Measurement of cell-mediated cytotoxicity (CMC) 1 directed to cell surface antigens has been facilitated by the elaboration of a relatively simple reproducible test in which ~lCr is released from labeled target cells (1-5). Other investigators have examined certain facets of the test (6-18) and have applied the technique to studies of tumor allograft systems (3-5, 16, 19, 20) and soluble antigens bound to the surface membrane of target cells (18,(21)(22)(23)(24)(25)(26).The purpose of this study was to detect the appearance of cytotoxic lymphocytes (CL) in spleens and draining lymph nodes of rats grafted with allogeneic skin b y means of a modified 51Cr release assay and to quantitate CMC during rejection and prolonged survival of allografts induced by enhancing antibodies. With 5~Cr-labeled embryonic fibroblasts (EFB) as target cells derived from donor strain rats that were the source of immunizing skin grafts, and with cytotoxic lymphoid cells from sensitized recipients, the level of cellular immunity could be determined in recipients of skin allografts.
Materials and MethodsAnlmals.--150-2OO-g adult and neonatal Lewis (Le) rats were both used as skin-graft recipients and donors of aggressor cells for assays of CMC in vitro. Brown-Norway (BN) rats were a source of skin allografts and of target cells for CMC assays. Both inbred strains were obtained from our own breeding colony or from a commercial breeder (Simonsen Laboratories, Gilroy, Calif.). BN skin grafts, 1.5 cm on edge, were applied to the nuchal region of 7-8-day-old Le neonates or to the axillae of adult Le rats. The latter were either normal, injected intraperitoneally with 5 mg of cyclophosphamide 2 daily beginning 4 days before grafting, or neonatally thymectomized 2448 hr after birth and used 100 or more days later. Completeness of * This is publication No. 573 from