Leshem B, Cohen I, Sherman L, Brautbar C, Kedar E
Lautenberg Center for General and Tumor Immunology, Hebrew University, Jerusalem, Israel.
J Immunol Methods. 1988 Jun 28;111(1):131-5. doi: 10.1016/0022-1759(88)90069-5.
We describe here a simple procedure, by which HLA class II antigens can be accurately and reliably identified in those patients where there is minimal or absent expression of HLA-DR,DQw antigens on B cells, or when the total number of leukocytes recovered from the patients do not permit reliable typing. Ficoll-Hypaque-separated peripheral blood mononuclear leukocytes, fresh or cryopreserved, were activated by PHA and then propagated in IL-2-containing medium until enough cells for typing were obtained (usually 7-14 days). At this stage, the cultured cells were shown to be primarily T cells (greater than 90% CD3+). Since the activated T cells propagate in the presence of IL-2, even a small number (10(4] of fresh or cryopreserved patients' cells suffice for this protocol. To date we have been able to successfully HLA-DR,DQw type 34/34 bone marrow transplantation candidates and 12/12 long-term dialysis patients, who were untypable using fresh cells. HLA-DR,DQw antigens on activated T cells from normal individuals were identical to those found on their uncultured B cells. In addition, class I antigens that were undetectable on the uncultured cells of one patient could be identified on activated T cells. The HLA antigens identified on the patients' activated T cells were confirmed by phenotypic analysis of cells from family members.
我们在此描述一种简单的方法，通过该方法可在那些B细胞上HLA - DR、DQw抗原表达极少或缺失的患者中，或者在从患者体内回收的白细胞总数不足以进行可靠分型的情况下，准确且可靠地鉴定HLA - II类抗原。经Ficoll - Hypaque分离的外周血单个核白细胞，无论是新鲜的还是冷冻保存的，均用PHA激活，然后在含IL - 2的培养基中传代培养，直至获得足够数量用于分型的细胞（通常为7 - 14天）。在此阶段，培养的细胞显示主要为T细胞（大于90% CD3 +）。由于激活的T细胞在IL - 2存在的情况下进行增殖，即使是少量（10⁴）新鲜的或冷冻保存的患者细胞也足以用于此方案。迄今为止，我们已成功地对34/34例骨髓移植候选者以及12/12例长期透析患者进行了HLA - DR、DQw分型，这些患者使用新鲜细胞无法分型。正常个体激活的T细胞上的HLA - DR、DQw抗原与未培养的B细胞上发现的抗原相同。此外，在一名患者未培养的细胞上无法检测到的I类抗原，在激活的T细胞上可以被鉴定出来。通过对家庭成员细胞的表型分析，证实了在患者激活的T细胞上鉴定出的HLA抗原。
