Relationship Of A Novel Murine Thymus Regression Related Protein IH1 With Androgen Receptor & Identification And Functional Study On Regulatory γδT Cells (Foxp3~+)

The novel thymus regression related protein,IH1,was first isolated from murine thymus,with a down-regulated expression manner in aging mice.IH1 had a cytoplasmic distribution with predicted molecular weight as 28kDa.It had the highest expression level in CD4^-CD8^-(DN) thymocytes,which were followed by CD4⁺,CD4⁺CD8⁺(DP) and CD8⁺ thymocytes.The eukaryotic cell expressed IH1 protein had a larger molecular weight identified in SDS-PAGE and the following Western blot analysis.As a consequence,we constructed expression vectors of the N- and C- terminal part of IH1.We found that the modification of the C-terminal part must have resulted in the molecular weight discrepancy. In the meanwhile,the rat counterpart of IH1 had 99.2%amino acid sequence homology with the murine one.It was called small androgen receptor interacting protein.Androgen has been shown to be involved in thymus involution.The expression pattern of androgen receptor(AR) had been explored by immunohistological assay and semi-quantitative realtime polymerase chain reaction.The results indicated the expression of AR in the thymocytes,with the thomocyte subpopulation DN the highest expression level.There were also AR expressions in other subpopulations of thymocytes as well as in some thymic epithelial cells.The existence of functional AR was further confirmed by in vitro thymocytes proliferation test.Our preliminary study showed a potential interaction between IH1 and AR.As a consequence,the scientific problem to be sloved in the first part of my thesis was whether IH1 could modulate the function of AR.Therefore,we studied the relation between IH1 and AR. By employing AR-responsive reporter plasmid,it was found that the expression level of IH1 could affect the transcriptional activity of AR.Subsequently,we did pull down, co-immunoprecipitation(CoIP),intracellular co-localization,and fluorescent resonance energy transfer(FRET) assay to investigate the potential interaction of IH1 and AR. Although AR could be pulled down by prokaryotic expressed GST-IH1 fusion protein, which could be served as an evidence of in vitro interaction of IH1 and AR,the eukaryotic intracellular interaction of IH1 and AR was not proved.Besides,when testosterone was absent,IH1 and AR had the same intracellular localization;however,upon testosterone treatment,AR gradually translocated to the nuclei,while IH1 still stayed in the cytoplasma. At last,FRET was used to study the possible dynamic interaction of IH1 and AR following testosterone treatment.The results did not support any direct interaction between IH1 and AR or conformational changed,testosterone binded AR.IH1 could promote the transcriptional activity of AR.Hence,we presumed that IH1 may be an AR coregulator,in spite of the lack of direct interaction between the two of them. However,the specific mechanisms demand further research.In conclusion,the first part of the thesis discussed the correlation of IH1 and AR in the thymus.Our results provided important data for the elucidation of the mechanism of IH1 and AR functioning.Regulatory T cells(Treg) play a critical role in the homeostasis mainteinance of the immune system.The best characterized Treg is of CD4⁺CD25^high phenotype,with Foxp3 expression as its specific marker.Whereas the vast majority of T cells express the T-cell receptor(TCR) composed ofαβheterodimer,a smaller population expressesγδTCR.A lot of evidences showed thatγδT cells have immunosurveillant activities and immunoregulatory effects as well.We chose two experimental systems for the research ofγδT regulatory cells.They were the murine system and the human system.In the murine system,we found that TGF-βcould enhance Foxp3 expression in murine splenocyte- and intestinal IEL- derivedγδT cells.The TGF-βinduced murineγδT cells mediated a potent immunosuppressive effect on anti-CD3 stimulated T cell activation and proliferation.These results indicated the existence of a TCRγδ⁺Foxp3⁺ T cell subpopulation that had immunoregulatory effects.The in vivo experiment is now underway in order to validate the existing regulatory effects found in vitro.In the human system,we also found the existence of Foxp3⁺γδT cells in PBMC from healthy donors and tumor patients.Besides,bothδ1 andδ2 subsets contain Foxp3⁺ cells, suggesting that Foxp3⁺ phenotype was not restricted to a certainδchain related subset.Of those Foxp3⁺γδT cells,some could express IL-2 receptorαchain(CD25).However,no IL-7 receptorαchain(CD127) was found to be expressed on their surfaces.The proportion of Foxp3⁺ phenotype was not significantly increased following TGF-βexposure.The study on the function of human Foxp3⁺γδT cells is now in process.In conclusion,in the second part of my thesis,we had studied the phenotype and function of Foxp3⁺γδT cells.Our results suggested the existence of murineγδT regulatory cells and provided clues and basis for the futher research on humanγδT regulatory cells.