Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization

It is currently not possible to predict which epitopes will be recognized by T cells in different individuals. This is a barrier to the thorough analysis and understanding of T-cell responses after vaccination or infection. Here, by combining mass cytometry with combinatorial peptide–MHC tetramer staining, we have developed a method allowing the rapid and simultaneous identification and characterization of T cells specific for many epitopes. We use this to screen up to 109 different peptide–MHC tetramers in a single human blood sample, while still retaining at least 23 labels to analyze other markers of T-cell phenotype and function. Among 77 candidate rotavirus epitopes, we identified six T-cell epitopes restricted to human leukocyte antigen (HLA)-A*0201 in the blood of healthy individuals. T cells specific for epitopes in the rotavirus VP3 protein displayed a distinct phenotype and were present at high frequencies in intestinal epithelium. This approach should be useful for the comprehensive analysis of T-cell responses to infectious diseases or vaccines.

[1]  R. Ward.  Mechanisms of protection against rotavirus infection and disease. , 2009, The Pediatric infectious disease journal.

[2]  Yuzhang Wu,et al.  Identification of an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope in rotavirus VP6 protein. , 2006, The Journal of general virology.

[3]  C. Fiocchi,et al.  Isolation of Human Intestinal Mucosal Mononuclear Cells , 1996, Current protocols in immunology.

[4]  Karen Sachs,et al.  Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators , 2012, Nature Biotechnology.

[5]  Jelle Matthijnssens,et al.  Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments , 2008, Archives of Virology.

[6]  T. Schumacher,et al.  Design and use of conditional MHC class I ligands , 2006, Nature Medicine.

[7]  K. Ley,et al.  Role of beta7 integrins in intestinal lymphocyte homing and retention. , 2009, Current molecular medicine.

[8]  S. M. McDonald,et al.  Complete genome sequence analysis of candidate human rotavirus vaccine strains RV3 and 116E. , 2010, Virology.

[9]  D. Speiser,et al.  High Frequencies of Naive Melan-a/Mart-1–Specific Cd8+ T Cells in a Large Proportion of Human Histocompatibility Leukocyte Antigen (Hla)-A2 Individuals , 1999, The Journal of experimental medicine.

[10]  Sean C. Bendall,et al.  Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum , 2011, Science.

[11]  Ton N Schumacher,et al.  Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers , 2009, Nature Methods.

[12]  Sean C. Bendall,et al.  Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. , 2012, Immunity.

[13]  Yuzhang Wu,et al.  A naturally processed epitope on rotavirus VP7 glycoprotein recognized by HLA-A2.1-restricted cytotoxic CD8+ T cells. , 2009, Viral immunology.

[14]  G. Kang,et al.  Rotavirus vaccination in developing countries. , 2012, Current opinion in virology.

[15]  Mario Roederer,et al.  A new “Logicle” display method avoids deceptive effects of logarithmic scaling for low signals and compensated data , 2006, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[16]  Morten Nielsen,et al.  Prediction of epitopes using neural network based methods. , 2011, Journal of immunological methods.

[17]  Alessandro Sette,et al.  The Immune Epitope Database 2.0 , 2009, Nucleic Acids Res..

[18]  J. Altman,et al.  Immediate Early Effector Functions of Virus-Specific CD8+CCR7+ Memory Cells in Humans Defined by HLA and CC Chemokine Ligand 19 Tetramers1 , 2003, The Journal of Immunology.

[19]  Jason S. Mitchell,et al.  Control of α4β7 Integrin Expression and CD4 T Cell Homing by the β1 Integrin Subunit , 2010, The Journal of Immunology.

[20]  Evan W. Newell,et al.  Simultaneous detection of many T-cell specificities using combinatorial tetramer staining , 2009, Nature Methods.

[21]  A. Bertoletti,et al.  Sources of diversity in T cell epitope discovery. , 2011, Frontiers in bioscience.

[22]  T. Schumacher,et al.  Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7 , 2008, Proceedings of the National Academy of Sciences.

[23]  B. Prasad,et al.  Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb. , 2002, The Journal of clinical investigation.

[24]  J. Altman,et al.  A robust method for production of MHC tetramers with small molecule fluorophores. , 2007, Journal of immunological methods.

[25]  Mark M. Davis,et al.  Interrogating the repertoire: broadening the scope of peptide–MHC multimer analysis , 2011, Nature Reviews Immunology.

[26]  Antonio Lanzavecchia,et al.  Central memory and effector memory T cell subsets: function, generation, and maintenance. , 2004, Annual review of immunology.

[27]  G. Gao,et al.  Germ Line-governed Recognition of a Cancer Epitope by an Immunodominant Human T-cell Receptor* , 2009, The Journal of Biological Chemistry.

[28]  Morten Nielsen,et al.  NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8–11 , 2008, Nucleic Acids Res..

[29]  Marion Pepper,et al.  Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. , 2007, Immunity.

[30]  A. Sewell,et al.  Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers , 2009, Journal of immunological methods.

[31]  Philip J. R. Goulder,et al.  Phenotypic Analysis of Antigen-Specific T Lymphocytes , 1996, Science.

[32]  Erin F. Simonds,et al.  A platinum‐based covalent viability reagent for single‐cell mass cytometry , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[33]  O. Ornatsky,et al.  Multiple cellular antigen detection by ICP-MS. , 2006, Journal of immunological methods.

[34]  B. Walker,et al.  Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. , 2003, The Journal of clinical investigation.