Hensel N, Agarwala V, Jiang Y Z, Mavroudis D, Molldrem J, Barrett A J
Bone Marrow Transplantation Unit, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1652, USA.
Bone Marrow Transplant. 1999 Jan;23(1):71-8. doi: 10.1038/sj.bmt.1701528.
Helper (HTLPf) and cytotoxic (CTLPf) lymphocyte precursor frequency assays are increasingly used in bone marrow stem cell and organ transplant compatibility testing. Current techniques require large cell numbers and radioisotopes. To improve the technique, we developed a miniaturized fluorescent read-out combined HTLPf/CTLPf limiting dilution assay. The assay requires only 5 x 10(6) stimulators, 2 x 10(6) responders and 0.24 x 10(6) target cells in Terasaki plates (40 microl/well). For the HTLPf, culture supernatants from each well were assayed for IL-2 production. The IL-2-dependent proliferation of the mouse 9.12 cell line was detected by a semi-automated fluorescent dye technique. After addition of rhIL-2 (recombinant human IL-2) on days 3 and 7, CTLPs were detected on day 10 by measuring the lysis of dye-labeled targets. Results were comparable to standard radioisotope-based techniques. The assay had a coefficient of variation of approximately 30%. The assay detected helper CD4 cells, pure cytotoxic CD8, helper CD8 cells and helper/cytotoxic CD8 cells. Discrimination was demonstrated between HLA-matched related and non-related pairs. The ease of testing and small cell numbers required should facilitate further evaluation of HTLPf and CTLPf for compatibility testing in unrelated donor transplantation and monitoring immune responses following adoptive transfer of lymphocytes.
辅助性（HTLPf）和细胞毒性（CTLPf）淋巴细胞前体频率测定在骨髓干细胞和器官移植相容性检测中越来越常用。目前的技术需要大量细胞和放射性同位素。为改进该技术，我们开发了一种小型化荧光读数结合的HTLPf/CTLPf有限稀释测定法。该测定法在板田氏培养板（40微升/孔）中仅需5×10⁶个刺激细胞、2×10⁶个反应细胞和0.24×10⁶个靶细胞。对于HTLPf，检测各孔培养上清液中的白细胞介素-2产生情况。通过半自动荧光染料技术检测小鼠9.12细胞系依赖白细胞介素-2的增殖。在第3天和第7天添加重组人白细胞介素-2（rhIL-2）后，在第10天通过测量染料标记靶细胞的裂解来检测细胞毒性T淋巴细胞前体。结果与基于标准放射性同位素的技术相当。该测定法的变异系数约为30%。该测定法可检测辅助性CD4细胞、纯细胞毒性CD8细胞、辅助性CD8细胞和辅助/细胞毒性CD8细胞。在HLA匹配的亲属和非亲属配对之间显示出区分。检测的简便性和所需细胞数量少应有助于进一步评估HTLPf和CTLPf在无关供体移植相容性检测以及淋巴细胞过继转移后免疫反应监测中的应用。
