| It is now evident that CD4+CD25+Foxp3+ regulatory T cells(Treg) represent a distinct lineage of T lymphocytes that plays a central role in controlling immune responses,and in promoting and maintaining self tolerance.In addition,Treg cells are.implicated in various immune evasion mechanisms used by cancers.Several studies have reported increased numbers of Treg cells in tumor tissue and/or peripheral blood of patients with various cancers,including breast,ovarian,gastric and lung cancers,lymphoma and melanoma associated with poor prognosis and survival of patients with ovarian cancer.Direct evident for the dominant role of Treg cells in immune evasion mechanisms has recently been provided in various experimental tumor models,where physical depletion of these cells or modulation of their function was the cause of therapeutic efficacy.Tumors employ an intricate set of direct and indirect active mechanisms to evade the immune system.These mechanisms include the loss or alterations of the antigen-presenting pathway,alterations in death receptor signaling,and production of an immunosuppressive cytokine milieu.Importantly,tumor cells can either directly recruit Treg cells into the tumor and/or induce immature myeloid DCs to secret TGF-βand/or IL-10 for conversion of CD4+CD25-na(?)ve T-cells into Treg cells within the tumor microenvironment. Tumor-induced expansion of Treg cells is an obstacle to successful cancer immunotherapy. Because of their dominant role in tumor immune evasion mechanisms,Treg cells have become the target of intense studies for therapeutic purpose.Disrupting Treg suppression in combination with cancer vaccines presents an important therapeutic approach with a great likelihood of success in the clinic.At present,various strategies were brought about for targeting Treg cells,mainly including two categories:physical depletion of Treg cells using anti-CD25 mAbs and IL-2 toxins,and inhibiting Treg cell suppressive function and reducing effector T cell susceptibility to suppression by targeting cell-surface suppressive molecules on Treg cells.However,the potential benefit of Treg cells depletion through the interleukin- 2 receptor is lost by the concurrent elimination of activated effector lymphocytes and possibly by the de novo induction of Treg cells replenishment.In theory, the functional inactivation of Treg cells will maintain them at high numbers in tumors and avoid their replenishment from the peripheral lymphocyte pool,which has the capacity to further suppress the effector lymphocyte anti-tumor response.Foxp3 represents a lineage-specific marker for Treg,and its detection is widely used to identify these cells in vivo.The importance of Foxp3 in the development and function of Treg is demonstrated by experiments of nature in which spontaneous mutations of the Foxp3 gene cause severe X-linked autoimmune lymphoproliferative disorders.The scurfy mutation in mice,which is associated with a null mutation of the Foxp3 gene,results in complete loss of Treg,autoimmunity,and premature death.Similarly,mutations of FOXP3 in humans are responsible for a disease-called immuno dysfunction polyendocrinopathy enteropathy X-linked syndrome(IPEX)-in which the impairment of Treg function is associated with several autoimmune disorders such as enteropathy and type 1 diabetes. That Foxp3 is not only a marker of murine Treg but that it is also necessary for their function is a broadly accepted concept.In theory,silencing Foxp3 expression in Treg cells could lead to functional reversion of Treg cells.Owing to the intracellular localization of Foxp3,however,it is not readily accessible using traditional methods of in vivo targeting.The recent emergence of RNAi offers a novel opportunity for post-transcriptional suppression of foxp3 production in vivo. Several traits of RNAi technologies,in their current state,bode well for the potential applicability of this tool toward in vivo suppression of Treg cells activity.First,the transient nature of knockdowns mediated by pre-synthesized small interfering RNAs, would be advantageous in a clinical setting,allowing for the suppression of regulatory cells, only as long as therapeutically necessary.Second,the greatest advantage of this technique lies in its ability to repress regulatory pathways without depleting effector lymphocytes. However,the main obstacle to developing siRNA for Foxp3 silence is delivering it in vivo to Treg cells.Although transfection can deliver siRNAs locally,a systemic method to deliver siRNA to specific cells via cell-surface receptors would provide a means to introduce siRNA into desired cells to achieve maximal therapeutic benefit,decrease the amount of drug required and avoid nonspecific silencing and toxicity in bystander cells. Recently,song et al designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells.The fusion protein was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody,siRNA bound to fusion protein induced silencing only in cells expressing HIV-1 envelope.In this paper,we designed a mouse CD3-specific single-chain antibody(scFv) fragment-protamine fusion protein(2C11scκtp)for delivering Foxp3-specific siRNA to Treg cells.It was demonstrated that 2C11scκtp could specifically deliver siRNA to CD3+T lymphoma cells and mouse primary T cells in vivo and in vitro.Moreover,Foxp3-specific siRNA delivered by 2C11scκtp efficiently silenced Foxp3 expression in Treg cells. Importantly,Treg cells with down-regulated Foxp3 expression proliferated and secreted Th1-biased cytokines in response to TCR stimulation,which could not more inhibit na(?)ve T cell proliferation.Finally,siRNA delivered by 2C11scκtp do not