug-induced tolerance recently established by us can regularly induce a long-g heart allograft tolerance without development CAV or intragraft mRNA accumulation of Th1 or Th2. Less

最近，我们描述了一种药物（环磷酰胺（CP）加丁硫丹（BU））诱导的小鼠皮肤同种异体移植物耐受性，可以克服完全H-2不匹配的屏障。利用这种方法，我们研究了该方案是否能延长同种异体心脏移植后的生存期，并抑制移植后同种异体心脏移植血管病变（CAV）的发展。该方法的组成为：第0天静脉给药1 × 108异体脾细胞，第2天腹腔注射200 mg/kg CP和30 mg/kg BU，第3天静脉注射同一品系小鼠1 × 107异体骨髓细胞。第28天进行心脏移植（HG）。外周血嵌合分析采用细胞术（FCM），移植耐受性评估采用心脏移植后固定皮移植的特异性接受度。用流式细胞术测定某些Vb家族的频率，以评估供体反应性T细胞的缺失。在移植后的不同时间进行更多的应力分析（elastica van Gieson）。采用rt - pcr方法分析心脏移植组织中Th1 （IL-2、IFN-γ）和Th2 （IL-4、IL-10）细胞的表达水平。在一个完全MHC不匹配的B10组合中。D2 （H-2d, IE+）→B10 (H-2b, IE-)，长期观察到稳定的多系混合嵌合，并且在受体B10小鼠的外周，IE反应的Vb11 + T细胞特异性减少。B10。D2移植物以供体特异性的方式被永久接受，并且移植后没有发生CAV。移植耐受的诱导是通过对供体株第二次固定皮移植的特异性接受来证实的。在供体B10。D2心脏移植物无Th1 （IL-2、IFN-γ）和Th2 （IL-4、IL-10）细胞因子积累。这些结果表明，我们最近建立的药物诱导耐受可以有规律地诱导长时间的同种异体心脏移植耐受，而不会发生CAV或Th1或Th2 mRNA的积累。最近，我们描述了一种药物（环磷酰胺（CP）加丁硫丹（BU））诱导小鼠皮肤同种异体移植物耐受，可以特别克服完全H-2不匹配的屏障。利用这种方法，我们研究了该方案是否能延长同种异体心脏移植后的存活时间和抑制移植后同种异体心脏移植血管病变（CAV）的发展。该方法的组成为：第0天静脉给药1 × 108异体脾细胞，第2天腹腔注射200 mg/kg CP和30 mg/kg BU，第3天静脉注射同一品系小鼠1 × 107异体骨髓细胞。第28天进行心脏移植（HG）。外周血嵌合分析采用细胞术（FCM），移植耐受性评估采用心脏移植后固定皮移植的特异性接受度。用流式细胞术测定某些Vb家族的频率，以评估供体反应性T细胞的缺失。在移植后的不同时间进行Hi分析（elastica van Gieson）。采用rt - pcr方法分析心脏移植组织中Th1 （IL-2、IFN-γ）和Th2 （IL-4、IL-10）细胞的表达水平。在一个完全MHC不匹配的B10组合中。D2 （H-2d, IE+）→B10 (H-2b, IE-)，长期观察到稳定的多系混合嵌合，并且在受体B10小鼠的外周，IE反应的Vb11+ T细胞特异性减少。B10。D2移植物以供体特异性的方式被永久接受，并且移植后没有发生CAV。移植耐受的诱导是通过对供体株第二次固定皮移植的特异性接受来证实的。在供体B10。D2心脏移植物无Th1 （IL-2、IFN-γ）和Th2 （IL-4、IL-10）细胞因子积累。这些结果表明，我们最近建立的药物诱导耐受可以有规律地诱导长时间的同种异体心脏移植耐受，而不会发生CAV或Th1或Th2 mRNA的积累。少

项目成果

Zhang, Q-W., Y.Tomita, G.Matsuzaki, S.Okano, I.Shimizu: "Induction of heart allograft tolerance without development of post-transplant cardiac allograft vasculopathy in chimerism-based drug-induced tolerance"Transplantation. (In press).

张，Q-W.，Y.Tomita，G.Matsuzaki，S.Okano，I.Shimizu：“在基于嵌合体的药物诱导耐受中诱导心脏同种异体移植耐受，而不发生移植后心脏同种异体移植血管病变”移植。

Zhang, Q-W., Y.Tomita, G.Matsuzaki, T.Uchida, et al.: "Chronic rejection of H-2 matched heart allografts : Early emergence of vasculopathy, alloantibody and accumulation of IFN-g and IL-1O mRNA"Transplantation. 14. 143-152 (2001)

张，Q-W.，Y.Tomita，G.Matsuzaki，T.Uchida，等人：“H-2 匹配心脏同种异体移植物的慢性排斥：血管病变的早期出现、同种抗体以及 IFN-g 和 IL-1O mRNA 的积累”

Yoshikawa M,Tomita Y, et al: "Inability of cyclophosphamide-induced tolerance to permit engraftment of pluripotent stem cell contained in moderate number of syngeneic bone marrow cells."Immunobiology. 201. 552-567 (2000)

Yoshikawa M、Tomita Y 等人：“环磷酰胺诱导的耐受性无法使中等数量的同基因骨髓细胞中包含的多能干细胞植入。”免疫生物学。

Tomita. Y., Q-W. Zhang, T. Uchida, M. Yoshikawa, I. Shimizu: "A technique of cervical aortic graft transplantation in mice"J Heart Lung Transplantation. 20. 699-702 (2001)

富田。

Zhang, Q-W., Y. Tomita, G. Matsuzaki, T. Uchida, et. al: "Chronic rejection of H-2 matched heart allografts : Early emergence of vasculopathy, alloantibody and accumulation of IFN-g and IL-10 mRNA"Transplantation. 14. 143-152 (2001)

张，Q-W.，Y. Tomita，G. Matsuzaki，T. Uchida，等。