Influence of Inflammatory Factors Produced during Graft-Versus-Host Disease on Immunological Properties of Mesenchymal Stem Cells (MSC).
Authors: Tarte Karin , Monnier Delphine , Tribut Olivier , Dulong Joelle , Bonnaventure Céline , Ame-Thomas Patricia , Bescher Nadège , Fest Thierry , Lamy Thierry , Semana Gilbert 
Published: October 2019

Abstract
MSC have been demonstrated to display non-HLA restricted immunosuppressive capacities in vitro and in vivo. Encouraging clinical results have been obtained using MSC infusion for the treatment of steroid-resistant acute graft-versus-host disease (aGVHD). However, very few data are available concerning the capacity of MSC to respond to inflammatory factors that are highly expressed during aGVHD or the influence of these factors on MSC outcome and immunological properties. In particular, the modulation, by environmental factors, of the production of the immunosuppressive enzyme indoleamine-2,3 dioxygenase (IDO) by MSC remains unexplored. The aim of our study was thus to evaluate the influence of the main inflammatory factors produced during GVHD on immunological properties of human clinical-grade MSC.
We first confirmed that MSC expressed functional INFγR1 and TNFR1 since IFNγ and TNFα induced a broad set of genes, including those coding for the inflammatory chemokines CXCL9 and CXCL10. During GVHD, a disruption of gastro-intestinal tract integrity occurs and is associated with the release of various microbial compounds that are sensed by Toll Like Receptors (TLR). Using quantitative RT-PCR, we performed an exhaustive analysis of TLR expression on MSC. Whereas TLR2, TLR5, and TLR9 were essentially absent, TLR3 and TLR4, were highly expressed on human clinical-grade MSC. Accordingly, stimulation of MSC by poly(I:C) or LPS induced expression of CXCL9 and CXCL10. Lastly, we focused our work on CD40/CD40L interaction since a high proportion of CD4posT lymphocytes is activated during GVHD and expresses CD40L. We demonstrated that CD40 was present at the beginning of clinical-grade MSC culture and was significantly lost, both at RNA and protein level, after several cell passages. We also provided some evidence that CD40 was uniformly and highly expressed in vivo on CD45negCD14negCD73pos bone marrow cells that were previously shown to be highly enriched for MSC. Importantly, stimulation of MSC by trimeric CD40L was associated with a strong activation of NF-κB signalling pathway and the induction of IL8 gene expression.
We then assessed the functional consequences of MSC stimulation by inflammatory factors on their immunological properties. IFNγ and TNFα, unlike TLR ligands and CD40L, induced the expression of relevant immunological markers such as HLA-I, HLA-II, CD54, CD106 and CD40. CD80 and CD86 were not inducible. Despite these phenotypic modifications, MSC did not become immunogenic and maintained their immunosuppressive properties. However, TNFα decreased and IFNγ increased this inhibitory effect. Interestingly, whereas IDO RNA was induced by TNFα and TLR ligands, IFNγ was the only inflammatory mediator able to induce IDO activity, as evaluated by HPLC. TNFα and LPS exerted a synergistic action with IFNγ for IDO activation.
In conclusion, MSC express functional key receptors involved in inflammatory response. Their activation modulates properties of MSC into opposite ways: taking part of the inflammatory process through secretion of inflammatory chemokines, or blocking T-cell proliferation through induction of IDO. Such balance should be further studied to favour the development of new clinical trials involving MSC for treatment of aGVHD.