Anja U. Engela

On the interactions between mesenchymal stem cells and regulatory T cells for immunomodulation in transplantation

Experimental studies have established the use of mesenchymal stem cells (MSC) as a candidate immunosuppressive therapy. MSC exert their immunomodulatory function through the inhibition of CD4+ and CD8+ T cell proliferation. It is unknown whether MSC impair the immunosuppressive function of regulatory T cells (Treg). In vitro and in vivo studies suggest that MSC mediate their immunomodulatory effects through the induction of Treg. In this review we will focus on the interactions between MSC and Treg, and evaluate the consequences of these cellular interplays for prospective MSC immunotherapy in organ transplantation.

Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Fourth Meeting: Lessons Learned from First Clinical Trials

The Fourth Expert Meeting of the Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Consortium took place in Barcelona on October 19 and 20, 2012. This meeting focused on the translation of preclinical data into early clinical settings. This position paper highlights the main topics explored on the safety and efficacy of mesenchymal stem cells as a therapeutic agent in solid organ transplantation and emphasizes the issues (proper timing, concomitant immunossupression, source and immunogenicity of mesenchymal stem cells, and oncogenicity) that have been addressed and will be followed up by the MiSOT Consortium in future studies.

Mesenchymal Stem Cells Induce an Inflammatory Response After Intravenous Infusion

Mesenchymal stem cells (MSCs) have potent immunosuppressive effects in vitro and are considered as a therapeutic option for autoimmune disease and organ transplantation. While MSCs show beneficial effects on immune disease progression and transplant survival in animal models, the immunomodulatory mechanisms involved are largely unknown. In the present study, we show that intravenously infused C57BL/6- green fluorescent protein (GFP) MSCs home to the lungs in C57BL/6 recipient mice and induce an inflammatory response. This response was characterized by increased mRNA expression of monocyte chemoattractant protein-1 (MCP1), IL1-β, and TNF-α and an increase in macrophages in lung tissue 2 h after MSC infusion. Simultaneously, serum levels of proinflammatory IL6, CXCL1, and MCP1 protein increased, demonstrating systemic immune activation after MSC infusion. In liver tissue, no C57BL/6-GFP MSCs were detected, but MCP1 and TNF-α mRNA levels peaked 4 h after MSC infusion. The expression of the anti-inflammatory cytokines TGF-β, IL4, and IL10 was only marginally affected. Nevertheless, 3 days after MSC infusion, animals developed a milder inflammatory response to lipopolysaccharides. Our results suggest that the in vivo immunomodulatory effects of MSCs originate from an inflammatory response that is induced by the infusion of MSCs, which is followed by a phase of reduced immune reactivity.

Immunological Aspects of Allogeneic and Autologous Mesenchymal Stem Cell Therapies

Mesenchymal stem cells (MSCs) have potential for therapeutic application as an immunomodulatory and regenerative agent. The immunogenicity and survival of MSCs after infusion are, however, not clear and evidence suggests that allogeneic but also autologous MSCs disappear rapidly after infusion. This may be associated with the susceptibility of MSCs to lysis by natural killer (NK) cells, possibly a result of culture-induced stress. In the present study we examined whether NK cell-mediated lysis of MSCs could be inhibited by immunosuppressive drugs. Human MSCs were isolated from adipose tissue and expanded in culture. Peripheral blood mononuclear cells were activated with interleukin (IL)-2 (200 U/ml) and IL-15 (10 ng/ml) for 7 days. CD3(-)CD16(+)CD56(+) NK cells were then isolated by fluorescence-activated cell sorting and added to europium-labeled MSCs for 4 hr in the presence or absence of immunosuppressive drugs. Lysis of MSCs was determined by spectrophotometric measurement of europium release. Nonactivated NK cells were not capable of lysing MSCs. Cytokine-activated NK cells showed upregulated levels of granzyme B and perforin and efficiently lysed allogeneic and autologous MSCs. Addition of tacrolimus, rapamycin or sotrastaurin to the lysis assay did not inhibit MSC killing. Furthermore, preincubation of activated NK cells with the immunosuppressive drugs for 24 hr before exposure to MSCs had no effect on MSC lysis. Last, addition of the immunosuppressants before and during the activation of NK cells, reduced NK cell numbers but did not affect their capacity to lyse MSCs. We conclude that the immunosuppressive drugs tacrolimus, rapamycin, and sotrastaurin are not capable of inhibiting the lysis of allogeneic and autologous MSCs by activated NK cells. Other approaches to controlling lysis of MSCs should be investigated, as controlling lysis may determine the efficacy of MSC therapy.

