Aaron Silva
BME PhD Proposal Presentation

Date: 2025-07-31
Time: 9:30
Location / Meeting Link: U.A. Whitaker Building, Room 1103 / https://gatech.zoom.us/j/93258950657

Committee Members:
Gabe A. Kwong, PhD (Advisor) Cheng Zhu, PhD M.G. Finn, PhD Felipe Quiroz, PhD Yonggang Ke, PhD


Title: DNA BARCODES TO DETECT, ISOLATE, AND SPECIFY ANTIGEN-SPECIFIC T CELLS

Abstract:
T cells play a critical role in adaptive immunity responding to a variety of peptide antigens presented on major histocompatibility complex (MHC) molecules through their T cell receptor (TCR). In addition to their importance in protective immunity, T cells are being used as living drugs in adoptive cellular therapies (ACT). Commonly, to study and isolate antigen-specific T cells, recombinant peptide MHC (pMHC) is fluorescently multimerized to be analyzed and sorted by flow cytometry. This presents three issues; (1) the high cost of flow cytometry machines, (2) fluorescent tags due to spectral overlap cannot cover the large library of TCRs, and (3) pMHC continual binding to TCR leads to T cell activation or cell death, impacting cell phenotype. A variety of tools have been developed to overcome these issues; many of them using DNA barcodes to label T cells. My proposal seeks to use DNA barcodes for detection and isolation of AgS T cells that would overcome some of the issues presented by flow cytometry. In this proposal, we will develop a low-cost method for AgS T cell detection that does not require flow cytometry. This approach uses a loop mediate isothermal amplification (LAMP) DNA barcode conjugated to pMHC multimers to label and detect rare AgS T cells. For low-cost detection, we will measure T cell presence with lateral flow assay that results in a positive test result when Cas12a detects the presence of LAMP BC and collaterally cleaves a DNA reporter (Aim 1). Additionally, we will develop a scarless, multiplexed isolation technique for AgS T cell sorting that does not impact T cell phenotype and could sort five different AgS T cells simultaneously. We will use pMHC monomer DNA gates to isolate chimeric antigen receptor (CAR) T cells for in vivo tumor regression studies (Aim 2). Finally, we want to specify the phenotype of AgS T cell that we isolate using magnetic assisted cell sorting (MACS) for large scale applications. We will adapt DNA gated sorting (DGS) to create a set of DNA gates that can isolate AgS T cells based on the expression of combination of two surface markers. We will adapt this system to isolate TSCM (CD45RA+, CD62L) CAR T cells for in vivo tumor regression studies (Aim 3). Successful completion of these aims will provide new technologies to detect and isolate AgS T cells that can be used in a variety of immunotherapeutic applications.