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Aim To identify target cells with high levels of Immunoglobulin Transcript 3 (ILT3) ligand on their membrane in order to identify the signaling mechanism of this strongly inhibitory protein. Methods We used the U2O osteosarcoma cell line which binds ILT3.Fc on 100% of the cell population. Cells were pre-incubated for 24 to 48 h with 50 μg/ml of ILT3.Fc or left untreated. Next, the cells were washed twice with cold PBS and fixed in 80% ethanol at 4C overnight. Cells were washed once before staining with Propidium Iodide/RNase Staining Buffer (BD Pharmingen) according to the manufacturer’s protocol. Cell doublets were gated out based on pulse width versus pulse area. The data was accumulated and analyzed by FACS using Cell Quest software. Cell growth in the presence of 0, 12.5, 25, and 50 μg/ml of ILT3.Fc was monitored at 24 h interval using the trypan blue exclusion method. The wound healing scratch assay was used according to the protocol described in PMID: 17406593 to assess the effect of ILT3.Fc on cell migration. Western Blot analysis of cells treated or not treated with ILT3.Fc was performed using antibodies to various kinases. Results We have discovered that cells from the U2OS cell line bind ILT3.FITC which inhibits their growth. Cell cycle analysis using Propidium iodide staining showed that ILT3.Fc induced cell cycle arrest in the transition from G1 to S phase.Wound healing cell culture experiments demonstrated inhibition of this process up to 48 h of U2OS exposure to ILR3.Fc, confirming morphological observation on apoptotic changes of the cells. Western blot analysis of PI3K/AKT and MAPK/ERK showed that ILT3.Fc induced dephosphorylation of these proteins inhibiting their signaling pathway. Conclusions Binding of ILT3.Fc to its ligand on the membrane of malignant cells blocks cell cycle progression and inhibits cell migration. These studies suggest that ILT3.Fc may serve as a potent inhibitor of tumor cell growth for cancer therapy. This opens new perspectives to the increasing immunotherapy field of check point blockade through ligand or receptor inhibition. [ABSTRACT FROM AUTHOR]

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8+ Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation. [ABSTRACT FROM AUTHOR]

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]