Single-cell level identification of antigen-specific T cells with dCODE Dextramer®

dCODE Dextramer® (HiT) Reagents Allow Multiplex Screening of Large Epitope Panels

Immudex, internal data 2020

Background

dCODE Dextramer® (HiT) carries a unique DNA barcode, specific for the MHC-peptide complex displayed on the Dextramer® (Fig. 1).

The MHC-peptide specificity can be identified by PCR and sequencing of the attached DNA barcode.

Fig.1 dCODE Dextramer® (HiT)

Study Description

Goal: Detect antigen-specific T-cell populations in a human PBMC sample using a multiplexed panel of dCODE Dextramer® (HiT) reagents.

  1. Healthy donor PBMC sample (haplotype A*02:01, A*29:02, B*35:01, B*57:01) stained with a pool of 56 MHC I dCODE Dextramer® (HiT) reagents displaying different viral and cancer epitopes. Each MHC-peptide specificity was made in duplicates, each with a different DNA barcode label.
  2. Following staining, dCODE Dextramer®-positive and -negative cells were individually sorted by flow cytometry.
  3. DNA barcodes bound to sorted cells were amplified by qPCR and sequenced. Specific enrichment of dCODE Dextramer®-positive cell populations was determined comparing with dCODE Dextramer®-negative cells
  4. Positive antigen-specific populations were confirmed by flow cytometry.

Results

dCODE Dextramer® (HiT) reagents with MHC alleles matching the donor’s haplotype detected four antigen-specific T-cell populations with a signal higher than the threshold (Fig.2). Results were confirmed by flow cytometry, which enabled identifying the same antigen-specific T-cell populations (Fig.3).

Fig. 2. Detection of T-cell responses by PCR and NGS. A threshold was set at a value of 2 (two-fold higher than the negative control signal) to identify positive antigen-specific T cells.
Asterisks (*) indicate antigen-specific T cells detected with a fold increase higher than 2 that were further validated by flow cytometry.

Fig. 3. Flow cytometry validation of the antigen-specific T cells detected by PCR and NGS. ​

Conclusions

  • dCODE Dextramer® (HiT) technology enables the generation of highly multiplexed antigen specificity data in a single experiment, allowing high-throughput epitope discovery and efficient neoantigen screening
  • Following identification, positive hits can be validated by flow cytometry and further analyzed using single-cell platforms for multi-omics analysis

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