Cytomegalovirus (CMV) has been implicated as an exacerbating agent in the development of transplantation-associated arteriosclerosis (TxAA), a major limitation in long-term cardiac allograft survival for which no satisfactory medical intervention is yet available. In an effort to define mechanisms explaining this association, we propose that CMV- infected graft endothelia can initiate a host immune activation cascade, and that the consequent localized release of cytokines can raise proximal uninfected endothelium to a state of enhanced alloimmunogenicity, ideally poised for immune attack by circulating host cellular immune components. Thus the experiments described in this proposal are designed to test the hypotheses that 1) CMV-infected endothelial cells (EC) can stimulate the generation of allogeneic and autologous cytolytic T cells (CTL) which exhibit promiscuous lytic activity against uninfected EC, 2) that these interactions occur by non-traditional mechanisms which are highly dependent upon costimulatory signals, 3) that uninfected endothelia activated by cytokines elaborated by CMV-responsive T cells can serve as a substrate for T cell transmigration and/or cytolysis, and 4) that these events can occur to a significant extent in the presence of clinically relevant concentrations of immunosuppressive agents. CTL precursor frequencies will be determined for populations of in vitro-stimulated T cells by measurement of radiolabel release from 51Cr-labelled infected or uninfected autologous or allogeneic EC targets. To determine the contribution of HLA and adhesion molecules in these responses, stimulator and/or target populations will be depleted of HLA class I and/or HLA class II-positive cells immunomagnetically or by fluorescence-activated cell sorting, or assayed for cytolysis in the presence of blocking antibodies. immunofluorescence flow cytometry will be used to identify responsive T cell subsets as naive or memory, alpha/Beta or gamma/delta, as well as by Vbeta type. Finally, to simulate CMV-triggered cytokine- mediated events which may occur at the graft/host interface, endothelial monolayers will be activated by culture beneath trans-well inserts containing T cells in combination with CMV-infected EC, then tested for their ability to promote trans-endothelial T cell migration and their susceptibility to CTL-mediated lysis in the absence or presence of various concentrations of clinically relevant immunosuppressive agents. Results of these studies will elucidate aspects of protective and/or pathogenic immune responses to this virus, potentially identify heretofore undescribed pathways of immune activation, and help elucidate the role of CMV in the development of transplantation-associated arteriosclerosis.
