There are four effector molecules that are candidates for involvement in the lytic function expressed by CD8+ cytotoxic T lymphocytes (CTLs): perforin, TNF-alpha, TNF-Beta, and IFN-gamma. In order to assess definitively the role of each of these molecules in CTL function, we propose to create cloned CTL lines that lack the perforin gene, and cloned CTL lines that lack the perforin gene plus one of the other three candidate genes (TNF-alpha, TNF-Beta or IFN-gamma). We propose to do this by the process of targeted gene disruption via homologous recombination. We will use the same process with ES cells to generate an inbred strain of mice completely lacking perforin genes. Such mice will also be a source for generation of perforin-less (P0) CTL clones. In preliminary work, we have produced mosaic mice from ES cells with one perforin allele disrupted. These mice are now being mated to produce a perforin-less mouse strain. This work will have the following principal aims: 1. Short range: (a) Generate cloned CTL lines with both copies of the perforin gene disrupted. This will provide a definitive answer to the question of whether perforin per se is required for classically defined CTL killing in vitro. (b) Generate additional ES cells with one perforin allele disrupted. 2. Intermediate range: (a) Use the perforin-less (P0) cloned CTL lines for the study of alternate pathways of CTL killing in the unambiguous absence of perforin as a lytic mechanism. P0 CTL lines will be used as starting material for the systematic elimination of TNF-alpha, TNF-Beta and IFN-gamma genes. (b) Produce inbred mice with both perforin alleles disrupted. 3. Long range: Use the inbred lines of mice homozygous-defective for the perforin gene to analyze the role of perforin in three CD8+T cell functions in vivo: graft rejection, viral clearance and tumor control.
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