DNA-gated Cytometry for Scarless Isolation of Antigen-specific T cells

Introduction:: ntroduction: Antigen(Ag)-specific CD8 T cells are key players in adaptive immunity and have transformative applications as cell therapies. Yet isolating these cells by fluorescence-based cytometry is challenging because of their receptor diversity and rarity. We develop DNA-gated cytometry for scarless and multiplexed sorting of Ag-specific CD8 T cells to overcome current challenges. DNA gated sorting (DGS) comprises 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 (Figure 1A). Unlike cell isolation with pMHC tetramers, our approach results in reversible clustering of pMHCI such that DGS isolation results in ‘scarless’ CD8 T cells to prevent TCR activation. These advances will be validated using CD8 T cells from transgenic mice (OT1, Pmel, P14), viral infection models (LCMV), and healthy human samples (EBV, CMV, Influenza). We anticipate that the success of our proposal will enable numerous applications spanning fundamental studies to cell therapies.


Materials and Methods:: Materials and Methods: C-terminal cysteine modified pMHCI molecule was conjugated to amine- and Cy5-modified target probe (TP) DNA (Integrated DNA Technologies) through hydrazone chemistry (pMHCI-TP-Cy5). Sorting using pMHCI-DNA was tested using splenocytes from P14 transgenic mouse model. Efficiency of sorting was validated through flow cytometry. Splenocytes were CD8+ T cell sorted (Miltenyi Biotec). CD8+ T cells were activated using CD3/CD28 activation beads (Miltenyi Biotec) and cultured for 3 days in 30 IU/mL IL-2 at 37° C at 5% CO₂ in mouse T cell media.


Results, Conclusions, and Discussions:: Results and Discussion: Sorting with Gp33-D^b pMHCI-TP-Cy5 increased Gp33+ T cell population from 28.7% to 95.5% (Figure 1B). Sorted Gp33+ T cell had similar percentage of pMHC-TP-Cy5 on the cell surface with three-fold higher Cy5 MFI over unsorted (Figure 1C). After sorting, Gp33+ T cells were cultured for three days after which were stained for expression of activation makers CD69 (early-activation) and CD25 (late-activation). Sorting resulted in no significant expression of CD69 and CD25 over MACS sorted CD8+ T cells signifying that the pMHC-TP sorting does not impact T cell product (Figure 1D).

Conclusion: We demonstrate an approach to capture and elute Ag-specific T cells using pMHCI-TP without significant impact on T cell product. Looking forward, we envision that we can use pMHC-TP to sort out defined Ag-specific T cells to manufacture defined T cell therapy products.


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