trigger potentially toxic interferon responses.Part One:Cloning and construction of 2C11sCκand 2C11sCκtp fusion gene1.Cloning of 2C11sCκand 2C11sCκtp fusion gene.Antibody variable segments VH and VL were directly amplified from total mRNA of 2C11-145 hybridoma cells producing hamster anti-mouse CD3 antibody by one-step RT-PCR method.The sequences of VH and VL were verified by amplicon sequceing.After that,overlapping PCR was performed to generate 2C11 scFv.The 2C11 scFv was fused to human Cκchain and/or protamine gene to form 2C11sCκand 2C11sCκtp by second overlapping PCR.The PCR products of 2C11sCκand 2C11sCκtp were cloned into pGEM-Teasy vector to generate pGEM-2C11sCκand pGEM-2C11sCκp,and sequence verified.2.Construction of expression vector containing 2C11sCκand 2C11sCκtp gene. 2C11sCκand 2C11sCκtp gene removed from pGEM-2C11sCκand pGEM-2C11sCκtp were doublely digested by Hindâ…¢and EcoRâ… endonucleases and cloned into pcDNA3.1(+) expression vector previously digested with the same enzyme to creat pcDNA3.1-2C11sCκand pcDNA3.1-2C11sCκtp,and sequence verified.Part Two:Expression and purification of 2C11sCκand 2C11sCκtp fusion protein1.Expression of 2C11sCκand 2C11sCκtp fusion protein.The pcDNA3.1-2C11sCκand pcDNA3.1-2C11sCκtp plasmids were transfected into CHO cells by the use of Lipofactamine 2000 according to the manufacturer's protocol.After 72 hours,the supernatants were collected and assayed for fusion expression by flow cytometry.After one round screening,we identified two CHO cell clones which highly expressed 2C11sCκand 2C11sCκtp protein respectively.2.Purification of 2C11sCκand 2C11sCκtp protein.Supernatants were collected, and 2C11sCκand 2C11sCκtp protein were purified by Ni++ chromatography.SDS-PAGE and western-blotting assays confirmed that purified 2C11sCκand 2C11sCκtp proteins were homogeneous,of which molecular mass were right.In vitro bind assays showed that 2C11sCκand 2C11sCκtp proteins were capable of effectively binding to CD3 molecule on lymphoma cell line YAC-1 or freshly isolated mouse lymph node cells.Part Three:Identification of potent and specific siRNAs for mouse Foxp3 gene1.Construction of cell lines stably expressing mouse Foxp3 gene.Because mouse T cells can not be easily transfected by conventional transfection methods such as liposome transfection,we need to construct a cell line stably expressing mouse Foxp3 gene and easily transfected.HELA cells were infected with MIGR1-MFoxp3 retrovirus, which expressed mouse Foxp3 and GFP gene.As a control,Hela cells were infected with MIGR1 retrovirus,which only expressed GFP gene.As shown in semi-quantitative PCR, real-time PCR and western-blotting assays,MIGR1-MFoxp3 retrovirus-infected HELA cells(HELA-MF cell) expressed high level of mouse Foxp3 mRNA and protein, compared with MIGR1 retrovirus-infected HELA cells(HELA-MR cell).Thus, HELA-MF cells can act as an effective platform for screening mouse Foxp3 gene-specific siRNA.2.Identification of potent and specific siRNAs for mouse Foxp3 gene.We synthesized 3 siRNA from QIAGEN Corporation.After introduced into HELA-MF cells, siRNA A was shown to most potently and specifically silence mouse Foxp3 expression at the same concentration as siRNA B and siRNA C.Part Four:Delivering siRNA by 2C11sCκtp protein1.2C11sCκtp protein specifically delivered FAM-labeled siRNA to CD3+T lymphoma cells in vitro,and mouse T lymphocytes in vivo or in vitro.The results showed that 2C11sCκp protein could bind to mouse CD3 molecule and FAM-labeled siRNA,and specifically delivered siRNA to CD3+ YAC-1 and EL-4 lymphoma cell or freshly isolated mouse lymph node T cells,but not human CD3-expressing Jurkat lymphoma cells in vitro.Also,when complexes of 2C1sCκtp protein and FAM-labeled siRNA injected to C57BL/6 mice,FAM fluorescence could be easily detected in CD3+ T cells,but not CD14+ mononuclear cells,CD19+ B cells and CD11c+ DC by flow cytometry.2.Foxp3-specific siRNA delivered by 2C11sCκtp protein silenced Foxp3 expression in TGF-β-induced or naturally occurring Treg cells.Real-time PCR and Foxp3 staining by Foxp3-specific antibody showed that Foxp3-specific siRNA A delivered by 2C11sCκtp protein,but not 2C11sCκprotein significantly inhibited Foxp3 expression in TGF-β-induced or naturally occurring Treg cells at mRNA and protein level in vitro.Part Five:Phenotype and function alteration of Foxp3-silenced Treg cells1.Phenotype of Foxp3-silenced Treg cells.The results showed that Treg cells with low Foxp3 expression down-regulated CD25,CLTA4 and GITR expression and up-regulated IL-2,IL-7Rαand PDE3B expression,which genes were modulated by Foxp3 positively or negatively respectively.1.Function of Foxp3-silenced Treg cells.When stimulated by plate-coated anti-CD3 and CD28 mAbs,Foxp3-silenced Treg cells vigorously proliferated and secreted large quantities of IL-2,IFN-γand TNF-αcytokines,indicating biased toward Th1 immune response.Part Six:Interferon responses associated with application of siRNAThe in vitro and in vivo assays demonstrated that siRNA delivered by 2C11sCκtp protein did not trigger IFN-αand inflammatory cytokine TNF-αproduction,indicating no induction of nonspecific immune responses.Conclusion2C11sCκtp fusion protein with mouse CD3 targeting capacity and nucleic acid-binding activity can effective deliver Foxp3-specific siRNA to mouse CD3+ T cells, which resulted in efficient Foxp3 gene silence.Treg cells with down-regulated Foxp3 expression were reversed phenotypically and functionally,which have a tendency toward Th1 fate.In summary,our study may provide a promising approach for inhibiting Treg cells function and improving tumor vaccine efficacy. |
PDF Full Text Request