Dataset Information

Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity.

ABSTRACT: Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45+HLA-DR-CD123+). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16lowFc?RIhighCD244high (88.5 ± 1.2%), (II) CD16highFc?RIhighCD244high (9.1 ± 0.4%), (III) CD16lowFc?RIlowCD244low (2.3 ± 1.3), and (IV) CD16highFc?RIlowCD244low (0.4 ± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I-III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I-II had significant increases in CD203c expression, whereas PBG III-IV remained unchanged compared with media-alone conditions. Thus, PBG III-IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.

SUBMITTER: Vivanco Gonzalez N

PROVIDER: S-EPMC7653073 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity.

Vivanco Gonzalez Nora N Oliveria John-Paul JP Tebaykin Dmitry D Ivison Geoffrey T GT Mukai Kaori K Tsai Mindy M MM Borges Luciene L Nadeau Kari C KC Galli Stephen J SJ Tsai Albert G AG Bendall Sean C SC

iScience 20201022 11

Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45<sup>+</sup>HLA-DR<sup>-</sup>CD12 ...[more]

PMID: 33205028

Similar Datasets

Project description:Rationale: Monocytes are key effectors of the mononuclear phagocyte system (MPS), playing critical roles in regulating tissue homeostasis and coordinating inflammatory reactions, including those involved in chronic inflammatory diseases such as atherosclerosis. Monocytes have traditionally been divided into two major subsets termed conventional (cMo) and patrolling (pMo) monocytes but recent systems immunology approaches have identified marked heterogeneity within these cells, and much of what regulates monocyte population homeostasis remains unknown. We and others have previously identified LYN tyrosine kinase as a key negative regulator of myeloid cell biology, however LYN’s role in regulating specific monocyte subset homeostasis has not been investigated. Objective: We sought to comprehensively profile monocytes in order to elucidate the underlying heterogeneity within monocytes and dissect how Lyn deficiency affects monocyte subset composition, signaling, and gene expression. We further tested the biological significance of these findings in a model of atherosclerosis. Methods and Results: CyTOF analysis of monocyte subsets and signaling pathway activation patterns in cMos and pMos revealed distinct baseline signaling profiles and far greater heterogeneity than previously described. Lyn deficiency led to a selective expansion of pMos and alterations in specific signaling pathways within these cells, revealing a critical role for LYN in pMo physiology. LYN’s role in regulating pMos was cell-intrinsic and correlated with an increased circulating half-life of Lyn-deficient pMos. Furthermore, single cell RNA sequencing revealed marked perturbations in the gene expression profiles of Lyn-/- monocytes with upregulation of genes involved in pMo development, survival, and function. Lyn deficiency also led to a significant increase in aorta-associated pMos and protected Ldlr-/- mice from high-fat diet induced atherosclerosis. Conclusions: Together our data identify LYN as a key regulator of pMo development and a potential therapeutic target in inflammatory diseases regulated by pMos.

Project description:BackgroundAutoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more severe disease and poor prognosis compared to ACPA-negative patients. However, the immune cell composition associated with antibody-positive/negative disease is incompletely defined. Mass cytometry (MC) is a high-dimensional technique offering new possibilities in the determination of the immune cell composition in rheumatic diseases. Here, we set up a broad phenotyping panel to study the immune cell profile of early untreated RA to investigate if specific immune cell subsets are associated with ACPA+ versus ACPA- RA.MethodsFreshly obtained PBMCs of early, untreated RA patients (8 ACPA+ and 7 ACPA-) were analysed using a 36-marker MC panel, including markers related to various immune lineages. Data were processed using Cytosplore for dimensional reduction (HSNE) and clustering. Groups were compared using Cytofast. A second validation cohort of cryopreserved PBMCs obtained from early RA patients (27 ACPA+ and 20 ACPA-) was used to confirm MC data by flow cytometry (FC). FC data were processed and analysed using both an unsupervised analysis pipeline and through manual gating.ResultsMC indicated no differences when comparing major immune lineages (i.e. monocytes, T and B cells), but highlighted two innate subsets: CD62L+ basophils (p = 0.33) and a subset of CD16- NK cells (p = 0.063). Although the NK cell subset did not replicate by FC, FC replication confirmed the difference in CD62L+ basophil frequency when comparing ACPA+ to ACPA- patients (mean 0.32% vs. 0.13%; p = 0.01).ConclusionsAlthough no differences in major lineages were found between early ACPA+ and ACPA- RA, this study identified the reduced presence of activated basophils in ACPA-negative disease as compared to ACPA-positive disease and thereby provides the first evidence for a connection between activated basophils and ACPA status.

Dataset Information

Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity.

Publications

Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets