PhD Scientific Days 2025

Molecular Medicine III.

Isolation and Characterization of Tissue-derived Extracellular Vesicles from Mouse Lymph Nodes

Name of the presenter

Bodnár Bernadett Réka

Institute/workplace of the presenter

Institute of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary

Authors

Bernadett R Bodnár^1,2,3, Sayam Ghosal^1,3, Brachyahu M Kestecher^1,2,3, András Försönits³, Nóra Fekete³, Edina Bugyik³, Zsolt I Komlósi³, Éva Pállinger³, Edit I Buzás^1,2,3, Xabier Osteikoetxea^1,3

1: HCEMM-SU Extracellular Vesicles Research Group, Budapest, Hungary
2: HUN-REN-SU Translational Extracellular Vesicle Research Group, Budapest
3: Institute of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary

Text of the abstract

Introduction:
Extracellular vesicles (EVs) are lipid bilayer-enclosed particles released by all cells and play key roles in immune regulation. While EVs derived from body fluids are well studied, tissue-derived EVs, especially from lymph nodes (LNs), remain poorly characterized due to technical limitations.

Aims:
This study aimed to develop a reproducible method for isolating and characterizing large (lEV) and small (sEV) EV subpopulations from murine LNs and to investigate the immunization-induced changes in their properties.

Methods:
Male C57BL/6 mice were immunized with complete Freund’s adjuvant (CFA) with or without ovalbumin (OVA). Inguinal and popliteal LNs were excised 9 days later. EVs were isolated using differential centrifugation and size-exclusion chromatography. EV morphology was evaluated by transmission electron microscopy (TEM), and Particle size distribution and concentration via nanoparticle tracking analysis. Protein and lipid contents were measured by BCA and SPV assays, respectively. EV surface markers were analyzed using bead-based flow cytometry.

Results:
OVA+CFA immunization significantly increased LN mass and altered cellular composition. OVA+CFA sEVs consistently showed higher particle counts and protein content compared to lEVs. Protein-to-lipid ratios were higher in sEVs, especially after CFA treatment. TEM confirmed intact vesicles with specific morphological features. Flow cytometry revealed immunization-specific changes in EV markers including CD45, CD146, and MHC class II, highlighting their immunomodulatory potential.

Conclusion:
This study establishes a protocol for LN-EV isolation and demonstrates that immunization influences the composition and surface markers of EVs. These findings support the role of tissue-derived EVs in immune regulation and their potential for immunotherapy and vaccine development.

Funding:
The project has received funding from the EU’s Horizon 2020 Research and Innovation Programme under grant agreement No. 739593 and the NKFIH 147023 OTKA-FK, 150767 Advanced, 151417 Excellence, 135637 OTKA-K grants, NVKP_16-1-2016-0004, EKÖP-2024-237, VEKOP-2.3.2-162016-00002, VEKOP-2.3.3-15-2017-00016, The Higher Education Excellence Program (FIKP) and the Therapeutic Thematic Programme TKP2021-EGA-23, also RRF-2.3.121-2022-00003 and 2019 − 2.1.7-ERA-NET-2021-00015.