Journal of Leukocyte Biology 49:90-97 (1991) © 1991 Wiley-Liss, Inc. Monovalent Fab Fragments of D7.5 Monoclonal Antibody Activate Intracellular Ca2 Mobilization and Secretion of

Previous studies have identified two mouse monoclonal antibodies, D7.5 and Gi.4, that each can cause secretion of cytolytic factors from natural killer (NK) cells and resting T cells in humans, rats, and hamsters. Toward elucidation of the molecular identity and the transmembrane signaling mechanism of the antibody-defined trigger molecules, we investigated 1) their surface density, 2) the crosslinking requirement for signaling, and 3) their ability to mobilize intracellular calcium ions. Equilibrium binding studies using monovalent Fab fragments of D7.5 showed the presence of approximately 20,000 trigger molecules per thymus cell and an antibody dissociation constant of 1.8 x 106 M. Thymus cells incubated in the presence of either intact or Fab fragments of D7.5 acquired the ability to lyse Yac-1 cells. Furthermore, the incubation media of thymocytes that were treated with either intact or Fab fragments of D7.5 contained effector-cell derived factors that could lyse Yac-1 cells. Flow cytometry measurements of changes in intracellular free calcium concentration ([Ca2 J) in thymocytes. A significantly elevated level of [Ca2 was observed at 60s after antibody addition and this response, which required the external calcium source, increased continuously during the 30 mm incubation. Further-more, dbCAMP plus theophylline did not alter the ability of D7.5 to induce calcium mobilization suggesting that the antibody-defined trigger may not use the signaling pathway involving breakdown of phosphatidylinositol. Key words: thymocyte activation, conformational triggers, transmembrane signaling, calcium mobilizatio