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Visualizing tolerance induction: behavior and function of regulatory T cells during the establishment of materno/fetal tolerance

Final Report Summary - VISTO (Visualizing tolerance induction: behavior and function of regulatory T cells during the establishment of materno/fetal tolerance)


We are interested in understanding the mechanisms that govern the establishment of immune tolerance. As a model system we have chosen to study materno/fetal tolerance, as pregnancy represents the most physiological model of immune tolerance. A fetus is inherently antigenic to its mother, yet is not rejected. The question of how fetus and placenta avoid rejection by the maternal immune system has intrigued immunologists and reproductive biologists alike for many years, yet important questions remain unanswered. The understanding of materno/fetal tolerance has implications for organ transplantation, autoimmune disease and cancer.
To better understand the establishment of materno/fetal tolerance we aimed at 1) visualizing and understanding when, where and how regulatory T cells are recruited during embryo implantation; 2) visualizing and understanding the relationship between regulatory T cells and other immune system cells in physiological and disease conditions; 3) identifying the molecular determinants for tolerogenic conditions during embryo implantation using a candidate gene approach.

One of the characteristic features of immune cells is their ability to migrate in order to reach the sites where their presence is needed. We therefore set up a method to study their recruitment and behaviour at the materno/fetal interface, the decidua. The decidua, in the uterine wall, represents the site where immune cells first encounter and get in contact with fetal derivatives during early pregnancy. We used intravital two-photon microscopy and fluorescent transgenic mouse lines, which allowed us to visualize regulatory T cells and dendritic cells, and to study their recruitment and their interactions both in the uterine wall and in the uterine draining lymph nodes. We further combined intravital two-photon microscopy with flow cytometry and immunohistochemistry.

1) Tregs at the materno/fetal interface
We found that numbers of regulatory T cells are subjected to dramatic changes already during the estrus cycle in mouse females in preparation of a possible pregnancy. Upon fecundation, regulatory T cells further increase in the uterus, both due to rapid proliferation and recruitment from the periphery. Only later on, regulatory T cells start to increase in the draining lymph nodes, suggesting a tolerogenic response occurring in two phases: a local one first, followed by a systemic response. Morphologically, regulatory T cells in the uterus show a different phenotype than the regulatory T cells in the lymph nodes, with a characteristic rapid upregulation of CTLA-4, an activation marker, and a down modulation of CD25. Interestingly, in the uterus of pregnant females, these cells do not seem motile when observed for long periods of time by two-photon microscopy. Typically, T cell activation and function require their arrest, suggesting that regulatory T cells in the uterus are functionally active to perform their tasks. Further analysis is needed to understand whether they mediate suppressive activity at this point during pregnancy.
Furthermore, we purified regulatory T cells from uteri and lymph nodes of pregnant and non-pregnant females and found that uterine regulatory T cells exhibit a different cytokine profile compared to regulatory T cells from lymph nodes. In particular, regulatory T cells from the uterus of pregnant females produce high levels of IL9. IL9 is a cytokine involved in the survival and activation of mast cells. We found that mast cell numbers in the uterine wall increased after implantation. These results suggest a possible crosstalk between regulatory T cells and mast cells mediated by IL9.

2) Regulatory T cell/dendritic cell interactions
We found that regulatory T cells and dendritic cells localize at different poles of the decidua upon implantation, suggesting their direct involvement in the process. We next generated transgenic animals that bear fluorescently labelled regulatory T cells and dendritic cells to study their interactions. Analysis of regulatory T cells and dendritic cells behaviour in the lymph nodes by intravital two-photon microscopy revealed that these cells establish dynamic interactions. The interactions at first are prevalently short lasting, the average motility of regulatory T cells is ~7 um/min. Over time, the number of long lasting interactions increases as pregnancy progresses, and regulatory T cell motility decreases to ~5um/min, suggesting an increased need for regulatory T cell activation and suppression activity.
Taken together our results suggest a model according to which regulatory T cells exhibit two different modalities of behaviour, depending on whether they operate in the uterus or in the draining lymph nodes. In the uterus they proliferate, and they are activated fast during early pregnancy already before implantation In the lymph nodes they are activated after implantation through the action of dendritic cells, and then enter proliferation.

3) Transcriptomic analysis of uterine microenvironment
Next, we analysed the transcriptomic profile of uterine microenvironment of pregnant females at different time points during early pregnancy (E4, E6, E8, E10, E11 and E12) and compared it with the transcriptomic profile obtained from the uterus of non pregnant females.
These type of genome-wide statistical analyses are time consuming and require the establishment of novel mathematical tools (in collaboration with Djamel Nehar and Adrien Six). Therefore, our analysis is still preliminary and incomplete. However, initial analysis reveal highly interesting trends: during the first 12 days of mouse pregnancy, three gene regulatory phases can be identified: an early, an intermediate and a late phase. Gene regulatory programs at E4 are closely related to the non-pregnant situation, while events in E6 and E8 cluster together, as well as events in E10, E11 and E12 cluster together. After fecundation, gene expression changes dramatically: first, stress activated signalling pathways, such as NF-kB and PI3K, are down-modulated. Next, immune response pathways, such as NFAT, are down-modulated, consistent with the establishment of materno-fetal tolerance. Finally, gene proliferation pathways, such as HMGB1, are upregulated. We finally identified 28 gene signatures that pregnancy shares with a tumor model (B16) we have previously analysed.

In summary, we have identified regulatory T cells from uterine origins as a novel and distinct population of regulatory T cells, which carry great potential in the treatment of recurrent spontaneous abortions. We further believe that the cross-comparison of the molecular features of early immune responses during pregnancy and tumor development will provide important new insights into the establishment of immune tolerance, with broad therapeutic implications.



The Marie Curie fellow, Maria Grazia Ruocco, has participated as a speaker and with posters to international congresses and has been invited as a seminar speaker to present her work in international renowned universities.

Maria Grazia Ruocco is currently training a PhD student at UPMC, Laura Florez, who joined the project as a Master student since Dicember 2012 and will defend her PhD thesis in September 2016. Laura Florez will participate to the 5th Annual YRLS Conference from the Young Researchers in Life Sciences Association, May 26-28, 2014 at the Pasteur Institute in Paris with an oral presentation or a poster.

Finally, we are currently working on a draft and anticipate to submit two manuscript for publication in high peer review journals by August 15.