DEPLETION OF BONE MARROW LYMPHOCYTES

我们成功地制定了一个切实可行的程序， 从骨髓同种异体移植物中去除> 99%的淋巴细胞， 保留了65-80%的克隆形成细胞。 通过使用逆流 离心淘洗，一种物理分离技术， 细胞大小和密度的差异，我们成功地消除了 急性GVHD作为高危患者治疗失败的原因 而不会显著影响植入。唯一的 阻碍其广泛应用的主要限制是 患者白血病复发率意外增加 慢性粒细胞白血病移植。 这一发现表明， 消除负责 移植物抗白血病效应;它表明，通过消除 大多数T细胞没有合理的方法来选择那些 淋巴细胞保留在移植物中，我们已经达不到我们的目标。 移植工程的目标。 在本申请中，我们提出 通过整合CCE和 单克隆抗体分离技术。 的组合 这些方法提供了优于单独使用任一种方法的优点。 我们 还建议详细探讨职能的基础， 在通过CCE分离的淋巴细胞亚群之间观察到差异。 我们假设这些区别有其基本参数 例如细胞周期阶段、活化状态和抗原先前历史 暴露（即幼稚与记忆）。 或许，他们的命运， 这些细胞群包括在骨髓移植物中时。 在 特别是，差异激活要求可能影响 CCE分离的淋巴细胞组分介导有益的或 有害影响。 将大量新鲜的 根据大小和密度分离细胞，同时 在表面标记的基础上进行正反选择 由单克隆抗体定义，为临床应用提供了一种现成的手段， 结果的应用。 在此基础上，我们确定了未来的目标 CCE与抗体介导的细胞分离的整合研究 以及理解尺寸/密度的功能意义 免疫活性细胞之间的异质性。

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.