The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA.
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.
Lucas
C
C
Department of Medicinal and Analytical Chemistry, Genentech Inc, South San Francisco, CA 94080.
Bald
L N
LN
Fendly
B M
BM
Mora-Worms
M
M
Figari
I S
IS
Patzer
E J
EJ
Palladino
M A
MA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D066298:In Vitro Techniques; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D008815:Mice, Inbred Strains; D010835:Phytohemagglutinins / Q000494:pharmacology; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D015291:Transforming Growth Factors / Q000502:physiology*
D000911:Antibodies, Monoclonal; D010835:Phytohemagglutinins; D011994:Recombinant Proteins; D015291:Transforming Growth Factors; D013755:Tetradecanoylphorbol Acetate