TRANSGENIC CONTROL OF PERFORIN GENE-EXPRESSION - FUNCTIONAL EVIDENCE FOR 2 SEPARATE CONTROL REGIONS

Perforin is a pore-forming effector molecule of CTL and NK cells. To c haracterize perforin gene expression and its transcriptional control m echanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murine perforin 5' flanking and promoter r egion in transgenic mice. Six out of seven transgenic lines expressed the perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Tissues not yet reported to contain perforin- expressing lymphocytes were identified. Transgene expression occurred in all cells that physiologically are able to express perforin, i.e., in T cells and NK cells, and in some T cells that normally may express little or no perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous perforin mRNA were co-expressed in t he lymphoid organs, as well as in the lung, the ileum, the oviduct/ute rus, and the bone marrow. At the single cell level, the perforin tag w as present on NK cells and on CD8(+), as well as on CD4(+) T cells. Al so targeted were Thy-1.2(+) gamma delta T cells, but not Thy-1.2(-) ga mma delta T cells, B cells, nor monocytes. During thymic T cell develo pment, transgene expression occurred in double negative (CD4(-)CD8(-)) thymocytes and was detected at all subsequent stages, but exceeded th e expression levels of the endogenous gene in the thymus. In conclusio n, the analyzed perforin 5' flanking and promoter region contains impo rtant cis-acting sequences that restrict perforin expression to T cell s and NK cells, and therefore provides a unique tool for manipulating T cell and/or NK cell-mediated immune responses in transgenic mice. On the other hand, the normal control of perforin gene expression involv es at least one additional negative control mechanism that was not med iated by the transgenic promoter and upstream region. This control res tricts perforin gene expression in thymically developing T cells and i n most resting peripheral T cells, but can be released upon T cell act ivation.