Mesenchymal Stem Cells and Solid Organ Transplantation

Mesenchymal stem cells are multipotent stem cells derived from various sources. This review describes their immunomodulatory effect on Tcells, B-cells, NK cells and dendritic cells and interactions with T-regulatory (CD4+/25high Foxp3 +) cells, the last being one of the most important mode of actions. These cells are “super-suppressive” cells which act in “cell-cell contact” or through release of cytokines and growth factors. The mechanism of immune-modulation is species specific. MSC has contributed significantly towards the evolution of “cell therapy” in transplantation immunobiology. Sparse data on clinical use of MSC in organ transplantation are available. Our experience is consistent with the prevailing notion that “cell therapy” with MSC in the lead will carry the torch of therapeutic avenues yet not explored. INTRODUCTION Mesenchymal stem cells (MSC) are multipotent stem cells (SC) derived from various sources. They were first isolated in 1974 from bone marrow (BM) by Friedenstein and Petrokova1. In undifferentiated state, they appear fibroblastoid and have small cell body with few long and thin cell processes. There are no specific markers to identify them; however, they are negative for hematopoietic cell markers like CD34/45/HLA-DR and express CD90/73/105 on their surface2-4. They have the plasticity to differentiate in to mesenchymal and non-mesenchymal cell types alike, both in vitro and in vivo2,4. Human adipose tissue derived MSCs (AD-MSC) are morphologically similar to their counterparts in BM; however, their proliferation and differentiation capacity is higher5. The International Society for Cellular Therapy has recommended the following minimum criteria for defining multi-potent human MSCs6,7 (i) adherence to plastic under standard culture conditions; (ii) positive for expression of CD105, CD73 and CD90 and negative for expression of the haematopoietic cell surface markers CD34, CD45, CD11a, CD19 or CD79a, CD14 or CD11b and histocompatibility locus antigen (HLA)DR; and (iii) under a specific stimulus, differentiation into osteocytes, adipocytes and chondrocytes in vitro. One of the most intriguing features of MSCs is that they escape immune recognition and can inhibit immune responses8. Because of their unique regenerative potential, MSCs exhibit potential for use in tissue regeneration and repair for conditions such as cardiac anomalies or injury, bone disorders and metabolic diseases. IMMUNOMODULATORY FUNCTIONS OF MSCS T CELL PROLIFERATION AND FUNCTION MSCs can suppress the T lymphocyte proliferation induced by alloantigens, mitogens and anti-CD3 and anti-CD28 antibodies in vitro, in humans, baboons and mice9-15. MSCs have a similar effect on memory MESENCHYMAL STEM CELLS AND SOLID ORGAN TRANSPLANTATION Trivedi HL1, Vanikar AV2 1Department of Nephrology, Transplantation Medicine and Immunology 2Department of Pathology, Laboratory Medicine and Transfusion Services and Department of Immunohematology G. R. Doshi and K. M. Mehta Institute of Kidney Diseases & Research Centre [IKDRC] – Dr. H.L. Trivedi Institute of Transplantation Sciences [ITS], Civil Hospital Campus, Asarwa, Ahmedabad, Gujarat, India. CellR4 2013; 1(2): 123-136 Corresponding Author: Trivedi HL, F.R.C.P.[C], DSc; e-mail: [email protected] and naive T cells as well as CD4+ and CD8+ T cells and this suppressive effect does not require major histocompatibility complex (MHC) restriction 10,15,16. Cell inhibition is believed to be due to soluble/growth factors like IFN-γ, interleukin [IL]-1β, Transforming growth factor (TGF)-β1 and hepatocyte growth factor (HGF) in humans9. Their immunomodulatory activity is believed to be through these growth factors and indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). The secretion of human leucocyte antigen-G5 [HLA-G5] by MSCs is reported to be essential for their following effects: suppression of T-cell and NK cell function, shift of the allogeneic T-cell response to a T-helper type 2 (Th2) cytokine profile and induction of CD4+CD25high forkhead box P3 (FoxP3+) regulatory T cells (Tregs)19,20. B CELL PROLIFERATION AND FUNCTION The major mechanism of B cell suppression by MSCs is attributed partly to the physical contact between MSCs and B cells and in part to the soluble factors released by MSCs; this leads to the blocking of B cell proliferation in the G0/G1 phase of the cell cycle with no apoptosis16,21,22. MSCs inhibit the proliferation of B cells activated with anti-immunoglobulin (Ig) antibodies, anti-CD40L antibody and cytokines (IL-2 and IL-4)22. MODULATION OF NATURAL KILLER (NK) CELLS Soluble factors such as TGF-β1 and PGE2 are believed to play a role in the MSC-mediated suppression of NK cell proliferation23. INTERACTION BETWEEN MSCS AND DENDRITIC CELLS (DCS) MSCs impair the differentiation of monocytes or CD34+ HSCs into DCs by inhibiting