During this research, we will investigate if, like T cells, HLA-G induces regulatory B cells (Bregs). These cells were recently described in animal and human autoimmune disease models in which they could play an important role in maintaining immune tolerance. Different phenotypes were attributed to this regulatory function of B cells, with IL-10 secretion being the major characteristic implicated in their function. Our project will be conducted with a recombinant soluble HLA-G protein which has already been produced, purified and validated in the laboratory 1. Differentiation of B cells into Bregs will be determined by flow cytometry using markers already described in the literature for these cells, such as: the CD32a/CD32b ratio, CD19high CD24high CD38high CD5+ CD23- CD1dhigh. This research will be continued by means of a transcriptome approach comparing the profiles of B cells with or without HLA-G treatment, which will make it possible not only to characterise the regulatory transcriptional profile in favour of B cell survival, but also to identify any new factors modulated by HLA-G in terms of the phenotype/function of B cells. The phenotypes of regulatory B cells identified will then be investigated in diseases in which HLA-G has been implicated, particularly in transplant or cancer patients.

Chronic rejection, in the form of bronchiolitis obliterans, remains the leading cause of death in lung transplant patients. The target of this chronic rejection is the bronchial epithelial cell (BEC), via the cell lesions involving its interaction with the dendritic cells of the airways and T cells in the recipient. Numerous in vitro and organ transplantation data suggest a state of tolerance associated with the expression of the HLA-G molecule, through the modulation of the functions of immune effector cells 2. In human lung transplants, we previously showed that the expression of HLA-G by BEC was correlated with lung graft stability 3. In order to reproduce the mechanisms as close as possible to reality, we propose to study a mixed reaction between APC and autologous T lymphocytes from lung transplant patients in a stable state, or experiencing chronic rejection in the presence of graft BEC. The potential role of the microenvironment (including the HLA-G molecule, and IL-15 implicated in rejection) of primary BEC cultures on monocyte differentiation in APC and on lymphocyte proliferation during the mixed reaction will be analysed. 

The study of the dialogue between the bronchial epithelial cells and the immune cells in the recipient is based on the hypothesis that one of the first levels of modulation is supposedly related to the ability of BEC directly to act as APC with regard to T lymphocytes. Secondly, BEC could affect the function of immune cells (dendritic cells and T lymphocytes) by producing a tolerogenic microenvironment, via the secretion of soluble factors. The implications of the HLA-G molecule in this microenvironment will be investigated. In order to achieve these objectives, we will work in partnership with INSERM respiratory pathophysiology unit U700 and the Service clinique de pneumologie at Hôpital Bichat. The ambitious nature of this project resides in the acknowledgement of local immunity in the lung transplant through the study of the immunological properties of the bronchial epithelial cell from the graft and its ability to control immune rejection. This project should thus make it possible to clarify the role of the bronchial epithelial cell as a major protagonist modulating immune response in the recipient. Lastly, in order to have a better understanding of the mechanisms regulating HLA-G expression, we will analyse HLA-G alleles both in the donor and the graft to evaluate their relevance in the context of graft acceptance or rejection. 

We recently demonstrated that the HLA-G inhibits the proliferation and differentiation of tumoural B cells (notably myelomatous CD138+ cells) 1, and also exerts an anti-osteoclastic activity. Our objective is to study the interactions between haematopoietic cells and mesenchymal stromal cells in co-culture tests using bone marrow specimens from myeloma patients compared with healthy individuals. The role of HLA-G and its receptors within these cell interactions and the benefit of its anti-osteoclastic activity will be evaluated through the use of blocking antibodies and recombinant proteins.

One of the pitfalls of cell therapy is the rejection of transplanted cells. In view of the tolerogenic properties of the HLA-G molecule, the therapeutic challenges are either the use of its immuno-tolerogenic properties in order to promote the implantation of transplanted cells, or the generation of cells expressing HLA-G for the tissue graft. In these contexts, we will conduct studies involving mesenchymal stems cells, and also induced pluripotent cells.

We propose to continue studying the functional consequences of HLA-G expression by mesenchymal stem cells (MSC) on the induction of immune tolerance in vitro and in vivo, i.e. by characterising the role of the HLA-G molecule in terms of the tolerogenic properties of MSC, in the context of their use, either in adjuvant tolerance-induction therapy, or in bone and skin repair. After having studied the role of HLA-G expressed by MSC on the function of T and NK cells 4, we will analyse antigen-presenting cells and B lymphocytes. Different aspects will be studied, such as the proliferative response, the secretion of cytokines and antibodies, and the induction of regulatory B cells and tolerogenic antigen-presenting cells. We will also study the influence of environmental factors, such as the presence of certain cytokines (IFN- and TNF- mimicking an inflammatory context or LIF, Progesterone and IL-10 mimicking a tolerogenic context) and hypoxic culture conditions on the expression and function of HLA-G by MSC. We have simultaneously entered into a partnership with the EFS at Hôpital Mondor (European REBORNE programme) based on the study of a murine xenotransplantation model consisting of an injection of human MSC in combination with biomaterials in immunocompetent mice. We will evaluate whether these cells are accepted and generate bone in vivo. The impact of HLA-G in the induction of this state of tolerance will be analysed through the use of anti-HLA-G blocking antibodies. Furthermore, our partnership with the Centre de Percy will continue with the aim of using allogeneic MSC originating from extraembryonic tissue in skin repair. As such, this partnership is also being implemented with the Centre des grands brûlés, at Hôpital Saint-Louis. 

​Nathalie ROUAS-FREISS, PhD

Team leader​

T​él : 33 (0)1 57 27 68 01

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Technician

Technician

Master 2 student

Wenjing DU - PhD student