Phosphatidylinositol hydrolysis in freshly isolated human T lymphocytes

Abstract

Antigen receptor-mediated activation of T and B lymphocytes results in activation of phospholipase C-γ isozymes with subsequent hydrolysis of membrane inositol phospholipids. As a method of screening autoimmune 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 resulting from surface receptor crosslinking. Using staphylococcal α-toxin, we permeabilized freshly isolated, purified human T lymphocytes to facilitate incorporation of [ 3H]myoinositol into membrane phospholipids. Aggregation of surface antigen receptors (TCR-CD3 complex and CD28 on T cells) with specific antibodies produced extensive ATP and Mg 2+-dependent hydrolysis of the membrane inositol phospholipids as measured by release of water soluble inositol phosphates. Anti-human CD3 antibody produced 18.5 ± 1.6 net % PI hydrolysis and anti-human CD28 antibody produced 4.6 ± 0.2 net % PI hydrolysis. Simultaneous anti CD3 CD28 crosslinking produced 30.8 ± 1.2 net % PI hydrolysis, an increase 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 (Na 3VO 4) was used as a positive control because it induces maximal protein tyrosine kinase-dependent PI hydrolysis in permeabilized cells. Na 3VO 4 consistently induced hydrolysis of > 50% of the membrane inositol phospholipid pool. These data indicate that costimulation of T cells with antibodies to CD3 and CD28 is synergistic and reinforces the importance of CD28 as an accessory T cell stimulus. This easy technique allows quick evaluation of the integrity of the early signaling cascade in lymphocytes as a screen for autoimmune and immunodeficiency diseases.