Using Mass Spectrometry for Mass T cell Epitope Discovery
CyTOF: A New Way to Profile Cells
Time of Flight Mass Cytometry (CyTOF) is a relatively new multiparametric technology. It far outpaces standard fluorescence-based flow cytometry in the number of parameters it can measure on a single cell at once.
In CyTOF, antibodies tagged with rare transition element isotopes label cellular antigens of interest. A time-of-flight mass cytometer then measures the amount of each label, as discussed previously. Earlier studies measuring 34 cell surface and intracellular proteins with this technology revealed high-dimensional complexity in the heterogeneity of human bone marrow and CD8+ T cell populations.
In a July 2013 article in Nature Biotechnology, Newell et al. pushed CyTOF and immunology a step further. They used CyTOF with peptide-MHC tetramers to measure the frequency of Rotavirus antigen-specific T cells in human peripheral blood mononuclear cells (PBMCs) and jejunal tissue.
Solving the Metal-Label Bottleneck with “Bar-Coding”
CyTOF can theoretically measure 100-200 parameters at once, depending on the instrument – far more than standard fluorescence-based flow cytometry. So far, though, only about 40 metal ions have been used for antibody labeling, and more metal-chelating technology is needed to use the CyTOF instrument’s full capacity.
The authors worked around this limit with a “bar-coding” method: each tetramer is labeled with a different combination of three out of ten metal ions, allowing for up to 120 distinct metal combinations.
Study Design
The authors set out to identify Rotavirus epitopes recognized by human CD8+ T cells, in the context of the MHC class I allele HLA-A*0201. Until this study, only two Rotavirus epitopes recognized by T cells had been identified, and little was known about the phenotypic and functional diversity of antigen-specific T cells for any given pathogen. Progress has been limited by the technical difficulty of accurately predicting epitopes and by the small number of cells available from human blood samples.
This method represents a major step forward for identifying more antigen-specific T cell epitopes and classifying these cells functionally. Using an MHC-prediction algorithm, the authors identified 77 candidate Rotavirus peptides that bind to HLA-A*0201. They added 32 positive and negative control tetramers, for a total of 109 labeled tetramers used to stain each sample at once. They combined this with 23-27 metal-chelated antibodies against cell surface and intracellular antigens, to phenotypically characterize the T cells. A specialized Matlab script analyzed the resulting high-dimensional mass spectrometry data from the PBMC and jejunal samples.
Key Findings
Across 17 healthy donors, the authors found CD8 T cell populations specific to two Rotavirus peptides, plus an average of 6-7 peptides from other viruses, including influenza, EBV, and CMV. They further characterized these antigen-specific T cell populations by their surface and intracellular marker expression.
CD8 T cells specific to six Rotavirus epitopes – including the two previously known ones – were repeatedly detected in PBMCs from at least two individuals. Of these, CD8 cells specific for a Rotavirus peptide from the VP3 protein were the most common among healthy donor PBMCs. They were also phenotypically distinct: an effector memory subtype, compared with the central memory phenotype typical of T cells specific to the other Rotavirus peptides. VP3-specific T cells were also uniquely present in jejunal tissue from obese patients who had undergone gastric bypass surgery.
In total, this methodology discovered at least 4 new Rotavirus peptides, along with unique characteristics of the different antigen-specific CD8 T cell populations.
Why It Matters
Combining CyTOF technology with tetramer “bar-coding” opens the door to a major expansion beyond fluorescence-based flow cytometry for identifying antigen-specific T cell populations. Vaccine development remains an ongoing goal for treating and preventing infectious disease and cancer. To support that goal, it matters not only to identify peptides that trigger a T cell response, but also to functionally characterize those T cells, so researchers can better promote the desired immune response.
Further Reading:
Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization. Newell EW, Sigal N, Nair N, Kidd BA, Greenberg HB, Davis MM. Nat Biotechnol. 2013 Jul;31(7):623-9. doi: 10.1038/nbt.2593. Epub 2013 Jun 9.
Cracking the code of human T-cell immunity. Harvey CJ, Wucherpfennig KW. Nat Biotechnol. 2013 Jul 9;31(7):609-10. doi: 10.1038/nbt.2626.
Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity. 2012 Jan 27;36(1):142-52. doi: 10.1016/j.immuni.2012.01.002.
Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Bendall SC, Simonds EF, Qiu P, Amir el-AD, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe’er D, Tanner SD, Nolan GP. Science. 2011 May 6;332(6030):687-96. doi: 10.1126/science.1198704.
Sanguine supplies research-grade human PBMCs for studies like this.
