Abstract MP49: Single Cell Multiplex Immunophenotyping Using Mass Cytometry And CITE-Seq Reveals Decreases In Circulating PD-1 + CD8 + Memory T Cells With Features Of Exhaustion In Human Hypertension

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Matthew R Alexander ◽  
Charles D Smart ◽  
Bethany L Dale ◽  
Fernando Elijovich ◽  
Cara Wogsland ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Single Cell ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Memory T Cells ◽  
Gene Set Enrichment Analysis ◽  
Mass Cytometry ◽  
Human Hypertension ◽  
Adaptive Immune Cells

Emerging evidence from animal models has demonstrated the importance of multiple innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets are altered in human hypertension. To test this, we performed high dimensional single cell profiling of human peripheral blood mononuclear cells using mass cytometry. Unsupervised computational analysis revealed a 40% decrease in CD8 + memory T cells in hypertensive individuals. Using Phenograph to identify subsets of these cells revealed a selective 60% decrease in PD-1 + CD8 + memory T cells in hypertension. This observation was confirmed in a validation cohort using flow cytometry in which PD-1 + CD8 + memory T cells were significantly decreased 44% in hypertensive compared to control individuals. To determine the phenotype of these PD-1 + CD8 + memory T cells, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) on four control and four hypertensive individuals. Using antibodies to identify PD-1 + and PD-1 - CD8 + memory T cells, gene set enrichment analysis of the coordinate single cell transcriptomic data revealed that PD-1 + cells exhibit over-representation of features of both immunologically active effector T cells and hypofunctional exhausted T cells. Thus, clustering analysis of PD-1 + CD8 + memory T cells was performed which demonstrated 4 distinct subclusters. One of these subclusters was decreased in hypertension and exhibited selective expression of multiple inhibitory receptors characteristic of exhausted T cells. At the protein level, this subcluster was marked by expression of the inhibitory receptor LAG3 and low levels of CD57. Combining these markers to identify PD-1 + LAG3 + CD57 - CD8 + memory T cells permitted identification of exhausted cells which demonstrated a significant 35% decrease in hypertensive compared to control individuals using flow cytometry. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1 + CD8 + memory T cells with features of exhaustion in human hypertension. These findings provide new insights into the pathogenesis of human hypertension including loss and/or re-invigoration of exhausted T cells.

scholarly journals Identification of human immune cell subtypes most responsive to IL-1β–induced inflammatory signaling using mass cytometry

2021 ◽  
Vol 14 (673) ◽  
pp. eabc5763 ◽  
Author(s):  
Hema Kothari ◽  
Corey M. Williams ◽  
Chantel McSkimming ◽  
Fabrizio Drago ◽  
Melissa A. Marshall ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Human Peripheral Blood ◽  
Effector Memory ◽  
High Morbidity ◽  
Cytokine Storm ◽  
Mass Cytometry ◽  
Myeloid Dendritic Cells

IL-1β is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1β blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4−CD8low/−CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1β. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16−CD56brightCD161− and CD16−CD56dimCD161+), and lineage− (Lin−) cells expressing CD161 and CD25. IL-1β also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1β stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1β–induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1β in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1β–neutralizing therapies.

scholarly journals Single-Cell Mass Cytometry on Peripheral Blood Identifies Immune Cell Subsets Associated with Primary Biliary Cholangitis

2020 ◽  
Author(s):  
Jin Sung Jang ◽  
Brian Juran ◽  
Kevin Y. Cunningham ◽  
Vinod K. Gupta ◽  
YoungMin Son ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Single Cell ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Memory B Cells ◽  
Primary Biliary Cholangitis ◽  
Mass Cytometry ◽  
Peripheral Immune System

