PHOSPHATIDYLINOSITOL HYDROLYSIS IN FRESHLY ISOLATED HUMAN T-LYMPHOCYTES

Antigen receptor-mediated activation of T and B lymphocytes results in activation of phospholipase C-gamma isozymes with subsequent hydrolys is of membrane inositol phospholipids. As a method of screening autoim mune or immunodeficient patients for early receptor signaling defects, we have developed a rapid technique for studying phosphatidylinositol (PI) hydrolysis in cultured cells and fresh clinical specimens result ing from surface receptor crosslinking. Using staphylococcal alpha-tox in, we permeabilized freshly isolated, purified human T lymphocytes to facilitate incorporation of [H-3]myoinositol into membrane phospholip ids. Aggregation of surface antigen receptors (TCR-CDS complex and CD2 8 on T cells) with specific antibodies produced extensive ATP and Mg2-dependent hydrolysis of the membrane inositol phospholipids as measur ed by release of water soluble inositol phosphates. Anti-human CD3 ant ibody produced 18.5 +/- 1.6 net % PI hydrolysis and anti-human CD28 an tibody produced 4.6 +/- 0.2 net % PI hydrolysis. Simultaneous anti CD3 /CD28 crosslinking produced 30.8 +/- 1.2 net % PI hydrolysis, an incre ase over either stimulus alone (p = 0.0013 two tailed t test). Isotype matched control antibodies produced 11.6 +/- 0.4% PI hydrolysis. The tyrosine phosphatase inhibitor orthovanadate (Na3VO4) was used as a po sitive control because it induces maximal protein tyrosine kinase-depe ndent PI hydrolysis in permeabilized cells. Na3VO4 consistently induce d hydrolysis of > 50% of the membrane inositol phospholipid pool. Thes e data indicate that costimulation of T cells with antibodies to CD3 a nd CD28 is synergistic and reinforces the importance of CD28 as an acc essory T cell stimulus. This easy technique allows quick evaluation of the integrity of the early signaling cascade in lymphocytes as a scre en for autoimmune and immunodeficiency diseases.