Sander S. Korevaar

Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells.

Background. The transcription factor FOXP3 has been identified as the molecule associated with the regulatory function of CD25+ T cells. Methods. To understand the biology of FOXP3+ T cells in allogeneic reactions, we measured FOXP3 mRNA expression levels in allostimulated CD25 bright+ cells and CD25 intermediate( int)/- cells and in peripheral blood mononuclear cells (PBMC). The effect of immunosuppressive drugs on FOXP3 expression was studied in mixed lymphocyte reactions (MLR) in the presence and absence of calcineurin inhibitors (CNI), &agr;CD25 mAb, and Rapamycin (Rapa), and analyzed in biopsies from cardiac allograft recipients during acute rejection by quantitative (Q)-PCR. Results. FOXP3 mRNA expression was restricted to the CD25 bright+ population that inhibited the proliferation of allostimulated CD25 int/- cells. In the MLR FOXP3 was readily induced after allostimulation. Kinetic examination of the MLR showed a 10–20-fold higher FOXP3 mRNA expression level after 5 days of culture. The CNI Cyclosporin and Tacrolimus, and &agr;CD25 mAb inhibited in vitro induced FOXP3 gene transcription (range 70%–90%), whereas Rapa did not inhibit the induction. After clinical heart transplantation the highest FOXP3 mRNA expression levels were measured in biopsies during acute rejection (P=0.03). Conclusions. The high FOXP3 mRNA levels during allogeneic responses in vivo and in vitro suggests that regulatory activities of CD25 bright+ T cells or the generation of these cells is an intrinsic part of activation. CNI and &agr;CD25 mAb in contrast to Rapa, did interfere with this immunosuppressive counter-mechanism and as a result might have an inhibitory effect to tolerance induction after transplantation.

Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells

There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft‐versus‐host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue‐derived mesenchymal stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6] or under control conditions, and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine‐2,3‐dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX‐2, involved in prostaglandin E2 production. Both conditions had a stimulatory, but differential, effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy.

Donor-derived mesenchymal stem cells suppress alloreactivity of kidney transplant patients.

Background. Human mesenchymal stem cells (MSC) have immunosuppressive capacities. Although their efficacy is currently studied in graft-versus-host disease, their effect on alloreactivity in solid organ transplant patients is unknown. In this study, the immunosuppressive effect of MSC on recipient anti-donor reactivity was examined before and after clinical kidney transplantation. Methods. Anti-donor reactivity was established in pretransplant and posttransplant mixed lymphocyte reactions (MLR) of 14 living-kidney donor-recipient pairs. MSC from donors and third-party controls were added to the MLR in a ratio of 1:5. Results. MSC were isolated from donor perirenal fat and showed multilineage differentiation potential and the capacity to inhibit lymphocyte proliferation. The immunosuppressive effect of MSC was dose dependent and mediated by cell-membrane contact and soluble factors, including interleukin-10 and indoleamine 2,3-dioxygenase. Donor-derived MSC significantly inhibited the recipient anti-donor reactivity before and 1 month after transplantation. This effect was independent of human leukocyte antigen background of MSC. Flow cytometric analysis showed that MSC inhibited the proliferation of CD4+ and CD8+ T-lymphocyte subsets in pretransplant and posttransplant donor-directed MLR, whereas MSC had no effect on B- or natural killer-cell proliferation. Conclusion. Donor MSC significantly inhibited the proliferation of alloactivated recipient T cells before and after kidney transplantation. We believe these findings should encourage MSC-based intervention in clinical organ transplantation.

Human Adipose Tissue-Derived Mesenchymal Stem Cells Induce Explosive T-Cell Proliferation

Mesenchymal stem cells (MSCs) inhibit the proliferation of allo-activated lymphocytes. This effect is primarily dependent on the secretion of anti-inflammatory factors by MSCs and is enhanced under inflammatory conditions. MSCs, however, also produce factors that can potentially activate resting immune cells. Full understanding of the behavior of MSCs under inflammatory and noninflammatory conditions is crucial when clinical application of MSCs is considered. Human adipose tissue-derived MSCs were cultured with nonactivated peripheral blood mononuclear cells (PBMCs) and the activation, proliferation, and function of PBMCs were examined. Seven days of coculture with autologous or allogeneic MSCs significantly increased the proliferation of PBMCs (3-fold). This effect was observed in both direct and transwell coculture systems. MSCs cocultured with PBMCs showed increased mRNA expression of the proinflammatory mediators interleukin-6 (IL-6), IL-8, tumor necrosis factor-α, the growth factors basic fibroblast growth factor and vascular endothelial growth factor-α, and the anti-inflammatory factor indoleamine 2,3-dioxygenase. After removal of MSCs, PBMCs showed a spectacular further increase in proliferation, with a maximum of 25-fold after 7 days. This increase in proliferation was not seen when PBMCs were kept in the presence of MSCs. The proliferating fraction of PBMCs largely consisted of CD4(+) T-cells with high CD25 expression and the proportion of CD127(neg)FoxP3(+) regulatory T-cells significantly increased from 5.0% to 8.5% of total CD4(+) T-cells. The expanded T-cells demonstrated normal responses to mitogen or alloantigen stimulation. The CD25(positive) fraction of these cells had immunosuppressive capacity. In conclusion, MSCs can stimulate the activation and proliferation of resting T-cells and generate regulatory T-cells. These findings are important when MSCs are applied in the clinic.

