Download to read offline
JoeH47PosterFinalDraft
- 1. Abstract Conventional Organ transplantationoften results in chronic organ rejection, even when donor and recipient are matched at the tissue level. Genetic variation between individuals gives rise to variable peptide antigens, which may be recognized as “non-self” by T cells in the context of the MHC. Thus, novel strategies are needed to overcome antigen induced organ rejection. One potential strategy is xeno-transplantation, which is the use of non-human tissues in transplantation. Preliminary studies have discovered that important antigens are conserved in human and in murine cells. However, one antigen, H47, is not detected on human cells although human cells possess and express the same “H47 antigen” as seen in mouse cells. Although the human gene which encodes H47 (VIMP) is transcriptionally expressed, one potential caveat is that the precursor H47 protein is not processed to the H47 peptide antigen in human cell systems. Current research is aimed at generating stable Transfectant cell lines, which over-express HuVIMP and murine H-47 in human cell systems. These cell lines will be subjected to a CML (T cell) killing assays at the Jackson Lab, Bar Harbor, ME. A better understanding of antigen processing variations between species can lead to a stronger understanding of cross species transplantation barriers, which may be valuable in augmenting development of novel xeno-transplantation strategies. T-Cell Xeno-Antigen Recognition Joseph Rovinsky B.S. Biotechnology Class of 2007 Principal Investigator: Dr. Peter Eden Science Department Introduction Minor Histocompatibility Antigens Proteins are constantly processed in the proteosome into peptide fragments known as Minor Histocompatibility antigens. These antigens vary based upon genomic differences between individuals within the same and across species. Antigenic variation may elucidate a T Cell mediated attack when presented by the MHC in normal immunosurveillance MHC I The Major Histocompatibility Complex I is a protein structure which presents antigenic peptides for surveillance by Cytotoxic (CD8) T-Cells. Cytotoxic T-Cells are specialized in there function to destroy cells which present antigens which are recognized as foreign or “non-self T-Cell /MHC Interaction(Figure 1) Each T-Cell is specific for one MHC isoform. The MHC is encoded by highly polymorphic genes, thus, the antigen peptides must be presented for review in the context of the specific MHC molecule. Figure 1. Antigen Processing from Precursor Protein NSF-RUI Grant Support Harvard Medical School Dr. Ye Yihong Dr. Tom Rapopor M.I.T. Broad Institute of Molecular Genetics Shannon Chilewski The Jackson Lab Dr. Derry Roopenian Greg Christianson Marywood University Dr. Michael Kiel - Conventional organ transplantation can result in chronic organ rejection due to antigen variations; even when donor/recipient matched at the MHC, thus new transplantation strategies needed - Alternative transplantation strategies are needed. One alternative is xeno- transplantation, the use of tissues from other species. However, T-cell recognition of “xeno-antigens” as “non-self” occurs, leading to organ rejection - Rules governing T-cell xeno-antigen expression, processing, presentation and recognition are not well understood - This project examines cross-species differences in transplantation antigen processing and presentation of a unique barrier antigen H47 - It was previously discovered that mouse T-Cells specific for H4 and H7 will recognize and kill human cells. H4 and H7 are thus conserved across species - When the experiment was performed for the H47 antigen, the murine T cells did not recognize several human lymphoid cell lines, and these cells lived - The goal of this project is to understand why H47 is not recognized - Results of this project may elucidate a better understanding of T-Cell xeno- antigen processing and presentation, in order to augment xenotransplantation strategies Experimental Rationale Figure 