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High-throughput single cell RNA-seq (scRNA-seq) has transformed our understanding of complex and heterogenous immune populations. New advances in scRNA-seq are expanding the number of molecules that can be profiled at the single cell level, such as oligo-conjugated antibody technologies that enable protein-expression profiling alongside mRNA. Although the ability to sequence the T-cell and B-cell immune repertoire can provide crucial insights into the complexities of the adaptive immune system, thus advancing discoveries in immunology and immuno-oncology, the ability to extract this information from single cells requires new technology to profile regions of the mRNA that are missed by conventional 3’ scRNA-seq. Here, we demonstrate a novel approach to extract immune-repertoire information, in addition to gene-expression information from the same cells, by using the BD Rhapsody™ Single-Cell Analysis system. In a single workflow, we profiled a panel of 400 mRNA targets and 40 protein markers, as well as the CDR3 region of T- and B-cell receptors in thousands of human PBMCs. After using the mRNA and protein information to robustly annotate different lymphocyte populations, we examined the T- and B-cell repertoires for differences in their clonal diversity. We were able to identify clonally expanded T cells and B cells and examine how their gene-expression profiles differ from that of nonexpanded cells. The study demonstrates the power of combining mRNA, protein, and immune-cell repertoire analysis at the single cell level to answer complex and important biological and clinical questions.

Insights Immunology 2021

Simultaneous mRNA, protein and immune-repertoire profiling of thousands of single cells

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Cancer is associated with concomitant myeloid cell responses that are characterized by an expansion of tumor associated myeloid cells including, myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs) and neutrophils (TANs). These myeloid derived cells are known to modulate immune responses that enhance cancer cell stemness, angiogenesis, metastasis, invasion and immune escape. The prevailing paradigm is that cancer interferes with hematopoiesis and skews the host system to generate cells of myeloid origin with tumor-promoting functions. Intriguingly, tumor derived factors can perturbate normal bone marrow (BM) hematopoiesis and promote extramedullary hematopoiesis in organs such as the spleen (SP). Advances in single-cell multiomic technologies now enable the analysis of high-dimensional protein and mRNA expression from thousands of cells simultaneously. Using the power of the BD Rhapsody™ Single-Cell Analysis System, we have carefully investigated melanoma associated extramedullary hematopoiesis in mice. To do this, we isolated four hematopoietic stem and progenitor cell populations simultaneously, including Lin- Sca1+ cKit+ (LSK), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocyte-erythrocyte progenitor (MEP) cells from the spleen and bone marrow of melanoma-bearing mice using the BD FACSMelody™ Cell Sorter followed by single-cell analysis using a BD® AbSeq panel for surface protein expression and single-cell whole transcriptome analysis using BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit. Using advanced analysis plugins in SeqGeq™ Software, we were able to analyze these hematopoietic stem and progenitor cells in this systemic immuno-suppressive myeloid environment. These advanced technologies are critical to uncover tumor mediated abnormalities in hematopoiesis.

A single-cell multiomics approach to study tumor-driven perturbations during hematopoiesis in mice