Susceptibility of human mesenchymal stem cells to tacrolimus, mycophenolic acid, and rapamycin.

Background. Mesenchymal stem cells (MSC) have multilineage differentiation and immunomodulatory capacities and are potentially useful for therapeutic applications, such as tissue regeneration and control of alloreactivity. MSC are present in most tissues including the transplantable organs. It is therefore unavoidable that MSC will be exposed to immunosuppressive drugs in a clinical transplantation setting. The molecular targets of these drugs are expressed in MSC, but the effect of their inhibition on MSC functioning is unknown. Methods. MSC were isolated and expanded from heart tissue and the effects of the calcineurin inhibitor tacrolimus, the cell cycle inhibitor mycophenolic acid (MPA), and the mammalian target of rapamycin inhibitor on MSC survival, proliferation, differentiation, and immunosuppressive capacity were examined. Results. Short-term exposure to the immunosuppressants did not induce toxicity or apoptosis in MSC, but high-dose tacrolimus induced toxicity after 7 days. MPA and rapamycin inhibited MSC proliferation at therapeutic doses. The immunosuppressants had differential effects on the differentiation capacity of MSC. Tacrolimus reduced the expression of troponin T type 2 and desmin during cardiomyogenic differentiation of MSC, whereas MPA decreased the deposition of calcified minerals during osteogenic differentiation. Rapamycin stimulated lipid production during adipogenic differentiation. Unexpectedly, MSC had adverse effects on the immunosuppressive efficacy of tacrolimus and rapamycin. There was no such effect of MSC on the function of MPA. Preincubation of MSC with tacrolimus increased the immunosuppressive capacity of MSC. Discussion. This study demonstrates that therapeutic concentrations of immunosuppressive drugs affect MSC function. MSC affect the efficacy of immunosuppressive medication. These findings are important for potential clinical use of MSC in combination with immunosuppressants.

Human mesenchymal stem cells are susceptible to lysis by CD8+ T cells and NK cells

There is growing interest in the use of mesenchymal stem cells (MSCs) to improve the outcome of organ transplantation. The immunogenicity of MSCs is, however, unclear and is important for the efficacy of MSC therapy and for potential sensitization against donor antigens. We investigated the susceptibility of autologous and allogeneic MSCs for lysis by CD8+ T-lymphocytes and NK cells in a kidney transplant setting. MSCs were derived from adipose tissue of human kidney donors and were CD90+, CD105+, CD166+, and HLA class I+. They showed differentiation ability and immunosuppressive capacity. Lysis of MSCs by peripheral blood mononuclear cells (PBMCs), FACS-sorted CD8+ T cells, and NK cells was measured by europium release assay. Allogeneic MSCs were susceptible for lysis by cytotoxic CD8+ T cells and NK cells, while autologous MSCs were lysed by NK cells only. NK cell-mediated lysis was inversely correlated with the expression of HLA class I on MSCs. Lysis of autologous MSCs was not dependent on culturing of MSCs in FBS, and MSCs in suspension as well as adherent to plastic were lysed by NK cells. Pretransplant recipient PBMCs did not lyse donor MSCs, but PBMCs isolated 3, 6, and 12 months after transplantation showed increasing lysing ability. After 12 months, CD8+ T-cell-mediated lysis of donor MSCs persisted, indicating there was no evidence for desensitization against donor MSCs. Lysis of MSCs is important to take into account when MSCs are considered for clinical application. Our results suggest that the HLA background of MSCs and timing of MSC administration are important for the efficacy of MSC therapy.

