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 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 alpha-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 (Na3VO4) was used as a positive control because it induces maximal protein tyrosine kinase-dependent PI hydrolysis in permeabilized cells. Na3VO4 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.
Berney
S M
SM
Department of Internal Medicine, University of Alabama at Birmingham 35294, USA.
Atkinson
T P
TP
D018106:CD28 Antigens / Q000502:physiology*; D017252:CD3 Complex / Q000502:physiology*; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D008213:Lymphocyte Activation; D010716:Phosphatidylinositols / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*
D018106:CD28 Antigens; D017252:CD3 Complex; D010716:Phosphatidylinositols; D011971:Receptors, Immunologic