Poloucková A, Vodvárková A, Kobylka P, Hrubá A, Gasová Z, Marinov I, Fales I, Sedlácek P, Kozák T, Starý J
Institute of Immunology, University Hospital Motol, Prague, Czech Republic.
Neoplasma. 2001;48(5):374-81.
The aim of this study was to establish a suitable method for in vitro T cell depletion in peripheral blood stem cell grafts for mismatched/haploidentical transplantation in children and adults with severe hematological disorders and for autologous transplantation in patients with autoimmune diseases refractory to conventional immunosuppressive treatment. Two different selection techniques have been used: CD34+ selection using immunoaffinity columns (CellPro Ceprate) followed by T cell depletion by E-rosetting or CD34+ selection using submicroscopic paramagnetic beads (CliniMACS device) with T cell depletion in a one step procedure. The mean purity and recovery of CD34+ cells and efficiency of T cell removal in the final product were compared. From March 1995 to December 1998 we prepared twelve allografts using Cell Pro system for eight children with high-risk hematological malignancies and six autografts for six patients with severe autoimmune diseases. From January 1999 to October 2000 we prepared fifteen allografts using CliniMACS system for ten children with high-risk hematological diseases and inborn metabolic disorders or primary immunodeficiences, five allografts for three adult patients with high-risk hematological malignancies and two autografts for two patients with autoimmune diseases. In allogeneic transplantation the median purity of CD34+ cells in the final products after CellPro and E-rosetting was 85.6% (55.3%-95.7%); median recovery was 24.8% (17%-35%), median transplanted doses of T cells per kilogram of body weight were 0.66x10(4) (0-2.8); in autologous transplantation the median purity of CD34+ was 92.6% (55.6%-96%), median recovery was 28% (22%-46.2%), median transplanted doses of T cells per kilogram of body weight were 0.39x10(4) (0.0-3.6). After CliniMACS technique the median purity of CD34+ cells was 94.87% (69.15%-99%),medianrecoverywas 58% (30%-79.6%), median transplanted doses of T cells per kg of body weight were 0.254x10(4) (0-14.15); in autologous transplantation the median purity of CD34+ was 94% (94%-94%, median recovery was 97.4% (95%-99.8%), median transplanted doses of T cells per kilogram of body weight were 0.87x10(4) (0.49-1.24). We consider both methods of CD34+ selection and T cell depletion suitable for peripheral blood stem cell processing before mismatched hemopoietic stem cell transplantation in patients without identical donor or before autologous transplantation for severe autoimmune diseases. However, magnetic separation using CliniMACS system results in higher levels of purity and recovery with efficient T cell depletion.
本研究的目的是建立一种合适的方法，用于严重血液系统疾病的儿童和成人进行错配/单倍体相合移植以及常规免疫抑制治疗无效的自身免疫性疾病患者进行自体移植时，对外周血干细胞移植物进行体外T细胞去除。使用了两种不同的分选技术：采用免疫亲和柱（CellPro Ceprate）进行CD34+分选，随后通过E花环沉降法去除T细胞；或采用亚微观顺磁珠（CliniMACS装置）进行CD34+分选，并在一步操作中去除T细胞。比较了最终产物中CD34+细胞的平均纯度和回收率以及T细胞去除效率。1995年3月至1998年12月，我们使用Cell Pro系统为8例高危血液系统恶性肿瘤患儿制备了12例同种异体移植物，为6例严重自身免疫性疾病患者制备了6例自体移植物。1999年1月至2000年10月，我们使用CliniMACS系统为10例患有高危血液系统疾病、先天性代谢紊乱或原发性免疫缺陷的患儿制备了15例同种异体移植物，为3例高危血液系统恶性肿瘤成年患者制备了5例同种异体移植物，为2例自身免疫性疾病患者制备了2例自体移植物。在异基因移植中，使用CellPro和E花环沉降法后最终产物中CD34+细胞的中位纯度为85.6%（55.3%-95.7%）；中位回收率为24.8%（17%-35%），每千克体重的中位移植T细胞剂量为0.66×10⁴（0-2.8）；在自体移植中，CD34+的中位纯度为92.6%（55.6%-96%），中位回收率为28%（22%-46.2%），每千克体重的中位移植T细胞剂量为0.39×10⁴（0.0-3.6）。采用CliniMACS技术后，CD34+细胞的中位纯度为94.87%（69.15%-99%），中位回收率为58%（30%-79.6%），每千克体重的中位移植T细胞剂量为0.254×10⁴（0-14.15）；在自体移植中，CD34+的中位纯度为94%（94%-94%），中位回收率为97.4%（95%-99.8%），每千克体重的中位移植T细胞剂量为0.87×10⁴（0.49-1.24）。我们认为，对于没有相同供体的患者进行错配造血干细胞移植之前或严重自身免疫性疾病患者进行自体移植之前，CD34+分选和T细胞去除的这两种方法均适用于外周血干细胞处理。然而，使用CliniMACS系统进行磁性分选可获得更高的纯度和回收率，并能有效去除T细胞。