Human adipose-tissue derived mesenchymal stem cells induce functional de-novo regulatory T cells with methylated FOXP3 gene DNA

Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft‐versus‐host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC‐induced CD4+CD25+CD127–forkhead box P3 (FoxP3)+ T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nTreg). Perirenal adipose‐tissue derived MSC (ASC) obtained from kidney donors induced a 2·1‐fold increase in the percentage of CD25+CD127–FoxP3+ cells within the CD4+ T cell population from allostimulated CD25–/dim cells. Interleukin (IL)‐2 receptor blocking prevented this induction. The ASC‐induced T cells (iTreg) inhibited effector cell proliferation as effectively as nTreg. The vast majority of cells within the iTreg fraction had a methylated FOXP3 gene Treg‐specific demethylated region (TSDR) indicating that they were not of nTreg origin. In conclusion, ASC induce Treg from effector T cells. These iTreg have immunosuppressive capacities comparable to those of nTreg. Their induction is IL‐2 pathway‐dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de‐novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.

Interaction between adipose tissue-derived mesenchymal stem cells and regulatory T-cells

Mesenchymal stem cells (MSCs) exhibit immunosuppressive capabilities, which have evoked interest in their application as cell therapy in transplant patients. So far it has been unclear whether allogeneic MSCs and host regulatory T-cells (Tregs) functionally influence each other. We investigated the interaction between both cell types using perirenal adipose tissue-derived MSCs (ASCs) from kidney donors and Tregs from blood bank donors or kidney recipients 6 months after transplantation. The immunomodulatory capacity of ASCs was not prejudiced by both Tregs from healthy donors and Tregs from graft recipients, indicating that ASCs were not targeted by the inhibitory effects of Tregs and vice versa. In addition, Tregs supported ASC function, as they did not alter the secretion of IFN-γ by immune cells and hence contributed to ASC activation and efficiency. ASCs exerted their suppressive role by expressing IDO, reducing levels of TNF-α, and by inducing the production of IL-10 in effector cells and Tregs. In conclusion, this study presents evidence that donor ASCs and acceptor Tregs do not impair each others function and therefore encourages the use of MSC therapy for the prevention of graft rejection in solid organ transplantation.

Mesenchymal stem cells control alloreactive CD8+CD28− T cells

CD28/B7 co‐stimulation blockade with belatacept prevents alloreactivity in kidney transplant patients. However, cells lacking CD28 are not susceptible to belatacept treatment. As CD8+CD28− T‐cells have cytotoxic and pathogenic properties, we investigated whether mesenchymal stem cells (MSC) are effective in controlling these cells. In mixed lymphocyte reactions (MLR), MSC and belatacept inhibited peripheral blood mononuclear cell (PBMC) proliferation in a dose‐dependent manner. MSC at MSC/effector cell ratios of 1:160 and 1:2·5 reduced proliferation by 38·8 and 92·2%, respectively. Belatacept concentrations of 0·1 μg/ml and 10 μg/ml suppressed proliferation by 20·7 and 80·6%, respectively. Both treatments in combination did not inhibit each others function. Allostimulated CD8+CD28− T cells were able to proliferate and expressed the cytolytic and cytotoxic effector molecules granzyme B, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α. While belatacept did not affect the proliferation of CD8+CD28− T cells, MSC reduced the percentage of CD28− T cells in the proliferating CD8+ T cell fraction by 45·9% (P = 0·009). CD8+CD28− T cells as effector cells in MLR in the presence of CD4+ T cell help gained CD28 expression, an effect independent of MSC. In contrast, allostimulated CD28+ T cells did not lose CD28 expression in MLR–MSC co‐culture, suggesting that MSC control pre‐existing CD28− T cells and not newly induced CD28− T cells. In conclusion, alloreactive CD8+CD28− T cells that remain unaffected by belatacept treatment are inhibited by MSC. This study indicates the potential of an MSC–belatacept combination therapy to control alloreactivity.

