Immune Cell Landscape of Patients With Diabetic Macular Edema by Single-Cell RNA Analysis

Purpose: We performed single-cell RNA sequencing (scRNA-seq), an unbiased and high-throughput single cell technology, to determine phenotype and function of peripheral immune cells in patients with diabetic macular edema (DME).Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from DME patients and healthy controls (HC). The single-cell samples were loaded on the Chromium platform (10x Genomics) for sequencing. R package Seurat v3 was used for data normalizing, clustering, dimensionality reduction, differential expression analysis, and visualization.Results: We constructed a single-cell RNA atlas comprising 57,650 PBMCs (24,919 HC, 32,731 DME). We divided all immune cells into five major immune cell lineages, including monocytes (MC), T cells (TC), NK cells (NK), B cells (BC), and dendritic cells (DC). Our differential expression gene (DEG) analysis showed that MC was enriched of genes participating in the cytokine pathway and inflammation activation. We further subdivided MC into five subsets: resting CD14++ MC, proinflammatory CD14++ MC, intermediate MC, resting CD16++ MC and pro-inflammatory CD16++ MC. Remarkably, we revealed that the proinflammatory CD14++ monocytes predominated in promoting inflammation, mainly by increasingly production of inflammatory cytokines (TNF, IL1B, and NFKBIA) and chemokines (CCL3, CCL3L1, CCL4L2, CXCL2, and CXCL8). Gene Ontology (GO) and pathway analysis of the DEGs demonstrated that the proinflammatory CD14++ monocytes, especially in DME patients, upregulated inflammatory pathways including tumor necrosis factor-mediated signaling pathway, I-kappaB kinase/NF-kappaB signaling, and toll-like receptor signaling pathway.Conclusion: In this study, we construct the first immune landscape of DME patients with T2D and confirmed innate immune dysregulation in peripheral blood based on an unbiased scRNA-seq approach. And these results demonstrate potential target cell population for anti-inflammation treatments.

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Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single cell sequencing

10.1101/2021.12.17.472326 ◽

2021 ◽

Author(s):

Zhibin Li ◽

chengcheng Sun ◽

Fei Wang ◽

Xiran Wang ◽

Jiacheng Zhu ◽

...

Keyword(s):

Single Cell ◽

Immune Cells ◽

Evolutionary Biology ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Mononuclear Cells ◽

Immune Cell ◽

Cell Types ◽

Genetic Regulatory Networks ◽

Peripheral Blood Mononuclear

Background: Immune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited. Methods: In this study, we compared the single-cell transcriptomes of 77 957 immune cells from 12 species using single-cell RNA-sequencing (scRNA-seq). Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells. Results: The results revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular cross-talks and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells. Conclusions: This study is the first to provide a comprehensive analysis of the cross-species single-cell atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders

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Single cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse immune cell subtypes including T-bet+ B cells

10.1101/2020.05.05.077362 ◽

2020 ◽

Cited By ~ 1

Author(s):

Roosheel S. Patel ◽

Joy E. Tomlinson ◽

Thomas J. Divers ◽

Gerlinde R. Van de Walle ◽

Brad R. Rosenberg

Keyword(s):

B Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Immune Cell ◽

Model Organisms ◽

Traditional Model ◽

Cell Type ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

ABSTRACTTraditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary (e.g. for host-pathogen interaction studies), but presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers. Here, we demonstrate the utility of single cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMCs) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: Monocytes/Dendritic Cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1- lymphocytes, and Basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Unexpectedly, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells; an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse. Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms, and form the basis for an immune cell atlas of horse peripheral blood.

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Dynamic changes in human single cell transcriptional signatures during fatal sepsis

10.1101/2021.03.01.21252411 ◽

2021 ◽

Author(s):

Xinru Qiu ◽

Jiang Li ◽

Jeff Bonenfant ◽

Lukasz Jaroszewski ◽

Walter Klein ◽

...