the response of the former to maturation signals, reducing the expression of co-stimulatory molecules and hampering the ability of the former to stimulate naive T cell proliferation and IL-12 secretion24-26. In addition, this inhibitory effect might be mediated via soluble factors and may be dose-dependent 25. MSCs isolated from human adipose tissue are more potent immunomodulators for the differentiation of human DCs than MSCs derived from the BM27. Trivedi HL, Vanikar AV 124 Figure 1. Immunomodulatory Role of Mesenchymal Stem Cell. Mesenchymal stem cell (MSC) has surface markers CD 54, CD 73, CD 90, CD 102, CD 105 and MHC-1. They suppress T lymphocyte proliferation through IFN-γ, interleukin(IL) 6, IL8, prostaglandin E2 and growth factors like SDF-1, VEGF and HGF. MSC secrete HLA-G which helps in suppressing NK and Tcell functions and shifting of T-cell responses to TH2 and T-regulatory cell-CD4+/25high) Fork-head Box p3 (FoxP3) activity. MSCs inhibit TNF secretion and promote IL-10 secretion, affecting dendritic cell [DC] (Immature[I], Mature [M] maturation and function resulting in shifting the immune response towards anti-inflammatory activity /tolerance. MSC inhibit IFN γ secretion from TH1 and NK cells and increase IL-4/IL-10 secretion from TH2 cells/Tregs, thereby promoting a TH1 to TH2 shift. In Ahmedabad, we generate T-regs (CD 4+/25high/127low/-) by co-culturing of AD-MSC with peripheral blood mononuclear cells (PBMC) by IL-2 supplementation. INDUCTION OF TREGULATORY CELLS (TREGS) MSCs may also modulate immune responses via the induction of Tregs. MSC can induce the generation of CD4+CD25+ cells displaying a regulatory phenotype (FoxP3+) in mitogen-stimulated cultures of peripheral blood mononuclear cells although the functional properties of these cells have not yet been elucidated18,28. Ge W et al29 reported that MSCs could induce kidney allograft tolerance by inducing the generation of CD4+CD25+FoxP3+ Tregs in vivo. Additionally, MSCs have been reported to induce the formation of CD8+ Tregs that are responsible for the inhibition of allogeneic lymphocyte proliferation14. The induction of Tregs by MSCs involves not only direct contact between MSCs and CD4+ cells, but also the secretion of soluble factors such as PGE2 and TGF-β130. Human gingiva-derived MSCs can induce IL-10, IDO, inducible NO synthase and cyclooxygenase 2 thereby serving as immunomodulatory components in the treatment of experimental inflammatory diseases31. A study has shown that the immunosuppressive effect of MSCs is mediated by the secretion of galectin-3, a protein known to modulate T cell proliferation, gene expression, cell adhesion and migration32. MSCs have also shown to prevent autoimmune B cell destruction and subsequent diabetes in NOD mice by inducing Tregs33. The effect of MSCs in the treatment of autoimmune diseases may be through the induction of de novo generation of antigen-specific CD4+CD25+FoxP3+ Tregs 34,35. However, a recent study reported that MSCs could sustain or suppress T-cell proliferation depending on their concentration, and a low MSC/T-cell ratio might support T cell proliferation36. In a recent study by Siod et al they found that MSCs could stimulate the activation and proliferation of resting T-cells and generate Tregs37. These data suggest that the culture conditions play an important role in the clinical application of MSCs36. MSC AND TRANSPLANTATION TOLERANCE Immunomodulatory role of MSCs in vitro and in vivo in experimental models has led to the evolution of “cell therapy” as a new branch for exploring therapeutic applicability in auto-immune disorders and allo-immune conditions which are otherwise not amenable to other therapeutic modalities. In pathological conditions, MSCs migrate preferentially into lymphoid organs, allografts, injured and/or inflammatory tissue sites after systemic transfusion, where they interact with the activated immune cells and modulate their function38,39. Mesenchymal stem cells and transplantation 125 Figure 2. Adipose Tissue Derived Mesenchymal Stem Cells And Transplant Tolerance. Adipose Tissue Derived Mesenchymal Stem Cells (AD-MSC) block the direct (donor antigen presenting cell [APC] and indirect (recipient APC) pathway of rejection by blocking the T-cell receptor site where antigen presenting cells interacts through MHC peptide. Thus activation and proliferation of T-cells is blocked and in indirect pathway, cyto-toxic T cells are blocked by generation of regulatory T-cells thus helping in preventing chronic rejection. Bartholomew et al13 first described the in vivo immunomodulatory properties of MSCs in a baboon model of skin transplantation. The therapeutic potential of MSCs in immunomodulation is being explored currently in several Phase I, II and III clinical trials40 many of which have recently been completed or are under way, as reported in the clinical trials