Department of Immunology, Faculty of Biology, University of Warsaw, Poland.
Nephrol Dial Transplant. 2010 Mar;25(3):710-7. doi: 10.1093/ndt/gfp586. Epub 2009 Nov 9.
A growing body of data shows that CD4(+)CD25(+) regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4(+)CD25(+) Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4(+)CD25(+) Tregs in vitro.
CD4(+)CD25(+) Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). Tregs were identified in MLR cultures by flow cytometry using anti-CD4, anti-CD25, anti-CTLA-4, anti-CD122, anti-GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay.
Although both rapamycin and cyclosporine A suppressed the induction of CD4(+)CD25(+) Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4(+)CD25(+) Tregs which expressed GITR, a negative regulator of Treg's suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF-beta1.
Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4(+)CD25(+) Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4(+)CD25(+) Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs.
越来越多的数据表明，CD4(+)CD25(+)调节性 T 细胞（Tregs）可以通过抑制对同种异体抗原的免疫反应来诱导移植耐受。然而，不同的免疫抑制剂药物在临床移植中使用时，均会对 CD4(+)CD25(+)Tregs 的产生和抑制能力产生实质性影响。本研究的目的是比较环孢素 A 和雷帕霉素对体外诱导和抑制人 CD4(+)CD25(+)Tregs 的作用。
在雷帕霉素（Treg-Rapa）或环孢素 A（Treg-CsA）存在的双向混合淋巴细胞反应（MLR）中诱导 CD4(+)CD25(+)Tregs。通过流式细胞术使用抗 CD4、抗 CD25、抗 CTLA-4、抗 CD122、抗 GITR mAb 和抗 PE-FOXP3 染色试剂盒，在 MLR 培养物中鉴定 Tregs。通过流式细胞术测定诱导的 Tregs 抑制外周血单个核细胞（PBMC）抗 CD3 Ab 触发增殖的能力来评估其抑制能力。通过酶联免疫吸附试验测定培养上清液中 TGF-β1 的浓度。
虽然雷帕霉素和环孢素 A 均抑制 MLR 中 CD4(+)CD25(+)Tregs 的诱导，但环孢素 A 培养的细胞抑制作用更为明显。另一方面，只有雷帕霉素显著降低了表达 GITR（Treg 抑制能力的负调节剂）的 CD4(+)CD25(+)Tregs 的百分比。重要的是，与 Treg-CsA 不同，Treg-Rapa 显示出显著的抑制活性，并能够抑制抗 CD3 Ab 激活的 PBMC 增殖。这种活性可能是由 TGF-β1 介导的。
与环孢素 A 不同，雷帕霉素不抑制 CD4(+)CD25(+)Tregs 的功能。这意味着雷帕霉素可以通过促进功能性 CD4(+)CD25(+)Tregs 的诱导，有助于移植耐受的发展。此外，我们的结果表明，雷帕霉素可以与功能性 Tregs 联合使用。
