Single cell detection of antigen-specific T cells by Cas12a of an LAMP DNA barcode

Introduction: Antigen-specific CD8 (AgS) T cells are key players in adaptive immunity that recognize fragments of antigens as peptides on major histocompatibility complexes (pMHC) through their T cell receptor (TCR). The presence of AgS can give insights into the strength of an immune response, the generation of immunity during vaccination, and the success of cancer immunotherapies. However, detecting AgS has several challenges as the affinity of TCR-pMHC interaction is order of magnitudes lower than those of antibodies, they are found at low frequency in peripheral blood, and expensive equipment or multi-day protocols are required. To overcome current challenges, we propose an assay for the sensitive, low-cost detection of AgS using DNA barcoded pMHC tetramers. To improve limit of detection of rare AgS, we are using loop mediated isothermal amplification (LAMP) to amplify a self-designed 200 nucleotide DNA barcode (BC) at a constant temperature, eradicating the need for an expensive thermocyclers. We conjugated this BC to streptavidin, which is conventionally used to create pMHC tetramers to stain AgS. To improve the sensitivity of this assay, we used crRNA-guided Cas12a for the collateral cleavage of DNA probes after initial recognition of the BC (Figure 1A). Currently, we validate this technology through the collateral cleavage of a fluorophore-quencher DNA probe after staining mouse AgS, but we foresee alternating between other DNA probes for the detection using low-cost methods (i.e. lateral flow assay).

Materials and

Methods: Here, we detect AgS T cells with pMHC tetramers conjugated to a BC that can be amplified using LAMP and detected using crRNA-guided Cas12a, which results in the cleavage of a DNA probe that is read using fluorescence. First, C-terminal cysteine modified streptavidin was conjugated to maleimide-modified Cy5 then to amine-modified BC through hydrazone chemistry (Stv-Cy5-BC). Splenocytes from C57BL/6 and P14 mouse were mixed to create mixtures of certain percentage or number of cells and stained with Db-Gp100 (non-cognate) or Db-Gp33 (cognate) tetramer made from Stv-BC. Additionally, to remove non-specific BC staining, CD8+ negative isolation and anti-PE isolation after staining with non-cognate or cognate pMHC tetramer made with Stv-PE was performed. Flow sorting was achieved by staining P14 transgenic splenocytes with anti-CD8 antibody and no, non-cognate, or cognate tetramer and sorted on CD8+ cells. LAMP of the cell mixture was performed at 65° C, which after crRNA-guided Cas12a detection of BC and cleavage of fluorophore-quencher DNA-probes was measured at 37° C at 556 nm for 120 minutes. Slope of the fluorescence was calculated using a MATLAB script that finds the slope over the increasing linear range of the curves.

Results, Conclusions, and Discussions: To determine if conjugating a 200 nucleotide BC impacts binding efficiency of pMHC tetramers, P14 splenocytes were stained with various molarities of Db-Gp33 tetramers made with Stv-Cy5 or Stv-Cy5-BC, resulting in a similar calculated KD, app between the two tetramers of 9.7 or 9.4, respectively (Figure 1B). As PBMCs contain a mixture of immune cells, we wanted to determine if a low percentage of CD8 T cells mixed in splenocytes could be detected over background staining. We found that only samples stained with cognate tetramer resulted in significant signal production over time compared to unstained cells (Figure 1C). Following, we wanted to see if the amount of signal produced over time is related to the amount of cells present (e.g. the amount of BC present). We see that more cells stained results in higher slope being present (Figure 1D). Finally, to create an sensitive assay, we tested the assay’s ability to detect a single AgS T cells, finding that we can detect single AgS using LAMP plus crRNA-guide Cas12a (Figure 1E). Collectively, our results show that we can stain and detect AgS using a sensitive, low-cost assay. Translating the read-out would provide a point-of-care test that requires no specialized instrumentation for rapid information about the state of AgS in an adaptive immune response

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