website of the United States sponsored by the National Institutes of Health [http://clinicaltrials.gov]. Because of their immunosuppressive properties, MSCs are believed to play a role in the maintenance of peripheral tolerance and the induction of transplantation tolerance, and they are considered potential candidates in cellular therapy for graft-versus-host disease (GVHD) and autoimmune diseases and in protecting solid-organ grafts from being rejected41. MSCs derived from umbilical cord, BM and occasionally adipose tissue are being tried or considered for clinical trials42. MSCs obtained from HLA-identical sibling donors, haplo-identical donors and third-party HLA-mismatched donors infused in 55 patients with steroid-refractory acute GVHD were shown to elicit a response in more than half the patients; the study showed that MSCs exerted their therapeutic effect in the case of both HLA-matched and HLA-unmatched donors. However, for GVHD, the use of MSCs is a double-edged sword, because the prevention of GVHD was associated with a high incidence of leukaemia relapse, which is the result of the non-specific immunosuppressive effect of MSCs on graft-versusleukaemia43,44. Liang et al45 reported that allogeneic MSC transplantation in patients with refractory SLE resulted in the amelioration of disease activity, improvement in the levels of serological markers and stabilization of renal function without the occurrence of serious adverse events. For solid organ transplantation, the beneficial effect of MSC-based immunosuppressive therapy is debatable. The application of calcineurin inhibitors (CNIs) would abrogate the immunosuppressive effect of MSC therapy. In addition, CNIs cause renal failure, hypertension and hyperglycaemia, and increase the risk of malignancy; therefore, efforts have been made to minimize the use of CNI in organ transplantation protocols. Embryonic, hematopoietic and mesenchymal stem cells have been successfully employed for tolerance induction in a variety of rodent and large-animal studies46-48. MSCs are not only able to evade the immune system, but they can also suppress immune responses directed against third-party cells, even inducing tolerance toward other tissues of the same origin when transplanted following intravenous infusion of MSCs13. This and other studies have further demonstrated that MSCs inhibit T-cell activation ex vivo10,11,13,15. A case report by Le Blanc et al51 suggested that systemic infusion of haplo-identical MSCs suppressed a grade IV GVHD in a 9-year-old child who had received a BM transplant (BMT). Ringden et al52 reported the administration of MSC with median dose of 1.0 ×106/kg to eight patients with steroid-refractory grades III-IV acute GVHD and one with extensive chronic GVHD52. No acute side-effects occurred after the MSC infusions. Six patients were treated once and three patients twice. Two patients received MSC from HLA-identical siblings, six from haplo-identical family donors and four from unrelated mismatched donors. Acute GVHD disappeared completely in six of eight patients. One of these developed cytomegalovirus gastroenteritis. Complete resolution was seen in gut (6), liver (1) and skin (1). Two died soon after MSC treatment with no obvious response. One of them had MSC donor DNA in the colon and a lymph node. Five patients were reported alive between 2 months and 3 years after the transplantation. Their survival rate was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSC during the same period (p = 0.03). One patient treated for extensive chronic GVHD showed a transient response in the liver, but not in the skin and he died of Epstein-Barr virus lymphoma. This study showed that MSC have very promising treatment for severe steroid-resistant acute GVHD. The underlying mechanism for this tolerizing phenomenon, including the involved target cells, is not yet known. Tolerance induction in the periphery is believed to be critical for the prevention of autoimmunity and maintenance of immune homeostasis. Central tolerance has been classically ascribed to clonal deletion of self-reactive T-cells in the thymus upon interaction with self-antigens. However, central tolerance is incomplete because not all self-antigens gain access to the thymus, and several self-reactive lymphocytes escape central deletion. In the past several years there has been growing evidence supporting this notion, revealing subpopulations of cells representing different arms of the immune system, as potential regulators of the immune system. These specific groups include T-cell subtypes (such as CD4+CD25+ T cells), as well as a unique fraction of DCs described as semimmature DCs, all of whom were shown to possess immune-modulating properTrivedi HL, Vanikar AV 126 ties. Therefore, “sentinels” in the periphery of the body are essential to maintain tolerance as well as immunity. These tolerogenic effectors, while