Molecular Technologies for Antigen-Based Immunity

Citation

Chour, William (2021) Molecular Technologies for Antigen-Based Immunity. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/z20t-nq62. https://resolver.caltech.edu/CaltechTHESIS:02192021-010538691

Abstract

The presence and proliferation antigen-specific T cells is a defining characteristic of an adaptive immune response against various disease types (autoimmune, cancer, and infectious). The use of Class I and Class II peptide-major histocompatibility complex (pMHC) reagents to identify such cells, however, is technically difficult and expensive, and it has been challenging to refine synthesis protocols for higher yield and more efficient assembly to accommodate large-scale applications. This achievement would enable high-throughput capture of corresponding T cell receptors (TCR), which may be further used in clinical applications such as adoptive cell transfer therapies. Overcoming this hurdle requires the development and integration of various molecular technologies and analytical methods.

Toward this end, the bulk of my thesis work, covered in Chapter 2, introduces these developments in the context of pMHCs, where the three subunits of each reagent are covalent linked together and expressed as a single protein. These single-chain trimer (SCT) technologies primarily consist of traditional DNA cloning and protein production techniques which have been streamlined for applications requiring output on the scale of 10²-10³ of reagents. This chapter serves as the foundation for much of the methodology discussed throughout the rest of my thesis, and thus should serve as a reference point. The generated constructs are also functionally validated here, and potential future research directions are outlined.

In Chapter 3, I explore the use of this technology in the context of COVID-19 to enumerate antigen specificity of the CD8+ T cell immune response. Class I SCTs were constructed to present peptides across several SARS-CoV-2 protein domains, using various HLA alleles to match haplotyped participant blood samples. These reagents were then used to capture SARS-CoV-2-specific T cells through flow and nanoparticle cytometry to demonstrate HLA-dependent, domain-dependent immune responses. Identified TCRs were cloned into T cells for confirmation of antigen specificity and functional cytotoxicity.

In Chapters 4 and 5, I explore potential pMHC applications in cancer antigen contexts, covering both tumor-associated and tumor-specific antigens. Through various collaborations across the west coast (UCLA, Parker Institute, Fred Hutchinson Cancer Research Center), I make use of the SCT platform to showcase new assays to discover and rank key tumor targets (Chapter 4). Finally, Chapter 5 is a reproduction of our lab’s published work concerning identification of antigen-specific CD8+ T cells from melanoma cancer patients.

In summary, the adaptation of SCTs in a high-throughput format allows for the rapid enumeration of antigen-specific T-cell receptor sequences. As demonstrated in the contexts of COVID-19 and cancer, this SCT platform enables subsequent downstream applications, such as single-cell, antigen-specific immunophenotypic mapping/analysis and target discovery for personalized immunotherapies.

Thesis Files