T-CELL STIMULATION AND CYTOKINE RELEASE INDUCED BY STAPHYLOCOCCAL-ENTEROTOXIN-A (SEA) AND THE SEAD227A MUTANT

Previous work demonstrated that human cytotoxic T cells activated by s uperantigens can lyse major histocompatibility complex (MHC) class II- positive target cells as well as MHC class II-negative tumour cells co ated with conjugates of monoclonal antibodies and superantigens. In or der to decrease MHC class II affinity, and therefore unwanted binding of the superantigen staphylococcal enterotoxin A (SEA) to MHC class II molecules, a point mutation was introduced into the SEA gene. This mu tation (SEAD227A) resulted in an approximately 3-log reduction of affi nity to human leucocyte antigen (HLA)-DR, but cytotoxicity mediated by this mutant superantigen towards antigen-labelled tumour cells is an efficient as cytotoxicity mediated by the native superantigen. We ther efore compared the T-cell activating potency of native and mutated SEA . Our data show that SEAD227A is 4- to 5-log less effective than nativ e SEA when activation of resting T cells is assayed in terms of blast formation, expression of cell surface activation markers and cytokine release. Furthermore, presenting either SEA or SEAD227A to MHC class I I-negative mononuclear cells by MHC class II-negative tumour cells did not result in significant blast formation of T cells, up-regulation o f CD25 or cytokine release. This suggests that lysis of MHC class II-n egative tumour cells is efficiently induced by monoclonal antibody tar geted superantigen, while activation of resting T cells requires addit ional co-stimulatory signals.