AbstractThe relationship between Primary Biliary Cholangitis (PBC), a chronic cholestatic autoimmune liver disease, and the peripheral immune system remains to be fully understood. Herein, we performed the first mass cytometry (CyTOF)-based, immunophenotyping analysis of the peripheral immune system in PBC at single-cell resolution. CyTOF was performed on peripheral blood mononuclear cells (PBMCs) from PBC patients (n=33) and age-/sex-matched healthy controls (n=33) to obtain immune cell abundance and marker expression profiles. Hiearchical clustering methods were applied to identify immune cell types and subsets significantly associated with PBC. Subsets of gamma-delta T cells (CD3+TCRgd+), CD8+ T cells (CD3+CD8+CD161+PD1+), and memory B cells (CD3-CD19+CD20+CD24+CD27+) were found to have lower abundance in PBC than in control. In contrast, higher abundance of subsets of monocytes and naïve B cells were observed in PBC compared to control. Furthermore, several naïve B cell (CD3-CD19+CD20+CD24-CD27-) subsets were significantly higher in PBC patients with cirrhosis (indicative of late-stage disease) than in those without cirrhosis. Alternatively, subsets of CD8+CD161+ T cells and memory B cells were lower in abundance in cirrhotic relative to non-cirrhotic PBC patients. Future immunophenotyping investigations could lead to better understanding of PBC pathogenesis and progression, and also to the discovery of novel biomarkers and treatment strategies.

scholarly journals Potent Anti-Tumor Activity of Bcma CAR-T Therapy Against Heavily Treated Multiple Myeloma and Dynamics of Immune Cell Subsets Using Single-Cell Mass Cytometry

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1859-1859 ◽  
Author(s):  
Yongxian Hu ◽  
Zhang Yanlei ◽  
Guoqing Wei ◽  
Chang alex Hong ◽  
He Huang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Immune Cell ◽  
Cell Mass ◽  
Mass Cytometry ◽  
Car T Cells ◽  
Car T Cell ◽  
Immune Cell Subsets ◽  
Car T

Background BCMA CAR-T cells have demonstrated substantial clinical activity against relapsed/refractory multiple myeloma (RRMM). In different clinical trials, the overall response rate (ORR) varied from 50% to 100%. Complete remission (CR) rate varied from 20% to 80%. Here we developed a BCMA CAR-T cell product manufactured via lentiviral vector-mediated transduction of activated T cells to express a second-generation CAR with 4-1BB costimulatory domain and evaluated the efficacy and safety, moreover, dynamics of immune cell subsets using single-cell mass cytometry during treatment were analyzed. Methods Our trial (ChiCTR1800017404) is a phase 1, single-arm, open-label single center study to evaluate the safety and efficacy of autologous BCMA CAR-T treatment for RRMM. Patients were subjected to a lymphodepleting regimen with Flu and Cy prior to CAR-T infusion. BCMA CAR-T cells were administered as a single infusion at a median dose of 3.5 (1 to 6) ×106/kg. MM response assessment was conducted according to the International Uniform Response Criteria. Cytokine-release syndrome (CRS) was graded as Lee DW et al described (Blood.2014;124(2):188-195). Phenotypic analysis of peripheral blood mononuclear cells (PBMCs), frozen BCMA CAR-T aliquots, phenotype and in vivo kinetics of immune cell subsets after CAR-T infusion were performed by single-cell mass cytometry. Results As of the data cut-off date (August 1st, 2019), 33 patients, median age 62.5 (49 to 75) years old were infused with BCMA CAR-T cells. The median observation period is 8.0 (0.7 to 18) months. ORR was 100% (The patient who died of infection at 20 days after CAR-T infusion were excluded). All the 32 patients achieved MRD negative in bone marrow by flow cytometry in 2 weeks after CAR-T infusion. Partial response (4 PR, 12.1%), VGPR (7 VGPR, 21.2%), and complete response (21 CR, 63.6%) within 12 weeks post CAR-T infusion were achieved. Durable responses from 4 weeks towards the data cut-off date were found in 28/33 patients (84.8%) (Figure 1a). All patients had detectable CAR-T expansion by flow cytometry from Day 3 post CAR-T cell infusion. The peak CAR-T cell expansion in CD3+ lymphocytes of peripheral blood (PB) varied from 35% to 95% with a median percentage of 82.9%. CRS was reported in all the 33 patients, including 4 with Grade 1, 13 with Grade 2 and 16 with Grade 3. During follow-up, 1-year progression-free survival (PFS) was 70.7% (Figure 1b) and overall survival (OS) was 71.7% (Figure 1c). Multivariate analysis of patients with PR and patients with CR+VGPR revealed that factors including extramedullary infiltration, age>60 years old, high-risk cytogenetics, late stage and CAR-T cell dose were not associated with clinical response (P>0.05). Single-cell mass cytometry revealed that the frequency of total T cells, CD8+ T cells, NK cells and CD3+CD56+ NKT cells in PB was not associated with BCM CAR-T expansion or clinical response. CD8+ Granzyme B+ Ki-67+ CAR-T cells expanded prominently in CRS period. As serum cytokines increased during CRS, non-CAR-T immune cell subsets including PD1+ NK cells, CD8+ Ki-67+ ICOS+ T cells expanded dominantly implying that non-CAR-T cells were also activated after CAR-T treatment. After CRS, stem cell like memory CAR-T cells (CD45RO+ CCR7- CD28- CD95+) remain the main subtype of CAR-T cells (Figure 1d). Conclusions Our data showed BCMA CAR-T treatment is safe with prominent efficacy which can overcome the traditional high-risk factors. We also observed high expansion level and long-term persistence of BCMA CAR-T cells contribute to potent anti-myeloma activity. Stem cell like memory CAR-T cells might be associated with long-term persistence of BCMA CAR-T cells. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The differential immune responses to COVID-19 in peripheral and lung revealed by single-cell RNA sequencing

