Abstract
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 CD56hi versus CD56dim defined 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.
Highlights
In response to infections or transformation, T and NK cells can directly kill target cells
We developed a mass cytometry panel to comprehensively interrogate cytotoxic molecule expression patterns, their relationship to T and NK cell lineages and the connection between T and NK differentiation stages and novel combinatorial patterns of cytotoxic molecule expression on a single-cell level (Table 1), and applied this approach to the analysis of PBMC from a cohort of healthy individuals
We identified T follicular helper-like cells (TFH) expressing CXCR5 +, regulatory T cells (Treg) based on Foxp3 expression, TH1 cells based on T-bet expression and naïve T cells based on CCR7, CD27, CD45RA (Fig. 1A, B)
Summary
In response to infections or transformation, T and NK cells can directly kill target cells This effector function can be exerted by the ligation of death receptors or by coordinated secretion of cytotoxic granules containing pore-forming proteins (perforin) and effector proteases (e.g., granzyme (Gzm) family, granulysin) (Voskoboinik et al, 2015). In that study, analysis of protein expression of cytotoxic molecules was limited to Gzm B and perforin These reports suggest that T cells may co-express different sets of cytotoxic molecules and that these patterns may reflect distinct specialized cytotoxic programs related to the state of T cell differentiation. Cytotoxic profiling has implications for the understanding of T cell function in infection, autoimmunity and tumor immunology
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