Combining next-generation sequencing and immune assays: a novel method for identification of antigen-specific T cells

PLoS One. 2013 Sep 19;8(9):e74231. doi: 10.1371/journal.pone.0074231. eCollection 2013.


In this study, we combined a novel sequencing method, which can identify individual clonotypes based on their unique T cell receptor (TCR) rearrangement, with existing immune assays to characterize antigen-specific T cell responses. We validated this approach using three types of assays routinely used to measure antigen-specific responses: pentamers which enable identification of T cells bearing specific TCRs, activation marker expression following antigen stimulation and antigen-induced proliferation to identify cytomegalovirus (CMV) specific clonotypes. In one individual, 8 clonotypes were identified using a pentamer reagent derived from the CMV pp65 protein. The same 8 clonotypes were also identified following sequencing of cells that upregulated an activation marker following incubation with an identical peptide derived from pp65. These 8 and an additional 8 clonotypes were identified using a more sensitive CFSE-based proliferation assay. We found clear sequence homology among some of the clonotypes identified, and the CDR3 region in one clonotype was identical to a previously published pp65-specific clonotype sequence. Many of these CMV-specific clonotypes were present at frequencies below 10(-5) which are undetectable using standard flow-cytometric methods. These studies suggest that an immune response is comprised of a diverse set of clones, many of which are present at very low frequencies. Thus, the combination of immune assays and sequencing depicts the richness and diversity of an immune response at a level that is not possible using standard immune assays alone. The methods articulated in this work provide an enhanced understanding of T cell-mediated immune responses at the clonal level.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antigens / immunology
  • Antigens, Viral / immunology
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism
  • Complementarity Determining Regions / chemistry
  • Complementarity Determining Regions / genetics
  • Complementarity Determining Regions / metabolism
  • Cytomegalovirus / immunology
  • Epitopes, T-Lymphocyte / immunology*
  • Flow Cytometry / methods
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Immunoassay / methods*
  • Immunophenotyping
  • Lymphocyte Activation / immunology
  • Phosphoproteins / immunology
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / metabolism
  • Receptors, Antigen, T-Cell, alpha-beta / genetics
  • Receptors, Antigen, T-Cell, alpha-beta / metabolism
  • T-Lymphocyte Subsets / immunology*
  • T-Lymphocyte Subsets / metabolism
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / metabolism
  • Viral Matrix Proteins / immunology


  • Antigens
  • Antigens, Viral
  • Complementarity Determining Regions
  • Epitopes, T-Lymphocyte
  • Phosphoproteins
  • Receptors, Antigen, T-Cell
  • Receptors, Antigen, T-Cell, alpha-beta
  • Tumor Necrosis Factor Receptor Superfamily, Member 9
  • Viral Matrix Proteins
  • cytomegalovirus matrix protein 65kDa

Grant support

Sequenta Inc. funded this research and had a role in the study design, data collection, analysis, decision to publish and preparation of the manuscript.