The key is to define your T cell populations of interest with correct gating strategies and to back up your T cell subset findings with functional analysis of these subsets. Overall, flow cytometry is an ideal way to visualize T cells in a heterogeneous sample. For example, defining a population as bearing a “surface phenotype resembling a central memory T cell, while secreting Th1 cytokines” is a safe and accurate way to go (see above Figure). When in doubt, let a cell’s actions lead its definition, and step back from pigeonholing via previously defined label.
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This is where flow cytometry cell sorting is advantageous, for sorting and functionally profiling T cell subsets (via a series of elispots or multiplex supernatant analysis of ex vivo cultures, for example) allows highly sensitive, multi-analyte profiling with far fewer cells than would be required for intracellular cytokine staining.Īnd what is a cell whose face certainly looks naïve (CD45RA+, CCR7+) yet is secreting IFN-g and TNF-a (as such cells have been found in human samples)? The winds of T cell differentiation ‘states’ or who is or is not a bona-fide Treg may change, but a functional profile will serve to anchor the biological relevance and potential role of your T cell population of interest on more solid ground.
When trolling these waters, it’s best to pair our T cell subset findings with functional profiling of the population of interest. Why T Cell Function Should Guide Your T Cell Analysis Some markers can change like the wind (it seems) and we must use caution to not underestimate the complex web of factors beyond mere ‘differentiation’ that impacts a T cell, causing it to express a given marker at a moment in time. However, forcing square pegs into the round holes present in many linear differentiation models (with stages in the process noted via marker changes) can serve to further cloud the T cell field, potentially leading to misleading claims about the actual status of a T cell in a given sample or group of patients. This, in turn, could allow better understanding of how the composite immune system functions in health and disease states (know your players, know the game) and also facilitate discovery of new therapeutic targets (á la anti-PD-1). By revealing more of the complex marker distribution on individual cells, we gain a clearer picture of the heterogeneity of this facet of the immune cell compartment as a whole. With this increase in parameter detection per sample, the subsetting of T cells in the literature has exploded.įor example, when considering one aspect of T cell biology, the naïve to memory differentiation post-antigen exposure, the field has transcended from an era of two subsets (naïve and memory) into the era of Central Memory, Transitional Memory, Effector Memory, Terminally Differentiated Effector Memory, etc.Īt first glance, these larger panels bring clarity to the T cell landscape. An antibody panel with more than 10-colors is no longer uncommon, and with the adaptation of the more recent Brilliant dyes, 14+ colors are now very feasible on many instruments. The Benefits And Caveats Of Advanced T Cell Antibody Panelsįlow panel sizes have expanded dramatically in the last 15+ years and continue to do so. Such gating strategies, when paired with CD3 inclusion, doublet exclusion, and appropriate live/dead gating, allow clear, accurate visualization of your T cell population of interest and enumeration of frequency in your sample. Single cell visualization of T cells in a heterogeneous sample is clearest when the defined T cell populations are determined with ‘rock-solid’ gating and data analysis strategies.įor example, detection of the total CD4 and CD8 T cell compartments (via CD3+ CD4+ and CD3+ CD8+ cells, respectively) is straightforward also, T cell populations that are clearly defined by surface antigen expression include antigen-specific (tetramer-binding) memory T cell clones and invariant Natural Killer T (iNKT) cells, a unique T cell subset discerned via binding to a CD1d-glycolipid loaded tetramer. With a large sensitivity range for fluorescent probes, >95% sampling efficiency, and the ability to sort populations of interest for further study, fluorescent-based cytometry remains a tool of choice for T cell analysis. It has been successfully used for many decades to accurately visualize and enumerate a variety of T cell subsets. Flow Cytometry is a remarkably powerful tool for the study of T cells.
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