Department of Medicine I, University of Wuerzburg, Würzburg, Germany.
Transplantation. 2011 Mar 27;91(6):597-604. doi: 10.1097/TP.0b013e3182090f67.
Inhibition of calcineurin (CnA) activity by cyclosporine A (CsA) is the mainstay in immunosuppressive therapy. CsA inhibits the phosphatase activity of the cytosolic phosphatase CnA and, therefore, prevents the dephosphorylation and subsequently nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT). However, CsA has multiple other targets within the cell and is, therefore, not specific. We developed a new approach to inhibit CnA/NFAT signaling. This synthetic peptide prevented CnA nuclear translocation in vitro. The purpose of this study was to demonstrate that this novel approach could potentially inhibit T-cell function in vitro and in vivo.
T-cell activation (Jurkat T cells, naïve rat T cells, and peripheral human T cells) was assessed by protein synthesis, interleukin (IL)-2 promoter activity, and IL-2 levels after T-cell activation. Immunohistological stainings for CnA were performed to investigate nuclear localization of CnA. The immunosuppressive effects in vivo of the synthetic peptide were investigated in rats with heterotopic transplanted hearts.
The nuclear localization signal peptide significantly decreased alloantigen-specific T-lymphocyte proliferation, IL-2 promoter activity, and IL-2 production (338% ± 27% vs. 149% ± 11%, n=8, P<0.05) in cultured T cells by inhibition of CnA nuclear translocation. The synthetic peptide also significantly decreased the number of graft infiltrating CD8 T lymphocytes. Moreover, treatment with the synthetic inhibitory inhibited acute graft rejection (5 ± 0.6 days vs. 12 ± 2 days, n=10, P<0.05).
Inhibition of nuclear translocation of CnA is a novel approach to inhibit the activation of the CnA/NFAT signaling cascade. Further studies have to demonstrate the long-term use of this principle in vivo.
环孢素 A（CsA）抑制钙调磷酸酶（CnA）的活性是免疫抑制治疗的主要方法。CsA 抑制细胞质磷酸酶 CnA 的磷酸酶活性，从而阻止转录因子活化 T 细胞核因子（NFAT）的去磷酸化和随后的核转位。然而，CsA 在细胞内有多个其他靶点，因此并不具有特异性。我们开发了一种抑制 CnA/NFAT 信号的新方法。这种合成肽可防止 CnA 在体外的核转位。本研究的目的是证明这种新方法有可能在体外和体内抑制 T 细胞功能。
通过蛋白质合成、白细胞介素（IL）-2 启动子活性和 T 细胞激活后 IL-2 水平来评估 T 细胞的激活（Jurkat T 细胞、幼稚大鼠 T 细胞和外周人 T 细胞）。进行 CnA 的免疫组织化学染色以研究 CnA 的核定位。通过对异位移植心脏的大鼠进行体内实验，研究合成肽的免疫抑制作用。
核定位信号肽通过抑制 CnA 的核转位，显著降低了同种抗原特异性 T 淋巴细胞的增殖、IL-2 启动子活性和 IL-2 的产生（培养的 T 细胞中为 338%±27%比 149%±11%，n=8，P<0.05）。该合成肽还显著减少了移植物浸润的 CD8 T 淋巴细胞的数量。此外，该合成肽的治疗抑制了急性移植物排斥（5±0.6 天比 12±2 天，n=10，P<0.05）。
抑制 CnA 的核转位是抑制 CnA/NFAT 信号级联激活的一种新方法。还需要进一步的研究来证明该原理在体内的长期应用。
