Atkuri Kondala R, Herzenberg Leonard A, Niemi Anna-Kaisa, Cowan Tina, Herzenberg Leonore A
Departments of Genetics and Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4547-52. doi: 10.1073/pnas.0611732104. Epub 2007 Mar 5.
Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%), the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.
尽管对原代淋巴细胞的研究几乎总是在维持大气氧水平（atmosO₂；20%）的二氧化碳培养箱中进行，但细胞在体内所遇到的生理氧水平（physO₂；5%）要低2至4倍。我们在此表明，在atmosO₂条件下培养原代T细胞会显著改变细胞内氧化还原状态（降低细胞内谷胱甘肽水平，增加细胞内氧化型谷胱甘肽水平），而在physO₂条件下培养则能维持细胞内氧化还原环境（细胞内谷胱甘肽/细胞内氧化型谷胱甘肽）接近其体内状态。此外，我们还表明，基于增殖指数和细胞产量，CD3/CD28诱导的T细胞增殖在atmosO₂条件下高于physO₂条件下。我们认为，这一明显矛盾的发现可能由另外两个关于CD3/CD28刺激的T细胞的发现来解释：（i）physO₂条件下细胞内一氧化氮（iNO）水平高于atmosO₂条件下；（ii）physO₂条件下CD69的峰值表达比atmosO₂条件下显著延迟且持续时间更长。由于体内高水平的细胞内NO和持续的CD69往往会下调T细胞反应，physO₂条件下较低的T细胞增殖反应可能反映了体内天然调节机制在体外的作用。因此，我们建议在atmosO₂条件下培养原代淋巴细胞时要谨慎，因为对非生理氧水平的必要适应可能会严重扭曲T细胞反应，尤其是在培养几天后。
