The central hypothesis of this application is that the survival of vascularized organ allografts is determined by a competition for activation between two different functional classes of alloantigen specific T cells that produce distinctive ensembles of cytokines. The experiments will focus on analysis of cytokine gene expression in heterotopic murine cardiac allografts during normal unmodified rejection and in animals treated by several protocols which result in long term graft survival. The broad, long term objective of these studies is to understand the cellular and molecular mechanisms by which allografts can survive in an immunocompetent host without continuous pharmacological immunosuppression.
The specific aims are to test four hypotheses. The first two are that l) cytokines previously identified with Thl type T cell clones, particularly IL-2 & IFNgamma, promote allograft rejection, while 2) those associated with Th2 type cells, particularly IL-4 & IL-1O, promote allograft survival. To test these hypotheses, the pattern of cytokine gene expression will be determined during rejection and in circumstances of specific allograft unresponsiveness and tolerance (lack of response to subsequent donor alloantigen challenge). Several methods to induce allograft unresponsiveness and tolerance will be examined including treatment with anti-CD4 mAb, a combination of anti-LFA-l/anti-ICAM-l, donor specific blood transfusion, anti-IFNgamma, and anti-IL-2R/anti-IL-2. Cytokine gene expression will be determined using a combined approach of in situ hybridization for cytokine mRNA and RT-PCR analysis. The frequency and histological distribution of cytokine mRNA containing cells will be correlated with the phenotype of the cellular infiltrate identified by immunohistochemical staining with a panel of mAb. Recipient mice will also be treated with neutralizing anti-cytokine mAbs to determine the effect on transplant survival and cytokine gene expression. The third specific aim is to test the hypothesis that T cells from allograft tolerant mice specifically recognize the allograft MHC antigens and express the Th2 type cytokines upon TCR stimulation. T cells isolated from allograft tolerant mice will be stimulated in vitro with alloantigen and anti-TCR Vbeta mAb and the expression of cytokine mRNA and protein production determined. Experiments will also determine whether any type 2 cytokine expressing T cells specifically recognize idiotypes on alloantigen specific Thl type cells. The final specific aim is to test the hypothesis that T cells from allograft tolerant mice can specifically inhibit rejection of second allografts which share MHC antigens with the primary allograft, due to the production of Th2 type cytokines in the second allograft. Both rejection of skin grafts by the primary cardiac allograft recipient animal and adoptive transfer of T cells from tolerant mice to irradiated syngeneic recipients, followed by cardiac allograft placement, will be examined to test this hypothesis.
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