An Optimized CFSE-based T-cell Suppression Assay to Evaluate the Suppressive Capacity of Regulatory T-Cells Induced by Human Tolerogenic Dendritic Cells
An Optimized CFSE-based T-cell Suppression Assay to Evaluate the Suppressive Capacity of Regulatory T-Cells Induced by Human Tolerogenic Dendritic Cells
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DOI：
10.1111/j.1365-3083.2010.02414.x
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发表时间：
2010-08-01
影响因子：
3.7
通讯作者：
ten Brinke, A.
中科院分区：
文献类型：
--
作者：
Boks, M. A.;Zwaginga, J. J.;ten Brinke, A.
在自身免疫性疾病或移植排斥反应中，预防或减少目前免疫激活的治疗是非常必要的。体外产生的致耐受树突状细胞(DC)被认为具有很强的细胞治疗这些疾病的潜力。耐受性DC介导的免疫抑制机制之一是诱导调节性T细胞(Treg)。因此，这种DC诱导Treg的效果将反映其耐受能力。由于目前还没有人诱导Treg的特定标记被描述，Treg只能通过功能来欣赏。因此，我们优化了一种体外抑制试验来筛选人DC诱导的Treg活性。IL-10产生的耐受性DC被用来诱导Treg，而Treg被证明能有效地抑制同种异体成熟DC(MDC)刺激的应答T细胞的增殖。我们的结果表明，在抑制实验中测得的IL-10 DC诱导的Treg的抑制能力随着iTreg剂量的增加而增加，随着T细胞刺激的抗原提呈细胞(APC)数量的增加而降低。降低共培养中反应性T细胞和刺激性MDC之间的比率明显改善了抑制试验的读出。此外，在抑制试验中，MDC诱导的T细胞被证明是必要的控制条件。总之，我们建议在抑制试验中同时滴定APC和iTreg，并在MDC启动的T细胞中加入阴性对照条件，以区分iTreg对T细胞的特异性和功能性抑制与可能的全身性抑制活动。
In autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are considered to have a strong potential as cellular therapy for these diseases. One of the mechanisms of immune suppression mediated by tolerogenic DC is the induction of regulatory T-cells (Treg). Consequently, the efficacy of such DC to induce Treg will reflect their tolerogenic capacity. Because no specific markers have been described for human induced (i)Treg yet, the Treg can only be appreciated by functionality. Therefore, we have optimized an in vitro suppression assay to screen for human DC-induced-Treg activity. IL-10-generated tolerogenic DC were used to induce Treg that were previously shown to effectively suppress the proliferation of responder T-cells stimulated with allogeneic mature DC (mDC). Our results show that the suppressive capacity of IL-10 DC-induced Treg measured in the suppression assay increases with the iTreg dose and decreases with higher numbers of antigen-presenting cells (APC) as T-cell stimulation. Lowering the ratio between responder T-cells and stimulator mDC present in the coculture clearly improved the read-out of the suppression assay. Furthermore, mDC-primed T-cells in the suppression assay were shown to be an essential control condition. In conclusion, we recommend titrations of both APC and iTreg in the suppression assay and to include a negative control condition with T-cells primed by mDC, to distinguish specific and functional suppression by iTreg from possible generalized suppressive activity.