The long-term goal of this project is to develop methods for induction of peripheral T cell tolerance that could improve prevention of graft rejection and graft-versus-host disease (GVHD) after human marrow transplantation. We propose that by understanding the mechanisms of immunosuppression by anti-CD3-epsilon antibodies in murine models, we will be able to exploit the use of anti-CD3-epsilon antibodies for achieving tolerance in man. Anti-CD3-epsilon F(ab')2 confers a """"""""competence to die"""""""" signal to antigen-activated, cycling T cells. T cell death requires both a """"""""competence to die"""""""" signal from the TCR or anti-CD3 F(ab')2, and FasL expression induced by prior antigen exposure, or the proximity of FasL+ cells. In mice transplanted with CD8+ TCR transgenic 2C cells specific for the Ld alloantigen, and OT-I cells that are not H2d-reactive, treatment with anti-CD3-epsilon F(ab')2 selectively depleted 2C cells and prevented manifestations of GVHD. Thus, anti-CD3-epsilon antibodies can induce selective immunosuppression by depletion of antigen-activated T cells. Anti-CD3-epsilon F(ab')2 delay but do not prevent rejection of skin grafts, indicating that their efficacy is limited. In vivo modulation of the TCR complex by anti-CD3-epsilon F(ab')2 may limit treatment efficacy and preclude clonal deletion, since activation-induced T cell death requires signaling above a critical threshold of TCRs engaged for sufficient time. We have generated a low avidity anti-CD3-epsilon mutant antibody which activates T cell death over a broader range of concentrations than wild type anti-CD3-epsilon antibody. We will test the hypothesis that low avidity anti-CD3-epsilon antibodies are more efficient at inducing depletion of activated T cells in vivo. Since low avidity peptides can induce T cell apoptosis without cytokine secretion, we have considered the hypothesis that low avidity anti-CD3-epsilon mAb may be unable to induce cytokine secretion, and may be safer in vivo. We present preliminary data that the CD3 and CD4 costimulation provides a positive signal rather than a death signal to pre-activated T cells. By using low avidity anti-CD3-epsilon F(ab')2 mutant antibodies and mutant mouse strains with fyn-/- and lck-/- peripheral T cells, we propose to test the hypothesis that the """"""""compentence to die"""""""" signal is transduced through the src tyrosine kinase Fyn rather than Lck.
Specific aims are: 1) Design low avidity CD3 ligands with improved immunosuppressive efficacy. 2) Select for low avidity CD3 ligands which induce apoptosis of antigen-activated T cell but no pathogenicity. 3) Define signaling pathways which activate T cell apoptosis in response to CD3 ligation.
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