Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number.
Engagement of the TCR initiates at least two transmembrane signaling pathways, the phosphatidylinositol pathway and a tyrosine kinase pathway. The T cell leukemic line Jurkat was used to study the relationship between the number of occupied TCR on the cell surface and the TCR-mediated activation of phosphatidylinositol-specific phospholipase C. We characterized a series of Ti beta-chain transfectants of the Jurkat mutant J.RT3-T3.5, in which surface expression of the TCR is limited by expression of the TCR beta-chain. Calibrated flow cytometry was used to determine the number of binding sites for anti-CD3 mAb on the surface of these cells, which was less than 1.2 x 10(3) to 1.2 x 10(4) sites/cell. In the presence of lithium chloride, the accumulation of inositol phosphates (InsP) in these cell lines in response to saturating concentrations of anti-CD3 mAb was proportional to the calculated surface TCR number. This result was consistent with dose-response studies using anti-CD3 mAb in Jurkat cells, in which ligand concentration, rather than number of binding sites, was limiting. Increase in intracellular free calcium concentration was a sensitive indicator of TCR engagement and correlated with the level of TCR expression, but less closely than did InsP levels. Induction of the early lymphocyte activation marker CD69 by anti-CD3 mAb also correlated with surface expression of TCR. In order to test whether limitation of this signaling pathway by TCR number may be relevant to signal transduction in the wild-type cell, we compared PLC activity in Jurkat cells during soluble anti-CD3 mAb-induced internalization of the TCR and also in response to immobilized mAb. The net accumulation of InsP per min decreased linearly with TCR number during the rapid phase of TCR internalization, confirming the limiting role of TCR number in this system. When internalization was prevented by immobilization of the stimulus, there was no decrease in the net accumulation of InsP per minute over time. In a Jurkat cell line transfected with the heterologous human muscarinic receptor, subtype 1, the InsP response to a muscarinic agonist was unaffected by TCR internalization, indicating that the distal phosphatidylinositol pathway was not affected by prolonged stimulation of the TCR. We conclude that transmembrane signaling through the TCR may be regulated by the number of surface TCR-ligand complexes. This observation has implications for transmembrane signaling in both mature T cells and thymocytes.
Graber
M
M
Howard Hughes Medical Institute, Department of Medicine, University of California, San Francisco 94143.
Bockenstedt
L K
LK
Weiss
A
A
D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology / Q000378:metabolism; D017252:CD3 Complex; D002118:Calcium / Q000378:metabolism; D015536:Down-Regulation; D004705:Endocytosis; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism*; D037181:Lectins, C-Type; D008213:Lymphocyte Activation; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology*; D014407:Tumor Cells, Cultured; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D002118:Calcium; D007295:Inositol Phosphates; D037181:Lectins, C-Type; D011948:Receptors, Antigen, T-Cell; D000099192:CD69 Antigens