PartⅠ:Construction, Cloning, And Expression Of A Human-Mouse Hybrid HLA-A2Dimer For Donor-Specific Tolerance Induction Part Ⅱ:Comparing The Phenotypic Consequences Of Singular And Concurrent Application Of IL-21and Rapamycin On Antigen Specific T Cell

Organ transplantation offers hope to a variety of patients with terminal functioning organs and tissues. However, rejection of transplants has proved to be a formidable challenge. Inducing tolerance to allografts is currently the holy grail of every transplantation immunologist. Current immune suppressive drugs aimed at maintaining tolerance to allografts have a major drawback of rendering the patient susceptible to infections and other side effects like malignancy and drug related toxicities with an overall rejection of the organ at some point. It has been shown that MHC-Ig dimers can induce suppression of alloresponsive T cells in a donor specific manner in vitro. Therefore, we set out to answer the question as whether these dimers will surmount rejection in vivo. To do this, we began by constructing, cloning, and expressing a human-mouse hybrid HLA-A2dimer. We employed overlap-PCR to join parts of two different already cloned plasmids to form the full length HLA-A2β2α1α2murinea3insert which was then cloned to pcDNA3.1to form pcDNA3.1HLA-A2β2α1α2murinea3. The IgG2bFc region was added by restriction digestion and ligation to form the plasmid pcDNA3.1HLA-A2β2α1α2murinea3IgG2bFc which was then transfected by electroporation to J558L cells. Screening was done using G418antibiotic for four weeks. Thereafter, ELISA was performed which confirmed that the transfection was successful. The dimer was employed in one way MLC experiments to determine its ability to block direct alloresponses in a donor-specific fashion in congenic mice. In our model, the donor mice are transgenic for HLA-A2while the recipient mice are transgenic for HLA-A24. Our MLC results showed that the proliferation of CD4+T cells reduced in the presence of the dimer. This was unexpected since we were interested in CD8+T cells of which we were unable to detect their proliferation status. This work is crucial for subsequent research aiming to answer the aforementioned question. Immunotherapies based on the adoptive transfer of naturally occurring or genetically redirected tumor-reactive T cells represent the best evidence of the therapeutic power of T cells. This however requires a great deal of persistence on the part of the adoptively transferred T cells in order to eliminate tumors. It is therefore inevitable to come up with ways to enhance the persistence of tumor reactive memory T cells if we are to combat cancer. Here, we assessed the ability, either singularly or concurrently, of the biological and pharmacological agents I1-21and rapamycin, respectively, to arrest the differentiation of antigen specific activated T cells so as to sparingly reach the effector cell stage and largely retain as well as expand the Tscm subset. In this way, the Tscm subset would provide a continuous reservoir for generating peptide specific tumor-reactive T cells that would eliminate a given tumor. We chose to work with I1-21and rapamycin since it has been suggested that these two have the ability to arrest T cell differentiation. Further, although there are other biological and pharmacological agents that have been suggested to be endowed with the capability to arrest T cell differentiation, we could access these two (i.e. rapamycin and I1-21) with much ease. In addition, no one has ever compared the phenotypic consequences of singular and concurrent application of I1-21and rapamycin before. We set out by obtaining PBLs from healthy donors which were then co-cultured with T2/AFP cells so as to generate AFP peptide specific tumor-reactive T cells. Controls,I1-21and/or rapamycin 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 flow cytometry for analysis. After six weeks of co-culture and analysis, we found that rapamycin had relatively higher Tscm subset cells compared to I1-21alone or rapamycin and I1-21together. However, the second week of co-culture proved to be the threshold time for expansion of the Tscm cells in the presence of I1-21and rapamycin for it is at this period that the concurrent application of these two agents exerted a tremendous expansion of the Tscm subset than any other experimental group at the same or any other given time. These results underscore the importance of augmenting immunotherapies by concurrent application of I1-21and rapamycin. In addition, our results provide immense knowledge in the search for effective immunotherapy strategies to fight cancer.