Hydrolysis of inositol phospholipids induced by stimulation of the T cell antigen receptor complex in antigen-specific, murine helper T cell clones. Requirement for exogenous calcium

Two murine, keyhole limpet hemocyanin-specific Th cell clones were studied for their ability to respond to antibody-mediated stimulation of the TCR complex or to Ag-pulsed accessory cells by hydrolyzing inositol phospholipids. Both clones were positive for the determinant expressed on the ε chain of CD3 that is recognized by the mAb, 145-2C11 (2C11 mAb); one clone also expressed the V(β)8 epitope of the α/β chains of the TCR recognized by the F23.1 mAb. Treatment of these cells with 2C11 or F23.1 mAb adsorbed onto polystyrene beads induced a time-dependent accumulation of inositol phosphates (IP). Keyhole limpet hemocyanin-pulsed accessory cells which expressed the appropriate MHC phenotype also induced IP accumulation, whereas no response was induced by medium-treated or MHC congenic accessory cells. The hydrolysis of inositol phospholipids induced by TCR perturbation depended upon the presence of exogenous Ca²⁺; Mg²⁺ did not substitute for Ca²⁺. Treatment of cells with ionomycin at concentrations up to 30 μM was unable to induce hydrolysis of inositol phospholipids, indicating that entrance of Ca²⁺ was itself insufficient to generate IP. Stimulated IP generation was rapidly blocked upon addition of EGTA to the incubation medium. Reducing the level of exogenous Ca²⁺ decreased the production of inositol mono-, bis-, and trisphosphate isomers similarly, suggesting that extracellular Ca²⁺ was required for the initiation of the hydrolysis rather than affecting phospholipase C affinity for its substrates. We concluded that activation of inositol phospholipid hydrolysis by perturbation of the TCR complex in the Th cell clones under investigation displays a Ca²⁺-dependent component which is likely to be proximal to IP generation.