CYCLIC-AMP SENSITIVE SIGNALING BY THE CD28 MARKER REQUIRES CONCOMITANT STIMULATION BY THE T-CELL ANTIGEN RECEPTOR (TCR CD3) COMPLEX/

We have previously demonstrated that activation of cAMP-dependent prot ein kinase (cAK) type I (cAKI, RI alpha(2)-C beta(2)) mediates the inh ibitory effects of cAMP on T-cell replication induced through the TCR/ CD3 complex. In the present study we have investigated the effect of c AMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa pro tein (pp100) and IL2 mRNA expression, induced through stimulation of t he TCR/CD3- and/or the CD28 molecules. Our results demonstrate that ty rosine phosphorylation of pp 100 stimulated by anti-CD3 is inhibited b y cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replicatio n. Combined stimulation with anti-CD3 and anti-CD28, which gives a syn ergistic response in T-cell replication, gave pp100 phosphorylation an d IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anti-CD3 and anti-CD28, the inhibitory effec t of cAMP on both T-cell replication and pp100 phosphorylation was com pletely abolished. The fact that pp 100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling f rom these receptors. In addition, our results are compatible with a mo del where cAMP, through activation of cAKI, eliminates both the PTK an d PKC activating capability of the T-cell receptor at a site(s) proxim al to PKC activation. Furthermore, the CD28 molecule which activates P TKs, enters the PTK cascade at a point distal to the target(s) for cAK I action. Therefore, during CD28 signalling PKC activation can be achi eved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP).