The consequence of mismatched buffers in purity checks when spectral cell sorting.

Although spectral flow cytometry has become a ubiquitous tool for cell analysis, the use of spectral cytometry on cell sorters requires additional considerations arising from the unique requirements of sorting workflows. Here, we show that care should be taken when ascertaining the purity of a sort on a spectral cell sorter, as the mismatch of buffers used for initial sample suspension and the buffers used for sort collection can affect the unmixing of the data, potentially giving rise to erroneous purity check results.

Evaluation of single-cell sorting accuracy using antibody-derived tag-based qPCR.

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.

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

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.

Co-staining with Fluorescent Antibodies and Antibody-Derived Tags for Cell Sorting Prior to CITE-Seq.

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.

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

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 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.

Flow cytometry analysis of protein expression using antibody-derived tags followed by CITE-Seq.

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.

Co-staining human PBMCs with fluorescent antibodies and antibody-oligonucleotide conjugates for cell sorting prior to single-cell CITE-Seq.

The dual interrogation of the transcriptome and proteome with single-cell resolution provides exquisite insights into immune mechanisms in health and disease. Here, we describe a cutting-edge protocol wherein we combine Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), a technique utilizing antibody-oligonucleotide conjugates (AOCs), with fluorescence-activated cell sorting to enrich rare cell populations. Our protocol incorporates co-staining of cells with both fluorescent antibodies and AOCs simultaneously for subsequent input into the cell sorting and CITE-Seq pipeline.

AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level.

By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 10 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling.

A Targeted Multi-omic Analysis Approach Measures Protein Expression and Low-Abundance Transcripts on the Single-Cell Level.

High-throughput single-cell RNA sequencing (scRNA-seq) has become a frequently used tool to assess immune cell heterogeneity. Recently, the combined measurement of RNA and protein expression was developed, commonly known as cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcripts but also nearly doubles the sequencing read depth required per single cell.

High-Dimensional Immunophenotyping with Fluorescence-Based Cytometry: A Practical Guidebook.

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.

Antigen-dependent versus -independent activation of invariant NKT cells during infection.

CD1d-reactive invariant NKT cells (iNKT) play a vital role in determining the characteristics of immune responses to infectious agents. Previous reports suggest that iNKT cell activation during infection can be: 1) solely driven by cytokines from innate immune cells, 2) require microbial Ag, or 3) require self-Ag. In this study, we examined the role of Ag receptor stimulation in iNKT cells during several bacterial and viral infections.

Distinct requirements for activation of NKT and NK cells during viral infection.

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells, NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However, whether similar mechanisms govern activation of these two cell types, as well as the significance of NKT cells for host resistance, remain unknown.

Bystander-activated memory CD8 T cells control early pathogen load in an innate-like, NKG2D-dependent manner.

During an infection the antigen-nonspecific memory CD8 T cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T cell receptor (TCR) signals that bystander-activated, cytotoxic CD8 T cells (BA-CTLs) receive in vivo and found evidence of limited TCR signaling.

Glycolipids that elicit IFN-γ-biased responses from natural killer T cells.

Natural killer T (NKT) cells recognize glycolipids presented by CD1d. The first antigen described, α-galactosyl ceramide (αGalCer), is a potential anticancer agent whose activity depends upon IFN-γ secretion. We report two analogs of αGalCer based on a naturally occurring glycosphingolipid, plakoside A.

Hepatic stellate cells function as regulatory bystanders.

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms, however, underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite, retinoic acid (RA), as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation.

Cardiolipin binds to CD1d and stimulates CD1d-restricted γδ T cells in the normal murine repertoire.

Cardiolipin (CL), a major phospholipid in bacterial cell walls, is sequestered from the immune system in mammalian mitochondria and is, therefore, a potential danger signal. Based on growing evidence that phospholipids constitute natural ligands for CD1 and that CD1d-restricted T cells recognize phospholipids, we hypothesized that CD1d binds and presents CL and that T cells in the normal immune repertoire respond to CL in a CD1d-restricted manner. We determined the murine CD1d-CL crystal structure at 2.

Cutting edge: the mechanism of invariant NKT cell responses to viral danger signals.

Invariant NK T (iNKT) cells influence the response to viral infections, although the mechanisms are poorly defined. In this study we show that these innate-like lymphocytes secrete IFN-gamma upon culture with CpG oligodeoxynucleotide-stimulated dendritic cells (DCs) from mouse bone marrow. This requires TLR9 signaling and IL-12 secretion by the activated DCs, but it does not require CD1d expression.

The surprising kinetics of the T cell response to live antigenic cells.

Cooperation between CD4(+) and CD8(+) T cells is required for the proper development of primary effector and memory CD8(+) T cells following immunization with noninflammatory immunogens. In this study, we characterized murine CD4(+) and CD8(+) T cell responses to male-specific minor histocompatibility (HY) Ags following injection of live male cells into females of the same strain. Male cells are rejected 10-12 days after transfer, coinciding with the expansion and effector function of CD8(+) CTLs to two H-2D(b)-restricted epitopes.

Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells.

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.

Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors.

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 (Valpha2) TCRalpha chains associated with a transgenic TCRbeta chain that the TRand CD25- CD4+ TCR repertoires are similarly diverse, yet only partially overlapping.

The CD8 population in CD4-deficient mice is heavily contaminated with MHC class II-restricted T cells.

In experiments to study the impact of deficiency in CD4+ T cell help on the magnitude of CD8+ cytotoxic T cell response to pathogens, it was noted that in CD4 gene knockout mice, the CD8 population made significant responses to several nominally major histocompatibility complex (MHC) class II-restricted epitopes in addition to the expected responses to MHC class I-restricted epitopes. A similar response by CD8+ T cells to class II-restricted epitopes was not observed in wild-type mice, or in mice that had been acutely depleted of CD4+ T cells just before the immunization. Coincident with this unexpected response to class II-restricted epitopes, it was also observed that the CD8+ response to the class I-restricted epitopes was consistently lower in CD4-/- mice than in wild-type mice.

Scurfin (FoxP3) controls T-dependent immune responses in vivo through regulation of CD4+ T cell effector function.

Scurfin, the protein product of the FoxP3 gene, is a forkhead-family transcription factor that negatively regulates T cell function. Mice carrying a loss-of-function mutation in FoxP3 (scurfy mice) present with fatal autoimmune-like disease caused by hyperresponsive CD4(+) T cells. Mice that overexpress scurfin (FoxP3 Tg mice) possess fewer mature T cells with reduced functional capabilities compared with normal littermate control mice.