Exploration of T-Cell Diversity Using Mass Cytometry
T-cell diversity is multifactorial and includes variability in antigen specificity, differentiation, function, and cell-trafficking potential. Spectral overlap limits the ability of traditional flow cytometry to fully capture the diversity of T-cell subsets and function. The development of mass cytometry permits deep immunoprofiling of T-cell subsets, activation state, and function simultaneously from even small volumes of blood. This chapter describes our methods for mass cytometry and high-throughput data analysis of T cells in patient cohorts. We provide a pipeline that includes practical considerations when customizing a panel for mass cytometry. We also provide protocols for the conjugation and titration of metal-labeled antibodies (including two T-cell panels) and a staining procedure. Finally, with the aim to support translational science, we provide R scripts that contain a detailed workflow for initial evaluation of high-dimensional data generated from cohorts of patients.
Key wordsMass cytometry CyTOF T cells Systems biology High-throughput analysis R Clustering Data processing
We thank Divij Matthew, University of Pennsylvania, USA, for his help with the titration figure, as well as Jacob Bergstedt, Lund University, Sweden, for his help with the R scripts.
- 2.Bengsch B, Ohtani T, Khan O, Setty M, Manne S, O'Brien S, Gherardini PF, Herati RS, Huang AC, Chang KM, Newell EW, Bovenschen N, Pe'er D, Albelda SM, Wherry EJ (2018) Epigenomic-guided mass cytometry profiling reveals disease-specific features of exhausted CD8 T cells. Immunity 48(5):1029–1045.e5. https://doi.org/10.1016/j.immuni.2018.04.026CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Good Z, Borges L, Vivanco Gonzalez N, Sahaf B, Samusik N, Tibshirani R, Nolan GP, Bendall SC (2019) Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells. Nat Biotechnol 37(3):259–266. https://doi.org/10.1038/s41587-019-0033-2CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Patin E, Hasan M, Bergstedt J, Rouilly V, Libri V, Urrutia A, Alanio C, Scepanovic P, Hammer C, Jönsson F, Beitz B, Quach H, Lim YW, Hunkapiller J, Zepeda M, Green C, Piasecka B, Leloup C, Rogge L, Huetz F, Peguillet I, Lantz O, Fontes M, Di Santo JP, Thomas S, Fellay J, Duffy D, Quintana-Murci L, Albert ML (2018) Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors. Nat Immunol 19(3):302–314. https://doi.org/10.1038/s41590-018-0049-7CrossRefPubMedGoogle Scholar