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1.Construction, Cloning, and Expression of a Human-mouse Hybrid HLA-A2Dimer for Donor-specific Tolerance Induction2.Comparing the Phenotypic Consequences of Singular and Concurrent Application of IL-21and Rapamycin on Antigen Specific T Cells in Vitro

Author: Victor Tunje Jeza
Tutor: WuXiongWen
School: Huazhong University of Science and Technology
Course: Immunology
Keywords: HLA-A2dimer Suppression Rejection Donor-specific and AlloresponseIl-21 Rapamycin concurrent application and cancer immunotherapies
CLC: R617
Type: PhD thesis
Year: 2013
Downloads: 5
Quote: 0
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Organ transplantation offers hope to a variety of patients with terminal functioning organs andtissues. However, rejection of transplants has proved to be a formidable challenge. Inducingtolerance to allografts is currently the holy grail of every transplantation immunologist. Currentimmune suppressive drugs aimed at maintaining tolerance to allografts have a major drawback ofrendering the patient susceptible to infections and other side effects like malignancy and drugrelated toxicities with an overall rejection of the organ at some point. It has been shown thatMHC-Ig dimers can induce suppression of alloresponsive T cells in a donor specific manner invitro. Therefore, we set out to answer the question as whether these dimers will surmountrejection in vivo. To do this, we began by constructing, cloning, and expressing a human-mousehybrid HLA-A2dimer. We employed overlap-PCR to join parts of two different already clonedplasmids to form the full length HLA-A2β2α1α2murineα3insert which was then cloned topcDNA3.1to form pcDNA3.1HLA-A2β2α1α2murineα3. The IgG2bFc region was added byrestriction digestion and ligation to form the plasmid pcDNA3.1HLA-A2β2α1α2murineα3IgG2bFc which was then transfected by electroporation to J558L cells.Screening was done using G418antibiotic for four weeks. Thereafter, ELISA was performedwhich confirmed that the transfection was successful. The dimer was employed in one way MLCexperiments to determine its ability to block direct alloresponses in a donor-specific fashion incongenic mice. In our model, the donor mice are transgenic for HLA-A2while the recipient miceare transgenic for HLA-A24. Our MLC results showed that the proliferation of CD4+T cellsreduced in the presence of the dimer. This was unexpected since we were interested in CD8+Tcells of which we were unable to detect their proliferation status. This work is crucial forsubsequent research aiming to answer the aforementioned question. Immunotherapies based on the adoptive transfer of naturally occurring or genetically redirectedtumor-reactive T cells represent the best evidence of the therapeutic power of T cells. Thishowever requires a great deal of persistence on the part of the adoptively transferred T cells inorder to eliminate tumors. It is therefore inevitable to come up with ways to enhance thepersistence of tumor reactive memory T cells if we are to combat cancer. Here, we assessed theability, either singularly or concurrently, of the biological and pharmacological agents Il-21andrapamycin, respectively, to arrest the differentiation of antigen specific activated T cells so as tosparingly reach the effector cell stage and largely retain as well as expand the Tscm subset. Inthis way, the Tscm subset would provide a continuous reservoir for generating peptide specifictumor-reactive T cells that would eliminate a given tumor. We chose to work with Il-21andrapamycin since it has been suggested that these two have the ability to arrest T celldifferentiation. Further, although there are other biological and pharmacological agents that havebeen suggested to be endowed with the capability to arrest T cell differentiation, we could accessthese two (i.e. rapamycin and Il-21) with much ease. In addition, no one has ever compared thephenotypic consequences of singular and concurrent application of Il-21and rapamycin before.We set out by obtaining PBLs from healthy donors which were then co-cultured with T2/AFPcells so as to generate AFP peptide specific tumor-reactive T cells. Controls, Il-21and/orrapamycin were applied in samples in24well plates. Samples were harvested on weeks one, two,four, and six and stained with anti-human CD3, CD8, CD44, and CD62L followed by flowcytometry for analysis. After six weeks of co-culture and analysis, we found that rapamycin hadrelatively higher Tscm subset cells compared to Il-21alone or rapamycin and Il-21together.However, the second week of co-culture proved to be the threshold time for expansion of theTscm cells in the presence of Il-21and rapamycin for it is at this period that the concurrentapplication of these two agents exerted a tremendous expansion of the Tscm subset than anyother experimental group at the same or any other given time. These results underscore theimportance of augmenting immunotherapies by concurrent application of Il-21and rapamycin. Inaddition, our results provide immense knowledge in the search for effective immunotherapystrategies to fight cancer.

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