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Single‐cell sorting (index sorting) is a widely used method to isolate one cell at a time using fluorescence‐activated cell sorting (FACS) for downstream applications such as single‐cell sequencing or single‐cell expansion. Despite widespread use, few assays are available to evaluate the proteomic features of the sorted single cell and further confirm the accuracy of single‐cell sorting. With this caveat, we developed a novel assay to confirm the protein expression of sorted single cells by co‐staining cells with the same marker using both antibody‐derived tags (ADTs) and fluorescent antibodies. After single‐cell sorting, we amplified the oligo of the ADT reagent as a surrogate signal for the protein expression using multiplex TaqMan™ qPCR on sorted cells. This assay is not only useful for confirming the identity of a sorted single cell but also an efficient method to profile proteomic features at the single‐cell level. Finally, we applied this assay to characterize protein expression on whole cell lysate. Because of the sensitivity of the TaqMan™ qPCR, we can detect protein expression from a small number of cells. In summary, the ADT‐based qPCR assay developed here can be utilized to confirm single‐cell sorting accuracy and characterizing protein expression on both single cells and whole cell lysate. [ABSTRACT FROM AUTHOR]

We report the development of an optimized 50‐color spectral flow cytometry panel designed for the in‐depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide‐spread implementation of such a panel across different cohorts and samples, we established a trimmed‐down 45‐color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome‐specific resolution in the context of a 50‐color unmixing matrix. [ABSTRACT FROM AUTHOR]

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE‐Seq) is a single‐cell phenotyping method that uses antibody‐derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single‐cell level. Despite the increased popularity of this technique to study cellular heterogeneity and dynamics, detailed methods on how to choose ADT markers and ensuring reagent performance in biological relevant systems prior to sequencing is not available. Here we describe a novel and easy‐to‐use multiplex flow proxy assay in which multiple protein markers can be measured simultaneously using a combination of ADT reagents and dye‐oligo conjugates by flow cytometry. Using dye‐oligo conjugates with sequences complementary to the ADT reagents, we can achieve specific binding and evaluate protein marker expression in a multiplex way. This quality control assay is useful for guiding ADT marker choice and confirming protein expression prior to sequencing. Importantly, the labeled cells can be directly isolated based on the specific fluorescence from dye‐oligo conjugates using a flow cytometry cell sorter and processed for downstream single‐cell multiomics. Using this streamlined workflow, we sorted natural killer cells and T cells efficiently using only ADT and dye‐oligo reagents, avoiding the possibility of decreased marker resolution from co‐staining cells with ADT and fluorescent antibodies. This novel workflow provides a viable option for improving ADT marker choice and cell sorting efficiency, allowing subsequent CITE‐Seq. [ABSTRACT FROM AUTHOR]

Author(s): Matthew A. Williams [1]; Aaron J. Tyznik [1]; Michael J. Bevan (corresponding author) [1] Although interleukin-2 (IL-2) was initially characterized as the primary T-cell growth factor following in vitro [...]

Although interleukin-2 (IL-2) was initially characterized as the primary T-cell growth factor following in vitro activation, less is known about its role in shaping T-cell responses to acute infections in vivo. The use of IL-2- or IL-2-receptor-deficient mice is problematic owing to their early development of autoimmunity, attributable to the central role of IL-2 in the generation, maintenance and function of CD4+CD25+ regulatory T cells. To bypass these inherent difficulties, we have studied the effect of IL-2 on T-cell responses to acute infections by adopting a mixed chimaera strategy in which T cells lacking the high-affinity IL-2 receptor could be studied in an otherwise healthy mouse containing a full complement of regulatory T cells. Here we show that although IL-2 signalling to pathogen-specific CD8+ T cells affects the number of developing effector and memory cells very little, it is required for the generation of robust secondary responses. This is not due to an altered T-cell-receptor repertoire development or selection, and does not reflect an acute requirement for IL-2 during secondary activation and expansion. Rather, we demonstrate a previously unappreciated role for IL-2 during primary infection in programming the development of CD8+ memory T cells capable of full secondary expansion. These results have important implications for the development of vaccination or immunotherapeutic strategies aimed at boosting memory T-cell function. [ABSTRACT FROM AUTHOR]

Naturally arising CD25+ CD4+ regulatory T cells (TR) play an important role in the prevention of autoimmunity. TCR specificity is thought to play a critical role in TR development and function, but the repertoire and specificity of TR TCRs remain largely unknown. We find by sequencing of TRAV14 (Vα2) TCRα chains associated with a transgenic TCRβ chain that the TR and CD25- CD4+ TCR repertoires are similarly diverse, yet only partially overlapping. Retroviral expression of TCRα genes in TCR transgenic RAG-deficient T cells revealed that a high frequency of TCRs derived from CD25+ but not CD25- CD4+ T cells confers the ability to rapidly expand upon transfer into a lymphopenic host. Thus, these data show that a large proportion of naturally arising TR have substantially more efficient interactions with MHC class II bound peptides from the peripheral self than CD25- T cells. [Copyright &y& Elsevier]

The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), a technique utilizing antibody-derived tags (ADTs) to profile mRNA and proteins simultaneously via sequencing, with fluorescence-activated cell sorting to enrich cell populations. Our protocol provides step-by-step guidance on co-staining cells with both fluorescent antibodies and ADTs simultaneously, instructions on cell sorting and an overview of the single-cell capture workflow using the BD Rhapsody™ system. This method is useful for in-depth single-cell characterization on sorted rare cell populations., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. Herein, we describe a simplified intracellular staining workflow for classification of circulating human T and NK cells with cytolytic potential. We suggest reagents for measuring cytolytic proteins and identification of cell subsets within conventional and unconventional T cells and NK cells., Competing Interests: G.V.B., N.K., S.R., S.J.W., and A.J.T. are employees of BD Biosciences. E.L. is a former employee of BD Biosciences., (© 2021 The Author(s).)

Recent technological advances have greatly diversified the platforms that are available for high-dimensional single-cell immunophenotyping, including mass cytometry, single-cell RNA sequencing, and fluorescent-based flow cytometry. The latter is currently the most commonly used approach, and modern instrumentation allows for the measurement of up to 30 parameters, revealing deep insights into the complexity of the immune system.Here, we provide a practical guidebook for the successful design and execution of complex fluorescence-based immunophenotyping panels. We address common misconceptions and caveats, and also discuss challenges that are associated with the quality control and analysis of these data sets.