Human Allogeneic Bone Marrow and Adipose Tissue Derived Mesenchymal Stromal Cells Induce CD8+ Cytotoxic T Cell Reactivity

Introduction For clinical applications, Mesenchymal Stromal Cells (MSC) can be isolated from bone marrow and adipose tissue of autologous or allogeneic origin. Allogeneic cell usage has advantages but may harbor the risk of sensitization against foreign HLA. Therefore, we evaluated whether bone marrow and adipose tissue-derived MSC are capable of inducing HLA-specific alloreactivity. Methods MSC were isolated from healthy human Bone Marrow (BM-MSC) and adipose tissue (ASC) donors. Peripheral Blood Mononuclear Cells (PBMC) were co-cultured with HLA-AB mismatched BM-MSC or ASC precultured with or without IFNy. After isolation via FACS sorting, the educated CD8+ T effector populations were exposed for 4 hours to Europium labeled MSC of the same HLA make up as in the co-cultures or with different HLA. Lysis of MSC was determined by spectrophotometric measurement of Europium release. Results CD8+ T cells educated with BM-MSC were capable of HLA specific lysis of BM-MSC. The maximum lysis was 24% in an effector:target (E:T) ratio of 40:1. Exposure to IFNγ increased HLA-I expression on BM-MSC and increased lysis to 48%. Co-culturing of PBMC with IFNγ-stimulated BM-MSC further increased lysis to 76%. Surprisingly, lysis induced by ASC was significantly lower. CD8+ T cells educated with ASC induced a maximum lysis of 13% and CD8+ T cells educated with IFNγ-stimulated ASC of only 31%. Conclusion Allogeneic BM-MSC, and to a lesser extend ASC, are capable of inducing HLA specific reactivity. These results should be taken into consideration when using allogeneic MSC for clinical therapy.

The effect of rabbit antithymocyte globulin on human mesenchymal stem cells

Mesenchymal stem cells (MSCs) possess immunomodulatory properties which are of key interest for their application in autoimmunity and transplantation. In transplantation, administration of MSCs has shown promising results in preclinical models and has recently moved to clinical trials. Therefore, it is important to study the interactions between MSCs and immunosuppressive drugs currently used in transplantation. We aimed to analyze the effect of rabbit antithymocyte globulin (rATG) MSCs. MSCs were obtained from perirenal fat of kidney donors and exposed to ranging doses of rATG (Thymoglobulin®, Genzyme; 0.5–100 μg/ml). Binding of rATG, effects on viability and susceptibility to be killed by cytotoxic lymphocytes as well as effects on their immunosuppressive potential of MSCs were tested. rATG binds dose‐dependently to MSCs. This binding was associated with slightly impaired viability after 48 and 72 h when compared with nonexposed MSCs. In contrast to nontreated MSCs, rATG preexposed MSCs were susceptible to be lysed by cytokine‐activated CD8+ cytotoxic cells and NKT cells. The capacity of MSCs to suppress the proliferation of anti‐CD3/CD28 activated CD4 and CD8 T cells were reduced by the presence of rATG in the culture. rATG reduces the viability and antiproliferative capacity of MSCs in a dose‐dependent manner and converts them into targets for CD8 T cells and NKT cell lysis.

Genetic variants of FOXP3 influence graft survival in kidney transplant patients.

FOXP3(+) regulatory T cells (Treg) play a role in controlling alloreactivity. It has been shown that short (GT)n dinucleotide repeats (≤(GT)15; S) in the promoter region of the FOXP3 gene enhance the promoter activity when compared to long (GT)n repeats (≥(GT)16; L). The present study retrospectively investigated the influence of this (GT)n FOXP3 gene polymorphism on renal allograft survival. A total of 599 consecutive first-time kidney transplant patients (median follow-up time 7.7 years) were subdivided according to their FOXP3 genotype into the S-genotype group (SG) and the L-genotype group (LG). The SG was superior to the LG in both general graft survival censored for death (logrank test, p=0.013) and graft survival following acute rejection (p=0.021). Multivariate analysis defined the (GT)n FOXP3 dinucleotide repeat polymorphism as an independent factor and confirmed an advantage for the SG in renal allograft survival (HR=0.67, 95% CI 0.48-0.94, p=0.02). This gene association study identified a beneficial effect of FOXP3 genetic variants on graft survival in kidney transplant patients.