ALLOANTIGEN-UNRESPONSIVENESS IS ASSOCIATED WITH EARLY TCR SIGNALING DEFECTS IN VIVO

Abstract

667 Intracellular signaling events that result in T cell unresponsiveness have recently been described in vitro. Studies of in vivo tolerance have been limited by the requirement for large numbers of clonally specific T cells. A detailed understanding of the biochemical events that occur in vivo after alloantigen recognition by T cells is however crucial for the future development of selective interventions modulating T cell activation in organ transplantation. To elucidate changes in intracellular signaling that characterize tolerancein vivo, we took advantage of mutant mice that express an alloantigen-specific TCR, 2C, as a transgene in >95% of peripheral T cells. The 2C TCR is restricted to Ld, a class I MHC molecule expressed in BALB/c mice. We have previously shown that 2C mice are extremely alloreactivein vitro and in vivo to BALB/c. We have also shown that the allorecognition is specific to the naturally occurring allopeptide p2Ca and the class I MHC molecule Ld. 2C mice transplanted with a vascularized BALB/c cardiac graft activate a sufficient number of T cells for biochemical analysis in vivo. Our current studies demonstrate that 2C mice can be rendered tolerant of vascularized BALB/c (H-2d) cardiac heart grafts. InFig 1., control, unmanipulated, 2C mice reject BALB/c cardiac grafts in 5-8 days whereas injection with semiallogeneic spleen cells (tolerized) confers alloantigen-specific tolerance. The tolerized state is long-lasting and associated with diminished in vitro alloresponsiveness.These tolerized mice show marked differences in early TCR-mediated signaling events compared to naive mice. Specifically, ZAP-70 phosphorylation is absent and Lck activity is diminished. The defects appear to be restricted to specific TCR-mediated signal transduction pathways as other proteins analyzed remain competent for signal transduction. We believe this is the first example of TCR-mediated signaling defects associated with in vivo alloantigen-specific tolerance.