CELLULAR IMMUNOLOGY 9, 1!%-210 (1973) Cytotoxic Immune Ceils with Specificity for Defined Soluble Antigens I. Assay with Antigen-Coated Target Cells19 * VOLKER SCHIRRMACHER 3 AND PIERRE GOLSTEIN 4 Detartment of Tumor Biology, Karolinska Institute, Stockholm, Sweden Received December 6. 1972 An in vitro cytotoxic system is described, in which immune cells specific for a given soluble antigen exert a specific cytotoxic effect on target cells to which this antigen has been covalently linked. The nature of the target cell is important in this system. When antigen-coated P 815-X2 mastocytoma cells and antigen-coated chicken red blood cells were incubated for several hours in culture medium at 37”C, the presence of membrane-bound antigen could still be demonstrated on the latter, but not on the former target cells. This might be the reason why antigen-specific target cell destruc- tion by specific immune cells was observed only with antigen-coated chicken red blood cells as target cells. The specificity of the cytotoxic effect was controlled in each experiment in a criss-cross way by using two non cross-reacting antigens both as immunogens and for coating the target cells. Specific cytotoxicity was demonstrable with both guinea pig and mouse immune cells and with different kinds of antigens: foreign proteins, hapten-heterologous protein conjugates and hapten-autologous protein conjugates. INTRODUCTION Immune cytotoxicity in vifro can be mediated by thymus-processed, T, cells (l-3), by non-thymus-processed, B, cells (4-6) or by macrophages (7-10). The structures responsible for the specificity of T cell mediated cytotoxicity are re- ceptors, specific for antigen, present on the surface of the T cells and very probably made by the cells that bear them (11). The structures responsible for the specificity 1 Abbreviations used : ars, arsanyl group ; ars-tyr, arsanyl-N-chloracetyl-1-tyrosine ; B cells, non-thymus-processed lymphocytes ; BDB, bis-diazotised benzidine ; BSA, bovine serum albumin; CRBC, chicken red blood cells ; DNP, dinitrophenyl group ; ECDI, 1-ethyl-3- (3-dimethylamino- propyl)carbodiimide-HCl ; GA, glutaraldehyde ; HGG, human gamma globulin ; KLH, keyhole limpet hemocyanine ; MSA, mouse serum albumin ; OA, ovalbumin ; PBS, phosphate-buffered saline ; PPD, purified protein derivative of turberculin ; R.I., Release Index ; RSA, rabbit serum albumin; SRBC, sheep red blood cells; sulf, sulfanyl group; T cells, thymus-processed lymphocytes. 2 This work was supported by the Deutsche Forschungsgemeinschaft (Fellowship Schi 46/Z) and the Swedish Cancer Society and it was conducted under Contract No. NIH-NCI-E-69-2005 within the Special Virus Cancer Program of the National Cancer Institute, National Institutes of Health, USPHS. 3 Present address: The London Hospital Medical College, University of London, Turner Street, London, El 2AD, Transplantation Immunology Unit, England. 4 Present address: Laboratoire d‘Immunologie des Tumeurs, Institut de Recherches sur les Maladies du Sang, HBpital Saint-Louis, 2, place du Dr. Fournier, 75-Paris 10, France. 198 C yri ht Q 1973 by Academic Press, Inc. A,?rf&ti f o reproduction in any form reserved.