Oliver Treacy

Immunogenicity of allogeneic mesenchymal stem cells

Mesenchymal stem cells (MSCs) inhibit proliferation of allogeneic T cells and express low levels of major histocompatibility complex class I (MHCI), MHCII and vascular adhesion molecule‐1 (VCAM‐1). We investigated whether their immunosuppressive properties and low immunophenotype protect allogeneic rat MSCs against cytotoxic lysis in vitro and result in a reduced immune response in vivo. Rat MSCs were partially protected against alloantigen‐specific cytotoxic T cells in vitro. However, after treatment with IFN‐γ and IL‐1β, MSCs upregulated MHCI, MHCII and VCAM‐1, and cytotoxic lysis was significantly increased. In vivo, allogeneic T cells but not allogeneic MSCs induced upregulation of the activation markers CD25 and CD71 as well as downregulation of CD62L on CD4+ T cells from recipient rats. However, intravenous injection of allo‐MSCs in rats led to the formation of alloantibodies with the capacity to facilitate complement‐mediated lysis, although IgM levels were markedly decreased compared with animals that received T cells. The allo‐MSC induced immune response was sufficient to lead to significantly reduced survival of subsequently injected allo‐MSCs. Interestingly, no increased immunogenicity of IFN‐γ stimulated allo‐MSCs was observed in vivo. Both the loss of protection against cytotoxic lysis under inflammatory conditions and the induction of complement‐activating antibodies will likely impact the utility of allogeneic MSCs for therapeutic applications.

Anti-donor immune responses elicited by allogeneic mesenchymal stem cells: what have we learned so far?

Mesenchymal stem (stromal) cells (MSCs) have potent anti‐inflammatory/immunosuppressive properties which underlie much of their therapeutic potential. This fact has led to the widely accepted belief that MSCs from genetically unrelated individuals (allogeneic (allo)‐MSCs) can be used therapeutically with equal efficacy to autologous MSCs and without triggering the donor‐specific immune responses that are typically associated with allo‐transplants. In this article, we critically review available experimental data to determine whether good in vivo evidence exists in support of the ‘immune privileged’ status of allo‐MSCs. We also examine published studies regarding the immunogenicity of allo‐MSCs following activation (‘licensing’) by inflammatory stimuli or following differentiation. Among the identified studies which have addressed in vivo immunogenicity of allo‐MSCs, there was substantial variability as regards experimental species, disease model, route of MSC administration, cell dose and stringency of the immunological assays employed. Nonetheless, the majority of these studies has documented specific cellular (T‐cell) and humoral (B‐cell/antibody) immune responses against donor antigens following administration of non‐manipulated, interferon‐γ‐activated and differentiated allo‐MSCs. The consequences of such anti‐donor immune responses were also variable and ranged from reduced in vivo survival of allo‐MSCs with accelerated rejection of subsequent allogeneic transplants to apparent promotion of donor‐specific tolerance. On the basis of these findings and on existing knowledge of allo‐antigen recognition from the field of transplant immunology, we propose that the concept of the immune privileged nature of allo‐MSCs should be reconsidered and that the range and clinical implications of anti‐donor immune responses elicited by allo‐MSCs be more precisely studied in human and animal recipients.

Chondrogenic Differentiation Increases Antidonor Immune Response to Allogeneic Mesenchymal Stem Cell Transplantation

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3(+) and CD68(+) infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.

Adenoviral Transduction of Mesenchymal Stem Cells: In Vitro Responses and In Vivo Immune Responses after Cell Transplantation

Adult mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential which makes them attractive targets for regenerative medicine applications. However, to date, therapeutic success of MSC-therapy is limited and the genetic modification of MSCs using viral vectors is one option to improve their therapeutic potential. Ex-vivo genetic modification of MSCs using recombinant adenovirus (Ad) could be promising to reduce undesired immune responses as Ad will be removed before cell/tissue transplantation. In this regard, we investigated whether Ad-modification of MSCs alters their immunological properties in vitro and in vivo. We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86. Moreover, Ad-transduction caused no significant changes in terms of pro-inflammatory cytokine expression, chemokine and chemokine receptor and Toll-like receptor expression. In addition, Ad-modification of MSCs had no affect on their ability to suppress T cell proliferation in vitro. In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues. Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.

