Interrogating T cell Reactivities Using Repertoire Sequencing
MONTAGNE-DISSERTATION-2020.pdf (12.18Mb) (embargoed until: 2024-05-01)
Montagne, Janelle Marie
MetadataShow full item record
Highly multiplexed technologies have enabled the precise characterization of T cells, including full transcriptomes, epigenomes, proteomes, and metabolomes. However, these approaches do not capture a crucial feature of T cells: their antigen specificities. T cells recognize precise molecular targets via their T cell receptors (TCRs). These targets are short peptides presented to T cells in the context of major histocompatibility complex molecules (pMHCs) on the surface of aptly named antigen presenting cells. The complexity of this TCR:pMHC interaction has greatly hindered the development of high-throughput T cell reactivity assays. We have therefore developed several tools to enable the large-scale analysis of T cells and their associated specificities. These tools include Framework Region 3 AmplifiKation Sequencing (“FR3AK-seq”), an ultra-efficient approach to TCR repertoire sequencing. FR3AK-seq enables the affordable and quantitative TCR repertoire analysis of many samples simultaneously. We used FR3AK-seq to analyze muscle-resident TCR repertoires from patients with myositis and controls and detected shared antigen-driven T cell responses. Additionally, we developed an in situ reverse transcription approach for adding sample-specific barcodes to TCR sequences prior to flow cytometric sorting and FR3AK-seq analysis. This enables the mixing of numerous samples prior to sorting, effectively streamlining the protocol, reducing cost, and adding phenotypic information to TCR repertoires. Finally, we designed a genetically encoded antigen library to increase the scale of specificities that can be interrogated in a single experiment. These approaches have the capacity to greatly enhance our understanding of human T cell responses in both health and disease.