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LFA-3 was purified with an intact (mLFA-3) or an enzymatically removed membrane-anchoring domain (sLFA-3). Gel filtration and sucrose gradient sedimentation showed sLFA-3 to be a single highly glycosylated polypeptide chain in solution, while mLFA-3 formed micelles of 8 LFA-3 monomers. 125I-mLFA-3 bound to Jurkat T leukemic cell surface CD2 with much higher avidity than sLFA-3. mLFA-3 binding had characteristics of a multivalent interaction with cell surface CD2 and had an avidity of 1.5 nM for Jurkat cells and 12 nM for resting T cells. Two CD2 mAbs tested did not block mLFA-3 binding: 9-1 and CD2.1. These mAbs were tested in combination with LFA-3 for their ability to activate T cells. The combination of mLFA-3 and CD2.1 mAbs induced a rapid increase in cytosolic free Ca2+ in Jurkat cells which was proportional to mLFA-3 occupation of CD2 sites. sLFA-3 showed no activity in the Ca2+ flux assay. The combination of mLFA-3 and the CD2.1 mAbs significantly stimulated proliferation of PBMC. The combination of mLFA-3 and 9-1 mAbs was weakly or not mitogenic for the same cells. The combination of CD2.1 and sLFA-3 at concentrations up to 480 nM was not consistently mitogenic. Therefore, monomeric LFA-3/CD2 interaction appears to have a relatively low affinity, while multimeric LFA-3 binds with high avidity. T cell activation by binding of LFA-3 to CD2 appears to require occupation of 10(4) to 10(5) CD2 sites, which is likely to occur during adhesion, but is rare in receptor systems with soluble ligands.

Type

Journal

The Journal of experimental medicine

Publication Date

02/1989

Volume

169

Pages

503 - 517

Addresses

Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115.

Keywords

B-Lymphocytes, T-Lymphocytes, Humans, Calcium, Phosphatidylinositols, Membrane Glycoproteins, Antigens, CD58, Receptors, Immunologic, Antigens, CD2, Antigens, Differentiation, T-Lymphocyte, Antigens, Surface, Lymphocyte Activation, Cell Adhesion, Dose-Response Relationship, Immunologic, Protein Conformation, Micelles, Solubility, Type C Phospholipases