The administration of soluble protein antigens via the oral route has been described as a means of inducing systemic immunological tolerance (oral tolerance). We had previously explored the mechanisms of oral tolerance induction with the use of ovalbumin (OVA) T cell receptor (TCR)- transgenic mice, and demonstrated the importance of IL-12 for the regulation of two mechanisms of oral tolerance, clonal anergy/deletion, and the generation of TGF--producing T cells. Thus, it was demonstrated that the administration of anti-IL-12 to intact OVA TCR-transgenic mice fed OVA, enhanced both TGF- production and apoptosis of antigen-specific T cells. To further understand the effect of anti-IL-12 treatment on oral tolerance, we conducted a series of studies on the role of various cytokines in TGF- production, both in vivo and in vitro. We demonstrated that the systemic administration of antibodies to IC-12 ovalbumin (OVA)-T cell receptor (TCR) transgenic mice fed high doses of OVA, followed by systemic OVA challenge, substantially enhances TGF- production after re- stimulation of peripheral T cells in vitro. Furthermore, we showed that na?ve (CD4+/MEL-14hi) OVA-TCR-T cells stimulated with OVA-pulsed dendritic cells in vitro produce 4-5 fold higher amounts of TGF- when cultured with anti- IL-12 or anti-IFN-. IL-4 was not required for TGF- production, however, it appeared to indirectly enhance TGF- production by promoting the growth of TGF- producing cells. Taken together, our findings demonstrate that IL-12 and IFN- are important negative regulators of TGF- production both in vivo and in vitro. They thus explain the ability of anti-IL-12 treatment to enhance oral tolerance as discussed above. In further studies carried out with Dr. Robert Seder (MIS/LCI/NIAID), the production of TGF- by T cells following primary and secondary stimulation in vitro was studied using T cells from TCR-transgenic, as well as IL-4 and IFN- knockout mice. It was determined that conditions favoring either high IL- 4 production (i.e., the presence of IL-4 in the priming cultures), or low IFN- production (i.e., culture with anti-IL- 12 and anti-IFN-) resulted in the emergence of TGF- producing T cells after a secondary in vitro stimulation. However, it was also determined that IL-4 was not absolutely required for TGF-- production, consistent with our prior findings. Finally, it was determined that IL-10, primarily by its ability to suppress IL- 12 production, and IL-7, primarily by its ability to induce IL-4 production in the T cell priming cultures, can enhance the differentiation of TGF--producing T cells, and that TGF- itself can prime T cells for their own production. This latter fact is important, as it suggests that the low level production of TGF- by one cell type, such as a T cell, could have dramatic effects on TGF- production by another cell, such as a B cell or antigen-presenting cell, the end result being an environment with high levels of TGF- that acts to suppress inflammation.
