DEPLETION OF BONE MARROW LYMPHOCYTES

我们已经成功地开发了一种实用的程序，一直以来 从异基因骨髓移植中去除99%的淋巴细胞，同时 保留65-80%的克隆形成细胞。通过使用逆流 离心洗脱是一种物理分离技术，它依赖于 细胞大小和密度的差异，我们已经成功地消除了 急性移植物抗宿主病是高危患者治疗失败的原因 在不显著影响嫁接的情况下，增加了人口。唯一的 阻碍其广泛应用的主要限制是 患者白血病复发发生率意外增加 移植治疗慢性粒细胞白血病。这一发现表明 细胞种群的消灭(S)负责 移植物抗白血病效应；它表明通过消除 大多数T细胞没有合理的选择手段 淋巴细胞被保留在移植物中，我们已经达不到我们的 嫁接工程的目标。在本申请中，我们建议 通过整合CCE和CCE来扩展我们的嫁接工程能力 基于单抗的分离技术。这两种技术的结合 与单独使用任何一种方法相比，这些方法都具有优势。我们 还提出了详细探讨功能划分的依据 CCE分离的淋巴细胞亚群之间存在差异。 我们假设这些区别是作为它们的基本参数 例如细胞周期阶段、激活状态和抗原先前病史 暴露(即天真与记忆)。因此，他们可能会关系到 当包括在骨髓移植中时，这些细胞群。在……里面 特别是，不同的激活要求可能会影响到 CCE分离的淋巴细胞组分用于调节有益或 有害的影响。能够将大量新鲜的 基于大小和密度的隔离细胞，同时 基于表面标记的正反向选择 由单抗确定，为临床提供了一种现成的手段 结果的应用。在此基础上，我们瞄准了我们的未来 CCE与抗体介导的细胞分离的整合研究 以及理解大小/密度的功能意义 免疫活性细胞间的异质性。

We have succeeded in developing a practical procedure that consistently removes > 99% of the lymphocytes from bone marrow allografts while sparing 65-80% of clonogenic cells. Through the use of counterflow centrifugal elutriation, a physical separation technique that relies on differences in cell size and density, we have successfully eliminated acute GVHD as a cause of treatment failure in a high risk patient population without significantly compromising engraftment. The only major limitation that precludes its widespread application is the unexpected increase in incidence of leukemic relapse in patients transplanted for chronic myelocytic leukemia. This finding suggests the elimination of the cell population(s) responsible for the graft-versus-leukemia effect; it indicates that by eliminating the majority of T cells without a rational means for selection of those lymphocytes to be retained in the graft, we have fallen short of our objective of graft engineering. In the present application we propose to expand our graft engineering capabilities in by integrating CCE and monoclonal antibody based separation technologies. The combination of these methods offers advantages over the use of either method alone. We also propose to explore in detail the basis for the functional differences observed between lymphocyte subpopulations isolated by CCE. We hypothesize that these distinctions have as their basis parameters such as cell cycle stage, activation state, and prior history of antigen exposure (i.e. naive vs memory). As such they may bear on the fate of these cell populations when included in a bone marrow graft. In particular, differential activation requirements may bear on the ability of CCE separated lymphocyte fractions to mediate beneficial or detrimental effects. The capacity to separate large numbers of freshly isolated cells on the basis of size and density, while simultaneously selecting both positively and negatively on the basis of surface markers defined by monoclonal antibodies, furnishes a ready means for clinical application of the results. On this basis we have targeted our future studies toward integration of CCE and antibody mediated cell separation and toward understanding the functional significance of size/density heterogeneity among immunocompetent cells.