The Development of Immunoreagents to Detect for a Cellular Immune Response
Date
2019-08-20
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Cell mediated immunity (CMI) is the adaptive T-cell-mediated defense mechanism against pathogens. Oftentimes, by assessing lymphocyte counts and/or cytokine profiles, the presence of an infection can be detected. While current diagnostic tests (e.g. interferon gamma release assay, delayed type hypersensitivity test) exist, many are not widely adopted due to the requirement of specific equipment for quantification, time restraints and/or false negative results. As such, the discovery of new biomarkers that can accurately detect and quantify a T-cell mediated, antigen specific immune response would be very valuable. We aimed to explore cellular immune responses (CIRs) through the direct detection of antigen specific Major Histocompatibility Complex II - T Cell Receptor (MHC II-TCR) interactions. We anticipated to create bioreagents that could enumerate TCR:MHC interactions, thereby giving possible insights of a previously activated CMI by targeting effector T-cells. To do this, superantigens (SAgs) (here: TSST-1) were split and fused to a reporter protein (YFP), allowing for specific TCR-pMHC interactions on antigen presenting cells (APCs) to be detected through a fluorescent marker. In the presence of a cognate pMHCII:TCR interaction, our split superantigens would be able to bind and reassemble, thereby quantifying the interaction using fluorescence. To further detect for sensitized T-cells, we engineered peptide-recombinant T cell receptor ligands (pRTLs) to be displayed on the surface of fluorescent E. coli using a pAIDA1 expression system. This system is intended to mimic the expression of MHC II on APCs. Interactions between the T-cells and pRTLs on the surface the E. coli can therefore be visualized using microscopy followed by flow cytometry analysis, whereby clustering of GFP-bacteria around effector T-cells would arise as a result of successful peptide display. Our results indicated no binding occurred between the individual C-domain of TSST-1 and T-cells. It was also revealed that our designed pRTL that were expressed on the surface of E.coli were unable to bind to T-cells, possibly due to complications with expressing the pRTL using a bacterial surface expression vector. Here, we describe the methodology of our approach, including construct design, protein production and analysis. We also explore existing technologies,and suggest future approaches and considerations that can be made to supplement the current research we have performed in this project. In conclusion, our developed biomarkers did not successfully bind to T-cells as we had anticipated, however, we did develop valuable insight regarding the use of SAg and pRTLs are potential bioreagents for the detection of a cellular immune response.
Description
Keywords
superantigens, molecular biology, immunology, bioengineering, split proteins, pAIDA1, surface display
Citation
Ma, C. W. Y. (2019). The Development of Immunoreagents to Detect for a Cellular Immune Response (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.