Cytotherapy, 2012; 14: 147–152 Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-g licensing MOÏRA FRANÇOIS1,2, IAN B. COPLAND3, SHALA YUAN2, RAPHAËLLE ROMIEU-MOUREZ2, EDMUND K. WALLER3 & JACQUES GALIPEAU2,3,4 1Department of Experimental Medicine, McGill University, Montréal, Canada, 2Lady Sir Mortimer B. Davis Jewish General Hospital and Lady Davis Institute for Medical Research, Montréal, Canada, 3Department of Hematology and Medical Oncology, and 4Department of Pediatrics, Emory University Winship Cancer Institute, Atlanta, Georgia, USA Abstract Human mesenchymal stromal cells (MSC) can suppress T-cell activation in vitro in an indoleamine 2,3-dioxygenase (IDO)-dependent manner. However, their clinical effects on immune ailments have been inconsistent, with a recent phase III study showing no beneﬁt in acute graft-versus-host disease (GvHD). We here tested the hypothesis that the banked, cryopreserved MSC often used in clinical trials display biologic properties distinct from that of MSC in the log phase of growth typically examined in pre-clinical studies. In freshly thawed cryopreserved MSC derived from normal human volunteers, we observed that MSC up-regulate heat-shock proteins, are refractory to interferon (IFN)-γ-induced up- regulation of IDO, and are compromised in suppressing CD3/CD28-driven T cell proliferation. Immune suppressor activity, IFN-γ responsiveness and induction of IDO were fully restored following 24 h of MSC tissue culture post-thaw. These results highlight a possible cause for the inefﬁcacy of MSC-based immunotherapy reported in clinical trials using cryopreserved MSC thawed immediately prior to infusion. Key Words: cryopreservation, immunosuppression, immunotherapy, indoleamine 2,3-dioxygenase, interferon-gamma, mesenchymal stromal cells Introduction One mechanism by which human bone marrow- derived mesenchymal stromal cells (MSC) sup- press T-cell proliferation in vitro is mediated by the induction of indoleamine 2,3-dioxygenase (IDO) (1,2). Because of their ability to inhibit activated T cells speciﬁcally, MSC have been the subject of phase I/II trials for their potential use as a cell-based immunotherapeutic for the treat- ment of autoimmune and alloimmune disorders (3–6). Clinical studies have demonstrated that the administration of autologous or allogeneic MSC is safe, and numerous phase II studies have suggested encouraging potency, particularly in the treat- ment of steroid-resistant graft-versus-host disease (GvHD) (7). However, therapeutic effectiveness of MSC is not universally observed (6), including the results of a large phase III trial of major histo- compatibility complex (MHC)-unmatched alloge- neic MSC for GvHD recently completed in the USA (8). The current model for MSC-based immuno- therapy is to expand cells ex vivo, cryogenically bank them until needed, then administer thawed MSC intravenously to the patient. Until now, the effect of cryopreservation recovery on the immunosup- pressive properties of MSC has not been investi- gated. With the present study, we demonstrate that freshly thawed MSC are unable to suppress T-cell proliferation in vitro, which correlates with impaired up-regulation of IDO in response to interferon (IFN)-γ. These observations provide some insight on the incon- sistencies between the pre-clinical and clinical utility of MSC for treatment of immune ailments. Correspondence: Jacques Galipeau, MD, Winship Cancer Institute, Emory University, Atlanta, Georgia, USA. E-mail: jgalipe@emory.edu (Received 29 May 2011; accepted 12 September 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.623691