Maria Grazia Cifone*, Luisa Cironi*, Angela Santoni# and Roberto Testi $#

*Department of Experimental Medicine, University of L'Aquila, #Department of Experimental Medicine, University of Rome "La Sapienza" and $Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata"

Corresponding author: Roberto Testi

Arachidonic acid (AA) release was observed following TCR/CD3 complex crosslinking in different tumor T cell lines as well as on purified peripheral T cells, in vivo. Direct in vitro measurement of enzymatic activity of TCR/CD3 stimulated Jurkat cell extracts on labeled vesicle substrates showed that TCR/CD3 crosslinking resulted in AA release from sn-1,2-diacylglycerol (DAG) vesicles, as detected by TLC analysis, suggesting DAG lipase activation following TCR/CD3 stimulation and DAG generation. On the contrary, no phospholipase A2 activation was observed in response to TCR/CD3 stimulation, since no lyso-phospholipids were generated in vitro from phosphatidylcholine or phosphatidylinositol (3,4) bisphosphate, as well as from phosphatidic acid vesicles. Moreover, 1-DAG lipase inhibitor RHC80267 completely blocked in vitro and in vivo TCR/CD3-dependent AA release, while not affecting TCR/CD3-dependent inositol (1,4,5) trisphosphate (IP3) generation. Importantly, evidence for further metabolism of released AA was obtained, since synthesis and release of cysteinyl leukotrienes (CLT), but not of leukotriene B4 or cycloxygenase products, could be detected by RIA in different T cell lines and peripheral blood T cells following TCR/CD3 crosslinking. Also, HPLC analysis revealed accumulation of leukotriene E4 in TCR/CD3 stimulated Jurkat cells. This was associated with translocation of 5-lipoxygenase from cytosol to the cell membranes. Finally, TCR/CD3-mediated CLT production was blocked by MK886, a specific inhibitor of 5-LO translocation and activation. Our data help define a further level in the fate of second messengers generated after TCR/CD3 triggering and suggests that additional mediators can play a role in the context of T cell activation.