Single-cell analysis identifies a KLF5–Neutrophil–Plasma cell axis driving immune suppression in POEMS syndrome

Abstract Introduction: POEMS syndrome is a rare clonal plasma cell disorder characterized by complex manifestations, marked heterogeneity, and delayed diagnosis. Its pathogenesis remains poorly understood (Isshiki et al., 2022), particularly regarding the phenotype of clonal plasma cells and their interactions with the immune microenvironment. Existing studies are largely limited to case reports or bulk-tissue analyses, with relatively scarce high-resolution data (Nagao et al., 2019). The immunomodulatory role of neutrophils in POEMS also remains uncharacterized. Single-cell technologies enable in-depth dissection of the immune ecosystem and may uncover novel mechanisms and therapeutic targets. Methods: Bone marrow samples from 9 POEMS and 7 monoclonal gammopathy of undetermined significance (MGUS) patients—including paired pre/post-treatment samples from 3 POEMS cases—underwent single-cell RNA sequencing (scRNA-seq). Analyses included marker identification, differential expression, GO/KEGG/GSVA enrichment, pseudotime trajectory, CellPhoneDB-mediated interaction, and UCell immune scoring. SCENIC identified neutrophil-specific transcription factors. KLF5 expression was validated by Western blot (WB). HL-60 cells were differentiated into neutrophil-like cells (dHL-60) and transduced for KLF5 overexpression or inhibited pharmacologically. Functional assays (CCK-8, Transwell, flow cytometry, WB, ELISA, immunofluorescence) assessed proliferation, migration, apoptosis, cytokine secretion, and NETs formation. NETs from KLF5-overexpressing dHL-60 cells were co-cultured with plasma cell lines (RPMI-8226, U266) to evaluate effects on proliferation, migration, vascular endothelial growth factor (VEGF) production, and tumorigenicity. Results: (1) Clonal plasma cells: scRNA-seq identified a unique cluster with MHC-IIlowSPP1highIGLV1-40highIGKV1-5highIGHV1-24high expression, marked by elevated metabolism and inflammatory signaling implicated in multisystem damage. (2) Immune microenvironment: Neutrophils were enriched and expressed VEGF, indicating roles in angiogenesis and immune suppression. Macrophages exhibited exhaustion with impaired antitumor function. Post-treatment samples showed reduced neutrophil abundance and partial immune recovery. (3) KLF5 activity in neutrophils: SCENIC identified KLF5 as a key transcription factor. Its expression was elevated in POEMS bone marrow (p = 0.0109). In vitro, KLF5 promoted dHL-60 proliferation (p = 0.0025), migration, VEGF (p = 0.0018), and IL-10 (p = 0.0019) secretion, suppressed apoptosis (p = 0.0049), and reduced IL-6 (p = 0.0120) and TNF-α (p = 0.0861). NETs formation was enhanced (p = 0.0077), and all effects were reversed by KLF5 inhibition (ML264). (4) NETs-mediated modulation of plasma cells: scRNA-seq of post-treatment plasma cells revealed downregulation of neutrophil activation and NETs-related pathways. NETs co-culture promoted plasma cell proliferation, migration, VEGF production and tumorigenicity, all of which were abrogated by DNase I. Conclusions: This study delineates the single-cell immune landscape of POEMS syndrome, identifying a clonally expanded plasma cell subset with distinct molecular and metabolic features. It reveals a neutrophil-driven immunosuppressive microenvironment, in which KLF5 enhances neutrophil function, VEGF production, and NETs release, thereby promoting plasma cell malignancy. The “KLF5–neutrophil–plasma cell” axis provides novel mechanistic insight into POEMS pathogenesis.