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

dCODE Dextramer® Reagents Identify Antigen-Specific Populations and Their TCR Clonotypes at Single-Cell Level

Adapted from Jacobsen, K. et. al. SITC 2019 poster P186

Background

dCODE Dextramer® (10x) reagents are DNA barcoded MHC Dextramer® reagents designed for use with 10x Genomics Feature Barcode protocol for Single-Cell Immune Profiling (Fig. 1). Each unique barcode is specific for the MHC-peptide displayed on the dCODE Dextramer®.

Fig.1 dCODE Dextramer®

Study Description

Goal:​ Detect antigen-specific T cells and their cognate T-cell receptors sequences in a human PBMC sample using a highly multiplexed panel of dCODE Dextramer® reagents.

  1. Healthy donor PBMC sample was stained with a pool of 50 different MHC I dCODE Dextramer® reagents displaying different viral and cancer epitopes
  2. dCODE Dextramer® positive cells were sorted by flow cytometry and loaded onto 10X Chromium controller
  3. Generation of three DNA libraries: 1) dCODE Dextramer® binders; 2) V(D)J sequences; 3) RNA expression. Each library sequenced by next-generation sequencing (Illumina)

 

Results

Four antigen-specific T-cell populations were identified: one for influenza (Flu), two for Epstein-Barr virus  (EBV-1 and EBV-2), one for MART-1 (Fig. 2). For each population, the paired clonal TCR sequences were directly obtained and quantified.
Multiple TCR clones were identified for each antigen antigen-specific T-cell population (Table 1). Highly expanded TCR clones were found in viral T-cell populations (EBV-1 and EBV-2). Fewer expanded TCR clones were found for MART-1.
To confirm whether the identified MART-1-specific T-cells represent a naïve population, it is possible to interrogate the available gene expression profile.

Fig. 2. Four antigen-specific T-cell populations identified after analysis of sequencing data

Fig. 2. Four antigen-specific T-cell populations identified after analysis of sequencing data

Specificity (MHC-peptide) Flu EBV-1 EBV-2 MART-1
Positive cells (number) 2594 1846 4472 187
Specific clones (number) 732 166 389 180
Most represented clone (number of cells) 110 1241 2373 8
Most represented clone (frequency) 4% 67% 53% 4%
Clone with >1 cell (number) 251 19 11 1
Clone with 1 cell (number) 481 147 345 179

Table 1. TCR clones for each antigen-specific population

Conclusions

  • dCODE Dextramer® technology enables the generation of highly multiplexed antigen-specificity data in a single experiment
  • Combining dCODE Dextramer® and 10x Chromium represents a powerful tool for deep phenotyping of immune relevant cells
  • Personalized dCODE Dextramer® libraries allow profiling of patients’ T cells and give a new understanding of T-cell immunity

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