2020 ◽  
Author(s):  
Gang Xu ◽  
Furong Qi ◽  
Hanjie Li ◽  
Qianting Yang ◽  
Haiyan Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Close Association ◽  
Suppressor Cells ◽  
T Cell Subsets ◽  
Peripheral Blood Mononuclear ◽  
Peripheral T Cells

Understanding the mechanism that leads to immune dysfunction induced by SARS-CoV2 virus is crucial to develop treatment for severe COVID-19. Here, using single cell RNA-seq, we characterized the peripheral blood mononuclear cells (PBMC) from uninfected controls and COVID-19 patients, and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DC) and increased monocytes resembling myeloid-derived suppressor cells (MDSC) which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8+ T cell subsets, compared to health controls. In contrast, the proportions of various activated CD4+ T cell subsets, including Th1, Th2 and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients' peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4 and CCL5 etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the patients' lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

Circulating immune cell profiles detected by mass cytometry differ significantly between patients with predominantly calcified and predominantly non-calcified coronary atherosclerosis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
K.A Kott ◽  
T Hansen ◽  
M De Dreu ◽  
S.T Vernon ◽  
T Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Biologically Active ◽  
Public Institution ◽  
Funding Source ◽  
Imaging Data ◽  
Mass Cytometry ◽  
Soft Plaque

Abstract Background/Introduction Inflammation is now a well-established component of the pathophysiology of coronary artery disease (CAD), but it is unknown whether atherosclerosis is associated with a distinct circulating immune cell profile. Mass cytometry time-of-flight (CYTOF) is a new precision technology which can be used to assess leukocyte populations comprehensively. Purpose To determine if patients with calcified and non-calcified (soft) coronary plaque have distinct circulating immune cell profiles when compared to healthy controls, and to assess whether this could be used to detect sub-clinical CAD. Methods Patients referred for a CT coronary angiogram were recruited; blood samples were collected and peripheral blood mononuclear cells (PBMCs) were isolated. Imaging data was analysed using a modified Gensini scoring system which incorporated plaque composition, with higher weighting given to soft plaque. The modified Gensini scores were then used to further segregate into calcified-predominant and soft-predominant disease groups. CYTOF analysis was performed on the PBMCs, with groups as outlined in Table 1. Results Age was significantly higher in the CAD+ group, but all other demographic features and risk factors did not differ between groups. Patients with predominantly calcified disease showed an increase in memory CD8 T cells (p=0.004), an increase in CD 39+ CD4 T cells (p=0.028), and a decrease in naïve CD8 T cells (p=0.005), which suggests an accumulated memory response in more quiescent disease. Patients with predominantly soft-plaque disease have higher pro-inflammatory monocyte populations (p=0.013) and proliferative CD4 T cell populations (p=0.011), suggesting acute innate and adaptive responses to biologically active plaque. Conclusions This pilot study has shown that further study should be pursued into the utility of CYTOF to identify sub-clinical CAD through differences in peripheral circulating immune cell profiles. Figure 1 Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): National Health and Medical Research Council of Australia, Heart Research Australia

scholarly journals High-dimensional single cell mass cytometry analysis of the murine hematopoietic system reveals signatures induced by ageing and physiological pathogen challenges