2. Overview of Project Goals 1 2 3 4 5 6 Figure 3. Sub-Cloning Results VIMP Myc- Digested with BamH1/KPN AIM I. Stable Transfection -Cell Lines: CEMDB/CIRDB Lymphoid Cells -Constructs: HuVIMP, Murine VIMP, H47A and H47B Mini-Genes -Effectene Transfectant Reagent -Previous T-Cell Killing assay conducted on cells transiently transfected and loaded with the various constructs/peptides. It is possible that only a small number of cells were transfected, preventing statistically significant T-Cell assay results. Stable transfectant cell lines will insure uniform expression. -Stable transfection “selects” based off of a selective pressure for cells that have only been transfected. All other cells will die when subjected to an antibiotic. AIM II. T-Cell Killing Assay -Previous CML (T-Cell Killing Assay) performed on transiently transfected cells, results were inconclusive (Figure 4) -Assay will be conducted in collaboration with the Jackson Lab, Bar Harbor, ME which specializes in this technique Current Research Aims 1. Effector: 21M/B10-A7 Target :CEMDB Transfected With HuVIMP 2. Effector: 21M/B10-A7 Target: CEMDB 3. Effector: 21M/B10-A7 Target: T2DB Loaded with mH47a 4. Effector: 21M/B10-A7 Target: T2DB Loaded with mH47b 5. Effector: 21M/B10-A7 Target: T2DB Loaded with Viral Peptides Dark Blue=50:1 Red= 25:1 Yellow= 5:1 Light Blue= 0.5:1 Transfected Cells-Expected to yield higher Percentage killed (3). Cells loaded with surface H47 effectively killed(4). Stable Transfection will insure uniform expression! Figure 4. Preliminary T-Cell Killing Assay Results Cell Lines (Suspension Cells) -Human Lymphoid Cells (CEMDB and CIRDB) -Cell Lines cultured in supplemented VcDMEM, 10^6 cells/mL Genetic Constructs -HuVIMP Myc-, H47a, H47b, Murine VIMP -Expression Vector: pCDNA3.1 TOPO Transfection -Effectene Reagent (Qiagen) Selective Pressure -250 mg Neomycin, suspended in DMSO will be added to cells in 60 x 60 mm -Cells will be split 48 hrs after addition -Sample of transfectant cells will be subjected to RT-PCR (Qiagen) using the appropriate primers in order to determine transfection success -After a two week culture, cells will be cryogenically preserved and shipped to the Jackson Lab, for further research Aim I. Stable Transfection Materials and Methods Effectors (T-Cells) -21M/B10-A7- T-Cell line that specifically recognizes and interacts with MHC isoform DB, expressed on the surface of CEM/CIR cell lines Targets (Cell Lines) -CEMDB/CEMDB Transfected with HuVIMP -C1RDB Transfected with Murine VIMP -T2DB Loaded with MH47A -T2DB Loaded with MH47B Chromium-51 Peptide Loading Procedural Overview -T2DB cells incubated in radioactive chromium. -Chromium binds to the MHC and can capture peptide antigens -Cell’s then incubated in peptides “Loaded”, which bind to the MHC CML Assay -Cells incubated for approx. 4 hours with T-Cells killing detected -Different Effector/Target ratios used; this assay performed at Jackson Lab Aim II. T-Cell Killing Assay Methods and Materials Future Research Acknowledgements HuVIMP 1.)Lambda HindIII 2.)VIMP Clone Digest 1 3.)VIMP Clone Digest 10 4.)VIMP Clone Digest 9 5.)Blank 6.)Blank VIMP Myc- successfully sub-cloned, removing Myc Tag Sequence Restriction enzyme digestion (BamH1/KPN) liberated VIMP fragment DNA Sequencing results confirmed VIMP sequence without Myc tag (Conducted at UCONN) pcDNA3.1 Figure 5. Cellular Location of HuVIMP The cellular location of HuVIMP was characterized in 2005 (Harvard) VIMP interacts with Derlin-1, p97 and E3 protein complexes in the synthesis of MHC I (Figure 5) Is there a difference between humans and other organisms at the cellular level which affects processing and presentation? Will potentially explore this possibility!!! Results of this research may elucidate novel information on antigen processing/presentation across species, a useful application in developing xeno- transplantation strategies. T-Cells recognize “foreign” antigens and mediate an immune attack Antigenic differences across species can lead to rejection!