This application explores the influence on immune function by the natural inhibitor of Fas and caspase-8 (FLICE) known as FLIP (FLICE-inhibitory protein). It also draws parallels between mice over-expressing FLIP and Fas-deficient lpr mice. The preliminary findings show that soluble Fas-ligand (FasL) can co-stimulate with anti-CD3 proliferation and IL-2 production by primary T-cells in a caspase-dependent fashion. Fas co-stimulation of T-cells also up-regulates ERK and NF-kappaB activities. This is likely mediated by FLIP via the physical association of FLIP with Raf-1 (the upstream regulator of the ERK pathway), and TRAF-1 and RIP (which connect with the NF-kappaB pathway). As such FLIP has a dual function of not only inhibiting Fas-mediated cell death through competition with caspase-8, it also signals positively to augment TCR signals.
Aim 1 examines whether FLIP cleavage by caspase-8 is actually required for it to augment ERK and NF-kappaB activities. FLIP contains two potential caspase cleavage sites. These have been mutated and the mutant FLIP will be stably transfected into T- and B-cell lines as well as retrovirally transfected into primary T-cells. The non-cleavable FLIP should compete with endogenous FLIP for binding to caspase-8 or to FADD at the DISC (Death-Inducer Signaling Complex) to decrease activation of ERK and NF-kappaB.
Aim 2 studies the reason for the depletion of CD8+ cells in FLIP-transgenic (Tg) mice, and whether this results from FLIP-induced increased TCR signaling and premature cell death selectively by CD8+ T-cells. The opposite scenario is considered in lpr mice that lack Fas expression. The TCR-Tg mouse OT-1 has been bred to FLIP-Tg and lpr mice and ovalbumin peptide (OVAp) will be used to monitor ERK and NF-kappaB activation, proliferation, in vivo cell cycling, and death.
Aim 3 examines whether FLIP over-expression or Fas deficiency cause retention of """"""""""""""""misselected"""""""""""""""" thymocytes, T-cells that survived positive selection but then do not encounter any proper peptide/MHC combination in the periphery, and are normally eliminated by apoptosis, but are retained in mice over-expressing FLIP or lacking Fas. Such """"""""misselected"""""""" CD8+ T-cells may be the source of the accumulating CD4-8- T-cells in lpr mice.
Aim 4 studies whether levels of FLIP expression determine which T-cells become memory T-cells. We explore a model in which FLIP levels decrease proportional to the intensity of cell cycling, making rapidly cycling T-cells sensitive to Fas-induced death. However, those T-cells which cycle less intensely will maintain FLIP levels and survive to become memory T-cells.
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