Keyword(s):

Single Cell ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Metabolic Pathways ◽

Mononuclear Cells ◽

Immune Cell ◽

Peripheral Blood Mononuclear ◽

Repeated Measures Design ◽

Blood Mononuclear Cells ◽

Fatal Sepsis

AbstractSystemic infections, especially in patients with chronic diseases, result in sepsis: an explosive, uncoordinated immune response that can lead to multisystem organ failure with a high mortality rate. Sepsis survivors and non-survivors oftentimes have similar clinical phenotypes or sepsis biomarker expression upon diagnosis, suggesting that the dynamics of sepsis in the critical early stage may have an impact on these opposite outcomes. To investigate this, we designed a within-subject study of patients with systemic gram-negative bacterial sepsis with surviving and fatal outcomes and performed single-cell transcriptomic analyses of peripheral blood mononuclear cells (PBMC) collected during the critical period between sepsis recognition and 6 hours. We observed that the largest sepsis-induced expression changes over time in surviving versus fatal sepsis were in CD14+ monocytes, including gene signatures previously reported for sepsis outcomes. We further identify changes in the metabolic pathways of both monocytes and platelets, the emergence of erythroid precursors, and T cell exhaustion signatures, with the most extreme differences occurring between the non-sepsis control and the sepsis non-survivor. Our single-cell observations are consistent with trends from public datasets but also reveal specific effects in individual immune cell populations, which change within hours. In conclusion, this pilot study provides the first single-cell results with a repeated measures design in sepsis to analyze the temporal changes in the immune cell population behavior in surviving or fatal sepsis. These findings indicate that tracking temporal expression changes in specific cell-types could lead to more accurate predictions of sepsis outcomes. We also identify molecular pathways that could be therapeutically controlled to improve the sepsis trajectory toward better outcomes.Summary sentenceSingle cell transcriptomics of peripheral blood mononuclear cells in surviving and fatal sepsis reveal inflammatory and metabolic pathways that change within hours of sepsis recognition.

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Immune Cell Profiling of Peripheral Blood as Signature for Response During Checkpoint Inhibition Across Cancer Types

Frontiers in Oncology ◽

10.3389/fonc.2021.558248 ◽

2021 ◽

Vol 11 ◽

Author(s):

Vinicius Araujo B. de Lima ◽

Morten Hansen ◽

Iben Spanggaard ◽

Kristoffer Rohrberg ◽

Sine Reker Hadrup ◽

...

Keyword(s):

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Immune Cell ◽

Large Fraction ◽

Response To Treatment ◽

Cell Phenotype ◽

Checkpoint Inhibition ◽

Color Flow ◽

Immune Profile

Despite encouraging results with immune checkpoint inhibition (ICI), a large fraction of cancer patients still does not achieve clinical benefit. Finding predictive markers in the complexity of the tumor microenvironment is a challenging task and often requires invasive procedures. In our study, we looked for putative variables related to treatment benefit among immune cells in peripheral blood across different tumor types treated with ICIs. For that, we included 33 patients with different solid tumors referred to our clinical unit for ICI. Peripheral blood mononuclear cells were isolated at baseline, 6 and 20 weeks after treatment start. Characterization of immune cells was carried out by multi-color flow cytometry. Response to treatment was assessed radiologically by RECIST 1.1. Clinical outcome correlated with a shift towards an effector-like T cell phenotype, PD-1 expression by CD8+T cells, low levels of myeloid-derived suppressor cells and classical monocytes. Dendritic cells seemed also to play a role in terms of survival. From these findings, we hypothesized that patients responding to ICI had already at baseline an immune profile, here called ‘favorable immune periphery’, providing a higher chance of benefitting from ICI. We elaborated an index comprising cell types mentioned above. This signature correlated positively with the likelihood of benefiting from the treatment and ultimately with longer survival. Our study illustrates that patients responding to ICI seem to have a pre-existing immune profile in peripheral blood that favors good outcome. Exploring this signature can help to identify patients likely to achieve benefit from ICI.

