Linking TCR sequence, structure, and function in the DO11.10 mouse model
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Thymus-maturated lymphocytes (T-cells) provide the immune system with the ability to appropriately detect and respond to diseases through a unique receptor. These T-cell receptors (TCRs) differentiate between self and foreign peptide antigens and form by hypervariable gene rearrangement and imprecise recombination, both of which can potentially generate upwards of 10¹⁸ unique TCRs in humans. Our work focused on characterizing the chicken ovalbumin (OVA) antigen specific parent hybridoma DO11.10 T-cell with (1) high-throughput sequencing, (2) structural analyses, and (3) TCR reconstitution into T-cells to determine the important factors linking TCR sequence, structure, and function. The repertoire of TCR sequences generated in mice immunized with OVA antigen were sequenced using a novel mouse TCR gene primer set. Using high-throughput sequencing, we determined the relative gene and hypervariable sequence frequencies in the repertoire. Based on our results in these experiments, no significant repertoire differences were found between mouse treatment groups. Additionally no sequences with similarity to previously studied OVA-specific T-cell clones were recovered. The antigen binding loops of TCRs have a high degree of structural similarity across diverse receptor sequences. Key residues in the complementarity determining, antigen-binding regions of TCRs form conserved canonical loop structures. Here we evaluated 249 TCRs and TCR-like antibodies to determine the prevalence of these key residues. Expanding on previous work, we found four new canonical class conformations and validated the key residues for these classes. Finally, we report our work to reconstitute engineered TCRs onto the TCR-deficient mouse 58 [superscript -/-] hybridoma line. We modified an existing antibody expression system to display the engineered TCRs in the hybridomas for activation assays. Expression of the TCR was found to be highly sensitive to the N-terminal signal sequence on each receptor chain. Additional work began to examine how these signal sequences alter stable expression and surface display of these TCRs. This work is important for the discovery of new methods and biological agents to target and respond to designed antigens, especially in the context of altering and engineering TCR specificity for therapeutic purposes.