As for most neoplasms, the pathogenesis and progression of B-cell malignancies is a dynamic process involving reciprocal interactions between malignant cells and surrounding components in the tumor microenvironment. In the case of certain B-cell lymphomas, neoplastic cells migrate to lymphoid sites and prompt the infiltration of non-malignant immune cells. The infiltration of cytotoxic lymphocytes correlates with tumor cell killing and good prognosis. However, most immune cell types in the tumor niche often become unresponsive or end up promoting tumor growth. In this study we present a sorting strategy using the BD FACSymphony™ S6 Cell Sorter that enables concurrent isolation of six cell populations from a tumor niche in a model of B-cell lymphoma. Downstream application of single-cell sequencing was used to unravel the respective roles of each cell subset for the tumor’s progression and/or evasion. The six cell subsets were isolated from two groups of mice that had been inoculated with an OVA-expressing B-cell lymphoma cell line and subsequently with either wild-type or BTLA-deficient OVA-specific CD8+ T cells. Prior to sorting, each tissue was labeled with a unique single-cell multiplexing antibody, allowing us to combine biological replicates (2 mice per group) for a higher throughput. After sorting, cells from both groups were pooled and loaded onto cartridges from the BD Rhapsody™ Single-Cell Analysis System, followed by library preparations for Next-Generation Sequencing. In addition to 34 surface proteins, marked with BD AbSeq antibodies, sequencing of 397 target mRNA transcripts enabled a detailed examination of the cellular responses that were triggered in the presence of the tumor. During cell analysis, various algorithms in SeqGeq™ Software v1.6 segregated the cells from each individual mouse and identified the sorted populations based on their specific phenotypic and molecular signatures. Importantly, because sequencing is biased toward detecting high-frequency proteins and mRNAs, the cell sorting aided more robust single-cell profiling of rare cell subsets within the sorted populations. These analyses also contributed to a better understanding of the conditions underlying the differences between the two mice groups. In sum, we demonstrated a scalable and high-throughput approach that enabled a comprehensive characterization of critical cell populations in tumor responses. Since these cell populations constitute potential targets for the treatment of B-cell lymphomas, this platform, notably through the analysis of protein expression, offers an opportunity for identification of new therapeutic targets. 

Leveraging the power of high-parameter cell sorting and single-cell multiomics to profile intratumoral immune cells in a modal of B-cell lymphoma

Obesity can weaken the body’s immune system and reduce its ability to fight off infections. • To understand the role immune cells play in obesity-related disorders, thousands of immune cells isolated from lymphoid organs and adipose tissue from a diet-induced obesity mouse model were examined using the BD Rhapsody TM , a high-throughput single cell analysis system. • DNA-barcoded universal antibodies from the BDR Mouse Single-Cell Multiplexing Kit (SMK) allowed us to combine a total of 8 samples into a single pool, significantly reducing experimental scale and cost while eliminating any potential batch effect. • The targeted immune-focused mRNA (~400 genes) and BDR AbSeq panel (30 DNA-barcoded antibodies) provided robust clustering of immune cell types and showed genes such as Pdcd1 and Tigit that are critical to functional immune responses. • Using this multiomics approach, we propose a model to explain certain obesity-related disease phenotypes.

Simultaneous analysis of mRNA and proteins in mouse immune cells using the BD® Mouse Single-Cell Multiplexing Kit and the BD® AbSeq Assay on the BD Rhapsody™ System

T-cell exhaustion is a dysfunctional state caused by persistent antigen stimulation mediated by chronic infections or cancer. Exhausted T cells are characterized by upregulation of inhibitory receptors, reduced frequency of long-lived memory cells and loss of effector function, including cytotoxic activity and cytokine production. Current immunotherapy approaches are focused on reinvigorating exhausted T cells through immune-receptor blocking antibodies. A deeper understanding of the exhausted states of primary infiltrating lymphocyte is critical for exploring personalized treatments. We have previously developed an in vitro culture system and companion multicolor flow cytometry panels to assess the expression of inhibitory receptors and differentiation markers, as well as inflammatory cytokine production over time by different subsets of CD4 + or CD8 + cells in response to chronic or transient stimulation. The same model was used in this study to address fundamental questions about the application of BD® AbSeq assay + scRNA-seq for deep cell characterization: 1) How can multiomic analysis be used to design cost-effective experiments profiling many samples using scRNA-seq? 2) Can multiomic analyses be used to look for interesting patterns of expression in kinetic analysis? 3) Does the combination of protein- and gene-expression analysis at the single cell level, mediated by computational analysis, enable comprehensive cell characterization and identification of targets that can be screened in follow-up, high-throughput flow cytometry-based assays?

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Simultaneous mRNA, protein and immune-repertoire profiling of thousands of single cells

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