Oberg H-H, Wesch D, Lenke J, Kabelitz D
Institute of Immunology, University Hospital Schleswig-Holstein, Michaelisstrasse 5, D-24105 Kiel, Germany.
Scand J Immunol. 2006 Sep;64(3):353-60. doi: 10.1111/j.1365-3083.2006.01825.x.
Naturally occurring regulatory T cells (Treg) suppress the activation of antigen-responsive T cells in a cell contact-dependent manner. In order to investigate the impact of soluble mediators and receptor-ligand interactions on the interplay between naive T cells and Treg, a reproducible suppressor cell assay which functions in the absence of additional feeder cells or antigen-presenting cells is mandatory. Here, we describe such a method which is suited to study the modulation of responder T cell/Treg interactions in vitro. Treg were isolated from negatively purified total human CD4+ T cells by positive selection using anti-CD25 monoclonal antibody (MoAb)-coated Dynabeads followed by a detachment step. The remaining CD4+ CD25- responder T cells were cocultured with CD4+ CD25+ Treg in the presence of T-cell Activation/Expansion Beads from Miltenyi Biotec pre-coated with anti-CD3 plus anti-CD28 monoclonal antibody (MoAb). The optimal concentration for coating was 5 microg/ml for both MoAb. At this concentration, strong proliferation of responder T cells was elicited which was almost completely suppressed by Treg at 1:1 cell ratios. When higher concentrations of anti-CD3/anti-CD28 MoAb were used for coating, Treg also showed some degree of proliferation. The optimized suppressor assay proved to be highly reproducible and was used here to confirm the partial or complete reversal of Treg-mediated T-cell suppression by some cytokines (IL-2, IL-15), soluble IL-6 receptor/IL-6 fusion protein and recombinant GITR-ligand. Furthermore, our data confirm that Treg do not need other cell types to suppress proliferation of CD4+ CD25- responder T cells.
天然存在的调节性T细胞（Treg）以细胞接触依赖的方式抑制抗原反应性T细胞的激活。为了研究可溶性介质和受体-配体相互作用对幼稚T细胞和Treg之间相互作用的影响，必须建立一种在没有额外饲养细胞或抗原呈递细胞的情况下仍能发挥作用的可重复抑制细胞测定法。在此，我们描述了一种适用于体外研究反应性T细胞/Treg相互作用调节的方法。通过使用抗CD25单克隆抗体（MoAb）包被的Dynabeads进行阳性选择，然后进行分离步骤，从阴性纯化的人总CD4 + T细胞中分离出Treg。将剩余的CD4 + CD25 - 反应性T细胞与CD4 + CD25 + Treg在预先包被有抗CD3加抗CD28单克隆抗体（MoAb）的Miltenyi Biotec公司的T细胞激活/扩增磁珠存在下共培养。两种MoAb的最佳包被浓度均为5μg/ml。在此浓度下，可引发反应性T细胞的强烈增殖，而在1:1细胞比例下，Treg几乎完全抑制了这种增殖。当使用更高浓度的抗CD3/抗CD28 MoAb进行包被时，Treg也表现出一定程度的增殖。优化后的抑制测定法被证明具有高度可重复性，并在此用于确认某些细胞因子（IL-2、IL-15）、可溶性IL-6受体/IL-6融合蛋白和重组GITR配体对Treg介导的T细胞抑制的部分或完全逆转。此外，我们的数据证实，Treg不需要其他细胞类型来抑制CD4 + CD25 - 反应性T细胞的增殖。
