(672b) Engineering DNA Gates for Extensible, Multiplexed Cell Sorting

Introduction:
The
activation and expansion of antigen-specific T cells is used
as a biomarker of disease, but our ability to simultaneously monitor these
complex polyclonal T cell responses is limited because the number of spectrally
distinct fluorophores with which we can label individual cell populations is
low. Here we introduce an extensible sorting and analysis platform called DNA
gated sorting (DGS) that uses orthogonal DNA strand displacement reactions coupled
to affinity agents to capture, isolate, and recover multiple target cell
populations from a complex biological sample (Fig. 1A). We use DNA
because the number of possible DNA sequences scales exponentially with length,
making it possible to design and multiplex large libraries of DNA gates at a
scale that potentially exceeds fluorophores. DGS therefore may improve our
ability to analyze host T cell dynamics in the context of disease or identify
new antigens through tetramer-guided epitope mapping.

Materials
and Methods: All
animal work was approved by GT and Emory IACUC. DNA sequences were purchased from IDT and conjugated to antibodies or
streptavidin expressing a C-terminal cysteine residue (StvC)
in house. To measure kinetics of strand displacement, DNA gates containing
a Cy5/Iowa Black quencher pair were incubated with a release probe to initiate
strand displacement, and fluorescence was monitored by a
plate reader or flow cytometry. For cell sorting, Ab-DNA gates tagged with
Dynabeads were used to
magnetically label target cells from mouse splenocytes.
Individual target populations were recovered by
addition of release probes.

Results
and Discussion: We designed DNA gates
consisting of orthogonal strand displacement reactions using a domain-based
approach and uniquely coupled DNA gates to antibodies by hydrazone
chemistry. Using Ab-DNA gates containing a fluorophore-quencher pair, we found
that strand displacement was completed within 5
minutes on the surface of cells with minimal crosstalk among our different
gates (Fig. 1B). To demonstrate DNA
gated sorting, we added magnetic beads to Ab-DNA gates for magnetic isolation
and recovery of CD8⁺ T cells from a mouse spleen (Fig. 1C). We benchmarked the sorting
efficiency of DGS against a commercial magnetic activated cell sorting (MACS)
kit and observed no significant difference in target cell purity, viability,
and yield between the two methods. We then extended DGS to sort primary CD19⁺
B cells, CD8⁺ T cells, and CD4⁺ T cells in parallel from
a mouse spleen with high purity and found that isolated CD8⁺ T cells
retain proliferative and killing potential (Fig. 1D). To test the ability of DGS to isolate antigen-specific T
cells during the course of an endogenous polyclonal immune response, we
infected mice with the model virus LCMV and achieved enrichments of ~30-50 fold
for CD8⁺ T cells specific for the LCMV-derived antigens NP205 and
GP276.

Figure 1. (A) DNA gates are uniquely mapped to
antibodies to capture target cells from a biological sample. Cells are sorted into separate populations by orthogonal strand
displacement reactions specific to each antibody-DNA gate pair. (B) Cells stained with initially
quenched Ab-DNA gates exhibit increased fluorescence after the initiation of
strand displacement. (C) Target
cells are magnetically labeled using Ab-DNA gates (upper). After strand
displacement, beads are released from the cell surface, enabling cell recovery
(lower). (D) Multiplexed DGS sorts
B220⁺ B cells, CD8⁺ T cells, and CD4⁺ T cells
from a mouse spleen with high purity.

Conclusions:
We
introduce a multiplexed cell sorting platform that
isolates cells using DNA gates with high purity. Through further expansion, DGS
may enhance analysis of cells where a large number of orthogonal channels are
required.