128-P: INHIBITION OF TCR SIGNALING BY RECOMBINANT ILT3Fc PROTEIN

Aim: Since ILT3Fc induces the upregulation of BCL6, DUSP10 and SOCS1 via downregulation of miRNA in T suppressor cells, we investigated the possibility that this molecule also inhibits TCR signaling and nuclear localization of transcription factors. Methods: Signaling studies were performed using Jurkat cells stimulated with CD3/CD28 monoclonal antibodies or PMA for various periods of time in the presence or absence of ILT3Fc. Western blots were used to analyze total cell lysates or nuclear fractions, using antibodies recognizing TCR signaling proteins or TCR-activated transcription factors. Results: The Jurkat cell line is similar to activated human T lymphocytes and shows high binding of ILT3Fc (>90%), representing an ideal tool for studying signaling events downstream of the ILT3 ligand. We found that micro RNA gene promoters can be activated by anti-CD3/CD28 mAbs in Jurkat cells and are inhibited by treatment with ILT3Fc. Mutations at the AP1 binding sites in the miR-21 and miR-155 gene promoters abolished reporter gene activity. ILT3Fc consistently inhibited PMA-induced reporter activities, demonstrating that AP1 is crucial to the inhibitory effect of ILT3Fc. Nuclear fractions of cell extracts obtained from activated Jurkat cells were probed for various subunits of the AP-1 protein complexes. ILT3Fc inhibited and delayed TCR signaling events such as cFOS and FOSB synthesis and translocation into the nucleus. Conclusions: These results indicate that the inhibitory activity of ILT3Fc is mediated through its effect of TCR signaling during the very early stages of T cell activation. This finding that ILT3Fc regulates miRNA transcriptionally by targeting TCR/AP1 signaling pathways indicates that this agent raises the threshold of TCR triggering inducing anergy. This phenomenon in conjunction with the capacity of ILT3Fc to inhibit several micro RNA explains the strong immunomodulatory activity of ILT3Fc, a reliable inducer of regulatory T cells and transplantation tolerance. [Copyright &y& Elsevier]