Moses R D, Orr K S, MacVittie T J, Gress R E
Experiemental Immunology, Branches, National Cancer Institute, Bethesda, MD 20892.
Transplantation. 1988 Aug;46(2):197-205. doi: 10.1097/00007890-198808000-00003.
We have developed a rhesus monkey model consisting of myeloablative total-body irradiation and T lymphocyte-depleted autologous bone marrow transplantation followed by MHC-mismatched heterotopic cardiac allograft implantation that has provided an opportunity to study the role of marrow T cells in cardiac allograft rejection. In order to assess quantitatively the effects of low numbers of residual marrow T cells following depletion, methods to deplete rhesus marrow extensively and to detect residual T cells following depletion at levels below the sensitivity of standard assays have been developed. A rhesus marrow limiting dilution assay has been developed that quantifies less than 1 T cell in 10(5) marrow cells and is superior to traditional detection methods by at least 3 logs. In a direct comparison of four T cell depletion methods, effective depletion has been achieved with complement-mediated cytotoxicity (C'MC), erythrocyte rosetting, and counterflow centrifugal elutriation (CCE), the latter with a simplified single-flow rate protocol. Median marrow T cell depletions of 2.1, 1.1, and 3.1 logs, and total nucleated cell losses of 40%, 61%, and 42% respectively, have been observed. A reported use of ricin A-chain-like toxins for the enhancement of C'MC was of low efficacy with rhesus peripheral blood T cell targets. CCE followed by C'MC has resulted in a median 4.8 logs depletion with residual marrow T cell contents less than 0.001%. Thus, C'MC, E-rosetting, and particularly CCE are effective methods of T cell depletion--and, when used in combination, extensively eliminate marrow T cells. A rhesus marrow limiting dilution assay detects residual T cells at these low levels. These techniques provide a basis for the quantitative study of the role of T cells in organ graft rejection following T lymphocyte-depleted autologous marrow transplantation.
我们已经建立了一种恒河猴模型，该模型包括清髓性全身照射和T淋巴细胞去除的自体骨髓移植，随后进行MHC不匹配的异位心脏同种异体移植植入，这为研究骨髓T细胞在心脏同种异体移植排斥反应中的作用提供了机会。为了定量评估去除后少量残留骨髓T细胞的影响，已经开发了广泛去除恒河猴骨髓以及检测去除后残留T细胞的方法，这些残留T细胞的水平低于标准检测方法的灵敏度。已经开发了一种恒河猴骨髓极限稀释分析方法，该方法可以定量10(5)个骨髓细胞中少于1个T细胞，并且比传统检测方法至少灵敏3个对数级。在对四种T细胞去除方法的直接比较中，补体介导的细胞毒性（C'MC）、红细胞玫瑰花结形成和逆流离心淘析（CCE）都实现了有效的去除，后者采用了简化的单流速方案。分别观察到骨髓T细胞中位数去除率为2.1、1.1和3.1个对数级，总有核细胞损失分别为40%、61%和42%。据报道，使用蓖麻毒素A链样毒素增强C'MC对恒河猴外周血T细胞靶标的效果不佳。CCE后再进行C'MC导致中位数去除率为4.8个对数级，残留骨髓T细胞含量低于0.001%。因此，C'MC、E玫瑰花结形成，尤其是CCE是有效的T细胞去除方法，并且联合使用时可广泛消除骨髓T细胞。恒河猴骨髓极限稀释分析可以检测到这些低水平的残留T细胞。这些技术为定量研究T淋巴细胞去除的自体骨髓移植后T细胞在器官移植排斥反应中的作用提供了基础。
