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DNA-gated cytometry for multiplexed sorting of antigen-specific CD8 T cells

Award Number: R01AI171892
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/20/2022
PERIOD OF PERFORMANCE END DATE: 05/31/2027

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DNA-gated cytometry for multiplexed sorting of antigen-specific CD8 T cells - PROJECT SUMMARY Ag-specific CD8 T cells express T cell receptors (TCRs) that recognize antigens in the form of processed peptides bound to major histocompatibility complex class I (MHCI) molecules. In healthy individuals, the CD8 TCR repertoire comprises approximately 106–108 different cell populations. Soluble pMHCI multimers are widely used to enumerate and isolate Ag-specific T cells by fluorescence activated cell sorting (FACS); however, FACS has limitations including low-throughput, high shear-stress damage (especially to rare cells), and low multiplexing depth due to limited number of fluorophores. For high-throughput cell sorting (>106 cells) such as for manufacturing T cell therapies, magnetic activated cell sorting (MACS) is commonly used but can only produce antibody-enriched or depleted cell fractions, and cells sorted by positive selection remain labeled with beads, preventing immediate downstream assays such as phenotyping by flow analysis. New approaches are needed for multiplexed, high-throughput and label-free isolation of Ag-specific T cells. This proposal will develop DNA- gated sorting (DGS) cytometry for multiplexed isolation of Ag-specific CD8 T cells. DGS comprise a molecular DNA circuit that couples a magnetic bead to pMHCI molecules through DNA hybridization, and that functions as a sorting ‘gate’ to capture, release, and recover Ag-specific T cells by toehold-mediated strand displacement. By using orthogonal DNA strand displacement reactions, a library of beads coated with different pMHCI antigens can simultaneously capture target cell populations en masse and each subpopulation can then be eluted by sequential strand displacement. In contrast to fluorophores, the number of possible DNA sequences to design strand displacement reactions scales exponentially by the length n of the DNA oligo (i.e., 4n), providing the possibility to extend this technology to isolate Ag-specific T cells at depths that is currently not possible. This proposal will also implement DGS with pMHCI monomers that multimerize when hybridized onto the bead to produce the required binding avidity for T cell capture, but after cell release, revert into monomers to dissociate from T cells resulting in label-free isolates. It will also implement light-induced peptide exchange to produce large pMHCI libraries to integrate with multiplexed DGS. Finally, this proposal will demonstrate an important application for the manufacturing of chimeric antigen receptor (CAR) T cells using virus-specific T cells to redirect them to tumor cells, using key benchmarks such as ex vivo functional assays (expansion, transduction efficiency, cytotoxicity) and in vivo therapy in mice bearing CD19+ cancer cells.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $791,000 )
2025	2025	GEORGIA TECH RESEARCH CORP	926 DALNEY ST NW	ATLANTA	GA	30318	FULTON	USA	Allergy and Infectious Diseases Research	001	4	5/30/2025	EXTENSION WITH OR WITHOUT FUNDS	$791,000
2025	2024	GEORGIA TECH RESEARCH CORP	926 DALNEY ST NW	ATLANTA	GA	30318	FULTON	USA	Allergy and Infectious Diseases Research	000	3	5/30/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $791,000

Issue Date FY: 2024 ( Subtotal = $395,500 )
2024	2024	GEORGIA TECH RESEARCH CORP	926 DALNEY ST NW	ATLANTA	GA	30318	FULTON	USA	Allergy and Infectious Diseases Research	000	3	5/13/2024	NON-COMPETING CONTINUATION	$395,500
														Subtotal = $395,500

Issue Date FY: 2023 ( Subtotal = $395,500 )
2023	2023	GEORGIA TECH RESEARCH CORPORATION	926 DALNEY ST NW	ATLANTA	GA	30332	FULTON	USA	Allergy and Infectious Diseases Research	000	2	5/12/2023	NON-COMPETING CONTINUATION	$395,500
														Subtotal = $395,500

Issue Date FY: 2022 ( Subtotal = $395,500 )
2022	2022	GEORGIA TECH RESEARCH CORPORATION	926 DALNEY ST NW	ATLANTA	GA	30332	FULTON	USA	Allergy and Infectious Diseases Research	000	1	6/20/2022	NEW	$395,500
														Subtotal = $395,500

Grand Total All Awards = $1,977,500

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DNA-gated cytometry for multiplexed sorting of antigen-specific CD8 T cells

Award Number: R01AI171892

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 06/20/2022

PERIOD OF PERFORMANCE END DATE: 05/31/2027

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