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Christos Nikolaou ◽  
Kerstin Muehle ◽  
Stephan Schlickeiser ◽  
Alberto Sada Japp ◽  
Nadine Matzmohr ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Plasma Cells ◽  
Cell Mass ◽  
Mass Cytometry ◽  
Preclinical Research ◽  
Hematopoietic System ◽  
Adaptive Immune Cells

Abstract Background Immune ageing is a result of repetitive microbial challenges along with cell intrinsic or systemic changes occurring during ageing. Mice under ‘specific-pathogen-free’ (SPF) conditions are frequently used to assess immune ageing in long-term experiments. However, physiological pathogenic challenges are reduced in SPF mice. The question arises to what extent murine experiments performed under SPF conditions are suited to analyze immune ageing in mice and serve as models for human immune ageing. Our previous comparisons of same aged mice with different microbial exposures, unambiguously identified distinct clusters of immune cells characteristic for numerous previous pathogen encounters in particular in pet shop mice. Results We here performed single cell mass cytometry assessing splenic as secondary and bone marrow as primary lymphoid organ-derived leukocytes isolated from young versus aged SPF mice in order to delineate alterations of the murine hematopoietic system induced during ageing. We then compared immune clusters from young and aged SPF mice to pet shop mice in order to delineate alterations of the murine hematopoietic system induced by physiological pathogenic challenges and those caused by cell intrinsic or systemic changes during ageing. Notably, distinct immune signatures were similarly altered in both pet shop and aged SPF mice in comparison to young SPF mice, including increased frequencies of memory T lymphocytes, effector-cytokine producing T cells, plasma cells and mature NK cells. However, elevated frequencies of CD4+ T cells, total NK cells, granulocytes, pDCs, cDCs and decreased frequencies of naïve B cells were specifically identified only in pet shop mice. In aged SPF mice specifically the frequencies of splenic IgM+ plasma cells, CD8+ T cells and CD4+ CD25+ Treg were increased as compared to pet shop mice and young mice. Conclusions Our study dissects firstly how ageing impacts both innate and adaptive immune cells in primary and secondary lymphoid organs. Secondly, it partly distinguishes murine intrinsic immune ageing alterations from those induced by physiological pathogen challenges highlighting the importance of designing mouse models for their use in preclinical research including vaccines and immunotherapies.

scholarly journals 661 Five immunotypic signatures identified in human glioblastoma correlate with tumor contact with the lateral ventricle, immune suppression, and patient outcome

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A698-A698
Author(s):  
Todd Bartkowiak ◽  
Sierra Barone ◽  
Madeline Hayes ◽  
Allison Greenplate ◽  
Justine Sinnaeve ◽  
...  
Keyword(s):  
Machine Learning ◽  
Patient Outcome ◽  
T Cells ◽  
Single Cell ◽  
Lateral Ventricle ◽  
Immune Cell ◽  
Cell Mass ◽  
Γδ T Cells ◽  
Survival Outcomes ◽  
Mass Cytometry

