Interleukin-10 (IL-10) causes suppression or down modulation of T cell, B cell, and antigen presenting cell (APC) functions. However, cellular IL-10 (cIL-10), derived from mammalian cells, also has immunostimulatory properties. Conversely, viral IL-10 (vIL-10), encoded by the BCRF I open reading frame of the Epstein Barr virus, is almost exclusively immunosuppressive. The structural and physiological basis for these key characteristics and differences have not been defined. The applicant's preliminary data now demonstrate that a defined structural change can convert vIL-10 to cIL-10-like activity, or cIL-10 to vIL-10-like activity, by interchanging a single amino acid between the viral and cellular ligands. This structural change correlates with IL-10 binding to the previously described IL-10R1 chain and may correlate with the function of a newly described IL-10R2 chain. The applicant hypothesizes that cIL-10 and vIL-10 differentially bind IL-10 receptors, activate second messenger pathways, and induce immunosuppressive or immunostimulatory responses. Dr. Bromberg also hypothesizes that IL-10R1 and IL-10R2 and/or their associated second messengers are differentially distributed on different cell types. He proposes that elucidation of these differences will increase understanding of how IL-10 regulates immunity and allow the manipulation of IL-10 responses for immunosuppressive therapy in transplantation.
The specific aims of the proposed research are: 1) Demonstrate the structural basis of the binding to and activation of the IL-10R complex by IL-10 ligand constructs. Binding and competition assays will be performed between various cIL-10 and vIL-10 constructs and IL-10R1 and IL-10R2 chains. Additional studies will assay second messenger pathways through Western blotting and electrophoretic mobility shift assays for JAK-STAT activation. 2) Demonstrate the immunologic activity of IL-10 constructs on T cell, B cell, and APC function. The differential distribution of IL-10R1 and IL-10R2 on T, B, and APC subsets will also be characterized. 3) Demonstrate the in vivo immunological activity of IL-10 constructs in a transplant model. Gene transfer of IL-10 constructs to cardiac allograft will be performed and the effect on graft survival and immune activation of T, B, and APC functions evaluated.
