Generation of H-2 Restricted Cytotoxic T Cells by Trinitrophenylated Proteins in Vitro: Specificity and Requirements

Abstract

Addition of TNBS-conjugated soluble proteins to cultures of murine spleen cells, of the H-2^k haplotype, led to the generation of MHC-restricted cytotoxic T cells. This phenomenon was effected by a wide range of proteins including ovalbumin, bovine γ-globulin, bovine serum albumin, human γ-globulin, human serum albumin (TNP-HSA), and keyhold limpet hemocyanin. Cytotoxic T lymphocytes (CTL) generated in such manner showed specificity for the hapten rather than for the protein carrier, in that they lysed cells bearing hapten coupled to either heterologous or homologous carriers. Spleen cell populations incubated with soluble HSA substituted to various degrees (8–40 moles TNP/mole HSA) displayed TNP on their surface as evidenced by a) C-mediated lysis, and b) spectrophotometric measurement of solubilized cell membranes. However, only those cells bearing HSA substituted at more than 26 moles TNP/mole HSA were susceptible to lysis by hapten-specific H-2 restricted CTL. Furthermore, TNP-HSA with lower substitution (<26 mole/mole) did not stimulate hapten-specific cytotoxic responses. These observations illustrate several features of target cell recognition by hapten-specific, H-2 restricted, cytotoxic T-cells: i) These CTL do not show specificity to the protein carrier. In this regard cytotoxic T cells differ from T cells responsible for delayed-type hypersensitivity (DTH). ii) Hapten and H-2 need not be covalently linked. iii) Hapten display on the cell surface, even in relatively large amounts, is not in itself sufficient. Rather, it seems that some stringent requirements of protein derivatization and/or distribution have to be met irrespective of whether the protein substituted is intrinsic to the cell membrane or exogenously added. These findings represent a reproducible system for further investigation of the exact molecular nature of the antigenic determinants recognized by CTL.