The Primary Research Of The Effect Of NEDD4L On CD4~+Foxp3~+ Regulatory T Cells

Objective:Regulatory T cell?Treg cell?that express the transcription factor Foxp3is a specialized subpopulation of T cell,including thymus-derived Treg?tTreg?and peripherally derived Treg?pTreg?.The main function of Treg is to suppress the proliferation of effector T cells and the production of inflammatory factors,establish autoantigen tolerance,regulate immune response and maintain the balance of immune system.Abnormal changes in the number or function of Treg can disrupt the homeostasis of immune system and lead to autoimmune diseases or cancer ultimately.Many studies have shown that the maintenance of differentiation,development and function of Treg are closely related to the TGF-?signaling.The neural precursor cells of E3 expressed developmentally down-regulated 4-regulated 4-like?NEDD4L?proteins degrade the type I receptor and Smad2/3 in the pathway of TGF-?signaling through the ubiquitin-proteasome system?UPS?to regulate TGF-?signaling.Therefore,this study analyzed the influence of NEDD4L on the percentage and phenotype of Treg by constructing a gene model of specific NEDD4L knockout in T cell of Foxp3^GFP mouse,and explored the potential value of NEDD4L in the field of autoimmune diseases and tumor therapy.Methods:1.Establishment of the gene model of specific NEDD4L knockout in T cell of Foxp3^GFP mouse.We generated FoxP3^GFPNEDD4L^f/f/f mice by crossing FoxP3^GFPFP mice and NEDD4L^f/f/f mice,next we generated wild type(WT:Lck-Cre^-/-FoxP3^GFPFP NEDD4L^f/f)mice and knockout type(KO:Lck-Cre^+/-FoxP3^GFPFP NEDD4L^f/f)mice by crossing FoxP3^GFPFP NEDD4L^f/f/f mice and Lck-Cre^+/-mice.The single cell suspension of the spleen from FoxP3^GFPFP mice was prepared for cell surface staining and nuclear staining.The expression levels of GFP and Foxp3 were detected and compared by flow cytometry to determine the consistency of the two expression levels.WT mice and KO mice were identified by Western Blot and polymerase chain reaction.2.Effects of NEDD4L on T lymphocytes in thymus.The 9-11 weeks old mice were weighed,and their thymus,spleen and mesenteric lymph nodes were isolated.The differences in morphology and weight of WT and KO mice were observed.The single cell suspension of thymus was prepared.The compartment of T cells,the percentage of Treg,and the MFI of Foxp3 gated on Treg were analyzed by flow cytometry.3.Effects of NEDD4L deletion in T cells on peripheral Treg.The single cell suspensions of spleen,peripheral blood,and mesenteric lymph node were prepared for detecting the frequency and number of peripheral CD4⁺T cells,CD8⁺T cells,and Treg,as well as the MFI of Foxp3 gated on Treg by flow cytometry.The expression levels of CD25,CD44,CD69,CD103,GITR,PD-1 and CTLA-4 on Treg were analyzed by flow cytometry.4.Effects of NEDD4L deficiency in T cells on Treg population after T cell activation in vitro.?1?The splenocytes suspensions were prepared and cells?1×10⁶/well?were cultured at37?,5%CO₂ for 72-96h in 96-well plate in the presence of anti-CD3/anti-CD28.Cells were then stained for flow cytometry analysis of the proportion of Treg and the MFI of Foxp3 after harvest.?2?CD4⁺T cells were MACS-sorted from splenocytes suspensions.Cells?5×10⁵/well?were cultured in 96-well plate for 72-96h in the presence of anti-CD3/anti-CD28.Cells were then stained for flow cytometry analysis of the frequency of Treg and the MFI of Foxp3 after harvest.?3?Na?ve CD4⁺T cells were MACS-sorted from splenocytes suspensions.Cells?3×10⁵/well?were cultured in 96-well plate for 72-96h in the presence of anti-CD3/anti-CD28,TGF-?and IL-2.Cells were then stained for flow cytometry analysis of the frequency of Treg and the MFI of Foxp3 after harvest.Results:?1?The gene model of specific NEDD4L knockout in T cell of Foxp3^GFP mouse was successfully constructed.WT mice and KO mice were accurately identified by polymerase chain reaction.The result of flow cytometry analysis showed that the expression level of GFP was consistent with the level of Foxp3 in Foxp3^GFP mouse.The expression of NEDD4L protein from the thymus of WT and KO mice was identified by Western Blot successfully.?2?There was no significant difference in body weight between WT and KO mice.There were no significant differences in morphology,weight and total number of cells from thymus,spleen and mesenteric lymph nodes between WT and KO mice.WT and KO mice showed similar frequencies and number of T cells from each stage and Treg,as well as the MFI of Foxp3 gated on Treg in thymus.?3?Mice lacking NEDD4L in T cells showed the increased proportion of peripheral Treg,the reduced frequency of CD8⁺T lymphocytes and the decreased number of T cells in spleen and mesenteric lymph nodes,the decreased percentage of CD4⁺T and CD8⁺T lymphocytes in peripheral blood,while no significant difference in MFI of Foxp3 gated on Treg.Mice lacking NEDD4L in T cells showed the increased expression levels of activation markers such as CD44,CD69,CD103 and Treg-associated markers such as GITR,PD-1 and CTLA-4.?4?NEDD4L deletion in T cells showed the increased proportion of Treg after splenic cells or CD4⁺T cells separated from spleen by magnetic beads activated in vitro,while no significant difference in the proportion of Treg in Treg differentiation in vitro.Conclusion:NEDD4L,the E3 ubiquitin ligase in UPS,is involved in regulating the percentage and phenotype of Treg outside the thymus;NEDD4L plays an important role in maintaining the balance between Treg and other T cells outside the thymus.