E. Dugast¹, L. Docherty², E. Kiss-toth², S. Pettré¹, R. Danger³, J. Soulillou¹, S. Brouard¹, J. Ashton-chess^4
1U643, INSERM, Nantes cedex 1/FRANCE, ²Department Of Cardiovascular Sciences, University of Sheffield, Royal Hallamshire Hospital, sheffield/UNITED KINGDOM, ³U643, Inserm, Nantes cedex 1/FRANCE, ⁴, TcLand Expression, nantes/FRANCE

Body: Introduction We previously identified Tribbles-1 as a blood and graft biomarker of chronic antibody-mediated rejection in kidney transplant recipients. Although Tribbles-1 is known to regulate MAPK signaling by interacting with certain MAPKKs, the functional role of Tribbles-1 in the immune system is still largely unknown. We previously screened different immune compartments and found Tribbles-1 to be principally expressed by peripheral blood cells. Within the myeloid lineage, Tribbles-1 was highly expressed by activated monocytes and dendritic cells, and within lymphocytes it was expressed at highest levels by resting B cells and CD4^posCD25^highCD127^low regulatory T cells. We aimed to explore the potential role of Tribbles-1 in regulatory T cells and to analyze any relationship it may have with the Treg master gene Foxp3. Methods We isolated CD4^posCD25^highCD127^low regulatory T cells and CD4+CD25- T cells by CD4 positive enrichment followed by FACS cells sorting and measured Tribbles-1 and Foxp3 mRNA by quantitative PCR. We also analyzed the regulation of Tribbles-1 and Foxp3 mRNA in regulatory T cells following 24 hours activation with IL-2 and anti-CD3 and anti-CD28 antibodies. Next, physical interaction between Tribbles-1 and Foxp3 proteins was analyzed in live cells by the Protein Complementation Assay in which HEK293 and HeLa cells were co-transfected with plasmids containing the Foxp3 and Tribbles-1 genes, each comprising a complementary fragment of GFP. Direct physical interactions between the two molecules leading to GFP fluorescence were analyzed by flow cytometry and microscopy. Finally, we studied the co-localization of Tribbles-1 and Foxp3 in regulatory T cells by fluorescent microscopy. Results Both Tribbles-1 and Foxp3 were expressed at significantly higher levels in regulatory T cells versus their CD4+CD25- counterparts (p<0.001). Moreover, within regulatory T cells, Tribbles-1 and Foxp3 mRNA levels correlated tightly (Spearman r = 1.0; p < 0.001). On the other hand, the regulation of Tribbles-1 and Foxp3 expression with an activating stimulus was different as Tribbles-1 was not regulated, contrary to Foxp3 which was up-regulated. Protein Complementation Assay revealed Tribbles-1 to physically interact with Foxp3 to the same extent as it interacts with the known and previously published Tribbles-1 binding partner MEK1. This interaction took place in the nucleus and had a tendency to decrease upon deletion of the Tribbles-1 N-terminal (important for nuclear localization) but not the C-terminal. We also found a co-localization of Tribbles-1 and Foxp3 in the nucleus of primary human regulatory T cells. Conclusion Overall our results show a relationship between Tribbles-1 and FOXP3 in terms of their expression, physical interaction and localization, suggesting a role for Tribbles-1 in regulatory T cells.

Disclosure: All authors have declared no conflicts of interest.