Prorgammable Immune Cytometry and Diagnostics

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Dahotre, Shreyas Narendra
Kwong, Gabriel A.
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Increasing our ability to broadly monitor T cell responses and sense disease biomarkers at depth throughout the course of a disease has provided clinicians with valuable new insights. For example, technologies that enable tracking of clonal expansion and contraction of T cells have revealed useful clinical biomarkers such as T cell counts for assessing disease burden and progression. Furthermore, the ability to manipulate these T cell responses has enabled new therapeutic approaches, including reprogramming T cells with chimeric antigen receptors for treatment of hematological malignancies. While these methods have revolutionized immunotherapy, detecting and modifying disease-specific T cells is still challenging because antigen-specific subsets are found at low frequency and current platforms such as flow cytometry lack the capacity to monitor more than a few clones at one time. By contrast, emerging analytical and therapeutic platforms based on DNA and RNA leverage the programmable properties of nucleic acids to enable ultrasensitive, multiplexed detection and selective gene modulation. In this thesis, I establish a framework whereby programmable platforms based on nucleic acids and pMHC are used to detect, isolate, and modulate T cell populations, providing scalable approaches that are not limited by biophysical constraints. First, I build a library of DNA barcoded peptide-MHC tetramers for multiplexed monitoring of viral-specific T cells during infection with single cell sensitivity. I then extend the use of DNA barcodes to develop Cas12a-based amplification approaches for detection of SARS-CoV-2 viral antigens and viral-specific T cells using lateral flow assays. To provide the ability to isolate antigen-specific T cell subsets, I next design and implement an extensible library of programmable DNA gates, comprising dynamic and orthogonal DNA strand displacement reactions, for sorting multiple T cell populations from a single sample. Finally, to modulate the activity of specific T cell subsets, I demonstrate that lipid nanoparticles conjugated with pMHC allows for functional mRNA delivery in vivo. This thesis describes new programmable technologies to track and modulate T cell responses to guide the development of a new generation of immune diagnostics and immunotherapies.
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