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Novel in vitro methods for the discovery of functional T-cell receptor epitopes from large peptide-coding libraries

University of British Columbia

Though it is understood that T-cells are a critical component of the immune system’s ability to destroy foreign invaders and attack cancerous cells, very little is known regarding the specific epitopes that are recognized by T-cells to carry out these functions. Generally, the epitopes mediating this immunity are short, contiguous peptides derived from antigenic proteins presented on major histocompatibility complex (MHC) molecules for inspection by T-cells. The ability to rapidly and deeply search peptide space to determine specific peptide epitopes that are naturally processed, presented, and capable of eliciting functional T-cell responses is a critical unmet need in the study of adaptive immunity. Here, I describe a novel method for deep T-cell epitope profiling that enables simultaneous in vitro interrogation of target cell populations encoding high-diversity minigene libraries with T-cell populations-of-interest. Targets eliciting T-cell reactivity are selectively isolated by fluorescence-activated cell sorting (FACS) and identified by deep amplicon sequencing. The approach was extensively validated using known murine T-cell receptor (TCR)/peptide-MHC pairs and it was shown that this method can unambiguously identify canonical minigenes from libraries of vastly more candidate antigens in parallel than would be feasibly tractable using conventional methods. The capability of this strategy was extended by applying a synthetic biology approach. Using pairs of immortalized natural killer (NK)-like effector cell lines and naturally tolerated target cell lines, I showed that fully reconstituting the TCR/CD3 complex in effectors and expressing relevant MHC-/minigene-coding sequences in targets is sufficient to re-direct the cytotoxicity of NK-like cell lines towards antigenic targets of recombinant TCR. These results provide indication that it should be possible to use an entirely synthetic framework for functionally screening recombinant TCR-of-interest against minigene libraries without the requirement to first isolate and expand primary T-cell clones or donor-derived antigen-presenting cells. The high-throughput T-cell antigen profiling methods described and validated in this thesis could allow investigators to generate TCR epitope data broader in scope than previously possible to better understand basic T-cell biology, develop better predictive models of T-cell reactivity, and rationally design T-cell-based immunotherapeutics for the treatment of cancer, infectious disease, and autoimmunity.

Text

2018-12-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/68080

Genome Science and Technology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/