ALLOANTIGEN-UNRESPONSIVENESS RESULTS IN DEFECTS IN INTRACELLULAR SIGNALING AND TRANSCRIPTIONAL ACTIVATION OF THE IL-2 PROMOTER.

Abstract

886 Intracellular signaling events that result in T cell unresponsiveness have been described in vitro, but the biochemical analysis of in vivo tolerance has been limited by a requirement for large numbers of clonally specific T cells. To clarify changes in intracellular signaling that characterize tolerance in vivo, we utilized mutant mice that express the 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 (H-2d) mice. We have shown that 2C mice can be rendered unresponsive to BALB/c by injection of live lymphocytes. The unresponsive state is functionally characterized by diminished mixed lymphocyte responsiveness, diminished IL-2 and IFNγ production and prolonged vascularized BALB/c cardiac allograft survival. Biochemically, the unresponsive state is characterized by an alteration in tyrosine phosphorylation of critical TCR-mediated intracellular signals. Two early TCR-mediated intracellular signaling events, Lck activity and Zap-70 phosphorylation appeared to be affected. We now demonstrate additional biochemical features of the anergic state. A predominant finding of the unresponsive state is deficient activation of the ZAP-70 substrate LAT (linker for activation of T cells). Diminished LAT activity may provide the link between our observations on a biochemical level (deficient early TCR-mediated signals) and the absence of proliferation and the prolonged cardiac allograft survival. Further, we now demonstrate distinct in vivo phenotypic characteristics of the unresponsive T cells suggesting that the induced unresponsive is associated with T cell anergy, rather than peripheral or central deletion. The predominant cell surface findings are early expression of the activation antigens CD69 and CD25 and late, sustained CD44 expression. To further characterize the intracellular signaling pathways associated with T cell anergy in vivo, we are now analyzing IL-2 DNA enhancer elements. Preliminary data suggests that there may be differential DNA binding activity of several transcription factors. We are now confirming this data and further delineating the specificity of the induced TCR-mediated signaling defects in anergic T cells in vivo. A detailed understanding of induced intracellular signaling defects associated with T cell anergy is clearly crucial for the future development of selective interventions modulating T cell activation in organ transplantation.