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Updated: May 26, 2026
Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry
Published on: June 26, 2018
Integrated single-cell transcriptomic analysis identifies PF4 + /PPBP + megakaryocyte-like granulocytes associated
Shaoqi Chen1, Yu Fan2, Miaotong Su2
1Department of Ultrasound, The First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, China.
Objectives:
Megakaryocytes (MKs) and low-density granulocytes (LDGs) are implicated in immune dysregulation and vascular pathology in autoimmune diseases (ADs), yet their precise subsets and pathological interactions remain poorly defined. We aimed to characterize MK and LDG subpopulations and elucidate their potential intercellular communication in ADs using single-cell transcriptomic analysis.
Methods:
Single-cell RNA sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells from 10 treatment-naive AD patients (4 pSS, 3 RA, and 3 SLE) and 3 healthy controls (HCs). MKs and LDGs were re-clustered to identify transcriptional subpopulations and interrogated for intercellular communication using CellChat. A distinct megakaryocyte-like granulocyte population was validated in an independent scRNA-seq dataset. Bulk RNA-seq (n = 139) and plasma ELISA assays were employed to support the associated molecular signatures. Crucially, flow cytometry of peripheral blood from AD patients (n = 5) and HCs (n = 4) was performed to provide protein-level validation of the identified megakaryocyte-like granulocytes.
Results:
MKs segregated into immune-active and platelet-generating subtypes, both exhibiting altered signaling in ADs. LDGs harbored a unique PF4 + /PPBP + megakaryocyte-like subpopulation with heightened interferon activity, proinflammatory signaling, and transcriptional signatures of increased neutrophil extracellular trap (NET) formation. Flow cytometry confirmed the presence of these granulocytes and showed a higher proportion in AD patients than in HCs. These granulocytes showed predicted communication with MKs via ITGB2-ICAM2 and APP-CD74 axes. Findings were consistently validated in an external scRNA-seq dataset and corroborated by bulk RNA-seq deconvolution and elevated plasma myeloperoxidase levels.
Conclusions:
We identify a potentially PF4 + /PPBP + megakaryocyte-like granulocyte subset associated with immune dysregulation in ADs. While flow cytometry provides protein-level evidence for this population, further mechanistic studies are required to fully elucidate its functional role in disease pathogenesis.
