Apoptosis of antigen-specific T lymphocytes upon the engagement of CD8 by soluble HLA class I molecules is Fas ligand/Fas mediated: Evidence for the involvement of p56lck, calcium calmodulin kinase II, and calcium-independent protein kinase C signaling pathways and for NF-κB and NF-AT nuclear translocation

The binding of soluble HLA class I (sHLA-I) molecules to CD8 on EBV-specific CTL induced up-regulation of Fas ligand (FasL) mRNA and consequent sFasL protein secretion. This, in turn, triggered CTL apoptosis by FasL/Fas interaction. Molecular analysis of the biochemical pathways responsible for FasL up-regulation showed that sHLA-I/CD8 interaction firstly induced the recruitment of src-like p56^lck and syk-like Zap-70 protein tyrosine kinases (PTK). Interestingly, p59^fyn was activated upon the engagement of CD3/TCR complex but not upon the interaction of sHLA-I with CD8. In addition, sHLA-I/CD8 interaction, which is different from signaling through the CD3/TCR complex, did not induce nuclear translocation of AP-1 protein complex. These findings suggest that CD8^- and CD3/TCR-mediated activating stimuli can recruit different PTK and transcription factors. Indeed, the engagement of CD8 by sHLA-I led to the activation of Ca²⁺ calmodulin kinase II pathway, which eventually was responsible for the NF-AT nuclear translocation. In addition, we found that the ligation of sHLA-I to CD8 recruited protein kinase C, leading to NF-κB activation. Both NF-AT and NF-κB were responsible for the induction of FasL mRNA and consequent CTL apoptosis. Moreover, FasL up-regulation and CTL apoptotic death were down-regulated by pharmacological specific inhibitors of Ca²⁺/ calmodulin/calcineurin and Ca²⁺-independent protein kinase C signaling pathways. These findings clarify the intracellular signaling pathways triggering FasL up-regulation and apoptosis in CTL upon sHLA-I/CD8 ligation and suggest that sHLA-I molecules can be proposed as therapeutic tools to modulate immune responses.