Transplantation has become the treatment of choice for end-stage failure of organ systems. This has been at the price of increased susceptibility to infection and risk of malignant neoplasia resulting from powerful but nonspecific immunosuppression. To overcome these problems, there is a need to develop methods that engender immunologic unresponsiveness to the graft while leaving the host defenses against infection and malignant transformation intact. Our approach is to modify the graft. We plan to reduce its capacity to activate the immune response, and to minimize its susceptibility to immunologic injury by reducing the expression of major histocompatibility complex (MHC) antigens within the graft. The MHC antigens (Class I and Class II) are the primary antigenic stimuli which trigger the immunologic rejection response. Our overall objective is to achieve a level of reduced MHC expression sufficient to allow graft and tissue acceptance. By measuring the alterations in the immune response caused by lowered levels of constitutive and inducible MHC Class II antigens, we will provide a biologic reference for the effectiveness of the oligonucleotide approach to MHC gene regulation. Four unique oligonucleotide approaches will be examined, all initially aimed at reducing MHC Class II expression: RFX-decoy; antisense to a key transcription factor CIITA; a triple helix forming oligo to the DRA promoter; and an optamer that blocks IFNgamma induction of MHC genes.
In Aim 1 a model gene transfection system for MHC Class II will be used to study the T cell proliferative response after reduction in Class II expression.
In Aim 2, the level of reduction in constitutive MHC Class II expression (achieved by oligos) required to produce a significant decrease in T lymphocyte response will be studied using measurements of individual cytokine producing cells.
Aim 3 examines the level of reduction in constitutive Class II expression necessary to reduce target cell injury and antibody mediated injury.
Aims 4 and 5 intend to develop antigen presenting cells (dendritic, endothelial, renal tubular) and determine the uptake, metabolism and activity of oligos in these relevant cells.
Aim 6 examines the ability of the most promising oligonucleotides to down regulate Class II expression and the alteration in T lymphocyte activation as a consequence of altering the immunogenicity of these graft derived antigen presenting cells. The goals of this project are to identify the oligonucleotide approach most effective in achieving inhibition of MHC Class II expression and minimizing immunologic responsiveness and susceptibility to target cell injury.
