Köller M D, Kiener H P, Aringer M, Graninger W B, Meuer S, Samstag Y, Smolen J S
Department Internal Medicine III, Division of Rheumatology, University of Vienna, Austria.
Clin Exp Immunol. 2003 May;132(2):225-31. doi: 10.1046/j.1365-2249.2003.02150.x.
Defects of T cell (Tc) proliferation have been demonstrated in several autoimmune diseases. Detailed mechanisms governing activation and proliferation of Tc are still not completely known. Here we show that under certain conditions human peripheral blood lymphocytes, once activated by anti-CD3, fail to respond to a subsequent restimulation via the Tc-receptor. Peripheral blood mononuclear cells (PBMC) were preactivated by anti-CD3 for 96 h following restimulation by anti-CD3, interleukin (IL)-2 and other mitogens. In control experiments unstimulated PBMC were incubated in medium alone. Immunophenotypes were analysed by flow cytometry. Cytokine production was determined by reverse transcription-polymerase chain reaction and intracellular signalling protein contents of Tc were compared by Western blotting. Furthermore, apoptosis was detected by terminal deoxyribose transferase-mediated deoxyuridine triphosphate nick end labelling assay. Unstimulated PBMC proliferate well after subsequent stimulation with anti-CD3, whereas IL-2 induces only limited proliferation. In contrast, preactivated cells respond only minimally to restimulation with anti-CD3, but IL-2 induces a marked proliferation. Both preactivated and unstimulated Tc respond well to restimulation by phytohaemagglutinin (PHA). In contrast, preactivated Tc show only a weak response to concanavalin A. Interestingly, when cells have been allowed to rest for 168 h, the responsiveness of preactivated Tc is restored. Immunoblots reveal that preactivated cells have a higher intracellular content of zeta-chain and p56lck. No differences are found concerning apoptosis after restimulation with anti-CD3 or the expression of ERK 1/2. The unresponsiveness to restimulation is due to an impairment of the transcription of the IL-2 gene and this defect is temporary. Despite the lack of proliferation, preactivated Tc phenotypically maintain an intermediate stage of activation. These data show how the same cell population can change its functional phenotype into a non-responder state.
T细胞（Tc）增殖缺陷已在多种自身免疫性疾病中得到证实。目前仍不完全清楚调控Tc激活和增殖的详细机制。在此我们发现，在某些条件下，人外周血淋巴细胞一旦被抗CD3激活，就无法对随后通过Tc受体进行的再刺激作出反应。外周血单个核细胞（PBMC）先用抗CD3预激活96小时，之后再用抗CD3、白细胞介素（IL）-2和其他有丝分裂原进行刺激。在对照实验中，未刺激的PBMC仅在培养基中孵育。通过流式细胞术分析免疫表型。通过逆转录-聚合酶链反应测定细胞因子的产生，并通过蛋白质免疫印迹法比较Tc的细胞内信号蛋白含量。此外，通过末端脱氧核糖核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法检测细胞凋亡。未刺激的PBMC在用抗CD3进行后续刺激后增殖良好，而IL-2仅诱导有限的增殖。相比之下，预激活的细胞对用抗CD3进行的再刺激反应极小，但IL-2可诱导显著的增殖。预激活的和未刺激的Tc对植物血凝素（PHA）的再刺激反应均良好。相比之下，预激活的Tc对刀豆蛋白A的反应较弱。有趣的是，当细胞静置168小时后，预激活的Tc的反应性得以恢复。免疫印迹显示，预激活的细胞中ζ链和p56lck的细胞内含量较高。在用抗CD3再刺激后，细胞凋亡或ERK 1/2的表达方面未发现差异。对再刺激无反应是由于IL-2基因转录受损，且这种缺陷是暂时的。尽管缺乏增殖，但预激活的Tc在表型上维持激活的中间阶段。这些数据表明同一细胞群体如何将其功能表型转变为无反应状态。