Role of Lentivirus‐Mediated Overexpression of Programmed Death‐Ligand 1 on Corneal Allograft Survival

To investigate the role of lentivirus‐mediated overexpression of programmed death‐ligand 1 (PD‐L1) on rat corneal allograft survival. A fully allogeneic rat cornea transplant model was used for in vivo studies. Lentiviral (LV) vectors are efficient tools for ex vivo genetic modification of cultured corneas. LV vector encoding for PD‐L1 (LV.PD‐L1) and LV vector encoding for eGFP (LV.eGFP, as control) were constructed and tested. PD‐L1 or eGFP expression was increased on corneal cells upon LV.PD‐L1 and LV.eGFP transduction, respectively. Both allogeneic controls and allogeneic LV.eGFP transduced corneas were uniformly rejected (MST: 13.8 ± 1.7 days and 12.3 ± 1.9 days, respectively). In contrast, allogeneic LV.PD‐L1 transduced corneas showed a high percentage (83%) of graft survival (MST > 30 days, n = 5, 15 days, n = 1). Graft opacity of PD‐L1 transduced corneas was present but was significantly reduced compared to control or eGFP expressing corneas. Flow cytometric analysis revealed that percentages of CD3+CD8+CD161+ and CD3+CD8+CD161– lymphocytes were decreased in animals receiving LV.PD‐L1 transduced corneas compared to animals grafted with LV.eGFP transduced corneas. Moreover, reduced expression of proinflammatory cytokines (IFN‐γ and IL‐6) in PD‐L1 transduced corneas compared to allogeneic controls was also observed. Local PD‐L1 gene transfer in cultured corneas is a promising approach for the prolongation of corneal allograft survival and attenuation of graft rejection.

Mesenchymal Stem Cell Therapy Promotes Corneal Allograft Survival in Rats by Local and Systemic Immunomodulation

Mesenchymal stem cells (MSCs) are being investigated extensively due to their ability to dampen immune responses. Here, we tested the ability of MSCs from three distinct sources to prolong rat corneal allograft survival. A fully allogeneic rat cornea transplant model (DA to LEW) was used. Recipient rats received 1 × 106 MSCs (syn [LEW], allo [DA] or third‐party [Wistar Furth]) intravenously 7 days before transplantation and again on the day of transplantation (day 0). A high percentage of untreated and syn‐MSC treated allografts were rejected (80% and 100%, respectively). Preactivation of syn‐MSCs with interferon gamma also failed to prolong allograft survival. Conversely, corneal allograft survival was significantly prolonged in allo‐MSC treated (90%) and third‐party MSC treated (80%) allograft recipients. Flow cytometric analysis revealed less infiltrating natural killer T cells in corneas of both allo‐ and third‐party MSC treated animals, coupled with a higher proportion of splenic CD4+Foxp3+ regulatory T cells, compared to controls. In the case of allo‐ and third‐party MSCs, results from a delayed‐type hypersensitivity assay clearly showed that hypo‐responsiveness was specific for corneal donor‐associated allo‐antigens. Thus, allo‐ and third‐party MSC treatment prolongs corneal allograft survival by suppressing peripheral immune responses and promoting an intragraft immunoregulatory milieu.

Donor Bone Marrow–derived Dendritic Cells Prolong Corneal Allograft Survival and Promote an Intragraft Immunoregulatory Milieu

Investigations into cell therapies for application in organ transplantation have grown. Here, we describe the ex vivo generation of donor bone marrow-derived dendritic cells (BMDCs) and glucocorticoid-treated BMDCs with potent immunomodulatory properties for application in allogeneic transplantation. BMDCs were treated with dexamethasone (Dexa) to induce an immature, maturation-resistant phenotype. BMDC and Dexa BMDC phenotype, antigen presenting cell function, and immunomodulatory properties were fully characterized. Both populations display significant immunomodulatory properties, including, but not limited to, a significant increase in mRNA expression of programmed death-ligand 1 and indoleamine 2,3-dioxygenase. BMDCs and Dexa BMDCs display a profound impaired capacity to stimulate allogeneic lymphocytes. Moreover, in a fully MHC I/II mismatched rat corneal transplantation model, injection of donor-derived, untreated BMDC or Dexa BMDCs (1 × 10(6) cells, day -7) significantly prolonged corneal allograft survival without the need for additional immunosuppression. Although neovascularization was not reduced and evidence of donor-specific alloantibody response was detected, a significant reduction in allograft cellular infiltration combined with a significant increase in the ratio of intragraft FoxP3-expressing regulatory cells was observed. Our comprehensive analysis demonstrates the novel cellular therapeutic approach and significant effect of donor-derived, untreated BMDCs and Dexa BMDCs in preventing corneal allograft rejection.

