Senescence and Mitochondrial Transfer Ability of Bone Marrow Mesenchymal Stromal Cells Influence Patterns of Alloreactivity after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However post-transplant disease recurrence and alloreactive complications (chiefly graft versus host diseases - GvHD) are still associated with severe morbidity and mortality. Proliferative advantage of transformed cells as well as immune and microenvironmental dysregulations contribute both to promoting relapse and escape from graft versus leukemia (GvL) effect. Bone marrow (BM) mesenchymal stromal cells (MSC) are well known for their role in hematopoiesis and immune modulation. MSCs have been shown able to transfer mitochondria (MT) to different cell populations, including immune effectors, in order to (re)establish a healthy oxidative metabolism within the recipient cell. Here we hypothesize that these mechanisms are highly active in BM microenvironment after allo-HSCT and may contribute to alloreactivity, modifying the immune environment. On this basis we stipulate that those intercellular exchanges are deeply impacted by MSCs' senescence, that could be deployed as a useful parameter to predict post-transplant outcomes.

To that end, we conducted a translational research, based on BM specimens prospectively collected at our Institution (NCT03357172, NCT03964922), involving patients undergoing allo-HSCT for high-risk MDS and AML and receiving fludarabine/busulfan-based conditioning regimens, anti-lymphoglobuline and cyclosporin/mycophenolate mofetil. MSC phenotype and functionality at the pre-transplant stage, MT capacity, immunomodulation towards T cells and phenotype were studied by multiparametric flow cytometry. Cell metabolism, in terms of oxygen consumption and extracellular acidification rate, was characterized with Seahorse XF technology. Percentages of senescent MSCs were determined with SA β-galactosidase test.

First, we compared phenotypic and metabolic characteristics of MSC from MDS/AML (N=29) patients with those retrieved from a cohort of healthy controls (N=8). We observed that patient MSCs were characterized by higher expression of CD157 and CD146 and lower expression of CD200 as compared to healthy MSCs. Pre-transplant senescence quantification highlighted a certain degree of heterogeneity within the MSC compartment in leukemic patients (median = 37.42% sd=16.30 for AML patient and m=11.74% sd=6.4 for HC). In an attempt to link clinical phenotypes to this heterogeneity, we observed that patients with high degree of MSC senescence in pretransplant samples (mean= 51.6% sd=16.6 cells/sample), developed more frequently acute GvHD, as compared to those with low degree of pre-transplant MSC senescence (mean= 23.8% sd=14.3) who experienced instead a higher incidence of disease relapse (p=0,004). Assuming that the link between senescence and alloreactivity could rely to different metabolic states and MT capabilities in MSCs, we analyzed these biological features in our samples. Indeed, we observed that low degree of MSC senescence was associated with higher MT to T cells, in particular to CD4+ cells, shifting their metabolism and phenotype toward an immunosuppressive state The characterization of cellular metabolism showed in highly senescent MSCs the presence of markers of mitochondrial dysfunction with high oxidative power, but reduced coupling efficiency of respiratory chain. This metabolic profile involved a high mitochondrial mass characterized by a high maximal respiration capacity compared to HC MSCs. The in vitro modulation of MSC senescence with nicotinamide administration restored healthy mitochondrial metabolism and improved coupling efficacy of oxidative phosphorylation, possibly impacting on the immunomodulation capabilities of this microenvironment component.

Here for the first time, we provide the evidence that MSC senescence plays an pivotal role in defining an altered immune state in transplanted AML possibly shifting the degree of alloreactivity from a high GvH/GvL response (high GvHD and low relapse) to a high immunosuppressive state (low GvHD and high relapse). These findings pave the way for implementing MSC senescence as a biomarker able to predict post-transplant outcomes and show new biological rationales to integrate donor-derived MSC products to better adjust GvH responses.