Hamaoka T, Katz D H
J Exp Med. 1974 Jun 1;139(6):1446-63. doi: 10.1084/jem.139.6.1446.
The studies presented here have focused on the important question of reversibility of inactivation of DNP-specific B lymphocytes induced by the DNP derivative of the copolymer of D-glutamic acid and D-lysine (D-GL). In so doing, we have analyzed the capacity of a strong T-cell stimulus, such as that provided by the allogeneic effect, and of gentle enzymatic treatment with trypsin to alter, prevent, or reverse the tolerance induced by DNP-D-GL. Under experimental conditions in which DNP-specific B lymphocytes were exposed first to the tolerogenic molecule, and rendered markedly unresponsive by such exposure either in vitro or in vivo, subsequent exposure to an allogeneic effect failed to appreciably reverse or alter the tolerant state. This contrasts directly with the capacity of DNP-D-GL to serve as a stimulus for DNP-specific B lymphocytes when the critical moment of specific binding occurs subsequent to the development of an allogeneic effect. In another series of experiments, the effects of enzymatic treatment with trypsin on the tolerant B-cell population were found to vary depending on the stage of tolerance at which such treatment was performed. Thus, when exposure of cells to DNP-D-GL for a relatively short time in vitro is carried out at low temperature (4 degrees C), the development of tolerance can be interceded by immediate trypsinization. In contrast, cells exposed to DNP-D-GL for longer periods of time and/or at 37 degrees C were not reversed to responsiveness by trypsinization. These data were interpreted to indicate that: (a) the effect(s) of trypsin in reversing (or preventing) tolerance at the cellular level does not depend necessarily on the susceptibility of the tolerogenic moiety to the action of the enzyme, and (b) the generation of the tolerance-inducing signal involves metabolic cellular processes that can be delayed somewhat by low temperature leaving such cells relatively more susceptible to intercedent manipulations such as trypsinization. Taken collectively, therefore, the evidence obtained in these studies reinforces the concept of central tolerance in B cells induced by DNP-D-GL as reflecting sub- or intracellular inactivating events.
此处展示的研究聚焦于由D-谷氨酸和D-赖氨酸共聚物（D-GL）的DNP衍生物诱导的DNP特异性B淋巴细胞失活的可逆性这一重要问题。在此过程中，我们分析了强T细胞刺激（如异基因效应所提供的刺激）以及用胰蛋白酶进行温和酶处理改变、预防或逆转由DNP-D-GL诱导的耐受性的能力。在实验条件下，DNP特异性B淋巴细胞首先暴露于致耐受性分子，并通过这种暴露在体外或体内显著变得无反应，随后暴露于异基因效应未能明显逆转或改变耐受状态。这与当特异性结合的关键时刻发生在异基因效应发展之后时，DNP-D-GL作为DNP特异性B淋巴细胞刺激物的能力形成直接对比。在另一系列实验中，发现用胰蛋白酶进行酶处理对耐受B细胞群体的影响取决于进行这种处理时的耐受阶段。因此，当在低温（4℃）下在体外将细胞暴露于DNP-D-GL相对较短时间时，立即进行胰蛋白酶处理可干预耐受性的发展。相反，暴露于DNP-D-GL更长时间和/或在37℃下的细胞不会通过胰蛋白酶处理恢复反应性。这些数据被解释为表明：（a）胰蛋白酶在细胞水平上逆转（或预防）耐受性的作用不一定取决于致耐受性部分对酶作用的敏感性，并且（b）耐受性诱导信号的产生涉及细胞代谢过程，低温可使其有所延迟，从而使此类细胞相对更易受到诸如胰蛋白酶处理等干预操作的影响。因此，总体而言，这些研究中获得的证据强化了由DNP-D-GL诱导的B细胞中枢耐受性反映亚细胞或细胞内失活事件的概念。
