REGULATION OF GENE EXPRESSION IN HUMAN T CELL BY INTERLEUKIN 12, 1, 18 AND TNF

已知IL-12可上调血管内皮细胞合成干扰素-γ 活化的T细胞；然而，IL-12对其他T细胞表达的影响 细胞因子的定义不那么明确。在这个项目中，我们正在研究 IL-12对多种细胞因子产生的影响，包括干扰素-γ， IL-2、IL-4、IL-10和IL-13，由纯化的正常人CD3 T细胞产生。 尽管静息T细胞在很大程度上对IL-12没有反应， 抗CD3激活的T细胞是高度IL-12反应的AS IL-12诱导STAT4介导的DNA结合的能力 活动。我们已经发现，纯化的人类T淋巴细胞的激活 固定化抗CD3单抗诱导肿瘤坏死因子-α和白介素2的快速表达 干扰素-g、IL-10和IL-10的mRNA水平逐渐升高 IL-13。用核糖核酸酶测定mRNA表达水平的变化 使用便于分析的引物模板集进行保护分析 多个细胞因子基因同时存在。我们发现IL-12 显著上调干扰素-g和白介素10的表达，下调 IL-2的产生。IL-12对IL-2产生的抑制作用 直接与IL-10的产生增加有关。此外，中和 IL-10活性与抗IL-10抗体联合应用可使IL-2产生正常化 在IL-12处理的T细胞中显示出抑制作用 IL-12是由IL-10介导的。因此，我们发现IL-12同时 上调干扰素-g和白介素10的产生，并通过直接 IL-10依赖的途径，反馈抑制IL-2的产生。事实是 IL-12对T细胞干扰素-γ和IL-2合成的不同调节作用 细胞研究表明IL-12并不能整体增强ALL的表达 Th1型淋巴因子。此外，IL-12上调的能力 IL-10的产生提供了一种限制IL-2依赖的机制 活化T细胞的克隆性扩增及一种新的细胞因子 IL-12可诱导的调控途径。在未来的研究中，我们 计划进一步确定IL-12对其他物质生产的影响 细胞因子，特别是Th2型细胞因子IL-4和IL-13通过 激活T细胞，并比较IL-12与其他药物的作用 巨噬细胞衍生的免疫调节细胞因子，尤其是IL-1β 和肿瘤坏死因子-α。我们还将进一步探讨功能后果 IL-12处理的T细胞产生IL-10的增加。结果是 这些研究将扩大我们目前对 IL-12对免疫效应细胞的影响。此信息可能在以下方面有用 在临床试验中解释IL-12诱导的任何治疗活性 目前正在进行中。它还可以提供对生理学的洞察 与大剂量IL-12治疗相关的某些毒性的基础。 此外，还分析了IL-12对细胞因子表达的影响。 体外实验可能有助于IL-12替代标志物的鉴定 生物活性可用于监测重组蛋白的效力 IL-12在人类受体中的表达。

IL-12 is known to up-regulate synthesis of interferon-gamma (IFN-g) in activated T cells; however, the effects of IL-12 on expression of other cytokines are less well defined. In this project, we are examining the effects of IL-12 on production of multiple cytokines, including IFN-g, IL-2, IL-4, IL-10 and IL-13, by purified, normal, human CD3+ T cells. Although resting T cells are largely IL-12-nonresponsive, anti-CD3-activated T cell blasts are highly IL-12-responsive as demonstrated by the ability of IL-12 to induce STAT4-mediated DNA-binding activity. We have found that activation of purified human T lymphocytes on immobilized anti-CD3 mAb induces rapid expression of TNF-a and IL-2 mRNA, and more gradual increases in mRNA levels for IFN-g, IL-10 and IL-13. Changes in mRNA expression levels were measured by RNase protection assay using primer template sets that facilitate analysis of multiple cytokine genes simultaneously. We have found that IL-12 markedly up-regulates expression of IFN-g and IL-10, and down-regulates production of IL-2. Inhibition of IL-2 production by IL-12 correlates directly with increased production of IL-10. Moreover, neutralization of IL-10 activity with anti-IL-10 antibodies normalizes IL-2 production in IL-12-treated T cells demonstrating that the inhibitory effects of IL-12 are IL-10-mediated. Thus, we have found that IL-12 simultaneously up-regulates production of IFN-g and IL-10, and, by a direct IL-10-dependent pathway, feedback inhibits production of IL-2. The fact that IL-12 differentially regulates synthesis of IFN-g and IL-2 in T cells demonstrates that IL-12 does not globally enhance expression of all Th1-type lymphokines. Furthermore, the ability of IL-12 to up-regulate production of IL-10 provides a mechanism for limiting the IL-2-dependent clonal expansion of activated T cells, and defines a novel cytokine regulatory pathway that is inducible by IL-12. In future studies, we plan to further define the effects of IL-12 on production of other cytokines, particularly the Th2-type cytokines IL-4 and IL-13 by activated T cells, and to compare the effects of IL-12 with that of other macrophage-derived immunoregulatory cytokines, most notably IL-1 beta and TNF-alpha. We will also further explore the functional consequences of increased IL-10 production in IL-12-treated T cells. The results of these studies will broaden our current understanding of the actions of IL-12 on immune effector cells. This information may be useful in interpreting any therapeutic activity induced by IL-12 in clinical trials that are now underway. It may also provide insight to the physiological basis for certain toxicities associated with high dose IL-12 therapy. Furthermore, analysis of the effects of IL-12 on cytokine expression in vitro may facilitate identification of surrogate markers of IL-12 bioactivity that could be useful in monitoring the potency of recombinant IL-12 in human recipients.