Ex Vivo Generation of Regulatory T Cells: Characterization and Therapeutic Evaluation in a Model of Chronic Colitis
Ex Vivo Generation of Regulatory T Cells: Characterization and Therapeutic Evaluation in a Model of Chronic Colitis
复制标题
DOI：
10.1007/978-1-60761-869-0_4
复制
发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Grisham, Matthew B.
中科院分区：
文献类型：
--
作者：
Karlsson, Fridrik;Robinson-Jackson, Sherry A.;Grisham, Matthew B.
天然存在的调节性 T 细胞（nTreg；CD4(+)CD25(+)Foxp3(+)）能够抑制在多种不同的自身免疫和慢性炎症疾病（如炎症性肠病、糖尿病和关节炎）动物模型中观察到的慢性炎症。探索 nTregs 如何以及在何处发挥体内抑制作用的一个主要限制是这些调节细胞的相对缺乏。尽管一些实验室已经描述了不同的方法来扩增流式纯化的nTregs或将常规/初始T细胞（CD4(+)Foxp3(-)）体外转化为表达Foxp3的“诱导”Tregs（iTregs；CD4(+)Foxp3(+)），但我们发现这些方法中有许多都有其自身的局限性。因此，我们试图开发一种相对简单的离体方法来产生大量表达 Foxp3 的 iTregs，可用于评估它们在体内慢性肠道炎症模型中的运输特性、抑制活性和治疗效果。我们提出了一个详细的方案，证明在 IL-2、TGF beta 和全反式视黄酸存在下，传统 CD4(+) T 细胞的多克隆激活诱导这些 T 细胞 >90% 转化为表达 Foxp3 的 iTregs，并在体外培养 4 天后促进增殖增加三到四倍。该方案适度增强了肠道归巢粘附分子 CCR9 的表面表达，但不增强 alpha(4)beta(7)。此外，我们提供的初步数据表明，这些 iTreg 在体外抑制 T 细胞激活方面明显更有效，并且在体内减轻慢性结肠炎方面与新鲜分离的 nTreg 同等有效。最后，我们报告该方案有可能从一个健康小鼠的脾脏中产生 30-4000 万个 iTreg。
Naturally occurring regulatory T cells (nTregs; CD4(+)CD25(+)Foxp3(+)) are capable of suppressing the chronic inflammation observed in a variety of different animal models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, diabetes, and arthritis. A major limitation in exploring how and where nTregs exert their suppression in vivo is the relative paucity of these regulatory cells. Although several laboratories have described different methods to expand flow-purified nTregs or convert conventional/naive T cells (CD4(+)Foxp3(-)) to Foxp3-expressing "induced" Tregs (iTregs; CD4(+)Foxp3(+)) ex vivo, we have found that many of these approaches are encumbered with their own limitations. Therefore, we sought to develop a relatively simple ex vivo method to generate large numbers of Foxp3-expressing iTregs that can be used to evaluate their trafficking properties, suppressive activity, and therapeutic efficacy in a mouse model of chronic gut inflammation in vivo. We present a detailed protocol demonstrating that polyclonal activation of conventional CD4(+) T cells in the presence of IL-2, TGF beta, and all trans retinoic acid induces >90% conversion of these T cells to Foxp3-expressing iTregs as well as promotes a three- to fourfold increase in proliferation following a 4-day incubation period in vitro. This protocol enhances modestly the surface expression of the gut-homing adhesion molecule CCR9 but not alpha(4)beta(7). Furthermore, we provide preliminary data demonstrating that these iTregs are significantly more potent at suppressing T-cell activation in vitro and are equally effective as freshly isolated nTregs at attenuating chronic colitis in vivo. Finally, we report that this protocol has the potential to generate 30-40 million iTregs from one healthy mouse spleen.