Creation of a Next-Generation MANAFEST Assay: A Proof-of-Concept

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Johns Hopkins University
T-cells recognize antigens by utilizing their T-Cell Receptors (TCRs), α/β heterodimers that bind to peptide:MHC complexes. The TCRβ chain is commonly used to track specific clonotypes because of its increased diversity due to the presence of the D gene, which is not present on the TCRα chain. Both the α and β chains are involved in antigen recognition and are necessary for the cloning of TCRs to be used in cell-based therapies, but the current processes for obtaining paired TCRs are time-consuming and expensive. MANAFEST, Mutation Associated NeoAntigen Functional Expansion of Specific T-cells, is an assay wherein peptide-stimulated T-cell culture is combined with bulk TCR sequencing to identify TCR Vβ CDR3 clonotypes. We aimed to optimize the current assay to create a next-generation MANAFEST that allows for antigen-specificity determination and the identification of paired TCRs in a single workflow. To develop this method, we utilized single cell sequencing coupled with the next-generation MANAFEST to both streamline the approach to obtaining paired TCRs and create a more-cost effective workflow. Our goal was to establish proof-of-concept for the development of a streamlined, high-throughput assay that is simultaneously able to identify antigen-specific T-cells and their corresponding TCRs, and we hypothesized that utilizing a single cell transcriptomic panel and TCR sequencing on peptide-stimulated cells would allow us to achieve this goal. We approached this hypothesis with a ten-day cell culture and peptide stimulation followed by single cell analysis and TCR sequencing on a high throughput platform that examined a panel of about 1,000 immune-related genes. We expected that we would be able to discover tumor specific TCRs by identifying the upregulation of genes associated with an activated T-cell signature. Indeed, our bioinformatics analysis revealed an upregulation of genes we anticipated to see, such as IFNγ, CCL3, and GZMB, and we were successfully able to identify the TCRs of the cells expressing these upregulated genes. In doing so, we addressed our central hypothesis—that utilizing a targeted, single cell transcriptomic panel coupled with TCR sequencing on peptide-stimulated T-cells can allow us to simultaneously identify both tumor reactive T-cells and their paired TCRs in a single workflow.
T-cell, T-cell receptor, TCR, Tumor identification, Oncology, Cancer, Neoantigens, MANAs, MANAFEST