The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA.
The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA.
复制标题
淋巴细胞激活过程中转化生长因子-β1 的自分泌产生。
作者：
C. Lucas;L. Bald;B. Fendly;M. Mora;I. Figari;E. Patzer;M. Palladino
我们描述了三种转化生长因子-β (TGF-β) mAb 的生产和表征，以及其中两种用于开发 TGF-β 1 特异性 ELISA 和研究体外免疫功能调节的用途。所有三种 mAb 均以高亲和力结合重组人 TGF-β 1 (rHuTGF-β 1)，并在免疫印迹中识别该分子的二聚体形式。 mAb 2G7 免疫沉淀 rHuTGF-β 1、TGF-β 2 和 rHuTGF-β 3，并在体外中和所有三种分子对水貂肺上皮样细胞 Mv1Lu 的生长抑制活性，表明具有共享的中和表位。 mAb 4A11 仅中和并免疫沉淀 rHuTGF-β 1，mAb 12H5 免疫沉淀 rHuTGF-β 1，但对 rHuTGF-β 1、TGF-β 2 或 rHuTGF-β 3 的生物活性没有影响。这些结果表明第二个中和表位可能是 TGF-β 1 所独有的。ELISA 是用 mAb 4A11 和12H5，范围为 0.63 至 40 ng/ml，即灵敏度为 0.63 ng/ml 或 63 pg/样品。该测定准确、精确且对活性 TGF-β 1 复合物具有特异性，但对非活性或潜伏 TGF-β 1 复合物无影响，并且不能与 TGF-β 2、rHuTGF-β 3、抑制素 A 和激活素 A 发生反应。从多个供体的人 PBMC 无血清培养物中获得的上清液中含有大量 TGF-β 1（3 至 15 ng/ml），仅在 pH 2 处理以转化潜伏后，才在 ELISA 中检测到。 TGF-β 变为活性形式。用重组人 IL-2 (rHuIL-2) 或 PHA-P/PMA 处理 PBMC 增强了潜在 TGF-β 1 的产生。mAb 4A11 和 2G7（但不是 mAb 12H5）增强了 PBMC 对 rHuIL-2/rHuTNF-α 和 PHA-P 的增殖反应，并增强了 rHuIL-2/rHuTNF-α 处理的 PBMC 向 LAK 细胞的发育。针对 COLO 靶细胞的细胞毒活性。这些发现表明，虽然PBMC分泌潜在的TGF-β1，但体外存在将潜在的TGF-β复合物转化为活性形式的机制，并且内源产生的TGF-β可以以自分泌方式调节免疫功能。
We describe the production and characterization of three mAb to transforming growth factor-beta (TGF-beta) and the use of two of them for the development of a TGF-beta 1-specific ELISA and for the study of the regulation of immune function in vitro. All three mAb bound recombinant human TGF-beta 1 (rHuTGF-beta 1) with high affinity and recognized the dimer form of this molecule in immunoblots. mAb 2G7 immunoprecipitated rHuTGF-beta 1, TGF-beta 2, and rHuTGF-beta 3 and neutralized the growth inhibitory activity of all three molecules in vitro on mink lung epithelial-like cells, Mv1Lu, indicating a shared neutralization epitope. mAb 4A11 neutralized and immunoprecipitated only rHuTGF-beta 1, and mAb 12H5 immunoprecipitated rHuTGF-beta 1 but had no effect on the bioactivity of either rHuTGF-beta 1, TGF-beta 2, or rHuTGF-beta 3. These results suggest that a second neutralization epitope may be unique to TGF-beta 1. The ELISA was developed with mAb 4A11 and 12H5, with a range of 0.63 to 40 ng/ml, i.e., a sensitivity of 0.63 ng/ml or 63 pg/sample. The assay is accurate, precise, and specific for the active but not the inactive or latent TGF-beta 1 complex and fails to react with TGF-beta 2, rHuTGF-beta 3, inhibin A, and activin A. Supernatants obtained from serum-free cultures of human PBMC from multiple donors contained significant quantities of TGF-beta 1 (3 to 15 ng/ml), which was detected in the ELISA only after pH 2 treatment to convert latent TGF-beta to the active form. Treatment of the PBMC with either recombinant human IL-2 (rHuIL-2) or PHA-P/PMA enhanced the production of latent TGF-beta 1. mAb 4A11 and 2G7, but not mAb 12H5 enhanced both the proliferative response of PBMC to rHuIL-2/rHuTNF-alpha and PHA-P and the development of the rHuIL-2/rHuTNF-alpha treated PBMC into LAK cells with cytotoxic activity against COLO target cells. These findings suggest that although PBMC secrete latent TGF-beta 1, mechanisms that convert the latent TGF-beta complex into an active form exist in vitro and that the endogenously produced TGF-beta can regulate immune functions in an autocrine fashion.