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. 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.

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. This method is useful for in-depth single-cell characterization on sorted rare cell populations.

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. 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.

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. 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 tissue biopsies and other 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.

Functional phenotyping of circulating human cytotoxic T cells and NK cells using a 16-color flow cytometry panel.

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.

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. For complete details on the use and execution of this protocol, please refer to Mair et al. (2020).

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 104 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. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020).

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. Furthermore, there is still a paucity of analysis tools to visualize combined transcript-protein datasets. Here, we describe a targeted transcriptomics approach that combines an analysis of over 400 genes with simultaneous measurement of over 40 proteins on 2 × 104 cells in a single experiment. This targeted approach requires only about one-tenth of the read depth compared to a whole-transcriptome approach while retaining high sensitivity for low abundance transcripts. To analyze these multi-omic datasets, we adapted one-dimensional soli expression by nonlinear stochastic embedding (One-SENSE) for intuitive visualization of protein-transcript relationships on a single-cell level.

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. We address common misconceptions and caveats, and also discuss challenges that are associated with the quality control and analysis of these data sets.

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. To test for Ag receptor signaling, Nur77(gfp) BAC transgenic mice, which upregulate GFP in response to Ag receptor but not inflammatory signals, were analyzed. iNKT cells in the reporter mice infected with mouse CMV produced IFN-γ but did not upregulate GFP, consistent with their reported CD1d-independent activation. However, two bacteria known to produce lipid Ags for iNKT cells induced GFP expression and cytokine production. In contrast, although Salmonella typhimurium was proposed to induce the presentation of a self-lipid, iNKT cells produced IFN-γ but did not upregulate GFP postinfection in vivo. Even in CD1d-deficient hosts, iNKT cells were still able to produce IFN-γ after S. typhimurium infection. Furthermore, although it has been proposed that endogenous lipid presentation is a result of TLR stimulation of APCs, injection of different TLR agonists led to iNKT cell IFN-γ but not increased GFP expression. These data indicate that robust iNKT cell responses to bacteria, as well as viruses, can be obtained in the absence of antigenic stimulation.

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. In this article, we show that, although both NKT and NK cells are activated via cytokines, their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient, whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results, GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection, consistent with their virtual lack of IL-12 production, whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo, NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV, whereas NK cells were still activated. In turn, splenic NK cell activation was more IL-18 dependent. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally, mice lacking NKT cells showed reduced control of MCMV, and depleting NK cells further enhanced viral replication. Taken together, our results show that NKT and NK cells have differing requirements for cytokine-mediated activation, and both can contribute nonredundantly to MCMV defense, revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses.

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. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL-mediated killing led to a significant defect in pathogen clearance. Together, these data suggest an innate role for memory CD8 T cells in the early immune response before the onset of a de novo generated, antigen-specific CD8 T cell response.

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. These compounds induce enhanced IFN-γ that correlates with detergent-resistant binding to CD1d and an increased stability of the lipid-CD1d complexes on antigen-presenting cells. Structural analysis on one of the analogs indicates that it is more deeply bound inside the CD1d groove, suggesting tighter lipid-CD1d interactions. To our knowledge, this is the first example in which structural information provides an explanation for the increased lipid-CD1d stability, likely responsible for the Th1 bias. We provide insights into the mechanism of IFN-γ-inducing compounds, and because our compounds activate human NKT cells, they could have therapeutic utility.

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-ß-dependent Foxp3(+) Treg induction but inhibits TGF-ß-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism, we investigated the ability of HSC to function as regulatory APC. Different from previous reports, we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function, HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast, HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells, whereas they greatly inhibited the Th17 differentiation. Furthermore, the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders, and therefore, by promoting Tregs and suppressing Th17 differentiation, they might represent key players in the mechanism that drives liver-induced tolerance.

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 2.3 Šresolution and established through additional lipid loading experiments that CL, a tetra-acylated phospholipid, binds to murine CD1d with two alkyl chains buried inside the CD1d binding groove and the remaining two exposed into the solvent. We furthermore demonstrate the functional stimulatory activity of CL, showing that splenic and hepatic γδ T cells from healthy mice proliferate in vitro in response to mammalian or bacterial CL in a dose-dependent and CD1d-restricted manner, rapidly secreting the cytokines IFN-γ and RANTES. Finally, we show that hepatic γδ T cells are activated in vivo by CD1d-bearing dendritic cells that have been pulsed with CL, but not phosphatidylcholine. Together, these findings demonstrate that CD1d is able to bind and present CL to a subset of CL-responsive γδ T cells that exist in the spleen and liver of healthy mice and suggest that these cells could play a role in host responses to bacterial lipids and, potentially, self-CL. We propose that CL-responsive γδ T cells play a role in immune surveillance during infection and tissue injury.

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. iNKT cells also produce IFN-gamma in response to mouse CMV infection. Their mechanism of mouse CMV detection is quite similar to that of CpG, requiring both TLR9 signaling and IL-12 secretion, while the need for CD1d expression is relatively minor. Consequently, iNKT cells have the ability to respond to a variety of microbes, including viruses, in an Ag-independent manner, suggesting they may play a broad role in antipathogen defenses despite their limited TCR repertoire.

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. Although anti-HY CD4(+) T cell responses are readily detectable day 5 posttransfer, CD8(+) responses are undetectable until day 10. The early CD4(+) response is not dependent on direct presentation of Ag by donor male cells, but depends on presentation of the male cells by recipient APC. The CD4(+) T cell response is required for the priming of CD8(+) T cell effector responses and rejection of HY-incompatible cells. Unexpectedly, HY-specific CD4(+) T cells are also capable of efficiently lysing target cells in vivo. The delay in the CD8(+) T cell response can be largely abrogated by depleting T cells from the male inoculum, and donor male CD8(+) T cells in particular suppress host anti-HY CD8(+) responses. These data demonstrate dramatic differences in host T cell responses to noninflammatory Ags compared with responses to pathogens. We explain the delayed CD8(+) response by proposing that there is a balance between cross-presentation of Ag by helper cell-licensed dendritic cells, on the one hand, and veto suppression by live male lymphocytes on the other.

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. 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.

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. Retroviral expression of TCRalpha 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.

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. Further experiments suggested that these two observations are linked and that the CD8 population in CD4-/- mice may contain a majority of T cells that were actually selected by recognition of MHC class II molecules in the thymus. These results have implications for understanding CD4 versus CD8 lineage commitment in the thymus, and for the practical use of CD4-/- mice as models of helper deficiency.