T cells play a vital role in the adaptive immune response against viral infections. Virus-specific T cells recognize infected cells through a heterodimeric surface receptor known as the αβ T cell receptor (TCR). The TCR recognizes a peptide epitope presented by a product of the major histocompatibility complex (MHC). All nucleated cells express class I MHC products, typically in association with one of thousands of “self” peptides. However, during a viral infection, “foreign” peptides derived from the expression of viral proteins would also be presented on the cell surface. T cells expressing TCRs that specifically recognize these viral peptide-MHC complexes (pMHC) can become activated and destroy the virus-infected cell. However, the immune system of many patients with chronic viruses has undergone a process of tolerance whereby the T cells are either non-existent or inactive. The ability to study the viral pMHC on infected cells would allow a greater understanding of anti-viral responses and the development of specific targeting molecules against these pMHC could lead to the generation of novel therapeutic interventions to control viral infections. The studies described here focus on these goals.Chapter 2 describes the characterization of two monoclonal antibodies that are specific for immuno-dominant hepatitis B virus epitopes, the core protein peptide 18-27 (Core18-27) and the envelope protein peptide 183-191 (Env183-191), both restricted by the human HLA-A*02:01 class I MHC. Both murine monoclonal antibodies exhibited specificity and high affinity for their respective epitopes. In addition, the antibodies were able to recognize pMHC generated exogenously by pulsing HLA-A*02:01+ T2 cells with the respective peptides, and also endogenous pMHC complexes produced by HBV infected cell lines. The antibodies were used further to quantify and visualize specific HBV pMHCs on the surface of various cell lines, allowing the study of how density and distribution of these pMHC ligands affected T cell activation.In Chapter 3, I sought to examine the use of soluble TCRs in comparison with a monoclonal antibody generated against the same antigen. To approach the question, the α and β chain genes of a TCR specific for the envelope protein peptide 183-191 (Env183-191) epitope were cloned from a T cell line and the extracellular domains were expressed in Escherichia coli. Characterization of the refolded, soluble TCR showed that only tetrameric forms bound specifically to Env183-191 pMHC in a surface plasmon resonance assay, or with peptide-pulsed target cells, consistent with the well-known low affinities of TCRs. To further explore the specificity of the TCR, I developed a sensitive bead-based avidity assay. Using this assay, a comparison between the TCR tetramer and the Env183-191 specific monoclonal antibody showed that although the tetrameric form of the soluble TCR had similar functional avidity for the Env183-191 pMHC as the monoclonal antibody, it required a higher density of pMHC ligands in order to achieve significant binding. Nevertheless, the assay revealed a difference in fine specificity of the TCR and the monoclonal antibody, consistent with evidence that the T cell clone could recognize a particular virus-variant (clade), which the antibody could not recognize. Finally in Chapter 4, I used the sensitivity of the avidity-based assay described in Chapter 3 to develop a novel method for the in vitro engineering of T cell receptors expressed in a yeast display system. In view of the limitations imposed by the low affinity of soluble TCRs, protein engineering is necessary to improve the monomeric affinity (a process called affinity maturation). Conventional methods for screening yeast-displayed libraries of TCRs had used tetrameric pMHC or commercially available dimeric MHC-Immunoglobulin fusion molecules. To improve the sensitivity of the screening strategy, the bead-based assay was applied to a yeast display system with model TCRs of varying affinities. The pMHC-coated beads were able to significantly stain lower affinity TCRs that had previously not been isolated using the tetrameric and dimeric reagents. Furthermore, the assay was used in the screening of a single-chain TCR library, isolating not only high affinity mutants but also intermediate affinity mutants that would not have been selected by the other reagents. In the absence of high affinity solutions within libraries, these intermediate affinity mutants can be potential leads with which further engineering can be done, thus increasing the success rate of TCR engineering. They could also serve as TCRs with improved activities in adoptive T cell therapies, which are known to benefit from such intermediate affinity TCRs.The molecular probes available to specifically target peptide-major histocompatibility complexes are relatively uncommon and thus the capacity to study pMHC distribution and quantities has been limited. The key problems have been due to the fact that the natural receptors for the pMHC, the T cell receptors, are difficult to express in soluble form and are of poor affinity. TCR-like antibodies against a specific pMHC provide one possible solution, but they are difficult to generate, and their specificities are often not adequate (e.g. they bind MHC epitopes such that the peptide contribution to binding varies). In this thesis, I describe the characterization and applications of two such antibodies and a soluble TCR that are able to specifically target HBV pMHCs. I also describe the development of a new screening assay that would facilitate yeast display protein engineering in the generation of high affinity TCRs specific for pMHC. These probes provide opportunities to study the details of viral peptide presentation and anti-viral immune responses, and they also provide potential molecular therapies, with targeted delivery capacities that would aid the treatment and resolution of viral infections.
【 预 览 】
附件列表
Files
Size
Format
View
Recognition of hepatitis B epitopes by T cell receptors and T cell receptor-like antibodies
PDF
download
878987797
028-85220240
