Antonio Galleu

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

Mesenchymal stromal cell apoptosis, induced in vivo by recipient cytotoxic cells, is required for immunosuppression and clinical responses. MSC sacrifice for immunosuppression Transfer of mesenchymal stromal cells (MSCs) induces immunosuppression, although the cells are undetectable shortly after transfer. The immunosuppressive mechanism of MSCs has been somewhat of a mystery, but Galleu and colleagues now suggest that MSC apoptosis is crucial. They observed in a mouse model of graft-versus-host disease that cytotoxic cells rendered the MSCs apoptotic shortly after transfer. Moreover, cells from patients that responded to MSC therapy had more cytotoxic activity against MSCs. These findings not only provide important mechanistic insight but also suggest that patients could be screened for responsiveness to MSC therapy before transfer. 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.

Patients with Early-Stage Myelodysplastic Syndromes Show Increased Frequency of CD4+CD25high+CD127low Regulatory T Cells

Regulatory T cells (Treg) have often been ascribed a role in the pathophysiology of several neoplastic diseases considering their potential ability to suppress anti-tumor immunity. This is particularly the case in myelodysplastic syndromes (MDS), which are clonal hematologic disorders characterized by marked immune dysregulation. We analyzed Treg frequencies in a cohort of 36 patients with early-stage MDS using a flow-cytometric approach based on the concomitant expression of CD25 and CD127. MDS patients showed a higher frequency of CD4+CD25high+CD127low Treg than healthy controls (1.51 vs. 1.14%), with no specific effect of patient- and disease-related factors. Our data point to impaired anti-tumor immunity in patients with MDS, even in the early stage, which has already been noted in other clonal disorders.

Enhancement of the immunoregulatory potency of mesenchymal stromal cells by treatment with immunosuppressive drugs

BACKGROUND AIMS Multipotent mesenchymal stromal cells (MSCs) are distinguished by their ability to differentiate into a number of stromal derivatives of interest for regenerative medicine, but they also have immunoregulatory properties that are being tested in a number of clinical settings. METHODS We show that brief incubations with rapamycin, everolimus, FK506 or cyclosporine A increase the immunosuppressive potency of MSCs and other cell types. RESULTS The treated MSCs are up to 5-fold more potent at inhibiting the induced proliferation of T lymphocytes in vitro. We show that this effect probably is due to adsorption of the drug by the MSCs during pre-treatment, with subsequent diffusion into co-cultures at concentrations sufficient to inhibit T-cell proliferation. MSCs contain measurable amounts of rapamycin after a 15-min exposure, and the potentiating effect is blocked by a neutralizing antibody to the drug. With the use of a pre-clinical model of acute graft-versus-host disease, we demonstrate that a low dose of rapamycin-treated but not untreated umbilical cord-derived MSCs significantly inhibit the onset of disease. CONCLUSIONS The use of treated MSCs may achieve clinical end points not reached with untreated MSCs and allow for infusion of fewer cells to reduce costs and minimize potential side effects.

TCRBV20S1 polymorphism does not influence the susceptibility to type 1 diabetes and multiple sclerosis in the Sardinian population.

Among the different T-cell receptor (TCR) BV20S1 polymorphisms, nucleotide substitution at position 524 results in the introduction of a stop codon, whose potential functional relevance is still unknown. We have recently showed in Sardinian subjects the most elevated allele frequency ever reported worldwide for this “null allele” (0.44). As this variant generates a gap in the TCR repertoire, this preliminary finding prompted us to further analyze the role of this polymorphism in the susceptibility to type 1 diabetes (T1D) and multiple sclerosis (MS), which are extremely common in this population. With this aim, we evaluated the influence of the TCRBV20S1 polymorphism by assessing it with the transmission disequilibirum test (TDT) in 652 T1D and 616 MS families, without detecting any significant difference. We conclude that the high frequency of this null allele in Sardinia is not directly related to the high incidence of these autoimmune diseases observed in this founder population.

Manufacturing mesenchymal stromal cells for the treatment of graft-versus-host disease: a survey amongst centers affiliated to the European Group of Blood and Marrow Transplantation

Highlights • Seventeen EBMT centers participated to a questionnaire on MSC manufacturing.• 88% of centers manufacture MSC from bone marrow and only 2 centers from umbilical cord.• Human platelet lysate has replaced bovine serum as culture medium supplement.• Release criteria extensively differ among centers.• The results highlight the need to harmonize MSC manufacturing.

