Mikhail Nosov

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.

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.

Macrophages behavior on different NIPAm‐based thermoresponsive substrates

Thermoresponsive materials and surfaces are widely used in cell culture applications. There is a lot of research work employing thermoresponsive materials with various structure and compositions. However, little is known about the immunological response to the thermoresponsive materials. Macrophage-like transformed murine cell line RAW264.7 was selected as it is a widely used standard model for immune activation analysis. This study proposes to compare the effects of thermoresponsive films with various compositions on macrophage cells. Thermoresponsive materials are a useful utility as a non-enzymatic harvesting system for tissue culture. As RAW264.7 cells are difficult to remove from the substrate by enzymatic methods we also explored the possibility to use thermoresponsive materials for the macrophage cultivation. Spin coating and solvent casting was used to produce films of N-isopropylacrylamide-based polymers from the nanometer to micrometer range. Successful cell adhesion and proliferation was highly dependent on the thickness and composition of the coating. RAW264.7 cells were successfully detached from the coatings upon temperature reduction. Furthermore, results indicate that the RAW264.7 cells remained inactivated as cell secreted cytokine remained at a low level and the surface receptor profile of RAW264.7 was not altered when cells were detached in this manner.