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Analysis of human glioma-associated co-inhibitory immune checkpoints in glioma microenvironment and peripheral blood

International Journal of Immunopathology and Pharmacology ◽

10.1177/20587384211056505 ◽

2021 ◽

Vol 35 ◽

pp. 205873842110565

Author(s):

Shaoping Shen ◽

Qiyan Wu ◽

Jialin Liu ◽

Liangliang Wu ◽

Rong Zhang ◽

...

Keyword(s):

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Immune Cell ◽

Checkpoint Inhibitors ◽

Myeloid Cell ◽

Immune Checkpoints ◽

Human Glioma ◽

Peripheral Blood Mononuclear ◽

Glioma Microenvironment

One biomarker for a better therapeutic effect of immune checkpoint inhibitors is high expression of checkpoint in tumor microenvironment The purpose of this study is to investigate the expression of immune checkpoints in human glioma microenvironment and peripheral blood mononuclear cells. First, single-cell suspension from 20 fresh high-grade glioma (HGG) specimens were obtained, and analyzed for lymphocyte composition, then six co-inhibitory immune checkpoints were analyzed at the same time. Second, 36 PBMC specimens isolated from HGG blood samples were analyzed for the same items. In GME, there were four distinct subtypes of cells, among them, immune cells accounted for an average of 51.3%. The myeloid cell population (CD11b+) was the most common immune cell identified, accounting for 36.14% on average; the remaining were most CD3+CD4+ and CD3+/CD8−/CD4− T lymphocytes. In these cells, we detected the expression of BTLA, LAG3, Tim-3, CTLA-4, and VISTA on varying degrees. While in PBMCs, the result showed that when compared with healthy volunteers, the proportion of NK cells decreased significantly in HGG samples ( p < 0.01). Moreover, the expression of BTLA, LAG3, and Tim-3 in CD45+ immune cells in PBMC was more remarkable in glioma samples. In conclusion, the CD11b+ myeloid cells were the predominant immune cells in GME. Moreover, some immune checkpoints displayed a more remarkable expression on the immune cells in GME. And the profile of checkpoint expression in PBMC was partially consistent with that in GME.

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The Chromatin Accessibility Landscape of Peripheral Blood Mononuclear Cells in Patients With Systemic Lupus Erythematosus at Single-Cell Resolution

Frontiers in Immunology ◽

10.3389/fimmu.2021.641886 ◽

2021 ◽

Vol 12 ◽

Author(s):

Haiyan Yu ◽

Xiaoping Hong ◽

Hongwei Wu ◽

Fengping Zheng ◽

Zhipeng Zeng ◽

...

Keyword(s):

Single Cell ◽

Peripheral Blood Mononuclear Cells ◽

Immune Cells ◽

Lupus Erythematosus ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Chromatin Accessibility ◽

Peripheral Blood Mononuclear ◽

Single Cell Sequencing ◽

Blood Mononuclear Cells

ObjectiveSystemic lupus erythematosus (SLE) is a complex autoimmune disease, and various immune cells are involved in the initiation, progression, and regulation of SLE. Our goal was to reveal the chromatin accessibility landscape of peripheral blood mononuclear cells (PBMCs) in SLE patients at single-cell resolution and identify the transcription factors (TFs) that may drive abnormal immune responses.MethodsThe assay for transposase accessible chromatin in single-cell sequencing (scATAC-seq) method was applied to map the landscape of active regulatory DNA in immune cells from SLE patients at single-cell resolution, followed by clustering, peak annotation and motif analysis of PBMCs in SLE.ResultsPeripheral blood mononuclear cells were robustly clustered based on their types without using antibodies. We identified twenty patterns of TF activation that drive abnormal immune responses in SLE patients. Then, we observed ten genes that were highly associated with SLE pathogenesis by altering T cell activity. Finally, we found 12 key TFs regulating the above six genes (CD83, ELF4, ITPKB, RAB27A, RUNX3, and ZMIZ1) that may be related to SLE disease pathogenesis and were significantly enriched in SLE patients (p <0.05, FC >2). With qPCR experiments on CD83, ELF4, RUNX3, and ZMIZ1 in B cells, we observed a significant difference in the expression of genes (ELF4, RUNX3, and ZMIZ1), which were regulated by seven TFs (EWSR1-FLI1, MAF, MAFA, NFIB, NR2C2 (var. 2), TBX4, and TBX5). Meanwhile, the seven TFs showed highly accessible binding sites in SLE patients.ConclusionsThese results confirm the importance of using single-cell sequencing to uncover the real features of immune cells in SLE patients, reveal key TFs in SLE-PBMCs, and provide foundational insights relevant for epigenetic therapy.

