Fernandez L A, MacSween J M
J Immunol Methods. 1987 Apr 16;98(2):271-7. doi: 10.1016/0022-1759(87)90015-9.
T cell colonies can be easily grown from peripheral blood, and are an index of cellular immunocompetence. Mitomycin-treated T cells are used as stimulator cells in mixed lymphocyte reactions and as feeder cells for growth of B cell colonies, the assumption being that mitomycin prevents proliferation of T cells. We tested this assumption by comparing the proliferation of mitomycin-treated T cells in response to stimulation with phytohemagglutinin (PHA) with that of untreated T cells in liquid cultures and in T cell colony assay. We found that incorporation of tritiated thymidine by cells from 11 healthy individuals pretreated with 25, 50 and 100 micrograms/ml mitomycin C was reduced to 13, 11 and 8%, respectively, of that of untreated cells when stimulated by an optimal concentration of PHA (10 micrograms/ml) in liquid cultures. However, parallel experiments with aliquots of the same cells showed that pretreatment with 25, 50 or 100 micrograms/ml mitomycin C merely reduced T cell colonies to 49, 45 and 45%, respectively, of untreated cells. In five additional experiments mitomycin 200 and 400 micrograms/ml reduced T cell colony numbers to 47 and 60%, respectively. Treatment of T cells with 9000 rad completely abolished T cell colony formation. Lower doses of radiation up to 6000 R did not abolish T cell colony formation, although it effectively blocked T cell proliferation to PHA in liquid cultures. 24 h preincubation of T cells with suboptimal doses of PHA and then treatment with mitomycin or radiation did not abolish T cell colony formation. T cells were recovered from the mitomycin-resistant T cell colonies and stimulated in liquid cultures with PHA, untreated or after exposure to 25 micrograms/ml mitomycin C. Incorporation of tritiated thymidine by the mitomycin-treated cells was reduced to 8% of the untreated controls. Our observations suggests: There may be inaccuracies in B cell colony assays using mitomycin-treated T cells because of significant T cell colony formation. There is a population of T cells in the peripheral blood of normal individuals which form colonies and are resistant to mitomycin.
T细胞集落很容易从外周血中培养出来，是细胞免疫能力的一个指标。丝裂霉素处理过的T细胞在混合淋巴细胞反应中用作刺激细胞，在B细胞集落生长中用作饲养细胞，假定丝裂霉素可阻止T细胞增殖。我们通过比较丝裂霉素处理过的T细胞在液体培养物中和T细胞集落试验中对植物血凝素（PHA）刺激的增殖情况与未处理的T细胞的增殖情况，来检验这一假定。我们发现，当在液体培养物中用最佳浓度（10微克/毫升）的PHA刺激时，来自11名健康个体的细胞，用25、50和100微克/毫升丝裂霉素C预处理后，其掺入氚化胸腺嘧啶核苷的量分别降至未处理细胞的13%、11%和8%。然而，用相同细胞的等分试样进行的平行实验表明，用25、50或100微克/毫升丝裂霉素C预处理仅使T细胞集落分别降至未处理细胞的49%、45%和45%。在另外五个实验中，200和400微克/毫升的丝裂霉素分别使T细胞集落数降至47%和60%。用九千拉德照射T细胞完全消除了T细胞集落形成。高达6000拉德的较低剂量辐射并未消除T细胞集落形成，尽管它有效地阻断了液体培养物中T细胞对PHA的增殖。用次最佳剂量的PHA对T细胞进行24小时预孵育，然后用丝裂霉素或辐射处理，并未消除T细胞集落形成。从耐丝裂霉素的T细胞集落中回收T细胞，并用PHA在液体培养物中刺激，PHA未处理或经25微克/毫升丝裂霉素C处理。经丝裂霉素处理的细胞掺入氚化胸腺嘧啶核苷的量降至未处理对照的8%。我们的观察结果表明：由于显著的T细胞集落形成，使用丝裂霉素处理过的T细胞进行B细胞集落试验可能存在不准确之处。正常个体外周血中有一群T细胞可形成集落且对丝裂霉素有抗性。
