Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.
The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56hiversus CD56dimdefined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.
Department of Microbiology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA.
Bertram
B
Bengsch
Institute for Immunology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA; Corporal Michael J. Crescenz Department of Veterans Affairs Medical Center, Philadelphia, PA 19104, USA.
Takuya
T
Ohtani
Institute for Immunology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA; Department of Medicine, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA.
Ramin Sedaghat
RS
Herati
Department of Pathology and Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands.
Niels
N
Bovenschen
Department of Medicine, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA; Corporal Michael J. Crescenz Department of Veterans Affairs Medical Center, Philadelphia, PA 19104, USA.
Kyong-Mi
KM
Chang
Department of Microbiology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA. Electronic address: wherry@mail.med.upenn.edu.
E John
EJ
Wherry
D019002:CD56 Antigen / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000502:physiology*; D000073883:CX3C Chemokine Receptor 1 / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D064368:Healthy Volunteers; D006801:Humans; D007156:Immunologic Memory; D007694:Killer Cells, Natural / Q000502:physiology*; D013058:Mass Spectrometry / Q000379:methods*; D052899:Pore Forming Cytotoxic Proteins / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D059010:Single-Cell Analysis; D018417:Th1 Cells / Q000276:immunology*
D019002:CD56 Antigen; D000073883:CX3C Chemokine Receptor 1; C095235:CX3CR1 protein, human; D052899:Pore Forming Cytotoxic Proteins
Cytotoxic; Granzymes and perforin; Mass cytometry; NK cell; T cell; T cell differentiation