Cereblon Controls T-lymphocyte Homeostasis And Activation

Background Emerging from its beginnings as a catastrophic teratogen,analogs derived from thalidomide,known as immunomodulatory drugs?IMiDs?,are now rapidly emerging for the treatment of cancer.One of the hallmarks of IMiD therapy is its association with T-lymphocytes activation.One of the anti-cancer mechanisms posited for this drug class is an emergent immunological response against endogenous tumor-associated antigens that restores functional immunosurveillance against developing tumor cells.Numerous barriers impede anti-tumor immunity with complexity that has largely evolved to keep autoimmune recognition in check.Using a zebrafish model,thalidomide was shown to suppress an E3-ubiquitin ligase substrate receptor known as cereblon,which subsequently results in abnormal limb development.Cereblon is conserved across all vertebrate species,but has no known involvement in immune regulation.The regulation of T cell and cereblon is still unknown.Our previous study is consistent with the work from other lab,which shows the Thalidomide could specifically inhibit the cereblon expression and Thalidomide may regulate the T cell immune function in cancer microenvironment.Objective Here,we demonstrate cereblon's physiological role in T-cell signaling and homeostasis using homozygous crbn germline knockouts mice.Contents The functional role of Thalidomide on T cell regulation has been widely studied.But the role of lenalidomide on immune regulation remains unknown.So we studied the lenalidomide first on T cells from human and mouse first.Using the cereblon knock-out mice,we observed the phenomenon of mouse after the knock-out,which including the limbs,the spleen,the cell numbers and the different expression of cereblon on different hematopoitic organs.We also studied how the immune cell distributed and the T cell proliferaton and cytokine production after the cereblon knock-out.We set up the graft versus host disease?GVHD?model to study the allo-antigen response of T cell after the cereblon knock-out and the signaling pathway of T cell.We also set up the T cell homeostasis model to study the functional role of cereblon on T cell apoptosis and the signaling pathway.Finally we set up the tumor model to study the anti-cancer ability of cereblon.Methods We use the qRT-PCR analyse the expression of cereblon mRNA;the flow cytometry to analyse the different distribution of T cells and the apoptosis ability of T cells;Cell Trace Violet to test the T cell proliferation;ELISA to test the different cytokine production of Th1?Th2?Th17 cytokines;Western-blot to test the different signaling on T cell activation and apoptosis;Fluo-4 method to test the calcium signaling in WT and CRBN^-/-T cells.To set up GVHD model,the T cell homeostasis model and the ouse tumor model to further study the different function of cereblon.Results We confirmed that the knockout ablates cerblon expression in tissues of hematopoietic origin.Peripheral blood and splenic lymphocytes with a na?ve phenotype are increased in number in deficient mice compared to wild-type C57BL6littermates with age.To explore the intrinsic threshold for activation of CRBN^-/-T-cells,in vitro studies and adoptive cell transfers were conducted into fully mismatched allogeneic host and sub-lethally-irradiated congenic mice.These studies show cereblon's role in regulating IL-2 production,apoptosis-associated protein expression and proliferation in response to T-cell receptor ligation and homeostatic expansion.For further study,we set up the GVHD model and the tumor model to learn the function of the cereblon on T cell regulation.Finally we showed the ablation of cereblon could enhance the anti-cacner ability in mouse model.Our result showed the cereblon gene could negatively regulate the T cell function.Conclusions Cereblon is a negative regulator of T cell function and homeostasis.Cereblon regulates T cells by inhibiting its proliferation and inducing apoptosis.The reason of the higher count of the CRBN^-/-lymphocytes is the elongation of the survival and the most important reason is the anti-apoptosis function of the CRBN^-/-T cell.Our study sets the stage for future development of cereblon-specific targeted therapy for potentiation of immunotherapy in malignant diseases and establishes a novel mode of sig naling regulation in T-cells.