Moretti L, Giuliodori L, Stramigioli S, Luchetti F, Baldi A, Sparaventi G
Divisione di Ematologia, USL 3, Pesaro, Italy.
Haematologica. 1992 Nov-Dec;77(6):463-9.
After marrow transplantation, the interaction of helper T lymphocytes from the donor with the patient alloantigens leads to cellular activation and release of IL-2 as initial events of the graft versus host reaction. A method for assessing the size of the pool containing allospecific helper T cells capable of producing IL-2 could be applied in the selection of better donors for marrow transplantation.
PBMC are added to replicate sets of wells each containing various amounts of EBV-LCL cells and PHA. After culture for some days the supernatant is removed from each well and added to IL-2 dependent CTLL-2 line. The proliferation of the CTLL-2 line is assessed by pulse labeling with 3H-thimidine. The precursor frequency of cell capable of producing IL-2 per ml/blood is estimated from the minimum X2 regression of the function of non-responding wells plotted as logarithmic function of the number of PBMC added per well.
Approximately 30-40% of PBMC are found to produce IL-2 under the following conditions in culture: the optimal PHA concentration is 1.25 micrograms/ml, the optimal number of stimulator EBV-LCL cells is 1 x 10(3) and 3 days of culture are required.
Here we report a rapid and quantitative technique of limiting dilution analysis that can estimate the frequency of peripheral blood mononuclear cells capable of secreting interleukin-2 following interaction with specific alloantigen.
骨髓移植后，供体辅助性T淋巴细胞与患者同种异体抗原相互作用，导致细胞活化并释放白细胞介素-2（IL-2），这是移植物抗宿主反应的初始事件。一种评估能够产生IL-2的同种特异性辅助性T细胞池大小的方法，可应用于骨髓移植更好供体的选择。
将外周血单个核细胞（PBMC）加入到多组复孔中，每组孔中含有不同数量的EB病毒转化的B淋巴细胞（EBV-LCL）和植物血凝素（PHA）。培养数天后，从每个孔中取出上清液，加入到依赖IL-2的CTLL-2细胞系中。通过用3H-胸腺嘧啶脉冲标记来评估CTLL-2细胞系的增殖。根据无反应孔的功能与每孔添加PBMC数量的对数函数关系的最小X2回归，估计每毫升血液中能够产生IL-2的细胞的前体频率。
发现在以下培养条件下，约30%-40%的PBMC可产生IL-2：PHA的最佳浓度为1.25微克/毫升，刺激细胞EBV-LCL的最佳数量为1×10(3)，培养3天。
在此我们报告一种快速定量的有限稀释分析技术，该技术可估计外周血单个核细胞与特异性同种异体抗原相互作用后分泌白细胞介素-2的频率。
