Manufacturing Mesenchymal Stromal Cells for the Treatment of Graft-versus-Host Disease: A Survey among Centers Affiliated with the European Society for Blood and Marrow Transplantation.

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients' stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

Bone marrow mesenchymal stromal cells induce nitric oxide synthase-dependent differentiation of CD11b+ cells that expedite hematopoietic recovery.

Bone marrow microenvironment is fundamental for hematopoietic homeostasis. Numerous efforts have been made to reproduce or manipulate its activity to facilitate engraftment after hematopoietic stem cell transplantation but clinical results remain unconvincing. This probably reflects the complexity of the hematopoietic niche. Recent data have demonstrated the fundamental role of stromal and myeloid cells in regulating hematopoietic stem cell self-renewal and mobilization in the bone marrow. In this study we unveil a novel interaction by which bone marrow mesenchymal stromal cells induce the rapid differentiation of CD11b+ myeloid cells from bone marrow progenitors. Such an activity requires the expression of nitric oxide synthase-2. Importantly, the administration of these mesenchymal stromal cell-educated CD11b+ cells accelerates hematopoietic reconstitution in bone marrow transplant recipients. We conclude that the liaison between mesenchymal stromal cells and myeloid cells is fundamental in hematopoietic homeostasis and suggests that it can be harnessed in clinical transplantation.

Impact of γ-chain cytokines on EBV-specific T cell cultures.

BACKGROUND: Recent preclinical adoptive immunotherapy studies in murine models prompt to employ "proper" rather than "as many as possible" antigen-specific T cells to gain better therapeutic results. Ideally, "proper" T cells are poorly differentiated in vitro, but retain the capacity to fully differentiate into effector cells in vivo, where they can undergo long-term survival and strong proliferation. Such requirements can be achieved by modifying culture conditions, namely using less "differentiating" cytokines than IL-2. METHODS: To evaluate this issue in human T cell cultures, we exploited a well characterized and clinical-grade protocol finalized at generating EBV-specific CTL for adoptive immunotherapy. In particular, we studied the impact of IL-7, IL-15 and IL-21 compared to IL-2 on different aspects of T cell functionality, namely growth kinetics, differentiation/activation marker expression, cytokine production, and short-term and long-term cytotoxicity. RESULTS: Results disclosed that the culture modifications we introduced in the standard protocol did not improve activity nor induce substantial changes in differentiation marker expression of EBV-specific CTL. CONCLUSIONS: Our data indicated that the addition of γ-chain cytokines other than IL-2 for the generation of EBV-specific T cell cultures did not produce the improvements expected on the basis of recent published literature. This fact was likely due to the intrinsic differences between murine and human models and highlights the need to design ad hoc protocols rather than simply modify the cytokines added in culture.

Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.

The immunosuppressive activity of mesenchymal stromal cells (MSCs) is well documented. However, the therapeutic benefit is completely unpredictable, thus raising concerns about MSC efficacy. One of the affecting factors is the unresolved conundrum that, despite being immunosuppressive, MSCs are undetectable after administration. Therefore, understanding the fate of infused MSCs could help predict clinical responses. Using a murine model of graft-versus-host disease (GvHD), we demonstrate that MSCs are actively induced to undergo perforin-dependent apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. When examining patients with GvHD who received MSCs, we found a striking parallel, whereby only those with high cytotoxic activity against MSCs responded to MSC infusion, whereas those with low activity did not. The need for recipient cytotoxic cell activity could be replaced by the infusion of apoptotic MSCs generated ex vivo. After infusion, recipient phagocytes engulf apoptotic MSCs and produce indoleamine 2,3-dioxygenase, which is ultimately necessary for effecting immunosuppression. Therefore, we propose the innovative concept that patients should be stratified for MSC treatment according to their ability to kill MSCs or that all patients could be treated with ex vivo apoptotic MSCs.

Unique regulatory properties of mesangial cells are genetically determined in the rat.

Mesangial cells are glomerular cells of stromal origin. During immune complex mediated crescentic glomerulonephritis (Crgn), infiltrating and proliferating pro-inflammatory macrophages lead to crescent formation. Here we have hypothesised that mesangial cells, given their mesenchymal stromal origin, show similar immunomodulatory properties as mesenchymal stem cells (MSCs), by regulating macrophage function associated with glomerular crescent formation. We show that rat mesangial cells suppress conA-stimulated splenocyte proliferation in vitro, as previously shown for MSCs. We then investigated mesangial cell-macrophage interaction by using mesangial cells isolated from nephrotoxic nephritis (NTN)-susceptible Wistar Kyoto (WKY) and NTN-resistant Lewis (LEW) rats. We first determined the mesangial cell transcriptome in WKY and LEW rats and showed that this is under marked genetic control. Supernatant transfer results show that WKY mesangial cells shift bone marrow derived macrophage (BMDM) phenotype to M1 or M2 according to the genetic background (WKY or LEW) of the BMDMs. Interestingly, these effects were different when compared to those of MSCs suggesting that mesangial cells can have unique immunomodulatory effects in the kidney. These results demonstrate the importance of the genetic background in the immunosuppressive effects of cells of stromal origin and specifically of mesangial cell-macrophage interactions in the pathophysiology of crescentic glomerulonephritis.

Increased HDAC1 deposition at hematopoietic promoters in AML and its association with patient survival.

Epigenetic changes play a crucial role in leukemogenesis. HDACs are frequently recruited to target gene promoters by balanced translocation derived oncogenic fusion proteins. As important epigenetic effector mechanisms, histone deacetylases (HDAC) have emerged as potential therapeutic targets. However, the patterns of HDAC1 localization and the role of HDACs in leukemia pathogenesis remain to be elucidated. Using ChIP-Chip analyses we analyzed HDAC1 deposition patterns at more than 10,000 gene promoters in a large cohort of leukemia patients and CD34+ controls. HDAC1 binding was significantly increased in AML blasts compared to CD34+ progenitor cells at 130 gene promoters whereas decreased binding was observed at 66 gene promoters. Distinct HDAC1 binding patterns occurred in AML subtypes with balanced translocations t(15;17), t(8;21) and inv(16). In addition, a more generalized signature was established, that revealed an AML specific pattern of HDAC1 distribution. Many of the HDAC1-binding altered promoters regulate genes involved in hematopoiesis, transcriptional regulation and signal transduction. HDAC1 binding patterns were associated with patients' event free survival. This is the first study to determine HDAC1 modification patterns in a large number of AML and ALL specimens. Our findings suggest that dyslocalization of HDAC1 is a common feature in AML. Importantly, HDAC1 modifications possess prognostic power for patient survival. Our findings suggest that altered HDAC1 localization is an explanation for the observed benefit of HDAC inhibitors in AML therapy.

Mesenchymal stem cells and innate tolerance: biology and clinical applications.

The properties of mesenchymal stem cells (MSC) have been widely investigated during the last decade, from their differentiation capacity to their immunosuppressive effect on any type of immune cell. These properties have been successfully harnessed for the treatment of inflammatory diseases such as graft versus host disease (GvHD). Different mechanisms have been proposed for their immunosuppressive properties, although it seems likely that they are used in concert. The inflammatory environment to which MSC are exposed plays a pivotal role in activating their functions. Conversely, the interplay of MSC with the immunoregulatory networks recruited during inflammation is fundamental to the delivery of immunosuppression. Since other types of terminally differentiated stromal cells share these properties, it is plausible that stemness is not a required feature. Therefore these functions may be involved in the physiological control of acute inflammation in various tissues. These notions highlight the importance of investigating the role of stromal cells as modulators of immune responses.