Evidence for a new HLA determinant that causes T cell activation without costimulation.

We describe the requirements for T cell activation by a mAb termed 4-10 that recognizes a novel determinant on MHC class I molecules. The determinant recognized by mAb 4-10 appears on resting T cells of all individuals tested (n greater than 30). Unlike previously described anti-class I mAb, 4-10 was shown to be directly mitogenic for T cells obtained from more than 20 normal donors. In order for 4-10 to exert its mitogenic effect on purified T cell populations it must be immobilized on a solid support. Immobilization of 4-10 can be circumvented if low numbers of adherent cells are added to the T cell cultures. mAb 4-10 preferentially activates the CD8+ T cell subset as judged by the fact that CD8+ T cells preferentially down-regulated their TCR after 4-10 activation and because CD4+ T cell activation with 4-10 requires approximately five times the concentration of mAb needed to reach comparable levels of activation observed with CD8+ T cells. We further observed that simultaneous cross-linking of class I and CD8 Ag by using 4-10 and anti-CD8 mAb almost completely abrogated the proliferative response of T cells when anti-CD8 was presented in immobilized form. In contrast, similar cross-linking with 4-10 and anti-CD4 diminished the response by about 20%. We also found that other anti-class I mAb were able to synergize in the activation of T cells with mAb 4-10 in a dose-dependent manner.

Effects of anagrelide on platelet cAMP levels, cAMP-dependent protein kinase and thrombin-induced Ca++ fluxes.

Anagrelide (BL-4162A, 6,7-dichloro-1,5-dihydroimidazo[2, 1-6] quinazolin-2[3H]one monohydrochloride hydrate) is a potent and broad spectrum inhibitor of platelet aggregation. Prior studies showed that anagrelide inhibited platelet cyclic AMP (cAMP) phosphodiesterase activity but did not appreciably elevate platelet cAMP levels. We examined the effects of anagrelide on washed human platelets and found that anagrelide caused significant elevation of cAMP levels. Anagrelide treatment also resulted in activation of the platelet cAMP-dependent protein kinase at anagrelide concentrations of 0.1 to 1 microgram/ml, which inhibited platelet aggregation but caused only small increases in platelet cAMP content. When whole platelets were incubated with radiolabeled phosphate, anagrelide increased phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. The pattern of protein phosphorylation stimulated by anagrelide treatment was similar to that observed when the platelets were treated with forskolin. Anagrelide also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets. The inhibition of increased intracellular Ca++ resulted from block of thrombin-induced mobilization of intracellular Ca++, as well as prevention of Ca++ influx through the plasma membrane. Anagrelide itself had no influence on inositol 1,4,5-trisphosphate-induced Caz5++ release from isolated platelet membrane vesicles. These studies suggest that anagrelide inhibits platelet phosphodiesterase activity in intact platelets resulting in an elevation in cAMP levels sufficient to activate the cAMP-dependent protein kinase and inhibit agonist-activated Ca++ fluxes.