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Immune modulation by lymph node stromal cell-derived extracellular vesicles

Periodic Reporting for period 1 - IMSTREV (Immune modulation by lymph node stromal cell-derived extracellular vesicles)

Reporting period: 2015-07-01 to 2017-06-30

The problem/issue being addressed and overall objectives
The gut mucosa represents the largest and most dynamic immunological environment, where immune cells continuously traffic and reside. Their major role is to clear harmful pathogens and to maintain tolerance against harmless commensal bacteria and food antigens. Regulatory T cells (Tregs), endowed with immune suppressive capacity, are the master regulators of mucosal tolerance. Therefore, they have attracted an increasing interest as potential therapeutic targets in chronic inflammatory diseases, such as ulcerative colitis or inflammatory bowel disease.
The aim of the proposal was to better understand the mechanism of peripheral tolerance, and based on previous observations showing that the gut‑draining mesenteric lymph nodes (mLN) harbor superior Treg‑inducing capacity (Cording, Muc Immunol, 2014), which could be attributed to LN stromal cells, namely to fibroblastic reticular cells (FRCs).
My goals included:
- the identification of tolerogenic molecules secreted from mLN FRCs,
- to reveal their intercellular transfer via FRC-derived microvesicles (MVs),
- and to understand the crosstalk between FRCs and dendritic cells (DCs) under tolerogenic circumstances.
Importance for the society
Gaining insight into the molecular mechanisms of peripheral tolerance can pave the way for future vaccine developments by identifying promising targets. Furthermore, my continuous interaction with researchers during conferences, inside my host institution, with my previous laboratory in Hungary and my future laboratory in Munich expands the cooperation between countries of the European Union.
Conclusion of the action
The results can be divided into two major parts, because the work plan was revised at the end of the first year (reported to the project organizer on 09-05-2016).
1. The tumor growth factor‑β1 (TGF‑β1) cytokine, known to play critical role in driving Treg differentiation, was upregulated in mLN-iFRCs, was associated with mLN-iFRC-derived MVs, and was shown as one of the major mediators of their Treg induction. These results have been accepted for publication at the European Journal of Immunology.
2. Resident DCs from the mLN were shown to harbor tolerogenic gene signatures and moderate Treg‑inducing capacity, which was stably maintained after transplantation to the skin-draining site, and suggests its imprinting driven by mLN-FRCs. I am aiming to publish these data in a high-impact scientific journal as a separate manuscript.
Detailed description of the performed scientific work including short summary of the results
The original plan aimed to identify soluble mediators shaping the tolerogenic properties of mLN‑iFRCs. The findings show that mLN‑iFRCs are key players of peripheral tolerance by supporting Treg‑induction via release of TGF‑β‑containing MVs.
The following steps of the original work plan have been completed:
- Characterization of subcellular structures within the supernatant of iFRCs.
- Analysis of the immune modulatory capacity (Treg-induction) of iFRC‑derived MVs.
- Generation of the list of factors delivered by iFRC‑derived MVs by using RNA‑Seq analysis.
- The functional validation of TGF‑β as a Treg‑inducing candidate in iFRC‑EVs.

After the usefulness of the cell lines in the originally planned experiments became questionable, I had to find another experimental system enabling to better understand the molecular mechanisms of peripheral tolerance and in parallel support my personal career development. The goal was then to understand the crosstalk between stromal cells and DCs under tolerogenic conditions. The results demonstrate that stromal cells instruct resident DCs of the mLN to keep tolerogenic properties.
The following steps were followed from the beginning of the second year to the end of the fellowship:
- Analysis of resident and migratory DC subsets in endogenous and transplanted LNs.
- Determination of the rate of DC replenishment in transplanted LNs.
- Establishment of a miniaturized Treg‑induction assay and assessment of the Treg‑inducing capacity of resident and migratory DCs isolated from transplanted LNs.
- Identification of gene signatures from resident and migratory DCs of endogenous and transplanted LNs.

Exploitation and dissemination of the results
The results are going to be published in “Open Access” journals. The data of the RNA‑Seq analysis will be shared through the “Gene Expression Omnibus” application. During the “HZI Progress Seminar Series”, the evaluation of the host group, and the meetings of the “Mucosal Immunology Group” (December 2015, July and September 2016, February 2017) I discussed my data with scientists of the field. In addition, I attended several national/international scientific conferences.
Progress beyond the state of the art
The project provided key observations in the field of stromal cell biology, which is an upcoming field of immunology. On one hand, the results suggest that stromal cells in the mLN modulate Tregs via release of MVs. On the other hand, the data reveal that stromal cells can also instruct incoming DCs to keep tolerogenic properties. Both results provide a better insight into the mechanism of peripheral tolerance. Disturbance in this well‑balanced homeostasis of the intestine might lead to the development of chronic immunologically mediated diseases, such as inflammatory bowel disease (IBD). The number of affected patients in Europe experienced a very steep increase in the last century. So far, a number of environmental, genetic, and microbiota‑related associations have been identified, however, high‑quality basic immunological studies are still needed to better understand the disease pathophysiology.
Potential impact on the career development of the fellow
Beside the promise of the translation of these basic research results into the development of novel drugs in the future, the project can positively influence my career development in many different aspects. Publication of the existing results will improve my chance to acquire third‑party funding. Moreover, I acquired numerous soft skills during the fellowship, which will help me in becoming a competitive and independent scientist. The development of the new work plan for the second year demanded my creativity and flexibility, from which I will benefit when establishing an own research group.
During the project period, I attended many international scientific conferences. Due to the connections established during these conferences and my willingness for mobility, I could easily find my next senior position at the Ludwig-Maximilian University of Munich. In addition, I became a member of the “Dendritic cells” as well as “T cells: subsets and function” study groups of the German Society for Immunology, helping me to cooperate with scientists in the same research field within Germany and the European Union. I will stay in close contact with colleagues of the host institute via the HZI Alumni network. Moreover, the project summary has been placed to the website of the host institute ( emphasizing that the results were obtained in the framework of a Marie Sklodowska Curie Individual Fellowship, and further improving the communication between the public and the scientific community.
Direct and indirect ways of Treg induction by mesenteric lymph node stroll cells.