HLA class I molecules perform a critical host defense function by binding foreign peptides derived from intracellular pathogens and displaying them on the cell surface in a form that can be recognized by specific cytotoxic T lymphocytes (CTL). Induction of HLA class I expression may be important because only newly-synthesized class I molecules bind peptides efficiently. We have shown previously that the cytokines tumor necrosis factor (TNF) or interferon (IFN) individually increase -- and together, increase synergistically -- expression of HLA class I molecules by activating transcription of the structural genes. My analysis of the HLA class I heavy chain promoter has defined by DNA elements mediating synergistic transcriptional activation by combined IFNs with TNF and has identified the IFN-gamma-induced and TNF-activated nuclear factors binding to these elements. Recently, we reported that TAP-1, which is a subunit of a putative transmembrane peptide pump that is required for efficient HLA class I cell surface expression, is also induced synergistically by this cytokine combination and, surprisingly, that is HLA class I. These results were obtained principally in cultured human endothelial cells (EC), which will remain the focus of the studies proposed here because the expression of HLA class I molecules by vascular EC is likely to be crucial in activating specific CTL at sites of inflammation, especially in allogeneic transplantation. The overall aim of this proposal is to determine the molecular basis cytokine synergy in the induction of HLA class I and TAP-1 expression.
My specific aims are: 1) fully identify and characterize the interactions of transcription factors that mediate cytokine synergy by using factor-specific antibodies in electromobility shift assays of DNA-binding proteins, by expressing transfected normal and mutated nuclear factors in the sense and antisense orientations, and by systematically altering the orientation and spacing of the previously-defined responsive elements, 2) identify the cytokine- responsive enhancer elements mediating the rapid and synergistic induction of TAP-1 by deletion analysis of promoter-reporter gene constructs and compare these elements those of HLA class I, and 3) determine the role of rapidly-induced TAP in the subsequent expression of HLA by delaying TAP expression with antisense DNA or by accelerating the expression of transfected HLA molecules. These studies will provide critical insights into the basic molecular mechanisms regulating HLA class I molecule expression, determine the role of inducible TAP-1 expression in supporting subsequently increased levels of HLA class I expression, and may well suggest ways to interrupt or encourage these interactions in pathological settings.