Effects of hypoxia on the immunomodulatory properties of adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived mesenchymal stem cells (ASC) are of great interest as a cellular therapeutic agent for regenerative and immunomodulatory purposes. The function of ASC adapts to environmental conditions, such as oxygen tension. Oxygen levels within tissues are typically much lower than under standard culture conditions and ASC used for therapy therefore encounter a change from normoxic to hypoxic conditions. The effect of hypoxia on the regenerative potential of ASC has been investigated in a number of studies. The effect of hypoxia on the immunomodulatory function of ASC, however, remains to be determined. In the present study the effect of hypoxic (1% oxygen) culture conditions on human ASC was examined. ASC showed no signs of toxicity under low oxygen levels and no major immunophenotypical changes were observed, apart from a down regulation of the marker CD105. Oxygen tension had no effect on the proliferation of ASC and colony forming unit efficiency remained the same under 1 and 20% oxygen. Under both oxygen levels ASC were capable of strong upregulation of the immunomodulatory molecules indoleamine 2,3-dioxygenase (IDO) and programed death ligand-1 upon stimulation with IFN-γ and TNF-α, and, in addition, IDO activity as measured by the accumulation of l-kynurenine was not affected under hypoxia. The ability of ASC to inhibit anti-CD3/CD28 stimulated CD4+ and CD8+ T cell proliferation was not hampered by hypoxia. The results of the present study demonstrate that the immunosuppressive capacity of ASC is maintained under hypoxic conditions. These findings are important for the therapeutic use of ASC and may be applied for the in vitro generation of ASC with improved functionality for therapeutic use.

Intragraft FOXP3 mRNA expression reflects antidonor immune reactivity in cardiac allograft patients

Background. Regulatory FOXP3+ T cells control immune responses of effector T cells. However, whether these cells regulate antidonor responses in the graft of cardiac allograft patients is unknown. Therefore, we analyzed the gene expression profiles of regulatory and effector T-cell markers during immunological quiescence and acute rejection. Methods. Quantitative real-time polymerase chain reaction was used to analyze mRNA expression levels in time-zero specimens (n=24) and endomyocardial biopsies (EMB; n=72) of cardiac allograft patients who remained free from rejection (nonrejectors; n=12) and patients with at least one histologically proven acute rejection episode (rejectors; International Society for Heart and Lung Transplantation [ISHLT] rejection grade >2; n=12). Results. For all analyzed regulatory and effector T-cell markers, mRNA expression levels were increased in biopsies taken after heart transplantation compared with those in time-zero specimens. Posttransplantation, the FOXP3 mRNA levels were higher in EMB assigned to a higher ISHLT rejection grade than the biopsies with grade 0: the highest mRNA levels were detected in the rejection biopsies (rejection grade >2; P=0.003). In addition, the mRNA levels of CD25, glucocorticoid-induced TNF receptor family-related gene, cytotoxic T lymphocyte-associated antigen 4, interleukin-2, and granzyme B were also significantly higher in rejecting EMB than in nonrejecting EMB (rejection grade ≤2). This increase in expression levels in relation to the histological rejection grade was only observed in patients who developed an acute rejection episode; the mRNA levels of nonrejectors remained stable irrespective of ISHLT rejection grade. Conclusions. These observations suggest that, after clinical heart transplantation, FOXP3+ T cells do not prevent acute rejection, but rather are a response to antidonor effector T-cell activity.

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.

The effect of the JAK inhibitor CP-690,550 on peripheral immune parameters in stable kidney allograft patients.

Introduction. CP-690,550 inhibits Janus kinase 3 (JAK3) which mediates signal transduction of receptors of the common γ-chain cytokines. These cytokines play key roles in lymphocyte function and homeostasis. As part of a phase 1 trial, we evaluated the effect of CP-690,550 on immune parameters. Material. Stable kidney transplant recipients (n=8) receiving mycophenolate mofetil and prednisolone were treated with CP-690,550, 30 mg twice daily orally for 29 days. Blood samples were collected on days 1 (before first dose), 15, 29 (end of treatment), and 57. Results. Two patients experienced minor infections (one urinary tract infection and one mild respiratory tract infection). Leukocyte counts remained stable, whereas a mean decrease in hemoglobulin of 8% was measured (P=0.01). CP-690,550 treatment for 29 days resulted in statistically significant changes in the number of circulating CD19+ B cells (P=0.05), CD3−CD16+CD56+ natural killer-cells (P<0.01), and CD4+CD25bright+ T cells (P=0.05; one-way analysis of variance). After CP-690,550 treatment on day 15 the number of B cells increased by a mean of 100%, (P=0.04), whereas those of natural killer cells and CD4+CD25bright+ T cells decreased by 65% (P=0.001) and 38% (P=0.03, t test), respectively, from pretreatment baseline. However, the regulatory capacities of the residual CD4+CD25bright+ T cells remained unchanged pre- and posttreatment. In addition, in the presence of CP-690,550, the interferon-γ production capacity of peripheral blood mononuclear cells was reduced by 39% (median) compared with predose baseline (P=0.01). Conclusions. These findings demonstrate the role of JAK3 in the homeostasis and function of select lymphocyte subpopulations. JAK3 inhibition may provide a novel mechanism for the modulation of allogeneic responses in patients after transplantation.