Fekete, N. | Search bioRxiv

bioRxiv2025

BeWo-derived extracellular vesicles downregulate IL-6Rα expression via miRNAs on CD4+ T lymphocytes

Regulatory T lymphocytes are essential for maternal immunotolerance. Their de novo differentiation in the placenta is regulated by local intercellular interactions involving primed uterine immune cells, fetal syncytiotrophoblasts, and the cytokine environment. Trophoblast-derived, HLA-G-positive extracellular vesicles (EVs) can bind to T lymphocytes, thereby influencing their differentiation and cytokine production. Thus, these EVs play a role in establishing and maintaining a tolerogenic environment. In our study, we used the BeWo choriocarcinoma cell line to model the effects of trophoblast-derived EVs. Large EVs derived from BeWo cells (BeWo-12.5K lEVs) reduce IL-6R expression on CD4+ T cells. This modifies the IL-6 pathway by downregulating the transcription factors STAT3 and NFKB1, and PIAS3, while upregulating STAT1. This may be caused by specific microRNAs (miRNAs), such as hsa-mir-92a-3p, hsa-mir-520f-3p, and hsa-mir-25-3p. These microRNAs are present in BeWo-12.5K lEVs and target the IL-6 pathway. BeWo-12.5K lEVs induce phenotypic and functional changes in T cells, enhancing the ratio of CD4+/CD25+ T cells that produce IL-10. Pregnancy-associated IL-6R downregulation has been demonstrated in clinical samples. Significantly lower levels of IL-6R were detected on circulating CD4+CD25+ T cells in healthy pregnant women than in healthy non-pregnant individuals. This finding reflects the in vivo significance of our in vitro studies. Our studies suggest that communication between maternal and fetal cells significantly influences the development and maintenance of local T cell polarity. The microRNA content of HLA-G+ 12.5K lEVs appears to be key to this process, as it alters the IL-6 pathway.

immunology↗

bioRxiv2025

Cholesteryl esters and high protein-to-lipid ratios distinguish Non-Vesicular Extracellular Particles from Extracellular Vesicles

Extracellular vesicles (EVs) are central to intercellular communication, yet the mechanisms underlying their biogenesis and diversity remain incompletely understood. Here, we integrate meta-analysis, advanced lipidomic, protein-to-lipid profiling, and super-resolution imaging to define the fundamental principles governing EV heterogeneity. Our meta-analysis of published transmission electron microcrographs across kingdoms reveals a highly conserved 110 nm average diameter and 200 nm upper size limit for intraluminal vesicles (ILVs), which are secreted as exosomes. Besides classical EV populations, we also characterize a distinct nanoparticle class: 167000 xg pellet of non-vesicular extracellular particles (167k-NVEPs), which exhibit a significantly higher protein-to-lipid ratio than 14000 xg pellet of large EVs (14k-lEVs) and 100000 xg pellet of small EVs (100k-sEVs), as measured by both biochemical assays and Raman spectroscopy. Lipid profiling demonstrates that 167k-NVEPs exhibit significant enrichment in cholesteryl esters and triacylglycerols, lipids typically associated with lipid droplets and the endosome/lysosome system. Analysis of lipid carbon-chain lengths reveals distinct signatures: 167k-NVEPs show pronounced enrichment at 16 and 18 carbons, while 100k-sEVs display enrichment at 32 and 34 carbons. This divergence indicates a potential connection to flexible biogenesis pathways. Marker heterogeneity across EV populations, confirmed by confocal and super-resolution microscopy, further underscores the limitations of relying on canonical tetraspanins for EV classification. Notably, 167k-NVEPs (likely exomeres) exhibit enrichment of Arf6 and CD63. Together, our findings provide compositional, biophysical, and molecular evidence supporting the formal recognition of 167k-NVEPs as a distinct class of extracellular particles and enabling exploring in disease biology and therapeutic delivery. Significance StatementExtracellular vesicles (EVs) are critical mediators of intercellular communication, yet their classification remains clouded by ambiguity in terms of their composition and biogenesis. This study resolves critical uncertainties through a cross-kingdom meta-analysis, establishing a conserved [~]110nm diameter and [~]200 nm upper size limit for intraluminal vesicles (ILVs), the precursors to exosomes. More significantly, we identify non-vesicular extracellular particles (167k-NVEPs) as a distinct class based on their unique sterol-rich lipidome, enrichment in lipids of 16 and 18 carbon chain length, elevated protein-to-lipid ratio, and functional cargo delivery. These features, alongside evidence of non-canonical origin and functional cargo delivery, establish NVEPs as a discrete class of extracellular particles.

cell biology↗