During an induced immune response, the BALB/c strain of laboratory mouse presents ovalbumin peptides by major histocompatibility complexes that stimulate responding T cells. This work first investigates the nature of ovalbumin peptide 323-339 presentation by the Class II MHC I-Ad in an in vitro setting, then focuses on the engineering of an individual T cell receptor (DO11) involved in ovalbumin response, and finally, pursues isolation of other responding T cell receptors with modern high throughput sequencing techniques.T cell activation requires formation of a tri-molecular complex between a major histocompatibility complex (MHC), peptide, and T cell receptor. In a common model system, the ovalbumin epitope 323-339 binds the murine class II MHC, I-Ad in at least three distinct registers. The DO11 T cell recognizes the least stable of these, as determined by peptide-MHC dissociation rates. Using synthesized peptides in combination with IL-2 secretion assays, we show that the alternate registers do not competitively inhibit display of the active register four. In contrast, this weak register is stabilized by the presence of n-terminal flanking residues active in MHC binding. DO11 is sensitive to the presence of wild type residues extending to at least the P-3 peptide position, and the transfer of flanking residues in the P-4 and P-2 positions to an I-Ad presented hen egg lysozyme epitope increases the activity of that epitope substantially. Further, we provide evidence that display of the active register is dependent on the stability of an adjacent, overlapping register three. When epitopes were inserted in a maltose binding protein carrier, activity positively correlated with stability of the adjacent register, which was modulated by P1 anchor residue substitutions. Collectively, these results illustrate the potential of flanking residue modification to increase the activity of weakly bound epitopes. This represents an alternative to substitution of anchor residues within a weakly bound register, which we show can significantly decrease the activity of the epitope to a T cell responder. In order to monitor the presentation of ovalbumin peptides, we have engineered a high affinity, multivalent single chain T cell receptor complex that exhibits specificity towards the OVA 323-339 peptide when displayed by I-Ad. DO11 was expressed as a single chain T cell receptor at levels in excess of 30 mg/L when oriented as beta variable region followed by alpha region separated by a flexible linker and incorporating previously identified solubility-inducing mutations. High affinity variants of DO11 were obtained from a randomized CDR3a; library displayed as pIII fusions by M13 bacteriophage and panned against the DO11-specific, pMHC-mimicking antibody KJ1-26. ScTCR dimers were created by introduction of a hexapeptide EE-epitope tag into the flexible (G4S)4 linker, followed by complexation with a specific monoclonal antibody. Tetramers were formed by further association of dimers with a fluorescently labeled anti-IgG1, and shown to specifically target peptide-loaded lymphoma cells in flow cytometry. The applicability of scTCR protein expression and phage display techniques is also explored with other TCRs and found to be generally applicable to those containing mouse TRBV13-2, a gene previously noted for its preferential usage in the responding repertoire in mouse autoimmune models. We also show that 172, a TCR isolated from a mouse experimental autoimmune encephalomyelitis model, can be expressed at high levels as a fusion with the short peptide IT9302, a peptide from the c-terminus of interleukin 10 shown to possess many of the immunosuppressive characteristics of its parent cytokine. We also report on the adaptation of phage display vector pMoPac24 to display fusion proteins with M13 major coat protein pVIII along with three previously identified display-optimized variants. We find that the production of phage particles containing these fusions and surface display is specific to the variant being expressed, but we demonstrate more efficient display for pVIII fusions as compared to pIII for DO11, as measured by inhibition of Interleukin 2 (IL-2) secretion during a 24-hour incubation with antigen presenting cells and specific peptide. The use of these vectors may be necessary for directed evolution of T cell receptors exhibiting low affinity interactions with their target peptide/MHC complexes. Finally, we report on the high throughput sequencing of mouse T cell receptor genes using pyrosequencing technologies. Alpha and Beta primer sets were designed to amplify cDNA from isolated mouse total RNA. The set was validated on T Cells isolated from mouse spleens, and used to identify T cell receptor genes from two clones, 3B6 and 4C4. A high throughput comparison of ovalbumin immunized mice and unimmunized controls is in progress. Identification of and subsequent engineering of T cell receptors involved in the ovalbumin response may provide further tools for future research on this murine system.
University of Minnesota Ph.D. dissertation. December 2012. Major: Chemical Engineering. Advisor: Jennifer A. Maynard. 1 computer file (PDF); ix, 123 pages.
Roy, Benjamin Michae.
Display of ovalbumin peptides by murine Class II major histocompatibility complexes and the engineering of responding T cell receptors.
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