Project 1: The immune response to oral antigen administration, including the development of oral tolerance, was explored with the use of ovalbumin (OVA) T cell receptor (TCR)-transgenic mice. Feeding high doses of OVA enhanced interferon-gamma (IFN-gamma) production in the Peyer's patches (PP), but induced tolerance in the peripheral lymphoid tissues (PLT) marked by suppressed proliferative and cytokine responses. Systemic administration of antibodies to interleukin-12 (anti-IL-12) simultaneous with antigen feeding modestly enhanced the degree of tolerance in the peripheral lymph nodes as shown by increased suppression of proliferative responses after in vitro re-stimulation, and secondary responses in the popliteal lymph node following in vivo challenge and in vitro re-stimulation. Systemic anti-IL-12 treatment was associated with augmented transforming growth factor beta (TGF-beta) production and T cell apoptosis in both PP and PLT. Cell mixing studies and proliferation assays in the presence of anti-TGF-beta provided evidence that the increased suppression of responses induced by anti-IL-12 was due primarily to the secretion of TGF-beta. These findings suggest that IL-12 negatively regulates two of the main mechanisms of oral tolerance, TGF-beta production and clonal deletion via apoptosis. Project 2: 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-beta production, both in vivo and in vitro. We demonstrated that the systemic administration of antibodies to IL-12 to ovalbumin (OVA)-T cell receptor (TCR) transgenic mice fed high doses of OVA, followed by systemic OVA challenge, substantially enhances TGF-beta production after re-stimulation of peripheral T cells in vitro. Furthermore, we showed that naive (CD4+/Mel-14hi) OVA-TCR-T cells stimulated with OVA-pulsed dendritic cells in vitro produce 4-5 fold higher amounts of TGF-beta when cultured with anti-IL-12 or anti-IFN-gamma. IL-4 was not required for TGF-beta production, however it appeared to indirectly enhance TGF-beta production by promoting the growth of TGF-beta producing cells. Taken together, our findings demonstrate that IL-12 and IFN-gamma are important negative regulators of TGF-beta production both in vivo and in vitro. They thus explain the ability of anti-IL-12 treatment to enhance oral tolerance as discussed above.
