Understanding the Immunomodulatory Effect of Mesenchymal Stem Cell Infused In Transplanted Patients with Steroid-Refractory GvHD
Authors: Dander Erica , Lucchini Giovanna , Vinci Paola , Introna Martino , Bonanomi Sonia , Balduzzi Adriana , Gaipa Giuseppe , Perseghin Paolo , Masciocchi Francesca , Capelli Chiara , Golay Josée , Algarotti Alessandra , Rambaldi Alessandro , Rovelli Attilio , Biondi Andrea , Biagi Ettore , D'Amico Giovanna 
Published: October 2019

Abstract
Abstract 2306
In the last few years the usage of third party mesenchymal stem cells (MSC) as therapy for steroid-refractory Graft versus Host Disease (GvHD) is constantly increasing and holds big promises. Nevertheless, at our knowledge, studies on MSC efficacy have been scarcely corroborated by biological analysis of patient response to cell infusion. Here, we report the immunological monitoring of 8 patients (7 male, 1 female; aged 4 to 33 years), with steroid-refractory GvHD (grade II to III), who received MSCs, between August 2009 and June 2010.
GvHD presented as acute in 6 cases and chronic in 2 cases. In 5 cases GvHD occurred as a single organ pathology (2 skin, 2 gut, 1 liver), while in 3 cases GvHD had multi-organ involvement (1 liver and oral mucosa, 1 skin and oral/ocular mucosa, 1 skin, gut and liver). All patients received 2 to 3 MSC infusions from third party donors aiming at 1 × 106/kg recipient body weight MSCs for each infusion. After MSC therapy, 2 patients showed complete response, 3 patients showed partial response, whereas 3 patients did not respond to MSC infusion.
To better comprehend the immunomodulatory effects of MSC infusions, we studied GvHD plasmatic markers, inflammatory cytokines and CD4+ T-cell subsets circulating in the peripheral blood (PB) of enrolled patients before MSC infusion and at day 7, 14 and 28 after cell therapy.
In accordance with clinical observations, in patients responding to MSC infusions, we observed a dramatic decrease of three validated GvHD plasmatic markers TNFRI, IL2Rα and elafin (Paczesny S et al. Blood 2009) to the mean levels of Healthy Donors (HD). In particular, at day 28 after therapy, TNFRI and IL2Rα levels decreased of 2 times (range=1.9-2.4 and range=1.4-2.8, respectively) and elafin levels decreased of 2.5 times (range=1.7-3.6). Partially responding patients showed a transient decrease of TNFRI, IL2Rα and elafin levels, while non responding patients showed stable or even increasing levels of all analysed markers.
Moreover, we investigated the effect of MSC infusion on lymphocyte counts. We demonstrated that patients responding to MSC infusion, oppositely to non responders, strongly decreased total and CD4+ lymphocyte counts in the PB (mean total T-cell Fold Decrease (FD)=11.85, range=1.3-116; mean CD4+ T-cell FD=12, range=1.5-116). Interestingly, after MSC infusion, CD4+ T-cell subsets changed significantly: Tregs increased and Th1 and Th17 populations decreased, and a new CD4+ cell subset balance was observed starting from day 7 after therapy. In particular, the mean FD of Th1/Treg ratio was 4.1 (range=4-4.2) and the mean FD of Th17/Treg ratio was 4.7 (range=3.3-6). Correspondingly, patient symptoms also gradually improved, suggesting an association between GvHD clinical course and CD4+ T-cell imbalance, reverted by MSCs in responding patients. In partially responding patients Th1/Treg and Th17/Treg showed a transient decreased and even slightly increased in the case of non responding patients.
In accordance with the decrease of Th1 CD4+ T cells in the PB of patients responding to MSC infusion, we observed a valuable decrease of IFNγ plasma concentrations (mean FD=48, range=30-65 in complete responders), which reached the levels typical of HD.
In summary, despite its limited size, the present study suggests that MSCs, upon infusion, are able to convert an inflammatory environment to a more physiological one, both at a cellular level, promoting the expansion of circulating Tregs, and at a molecular level, diminishing inflammatory cytokines. Further studies on a larger group of patients, clarifying the mechanisms of action used in vivo by MSC to tune ongoing allo-reactions, will be fundamental to provide the rationale for improving current clinical trials.

Disclosures:
No relevant conflicts of interest to declare.