PhD Scientific Days 2026

Poster Session 2.C - Molecular Medicine

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

Name of the presenter

Bodnár, Bernadett Réka

Institute/workplace of the presenter

Semmelweis University, Genetics, Cell- and Immunobiology Institute

Authors

Bernadett R Bodnár^1,2,3, Krisztina Shielnik¹, Sayam Ghosal^1,2, Brachyahu M Kestecher^1,2,3, András Försönits¹, Nóra Fekete¹, Edina Bugyik¹, Zsolt Komlósi¹, Éva Pállinger¹, Edit I Buzás^1,2,3, Xabier Osteikoetxea^1,2
1: Institute of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary
2: HCEMM-SU Extracellular Vesicles Research Group, Budapest, Hungary
3: HUN-REN-SU Translational Extracellular Vesicle Research Group, Budapest

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 (SEC). 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:
EU’s Horizon 2020 (No. 739593), (NKFIH) 147023 OTKA-FK, 150767 Advanced, 151417 Excellence, 135637 OTKA-K, NVKP_16-1-2016-0004, EKÖP-2024-237, VEKOP-2.3.2-162016-00002, VEKOP-2.3.3-15-2017-00016, FIKP and TKP2021-EGA-23, RRF-2.3.121-2022-00003, 2019 − 2.1.7-ERA-NET-2021-00015. SUPPORTED BY THE 2025-2.1.1-EKÖP-2025-00014 UNIVERSITY RESEARCH SCHOLARSHIP
PROGRAMME OF THE MINISTRY FOR CULTURE AND INNOVATION FROM THE SOURCE OF THE
NATIONAL RESEARCH, DEVELOPMENT AND INNOVATION FUND