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.

Rapid and Efficient Stable Gene Transfer to Mesenchymal Stromal Cells Using a Modified Foamy Virus Vector

Mesenchymal stromal cells (MSCs) hold great promise for regenerative medicine. Stable ex vivo gene transfer to MSCs could improve the outcome and scope of MSC therapy, but current vectors require multiple rounds of transduction, involve genotoxic viral promoters and/or the addition of cytotoxic cationic polymers in order to achieve efficient transduction. We describe a self-inactivating foamy virus vector (FVV), incorporating the simian macaque foamy virus envelope and using physiological promoters, which efficiently transduces murine MSCs (mMSCs) in a single-round. High and sustained expression of the transgene, whether GFP or the lysosomal enzyme, arylsulphatase A (ARSA), was achieved. Defining MSC characteristics (surface marker expression and differentiation potential), as well as long-term engraftment and distribution in the murine brain following intracerebroventricular delivery, are unaffected by FVV transduction. Similarly, greater than 95% of human MSCs (hMSCs) were stably transduced using the same vector, facilitating human application. This work describes the best stable gene transfer vector available for mMSCs and hMSCs.

Effects of maternal obesity on Wharton's Jelly mesenchymal stromal cells

We investigated whether maternal metabolic environment affects mesenchymal stromal/stem cells (MSCs) from umbilical cord’s Wharton’s Jelly (WJ) on a molecular level, and potentially render them unsuitable for clinical use in multiple recipients. In this pilot study on umbilical cords post partum from healthy non-obese (BMI = 19–25; n = 7) and obese (BMI ≥ 30; n = 7) donors undergoing elective Cesarean section, we found that WJ MSC from obese donors showed slower population doubling and a stronger immunosuppressive activity. Genome-wide DNA methylation of triple positive (CD73+CD90+CD105+) WJ MSCs found 67 genes with at least one CpG site where the methylation difference was ≥0.2 in four or more obese donors. Only one gene, PNPLA7, demonstrated significant difference on methylome, transcriptome and protein level. Although the number of analysed donors is limited, our data suggest that the altered metabolic environment related to excessive body weight might bear consequences on the WJ MSCs.

Umbilical cord as a source of immunomodulatory reagents

Abstract One of the main successes of cellular therapies in the last decade has been the generation of immunomodulating reagents for clinical use. Umbilical cord can be used to source such cell populations. In this chapter we will concentrate on describing the features of and the clinical experience with regulatory T-cells and mesenchymal stromal cells . Although relevant immunomodulating activity can also be ascribed to hematopoietic stem cells, that is the subject of another chapter of this book. Similarly, other populations have been isolated and demonstrated to exhibit immunosuppressive activity but, probably because of their small concentration and the difficulty in their expansion, they have not been developed any further.

Immune Tolerance in Hemopoietic Stem Cell Transplantation

Transplantation of allogeneic tissues is a procedure used in the treatment of a wide variety of conditions with differing pathogenesis and etiology. While the technique varies depending on the type of tissue, the underlying principle consists in the long-term replacement of the diseased or dysfunctional tissue with a healthy one. Hemopoietic stem cell transplantation (HSCT) is a special case study because the transplant is also associated with a number of major immunological events that affect both the recipient immune system and the adoptively transferred donor immune cells. Although still far to be completely understood, the immunological changes have several implications for the underlying disease as well as on the newly formed immune repertoire. In comparison to solid organ transplantation, allogeneic HSCT presents two major differences. The first is that both donor and recipient have the ability to potentially mount an immune response because the donor HSC preparation contains alloreactive precursors. The second is that, once achieved, transplantation tolerance does not require any long-term immunosuppressive treatment. Therefore, HSCT offers a unique opportunity to understand the underlying mechanisms accounting for the induction of drug-free control of immune-mediated rejection, and as a consequence to provide the basis for harnessing these mechanisms in the clinical setting. In this article we review how HSCT is employed as a treatment for various ailments, the underlying rationale, and summarize available evidence to explain its tolerance-inducing effect.