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Abnormal B-Cell and Tfh-Cell Profiles in Patients With Parkinson Disease

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000001125 ◽

2021 ◽

Vol 9 (2) ◽

pp. e1125

Author(s):

Rui Li ◽

Thomas Francis Tropea ◽

Laura Rosa Baratta ◽

Leah Zuroff ◽

Maria E. Diaz-Ortiz ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Parkinson Disease ◽

B Cell ◽

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Immune Cell ◽

Ex Vivo ◽

Cell Counts

Background and ObjectivesThere has been growing interest in potential roles of the immune system in the pathogenesis of Parkinson disease (PD). The aim of the current study was to comprehensively characterize phenotypic and functional profiles of circulating immune cells in patients with PD vs controls.MethodsPeripheral blood was collected from patients with PD and age- and sex-matched neurologically normal controls (NCs) in 2 independent cohorts (discovery and validation). Comprehensive multicolor flow cytometry was performed on whole blood leukocytes and peripheral blood mononuclear cells to characterize different immune subsets and their ex vivo responses.ResultsThe discovery cohort included 17 NCs and 12 participants with PD, and the validation cohort included 18 NCs and 18 participants with PD. Among major immune cell types, B cells appeared to be preferentially affected in PD. Proliferating B cell counts were decreased in patients with PD compared with controls. Proportions of B-cell subsets with regulatory capacity such as transitional B cells were preferentially reduced in the patients with PD, whereas proportions of proinflammatory cytokine-producing B cells increased, resulting in a proinflammatory shift of their B-cell functional cytokine responses. Unsupervised principal component analysis revealed increased expression of TNFα and GM-CSF by both B cells and T cells of patients with PD. In addition, levels of follicular T cells, an important B-cell helper T-cell population, decreased in the patients with PD, correlating with their B-cell abnormality.DiscussionOur findings define a novel signature of peripheral immune cells and implicate aberrant Tfh:B-cell interactions in patients with PD.

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Single-Cell RNA Sequencing of Peripheral Blood Reveals Immune Cell Signatures in Alzheimer’s Disease

Frontiers in Immunology ◽

10.3389/fimmu.2021.645666 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hui Xu ◽

Jianping Jia

Keyword(s):

Immune Response ◽

T Cells ◽

Single Cell ◽

Immune Cells ◽

Peripheral Blood ◽

Immune Cell ◽

Adaptive Immune Response ◽

Immune Repertoire ◽

Adaptive Immune ◽

Peripheral Immune Cells

The peripheral immune system is thought to affect the pathology of the central nervous system in Alzheimer’s disease (AD). However, current knowledge is inadequate for understanding the characteristics of peripheral immune cells in AD. This study aimed to explore the molecular basis of peripheral immune cells and the features of adaptive immune repertoire at a single cell level. We profiled 36,849 peripheral blood mononuclear cells from AD patients with amyloid-positive status and normal controls with amyloid-negative status by 5’ single-cell transcriptome and immune repertoire sequencing using the cell ranger standard analysis procedure. We revealed five immune cell subsets: CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and monocytes–macrophages cells, and disentangled the characteristic alterations of cell subset proportion and gene expression patterns in AD. Thirty-one cell type-specific key genes, comprising abundant human leukocyte antigen genes, and multiple immune-related pathways were identified by protein–protein interaction network and pathway enrichment analysis. We also found high-frequency amplification clonotypes in T and B cells and decreased diversity in T cells in AD. As clone amplification suggested the activation of an adaptive immune response against specific antigens, we speculated that the peripheral adaptive immune response, especially mediated by T cells, may have a role in the pathogenesis of AD. This finding may also contribute to further research regarding disease mechanism and the development of immune-related biomarkers or therapy.

