Ericsson P O, Orchansky P L, Carlow D A, Teh H S
Department of Microbiology, University of British Columbia, Vancouver, Canada.
J Immunol. 1996 Mar 15;156(6):2045-53.
In this study, we determined the functional and biochemical differences in naive and primed CD4 T cells that expressed a TCR specific for the pigeon cytochrome c (pcc) peptide presented by I-Ek MHC class II molecules. Naive CD4 T cells expressing the transgenic TCR were isolated from the peripheral lymphoid organs of transgenic mice and stimulated with pcc peptide and IL-2 for 10 to 14 days. After this culture period, the Ag-primed cells were quiescent, as judged by the lack of expression of the early activation marker CD69, low expression of CD25 (IL-2R), and failure to incorporate thymidine. The primed cells required 10-fold less peptide than naive cells to achieve the same degree of proliferation and for the induction of CD69. Primed cells also mobilized calcium more efficiently with regard to Ag dose and magnitude of the response. The biochemical signal-transduction events in naive and primed T cells were compared by stimulating them with different concentrations of pcc peptide presented by adherent Ek-transfected fibroblasts. It was found that tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK) in primed cells required 10-fold less Ag and occurred more rapidly and intensively. Interestingly, peptide stimulation induced tyrosine phosphorylation of phospholipase C (PLC)-gamma 1 exclusively in primed cells. RasGAP was also more efficiently tyrosine phosphorylated in primed cells. By contrast, Shc was tyrosine phosphorylated to the same extent in naive and primed cells. PI3Kp85 was not tyrosine-phosphorylated in naive and primed cells either before or after peptide stimulation. We propose that the higher sensitivity of the primed cells to Ag stimulation is most likely dependent, at last in part, on the more efficient activation of PLC-gamma 1, MAPK, and calcium-dependent pathways.
在本研究中，我们确定了表达针对由I-Ek MHC II类分子呈递的鸽细胞色素c（pcc）肽的TCR的初始和致敏CD4 T细胞在功能和生化方面的差异。从转基因小鼠的外周淋巴器官中分离出表达转基因TCR的初始CD4 T细胞，并用pcc肽和IL-2刺激10至14天。在此培养期后，根据早期激活标志物CD69的表达缺失、CD25（IL-2R）的低表达以及未能掺入胸苷判断，抗原致敏细胞处于静止状态。致敏细胞达到相同增殖程度和诱导CD69所需的肽量比初始细胞少10倍。就抗原剂量和反应强度而言，致敏细胞还能更有效地动员钙。通过用贴壁的Ek转染成纤维细胞呈递的不同浓度的pcc肽刺激初始和致敏T细胞，比较了它们的生化信号转导事件。发现致敏细胞中丝裂原活化蛋白激酶（MAPK）的酪氨酸磷酸化和激活所需的抗原量少10倍，且发生得更快、更强烈。有趣的是，肽刺激仅在致敏细胞中诱导磷脂酶C（PLC）-γ1的酪氨酸磷酸化。RasGAP在致敏细胞中也更有效地发生酪氨酸磷酸化。相比之下，Shc在初始和致敏细胞中的酪氨酸磷酸化程度相同。在肽刺激之前或之后，PI3Kp85在初始和致敏细胞中均未发生酪氨酸磷酸化。我们提出，致敏细胞对抗原刺激的更高敏感性很可能至少部分取决于PLC-γ1、MAPK和钙依赖性途径的更有效激活。
