The studies detailed in this application will examine the role of the granule thiol protease, dipeptidyl peptidase I (DPPI) in generation of cytotoxic T lymphocyte (CTL) effector functions and the evolution of in vivo alloimmune responses. DPPI has been found to be expressed at high levels in the specialized effector granules of CTL, natural killer cells, mast cells, and myeloid cells where the enzyme is responsible for post-translational processing and activation of the granule serine proteases expressed in these immune effector cells. Recent studies indicate the presence of specific inhibitors of DPPI during primary alloantigen stimulated T-cell responses not only prevents generation of the enzymatically active forms of the granzyme family of granule serine proteases expressed by CTL but also impairs CD8 (+) T-cell growth and differentiation of multiple cytotoxic effector functions. The proposed studies will examine the mechanisms whereby inhibition of DPPI or other DPPI-like protease(s) impair generation of not only granzyme dependent effector cell function but also appears to modulate other CD8 (+) T-cell responses not previously attributed to granzyme activity.
These specific aims will be addressed: 1) Determine whether DPPI activity or other DPPI-like thiol protease activities play roles in CD8 (+) T-cell growth and differentiation by: (a) assessment of the effects of DPPI-specific antisense oligonucleotides on CD8(+) T-cell growth and proliferation; and (b) characterization of DPPI-like thiol proteases that may play a role in CD8(+) T-cell growth and differentiation; (2) Test the hypothesis that impaired granzyme activation leads to diminished CD8(+) T-cell growth and differentiation by: (a) assessment of the potential toxicity of unprocessed granzyme proteins within DPPI inhibited CTLL-2 and CD8 (+) T-cells; (b) investigation of the role of DPPI-processed granzymes in regulating CD8(+) T-cell growth and differentiation; and (c) assessment of the effects of granzymes on cytokine responses by APC during MLC; (3) Generate DPPI deficient T-cells by targeted disruption of the murine DPPI gene; and (4) Assess the role of DPPI in generation of in vivo cytotoxic effector functions and alloimmune responses during graft versus host disease and organ allograft rejection. These studies should provide new insights into the regulation of CTL effector mechanisms and the role that such effector mechanisms play in complications of bone marrow and organ transplantation.
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