Culture expanded primary chondrocytes have potent immunomodulatory properties and do not induce an allogeneic immune response.

OBJECTIVE Allogeneic cell therapies, such as mesenchymal stromal cells (MSC), which have potent regenerative and anti-inflammatory potential are being investigated as a therapy for osteoarthritis (OA) and cartilage injury. Here we describe another potential source of regenerative and anti-inflammatory allogeneic cells, culture expanded primary chondrocytes (CEPC). In direct comparison to allogeneic MSC, we extensively assess the immunological interactions of CEPC in an allogeneic setting. METHODS Chondrocytes were isolated from rat articular cartilage and cultured in normoxic or hypoxic conditions. In vitro co-culture assays with allogeneic lymphocytes and macrophages were used to assess the immunomodulatory capacities of the chondrocytes, followed by immune response analysis by flow cytometry, ELISA and qPCR. RESULTS CEPC showed reduced induction of proliferation, activation and cytotoxic granzyme B expression in allogeneic T cells. Importantly, exposure to pro-inflammatory cytokines did not increase CEPC immunogenicity despite increases in MHC-I. Furthermore, CEPC had a potent ability to suppress allogeneic T cell proliferation, which was dependent on nitric oxide production. This suppression was contact independent in hypoxia cultured CEPC. Finally, chondrocytes were shown to have the capacity to modulate pro-inflammatory macrophage activity by reducing MHC-II expression and TNF-α secretion. CONCLUSION These data indicate the potential use of allogeneic chondrocytes in OA and cartilage defects. The lack of evident immunogenicity, despite exposure to a pro-inflammatory environment, coupled with the immunomodulatory ability indicates that these cells have the potential to evade the host immune system and suppress inflammation, thus potentially facilitating the resolution of OA induced inflammation and cartilage regeneration.

Corneal Immunosuppressive Mechanisms, Anterior Chamber-Associated Immune Deviation (ACAID) and Their Role in Allograft Rejection.

Corneal transplantation is the most frequently performed transplant procedure in humans. Human leukocyte antigen matching, while imperative for other types of organ transplants, is usually not performed before cornea transplantation. With the use of topical steroid immunosuppressants, which are subsequently tailed off to almost zero, most corneal transplants will not be rejected in recipients with low risk of graft rejection. This phenomenon has been described as immune privilege by Medawar many years ago. However, this immune privilege is relative and can be easily eroded, e.g. by postoperative nonspecific inflammation or other causes of corneal or ocular inflammation. Interestingly, corneas that are at high risk of rejection have a higher failure rate than other organs. Considerable progress has been made in recent years to provide a better understanding of corneal immune privilege. This chapter will review current knowledge on ocular immunosuppressive mechanisms including anterior chamber-associated immune deviation and discuss their role(s) in corneal allograft rejection. Ultimately, this evolving information will be of benefit in developing therapeutic strategies to prevent corneal transplant rejection.

Mesenchymal stem cell therapy to promote corneal allograft survival: current status and pathway to clinical translation.

Purpose of reviewThis article reviews the literature on the therapeutic potential of mesenchymal stem cells (MSCs) to prolong corneal allograft survival. Recent findingsTo date, only small numbers studies have investigated the MSC ability to modulate corneal allograft survival. Most reports have shown positive results, which is encouraging, however as different MSC-application strategies (time point of injection, cell number/number of injections, route of injection, MSC source, MSC licensing) have been employed in various animal models it is difficult to compare and validate the results. The MSC ability to promote graft survival has been attributed to their modulation of the recipient immune system, altering the Th1/Th2 balance, expanding Foxp3+ regulatory T cells, polarizing macrophages and inhibiting intra-graft infiltration of antigen presenting cells. More in depth analysis is required to elucidate the mechanism of MSC-immunomodulation in vivo. SummaryMSCs have shown the potential to modulate corneal allograft rejection in various models using MSCs from different species. In particular for high-risk patients with poor prognosis MSC therapy might be a promising approach to promote corneal allograft survival. First-in-man clinical trials with MSC will hopefully shed new light on MSC-mediated immunomodulation in vivo and contribute to the restoration of vision in patients receiving corneal allografts.