MORPHOLOGIC AND FUNCTIONAL-CHARACTERIZATION OF PERFORIN-DEFICIENT LYMPHOKINE-ACTIVATED KILLER-CELLS

Mice deficient in perforin, a key mediator of lymphocyte-mediated cyto lysis, have recently been generated using the gene knockout technique. CTL and NK cells derived from these mice have been shown to be defect ive in the granule-dependent cytolytic pathway. To investigate whether the granule-formation process has been altered in these perforin-defi cient cytotoxic cells, rendering them defective in using the other gra nule mediators, we have examined in the present study the morphologic and functional characteristics of perforin-deficient LAK cells. Perfor in-deficient LAK cells, similar to wild-type LAK cells, were shown to contain a large number of granules in their cytoplasm. By electron mic roscopy, the morphology of the granules present in these two cell popu lations appeared indistinguishable. The complete depletion of perforin in LAK cells derived from perforin gene-knockout mice was further con firmed by immunoelectron microscopy using anti-perforin antiserum. The expression of other cytolytic mediators, present either within the gr anules (granzymes A and B) or elsewhere (Fas ligand), appeared to be u nperturbed, as investigated using the reverse transcription-PCR techni que. Like the CTL and NK cells isolated from perforin-deficient mice, perforin-deficient LAK cells could lyse only target cells that express high levels of Fas molecule. Furthermore, these perforin-deficient LA K cells, similar to wild-type LAK cells and a CTL clone, were resistan t to perforin-mediated cytolysis.