BackgroundGlioblastomas make up more than 60% of adult primary brain tumors and carry a median survival of less than 15 months despite aggressive therapy. Immunotherapy, now standard of care for many peripheral solid tumors, offers an appealing alternative platform that may improve survival outcomes for patients with glioblastoma; however, predictive features that could inform responsiveness to different immunotherapeutic modalities remains to be elucidated. Recent studies have demonstrated that patients whose tumors show radiographic contact with the lateral ventricle have diminished survival outcomes compared to patients whose tumors do not contact the lateral ventricle. While greater immune infiltrate correlates with more favorable outcomes and more effectual responses to immunotherapy, the anti-tumor immune response in the ventricle is unknown. We hypothesized that ventricle contact may provide a uniquely immunosuppressive microenvironment within the brain that promotes tumor growth by suppressing anti-tumor immunity, that may be overcome with appropriate targeting strategies.MethodsPrimary glioblastoma tumors obtained in accordance with the Declaration of Helsinki and with institutional IRB approval (#131870) were disaggregated into single-cell suspensions. Radiographic contact with the LV was identified by MRI imaging and confirmed by a trained neurosurgeon. Multi-dimensional single-cell mass cytometry (CyTOF) then measured >30 immune parameters in thirteen immune subpopulations infiltrating human glioblastomas, including T cells, natural killer cells, B cells, microglia, peripheral macrophages, and myeloid-derived suppressors cells (MDSC). Computational machine-learning pipelines including Citrus, t-SNE, FlowSOM, and MEM identified key differences in the abundance and phenotypes of immune infiltrates.ResultsOn the basis of glioblastoma contact with the ventricle, we computationally identified consequential distinctions in the abundance of T cell, macrophage, and microglia subsets constituting five immunotype signatures among glioblastoma patients. Immunotypes associated with CD69+CD32+CD44+ peripheral macrophages and PD-1+TIGIT+ CD8 T cells correlated with ventricle contact, whereas immunotypes associated with enriched γδ T cells, B, NK cell, and tissue-resident microglial cells correlated with tumors distal to the ventricle. Further, immune infiltration in the tumor microenvironment correlated with patient outcome, with higher lymphocyte infiltrates correlating with more favorable outcomes, and immune exhaustion correlating with less favorable outcomes.ConclusionsSingle-cell mass cytometry in conjunction with the machine learning tools identified key differences in immune cell abundance between lateral ventricle contacting and non-contacting glioblastomas. These results provide key insights into the immune microenvironment of glioblastomas and elucidate several clinically actionable immunotherapeutic targets that may be used to optimize treatment strategies for glioblastomas based on ventricle contact status.Ethics ApprovalThis study was approved by Vanderbilt University’s Institutional Ethics Board, approval number 131870

scholarly journals Identification of Human Immune Cell Subtypes Most Vulnerable to IL-1β-induced Inflammatory Signaling Using Mass Cytometry

2020 ◽  
Author(s):  
Hema Kothari ◽  
Corey M. Williams ◽  
Chantel McSkimming ◽  
Mythili Vigneshwar ◽  
Eli R. Zunder ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
T Cell Subsets ◽  
Effector Memory ◽  
High Morbidity ◽  
Cytokine Storm ◽  
Mass Cytometry ◽  
Immune Cell Subsets

ABSTRACTIL-1β has emerged as a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19 and blockade of the IL-1 receptor (IL-1R) with Anakinra has entered clinical trials in COVID-19 subjects. Yet, knowledge of the specific immune cell subsets targeted by IL-1β and IL-1β-induced signaling pathways in humans is limited. Utilizing mass cytometry (CyTOF) of human peripheral blood mononuclear cells, we identified effector memory CD4 T cells and CD4−CD8low/-CD161+ T cells as the circulating immune subtypes with the greatest expression of p-NF-κB in response to IL-1β stimulation. Notably, CCR6 distinctly identified T cells most responsive to IL-1β. Other subsets including CD11c myeloid dendritic cells (mDCs), classical monocytes (CM), two subsets of natural killer cells (CD16−CD56brightCD161− and CD16−CD56dimCD161+) and a population of lineage−(Lin-) cells expressing CD161 and CD25 also showed IL-1β-induced expression of p-NF-kB. The IL-1R antagonist, Anakinra significantly inhibited IL-1β-induced p-NF-kB in the CCR6+ T cells and CD11c mDCs with a trending inhibition in CD14 monocytes and Lin−CD161+CD25+ cells. IL-1β also induced a rapid but much less robust increase in p-p38 expression as compared to p-NF-kB in the majority of these same immune cell subsets. Prolonged IL-1β stimulation greatly increased p-STAT3 and to a much lesser extent p-STAT1 and p-STAT5 in T cell subsets, monocytes, DCs and the Lin−CD161+CD25+ cells suggesting IL-1β-induced production of downstream STAT-activating cytokines, consistent with its role in cytokine storm. Interindividual heterogeneity and inhibition of this activation by Anakinra raises the intriguing possibility that assays to measure IL-1β-induced p-NF-kB in CCR6+ T cell subtypes could identify those at higher risk of cytokine storm and those most likely to benefit from Anakinra therapy.

