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Monovalent Fab Fragments of D7.5 Monoclonal Antibody Activate Intracellular Ca 2+ Mobilization and Secretion of Cytolytic Factors by Thymus Cells
Author(s) -
Peacock James S.,
Tan Jacqueline,
Guffee Judy,
SteinStreilein Joan
Publication year - 1991
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.49.1.90
Subject(s) - biology , intracellular , cytolysis , microbiology and biotechnology , monoclonal antibody , antibody , secretion , calcium , calcium in biology , biochemistry , cytotoxic t cell , immunology , chemistry , in vitro , organic chemistry
Previous studies have identified two mouse monoclonal antibodies, D7.5 and G1.4, 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 10 ‐6 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 ([Ca 2+ ] i ) in thymocytes. A significantly elevated level of [Ca 2+ ] i was observed at 60s after antibody addition and this response, which required the external calcium source, increased continuously during the 30 min incubation. Furthermore, 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.