The autocrine production of transforming growth factor-beta1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA.

Abstract

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.