scholarly journals Single cell analysis of blood mononuclear cells stimulated through CD3 and CD28 shows collateral activation of B and NK cells and demise of monocytes

2020 ◽  
Author(s):  
Nathan Lawlor ◽  
Djamel Nehar-Belaid ◽  
Jessica D.S. Grassmann ◽  
Marlon Stoeckius ◽  
Peter Smibert ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Single Cell ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Healthy Human ◽  
Effector Molecules ◽  
Blood Mononuclear Cells

AbstractImmune cell activation assays have been widely used for immune monitoring and for understanding disease mechanisms. However, these assays are typically limited in scope. A holistic study of circulating immune cell responses to different activators is lacking. Here we developed a cost-effective high-throughput multiplexed single-cell RNA-seq combined with epitope tagging (CITE-seq) to determine how classic activators of T cells (anti-CD3 coupled with anti-CD28) or monocytes (LPS) alter the cell composition and transcriptional profiles of peripheral blood mononuclear cells (PBMCs) from healthy human donors. Anti-CD3/CD28 treatment activated all classes of lymphocytes either directly (T cells) or indirectly (B and NK cells) but reduced monocyte numbers. Activated T and NK cells expressed senescence and effector molecules, whereas activated B cells transcriptionally resembled autoimmune disease- or age-associated B cells (e.g., CD11c, T-bet). In contrast, LPS specifically targeted monocytes and induced two main states: early activation characterized by the expression of chemoattractants and a later pro-inflammatory state characterized by expression of effector molecules. These data provide a foundation for future immune activation studies with single cell technologies (https://czi-pbmc-cite-seq.jax.org/).Graphical abstract

scholarly journals Interplay of Monocytes and T Lymphocytes in COVID-19 Severity

2020 ◽  
Author(s):  
Lindsey E. Padgett ◽  
Huy Q. Dinh ◽  
Serena J. Chee ◽  
Claire E. Olingy ◽  
Runpei Wu ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Coagulation Factor ◽  
Effector Memory ◽  
Sugar Residue ◽  
Monocyte Subsets ◽  
Mass Cytometry ◽  
T Lymphocyte Subsets

ABSTRACTThe COVID-19 pandemic represents an ongoing global crisis that has already impacted over 13 million people. The responses of specific immune cell populations to the disease remain poorly defined, which hinders improvements in treatment and care management. Here, we utilized mass cytometry (CyTOF) to thoroughly phenotype peripheral myeloid cells and T lymphocytes from 30 convalescent patients with mild, moderate, and severe cases of COVID-19. We identified 10 clusters of monocytes and dendritic cells and 17 clusters of T cells. Examination of these clusters revealed that both CD14+CD16+ intermediate and CD14dimCD16+ nonclassical monocytes, as well as CD4+ stem cell memory T (TSCM) cells, correlated with COVID-19 severity, coagulation factor levels, and/or inflammatory indicators. We also identified two nonclassical monocyte subsets distinguished by expression of the sugar residue 6-Sulfo LacNac (Slan). One of these subsets (Slanlo, nMo1) was depleted in moderately and severely ill patients, while the other (Slanhi, nMo2) increased with disease severity and was linked to CD4+ T effector memory (TEM) cell frequencies, coagulation factors, and inflammatory indicators. Intermediate monocytes tightly correlated with loss of naive T cells as well as an increased abundance of effector memory T cells expressing the exhaustion marker PD-1. Our data suggest that both intermediate and non-classical monocyte subsets shape the adaptive immune response to SARS-CoV-2. In summary, our study provides both broad and in-depth characterization of immune cell phenotypes in response to COVID-19 and suggests functional interactions between distinct cell types during the disease.One Sentence SummaryUse of mass cytometry on peripheral blood mononuclear cells from convalescent COVID-19 patients allows correlation of distinct monocyte and T lymphocyte subsets with clinical factors.

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