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COVID-19 Specific Immune Markers Revealed by Single Cell Phenotypic Profiling

Biomedicines ◽

10.3390/biomedicines9121794 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1794

Author(s):

Francesca Sansico ◽

Mattia Miroballo ◽

Daniele Salvatore Bianco ◽

Francesco Tamiro ◽

Mattia Colucci ◽

...

Keyword(s):

Viral Infection ◽

Single Cell ◽

Peripheral Blood ◽

Blood Donors ◽

Mononuclear Cells ◽

Cell Function ◽

Immune Cell ◽

Surface Markers ◽

Immune Functions ◽

Immune Markers

COVID-19 is a viral infection, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and characterized by a complex inflammatory process and clinical immunophenotypes. Nowadays, several alterations of immune response within the respiratory tracts as well as at the level of the peripheral blood have been well documented. Nonetheless, their effects on COVID-19-related cell heterogeneity and disease progression are less defined. Here, we performed a single-cell RNA sequencing of about 400 transcripts relevant to immune cell function including surface markers, in mononuclear cells (PBMCs) from the peripheral blood of 50 subjects, infected with SARS-CoV-2 at the diagnosis and 27 healthy blood donors as control. We found that patients with COVID-19 exhibited an increase in COVID-specific surface markers in different subsets of immune cell composition. Interestingly, the expression of cell receptors, such as IFNGR1 and CXCR4, was reduced in response to the viral infection and associated with the inhibition of the related signaling pathways and immune functions. These results highlight novel immunoreceptors, selectively expressed in COVID-19 patients, which affect the immune functionality and are correlated with clinical outcomes.

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The immune cell landscape of peripheral blood mononuclear cells from PNS patients

Scientific Reports ◽

10.1038/s41598-021-92573-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Qing Ye ◽

Chao Zhou ◽

Sisi Li ◽

Jingjing Wang ◽

Fei Liu ◽

...

Keyword(s):

T Cells ◽

Nephrotic Syndrome ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Immune Cell ◽

Immunological Mechanism

AbstractExisting research suggests that the human immune system and immune cells are involved in the pathogenesis of nephrotic syndrome, but there is still a lack of direct evidence. This study tried to analyze the profiling of immune cells in the peripheral blood of steroid-sensitive nephrotic syndrome (SSNS) patients and steroid-resistant nephrotic syndrome (SRNS) patients before and after standard steroid treatment to clarify the immunological mechanism of nephrotic syndrome patients. The number and proportion of CD4 + T cells in patients with nephrotic syndrome remained unchanged. However, there is an imbalance of Th1 and Th2 and an excessive increase of Th17 cells. The number of CD8 + T cells and the number of effector CD8 + T cells in them increased significantly, but only in SSNS, the number of activated CD8 + T cells increased, and the number of activated Treg cells decreased significantly. Nephrotic syndrome patients also have B cell disorder, and it is more prominent in SSNS patients. Compared with the normal control, only the number of B cells and plasmablast in SSNS patients increased significantly (Z = − 2.20, P = 0.028). This study also observed that transitional B cells decreased in both SSNS and SRNS patients, but SSNS patients' decrease was lower than in SRNS patients. Compared with normal controls, monocytes in patients with nephrotic syndrome decreased significantly. The main reason was that Non-classical Monocyte decreased, while Classical Monocyte increased slightly. The total number of NK cells did not change, but the internal cell subgroups' composition occurred. Changes, realized as CD56hi NK cells increased, CD56low NK cells decreased; and the above trend is more evident in SSNS patients. Patients with nephrotic syndrome have immune disorders, including T cells, B cells, Monocytes, and NK cells. It can be confirmed that immune factors are involved in the pathogenesis of the nephrotic syndrome.

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