The Study On Immune Function Of Ptd-Foxp3

Natural regulatory T cells (nTregs) suppress pathological and physiological immune responses, contributing to the maintenance of immunological self-tolerance and immune homeostasis. Foxp3, highly expressed in nTregs, is required for nTreg development and be necessary for mature Treg function as an essential transcription factor. Foxp3can interact with key transcription factors of other T cells such as Th1、Th17and has a close correlation with their differentiation, involving in the pathological processes of clinical immune diseases.HIV trans-activating transcriptional activator(Tat) has the ability to cross the plasma membrane and give rise to the intracellular delivery of therapeutic cargos without cytotoxicity. Tat derived peptide has been utilized in basic research and is an important drug carrier, providing a new tool for life science research.Objective:To study the biological function of PTD-Foxp3fusion protein and observe the effect of fusion protein on the differentiation of Tregs, Th1and Th17and the treatment of immune related diseases, which provides theoretical and experimental basis for ultimate application in the treatment of immune related diseases such as clinical organ transplantation and autoimmune diseases.Methods: (1) Human lymphoma cell line Jurkat, PBMCs and CD4+CD25-T cells, isolated from human peripheral blood, were treated with PTD-hFOXP3fusion protein. The proliferative capacity of fusion protein transduced CD4+CD25-T cells and the suppression of fusion protein transduced CD4+CD25T cells to activated CD4+CD25-T cells were analyzed by Cell Counting Kit-8(CCK-8); The mRNA levels of IL-2、INF-γ、IL-10、IL-4、 TGF-β and CTLA-4were analysed by realtime RT-PCR; enzyme-linked immunosorbent assay (ELISA) was used to detect the cytokine levels of IL-2, INF-y, IL-10and TGF-(3in the cell culture supernatant and the protein level of CTLA-4was detected by FCM; At the same time, the inhibition of PTD-hFOXP3on the two way mixed lymphocyte reaction was administrated by CCK-8; The binding ability of PTD-hFOXP3fusion protein to IL-2promoter was determined by chromatin immunoprecipitation (ChIP) assay for studying the mechanisms;(2) The effect of PTD-mFoxp3fusion protein on the differentiation of Th1and Th17in vitro was observed. Isolated CD4+T cells were induced into Th1or Th17with the administration of cytokines, then were treated by PTD-mFoxp3; The effect of PTD-mFoxp3fusion protein on the mRNA levels of INF-γ、T-bet、IL-17A and RORyt was detected by realtime RT-PCR; And FCM was used to detect the protein level change of INF-y and IL-17A; These assays could be used to evaluate the impact of PTD-mFoxp3on the two T cell subsets differentiation;(3) Delayed type hypersensitivity (DTH) modle mice, induced by ovalbumin (OVA) were treated by a subcutaneous injection of PTD-mFoxp3fusion protein; Local inflammatory responses were examined by ear swelling and histological analysis; The expression of INF-y by local ear tissues and ear draining lymph nodes cells was measured; The response ability of splenocytes to OVA was determined; The treatment effect of PTD-mFoxp3fusion protein on Th1mediated DTH was evaluated by these assays.Results:(1) PTD-hFOXP3-transduced CD44CD25T cells failed to proliferate and inhibited the proliferation of CD3/CD28antibodies activated CD4+CD25T cells; The results of RT-PCR、ELISA and FCM showed that PTD-hFOXP3-transduced CD4+CD25-T cells failed to produce IL-2and IFN-y, but produced large amounts of the cytokines IL-4, IL-10and TGF-β,in response to TCR stimulation in vitro, as well as high levels of CTLA-4; Meanwhile, PTD-hFOXP3could inhibited the MLR; ChIP assays demonstrated that PTD-hFOXP3can bind with the IL-2gene promoter and repress the expression of IL-2;(2) The recombinant cytokines microenvironment during CD4+T cells activation by CD3/CD28antibodies determined T cells differentiation through selective signal transducer and activator of transcription proteins to Th1or TH17in vitro; Under the Th1polarization conditions, the PTD-mFoxp3fusion proteins treatment downregulated the mRNA levels of IFN-y and T-bet, and the FCM results showed that the ratio of IFN-y+CD4+T cells also declined; Similarly, under the Th17polarization conditions, the production of IL-17A and RORyt on mRNA level and the protein level of IL-17A was downregulated by PTD-mFoxp3;(3) Administration of PTD-mFoxp3fusion protein by a subcutaneous injection significantly attenuated inflammatory reactions associated with DTH, including the ear swelling, the infiltration of inflammatory cells, and the IFN-y expression in the inflammatory site; PTD-mFoxp3also had a suppressive effect on ovalbumin-stimulated production of IFN-y by ear draining lymphocytes and splenocytes and proliferation activity of splenocytes.Conclusion:The PTD-hFOXP3fusion protein has the ability to convert CD4+CD25T cells to Treg like cells; PTD-mFoxp3inhibits the differentiation of Th1and Th17in vitro, and inhibited the development of delayed type hypersensitivity in DTH mice effectively; PTD-hFOXP3/mFoxp3affects the differentiation of T cells subsets and paves the way for the FOXP3application in clinical diseases treatment such as transplantation rejection、cancer、inflammation and autoimmune diseases.