Characterization of mutated transforming growth factor-β1 transformants and its modulatory effect on IgA isotype synthesis by murine B lymphocytes

Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine having both inhibitory and stimulatory effects on the differentiation of a variety of cell types. In the context of B cell differentiation, it has been shown that purified TGF-β1 increases IgA isotype switching by murine and human B cells in vitro. However, it has not been formally accepted whether TGF-β1 is actually an important inducer of IgA synthesis in vivo. In addition, it is difficult to evaluate the autocrine and paracrine effect because endogenous TGF-β1 is secreted not only in small amounts but in an inactive form. As a first step in understanding the role of TGF-β1 in induction of IgA synthesis in vivo, CHO cells were stably transfected with mutated TGF-β1 cDNA under the control of a metallothionein promoter, and the biological function of the expressed proteins was characterized in a variety of ways. TGF-β1-transfected CHO cells (TT-CHO cells) induced by zinc sulfate without any artificial activation were found to secrete at least 5,000 pg/ml as measured by ELISA. In addition, the secreted TGF-β1 adhered to soluble type II TGF-β1 receptors. A bioassay using Mv1Lu cells showed that the supernatant from TT-CHO cells retained not less than 8,000 pg/ml of TGF-β1, confirmed by a blocking experiment with anti-TGF-β1 antibody. These results indicate that the mutated recombinant TGF-β1 expressed from TT-CHO cells is fully biologically active. We were then interested in the effect of the produced rTGF-β1 on IgA isotype synthesis by mouse spleen B cells. The TT-CHO cell supernatant, in combination with rIL-2, substantially increased IgA isotype production. Finally, LPS-activated murine spleen B cells were transfected transiently with mutated TGF-β1 cDNA, resulting in IgA isotype induction. Our results suggest that B cell transfectants produce rTGF-β1 in an active form that can modulate its own differentiation in an autocrine fashion.