constitutively active in autoimmunity prevention, may play a pivotal role in maternal-fetal non-rejection, as well as in immune evasion of tumors and metastases. MSC EVADE ALLO-REJECTION The major limit to solid organ graft survival is Tcell recognition by the recipient of alloantigen (dominated by, but not confined to MHC/HLA antigens)53. There are two mechanisms mediating this powerful rejection response; “direct” recognition, involving recognition by recipient CD8+ or CD4+ T-cells of donor MHC class I and class II molecules; and “indirect” mechanisms involving recognition of peptides from the allogeneic tissue53. Recipient antigen presenting cells (APC) such as DC process alloantigen into peptides and present these to naive T cells on self-MHC molecules54. However there are notable exceptions to these allorejection processes; the fetal allograft evades rejection by the mother through a complex series of actions, similarly tissue which has limited lymphatic drainage is less prone to allorejection55,56. Interestingly tumor cells, whilst not allogeneic, are in many cases both “altered-self” and immunogenic but often actively modulate immune responsiveness to evade immune surveillance57. Thus mechanisms of tumor evasion of the immune system may provide insight into how allogeneic MSC are tolerated by the mismatched host. There is supporting evidence for the use of allogeneic MSC from both in vitro and in vivo studies that show MSC avoid normal allo-responses. A small number of invivo studies suggest that MSC play a role in enabling alloantigen tolerance. Koc et al58 showed no evidence of alloreactive T cells and no incidence of GVHD when allogeneic MSC were infused into patients with Hurler’s syndrome or metachromatic leukodystrophy. In a previous study by the same group59, autologous culture-expanded MSC were infused to breast cancer patients to investigate whether MSC would enhance the engraftment of peripheral blood stem cells after myeloablative therapy. Results showed rapid hematopoietic recovery and no signs of toxicity from MSC infusion. Horwitz et al60 reported that donor MSC contributed to bone remodelling after allogeneic stem cell transplantation (SCT) in 3 children with osteogenesis imperfecta, a rare genetic disorder of type I collagen. This is supported by data from Bartholomew et al61 who showed that invivo administration of allogeneic MSC prolonged 3rd party skin graft survival in animal models. Furthermore, Saito et al62 demonstrated that MSC undergoing differentiation to a cardiac phenotype were tolerated in a xenogeneic environment, retaining their ability to be recruited to the injured myocardium. More recent work by Aggarwal and Pittenger18 supported the feasibility of MSC-transplantation showing that MSC altered the phenotypes of specific immune cell subtypes thereby creating a tolerogenic environment. These reports suggest that transplantation of MSC could be beneficial in patients with various disorders requiring tissue regeneration, and provide evidence supporting the tolerance of allogeneic MSC by recipients. Data supporting the contention that MSC avoid allogeneic responses has also come from a large body of in vitro experiments, usually involving coculture or mixed lymphocyte reactions (MLR). Evidence from these studies indicates that the use of mismatched MSC does not provoke a proliferative T cell response in allogeneic MLR, thus suggesting an immunosuppressive role for MSC11,13,15,26,62-63. Le Blanc et al64 showed that MSC failed to elicit proliferation of allogeneic lymphocytes. Additionally, they demonstrated that MSC remained immunosuppressive even after IFN-γ stimulation. Krampera et al15 confirmed these findings, they showed that murine MSC lack MHC class II and inhibited T cell proliferation. Tse et al12 also showed that human MSC fail to elicit allogeneic T cell response in MLR even when MHC class II was upregulated. Consistent with these studies, Bartholomew et al13 showed that allogeneic baboon MSC suppressed the proliferative activity of lymphocytes in vitro and prolonged graft survival. These findings support the view that MSC can be transplanted between MHCincompatible individuals. Although these data show that successful use of allogeneic MSC in regenerative therapy is possible, such approaches are unlikely to be broadly acceptable until it is understood why MSC are not rejected. This question has been the subject of intense recent study and three candidate mechanisms are emerging. MSC appear to evade allogeneic rejection by (a) being hypoimmunogenic; (b) modulating T cell phenotype and (c) creating an immunosuppressive local milieu. These mechanisms are inter-related and will involve cell contact dependent and independent interactions. The challenge facing the field is to unravel the contribution of these diverse interactions. Mesenchymal stem cells and transplantation 127