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.

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.

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).

Innate immune induction and influenza protection elicited by a response-selective agonist of human C5a.

The anaphylatoxin C5a is an especially potent mediator of both local and systemic inflammation. However, C5a also plays an essential role in mucosal host defense against bacterial, viral, and fungal infection. We have developed a response-selective agonist of human C5a, termed EP67, which retains the immunoenhancing activity of C5a at the expense of its inflammatory, anaphylagenic properties. EP67 insufflation results in the rapid induction of pulmonary cytokines and chemokines. This is followed by an influx of innate immune effector cells, including neutrophils, NK cells, and dendritic cells. EP67 exhibits both prophylactic and therapeutic protection when tested in a murine model of influenza A infection. Mice treated with EP67 within a twenty-four hour window of non-lethal infection were significantly protected from influenza-induced weight loss. Furthermore, EP67 delivered twenty-four hours after lethal infection completely blocked influenza-induced mortality (0% vs. 100% survival). Since protection based on innate immune induction is not restricted to any specific pathogen, EP67 may well prove equally efficacious against a wide variety of possible viral, bacterial, and fungal pathogens. Such a strategy could be used to stop the worldwide spread of emergent respiratory diseases, including but not limited to novel strains of influenza.