Up-Regulation of Immune Tolerance Genes in Leukemic Mesenchymal Stromal Cells Is Induced By Acute Myeloid Leukemia Cells through an IFN-Gamma-Dependent Inflammatory Signaling
Authors: Ciciarello Marilena , Corradi Giulia , Simonetti Giorgia , Marconi Giovanni , Sangaletti Sabina , Colombo Mario Paolo , Martinelli Giovanni , Cavo Michele , Curti Antonio 
Published: October 2019

Abstract
Introduction. Mesenchymal stromal cells (MSCs) substantially contribute to the creation of hematopoietic niche by regulating hematopoietic stem cell (HSC) fate and have a unique immune-modulating capacity. In the leukemic milieu, the presence of MSCs constitutes a side effect, since MSCs not only favor leukemic cell survival, but they can also generate an immune-tolerant environment. Although recent findings have outlined a putative MSC role in hematological malignancy development, MSC-dependent mechanisms potentially supporting leukemia remain unclear. We hypothesize that leukemic cells can shape bone marrow (BM) MSCs by inducing functional changes, able to convert the BM microenvironment from hostile to permissive for leukemia.
Methods. We isolated acute myeloid leukemia (AML) cells and generated AML-MSCs from the BM of AML patients. Next, we set up AML-MSC/AML cell co-culture experiments and we investigated gene expression in AML-MSCs and AML cells before and after co-cultures.
Results. Our microarray data on BM isolated cells (AML patients, N=61; healthy donors, N=7) indicated Interferon(IFN)-γ as un up-regulated gene in almost 40% of AML samples. Furthermore, multivariate analysis, showed that IFN-g-positive AML patients had a better overall survival. Thus, we decided to deepen IFN-ϒ-dependent modifications in leukemic milieu through in vitro studies. In AML-MSC/AML cell co-culture experiments, we confirmed microarray data and we found that AML cells produced IFN-γ. We next demonstrated that indoleamine 2,3-dioxygenase (IDO)1 enzyme, a master regulator of MSC immune suppressive functions, is up-regulated in AML-MSCs after co-culture with IFN-γ-producing AML cells. Such effect was abrogated by adding to cell cultures an IFN-γ neutralizing antibody. Finally, we found that AML-MSCs, after co-culture with IFN-γ-producing AML cells, were able to induce regulatory T cell in a IDO1-dependent manner. To gain further insight in AML cell-dependent MSC modifications, we analyzed MSC expression of IFN-γ-stimulated genes (ISGs) such as Programmed death-ligand (PDL)-1 and Nitric Oxide synthase (NOS)-2 which are known to regulate immunity and promote tolerance. In particular, we tested the ISG expression in MSCs after co-cultures with IFN-ϒ positive or IFN-γ negative AML cells. We found that IFN-γ positive, but not IFN-γ negative AML cells, were able to induce PDL-1 and NOS2 in AML-MSCs. Thus, ISG expression profile in AML-MSCs after co-cultures with IFN-γ positive AML cells was similar as that observed in MSCs after exposure to recombinant IFN-γ.
Conclusions. Our data suggest that inflammatory signals produced by AML cells are able to modify MSC functions, thus favoring an immune-tolerant and leukemia supporting milieu. Overall, our results would likely contribute to unravel MSC-dependent mechanisms promoting leukemia and will help to provide novel applications for drugs already under experimentation (e.g. IDO-inhibitors, Checkpoint inhibitors) to translate into more effective therapies in AML patients.

Disclosures
Cavo: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.