Hale D A, Gottschalk R, Maki T, Monaco A P
Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Transplantation. 1998 Feb 27;65(4):473-9. doi: 10.1097/00007890-199802270-00004.
Posttransplant donor-specific bone marrow (BM) infusion in mice treated with antilymphocyte serum (ALS) induces specific unresponsiveness (tolerance) to skin allografts, which can be augmented by the adjuvant administration of chemotherapeutic immunosuppressive agents. The purpose of this study was to determine the optimal dose and timing of administration of sirolimus (rapamycin) to induce maximal skin allograft survival in ALS-treated, BM-infused recipients.
DBA/2 donor skin grafts were placed on B6AF1 recipients (class I- and II-disparate). Groups of recipient mice (n=10 each) received combinations of the following treatment protocols: ALS, 0.5 ml on days -1 and 2; BM, 25x10(6) donor-specific cells on day 7; sirolimus, 6, 12, 18, or 24 mg/kg at times indicated; and cyclosporine, 50 mg/kg at times indicated. The immune status of putatively tolerant animals was examined with mixed lymphocyte cultures, cell-mediated lympholysis assays (CML), and limiting dilution analyses.
When administered in conjunction with ALS/BM, a single dose of sirolimus (6 mg/kg) on days 21, 18, 14, 10, or 7 resulted in median skin graft survival times of 35, 26, 40, 46, and 103 days, respectively, versus a median survival of 27 days in mice given ALS and BM alone. The addition of cyclosporine to sirolimus (6 mg/kg) given on day 7 or days 7 and 10 did not significantly increase graft survival over that achieved with sirolimus alone. A single dose (18 or 24 mg/kg) of sirolimus administered on day 7 to ALS/BM-treated recipients resulted in 100% 200-day skin graft acceptance. Tolerant mice demonstrated nonspecific suppression of the mixed lymphocyte culture assays at 90 and 200 days and a nonspecific reduction of the CML assay at 50 days. By 200 days, the third-party CML response was restored, whereas donor-specific cell-mediated cytotoxicity remained suppressed. There was a donor-specific reduction in the number of alloreactive cytotoxic T lymphocyte clones by limiting dilution assay at 120 days. In vivo specificity of immunosuppression induced with this protocol was demonstrated by indefinite survival of second donor-specific skin grafts placed on putatively tolerant mice at day 90, whereas third-party skin grafts were rejected in 14 days.
A single dose of sirolimus (18-24 mg/kg) administered on day 7, within the context of an ALS/BM immunosuppressive regimen, reliably induces permanent skin allograft acceptance in this model. In vitro measures of immunocompetence demonstrated an early nonspecific suppression of the recipients immune status and later recovery of third-party immunoreactivity. In vivo testing indicates an operationally tolerant state that is donor-specific 90 days after treatment.
在用抗淋巴细胞血清（ALS）治疗的小鼠中，移植后给予供体特异性骨髓（BM）输注可诱导对皮肤同种异体移植物的特异性无反应性（耐受），化疗免疫抑制剂的辅助给药可增强这种耐受。本研究的目的是确定西罗莫司（雷帕霉素）的最佳给药剂量和时间，以在接受ALS治疗、BM输注的受体中诱导最大程度的皮肤同种异体移植存活。
将DBA/2供体皮肤移植到B6AF1受体（I类和II类不相容）上。每组受体小鼠（每组n = 10）接受以下治疗方案的组合：ALS，在第-1天和第2天给予0.5 ml；BM，在第7天给予25×10⁶个供体特异性细胞；西罗莫司，在指定时间给予6、12、18或24 mg/kg；环孢素，在指定时间给予50 mg/kg。通过混合淋巴细胞培养、细胞介导的淋巴细胞溶解试验（CML）和有限稀释分析来检查假定耐受动物的免疫状态。
当与ALS/BM联合给药时，在第21、18、14、10或7天给予单剂量西罗莫司（6 mg/kg），皮肤移植的中位存活时间分别为35、26、40、46和103天，而单独给予ALS和BM的小鼠中位存活时间为27天。在第7天或第7天和第10天给予西罗莫司（6 mg/kg）时添加环孢素，与单独使用西罗莫司相比，移植存活时间没有显著增加。在第7天给予ALS/BM治疗的受体单剂量（18或24 mg/kg）西罗莫司可导致100%的皮肤移植在200天被接受。耐受小鼠在90天和200天时显示混合淋巴细胞培养试验的非特异性抑制，在50天时CML试验非特异性降低。到200天时，第三方CML反应恢复，而供体特异性细胞介导的细胞毒性仍然受到抑制。通过有限稀释分析在120天时发现同种异体反应性细胞毒性T淋巴细胞克隆数量有供体特异性减少。在第90天，将第二个供体特异性皮肤移植到假定耐受的小鼠上可无限期存活，而第三方皮肤移植在14天内被排斥，证明了该方案诱导的免疫抑制在体内具有特异性。
在ALS/BM免疫抑制方案的背景下，在第7天给予单剂量西罗莫司（18 - 24 mg/kg）可在该模型中可靠地诱导永久性皮肤同种异体移植接受。免疫能力的体外测量显示受体免疫状态早期有非特异性抑制，后期第三方免疫反应性恢复。体内测试表明治疗90天后存在供体特异性的操作性耐受状态。
