Korevaar, S.S.

Mesenchymal stem cells derived from adipose tissue are not affected by renal disease (Article)

2012-10-01T00:00:00Z

Mesenchymal stem cells are a potential therapeutic agent in renal disease and kidney transplantation. Autologous cell use in kidney transplantation is preferred to avoid anti-HLA reactivity; however, the influence of renal disease on mesenchymal stem cells is unknown. To investigate the feasibility of autologous cell therapy in patients with renal disease, we isolated these cells from subcutaneous adipose tissue of healthy controls and patients with renal disease and compared them phenotypically and functionally. The mesenchymal stem cells from both groups showed similar morphology and differentiation capacity, and were both over 90% positive for CD73, CD105, and CD166, and negative for CD31 and CD45. They demonstrated comparable population doubling times, rates of apoptosis, and were both capable of inhibiting allo-antigen-and anti-CD3/CD28-activated peripheral blood mononuclear cell proliferation. In response to immune activation they both increased the expression of pro-inflammatory and anti-inflammatory factors. These mesenchymal stem cells were genetically stable after extensive expansion and, importantly, were not affected by uremic serum. Thus, mesenchymal stem cells of patients with renal disease have similar characteristics and functionality as those from healthy controls. Hence, our results indicate the feasibility of their use in autologous cell therapy in patients with renal disease.

Immunological aspects of allogeneic and autologous mesenchymal stem cell therapies (Article)

2011-12-01T00:00:00Z

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 (200U/ml) and IL-15 (10ng/ml) for 7 days. CD3-CD16+CD56+NK cells were then isolated by fluorescence-activated cell sorting and added to europium-labeled MSCs for 4hr 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 24hr 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 explosive T-Cell proliferation (Article)

2010-12-01T00:00:00Z

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-Î±, the growth factors basic fibroblast growth factor and vascular endothelial growth factor-Î±, 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 CD127negFoxP3+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 CD25positivefraction 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.

The Jak inhibitor CP-690,550 preserves the function of CD4 +CD25brightFoxP3+ regulatory T cells and inhibits effector T cells (Article)

2010-08-01T00:00:00Z

The Jak inhibitor CP-690,550 inhibits alloreactivity and is currently being investigated for prevention of allograft rejection after transplantation. In this study, we examined the effect of CP-690,550 on IL-2-mediated Jak/STAT5 phosphorylation by CD4+CD25brightFoxP3 +CD127-/low T cells (Treg) and CD4+CD25 neg effector T cells (Teff) in kidney transplant (KTx) patients. Phosphospecific flow cytometry was used to study the effect of CP-690,550 on IL-2-induced intracellular STAT5-phosphorylation. IL-2-induced phosphorylation of STAT5 (P-STAT5) in both Treg and Teff, which was significantly higher for CD4+CD25bright Treg (increased by 71%, mean) than for CD4+CD25neg Teff (increased by 42%). In the presence of 100 ng/mL CP-690,550, a clinically relevant exposure, IL-2-induced P-STAT5 was partially inhibited in CD4+CD25brightTreg (% inhibition; 51%), while almost completely blocked in Teff (%inhibition; 84%, p = 0.03). The IC50 was 2-3 times higher for Treg (104 ng/mL) than for Teff (40 ng/mL, p = 0.02). In the presence of CP-690,550, Treg exhibited additional suppressive activities on the alloactivated proliferation of Teff (56%, mean). In addition, CD4+CD25bright Treg from KTx-patients receiving CP-690,550 vigorously suppressed the proliferation of Teff (87%, mean). Our findings show that CP-690,550 effectively inhibits Teff function but preserves the suppressive activity of CD4+CD25bright regulatory T cells.

The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins (Article)

2010-08-01T00:00:00Z

Rabbit anti-thymocyte globulins (rATG) induce CD4+CD25 +forkhead box P3 (FoxP3+) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4+CD25+FoxP3+CD127 -/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25+ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4 +CD25+FoxP3+CD127-/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3 +T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3+ T cells. The rATG tacrolimus-induced CD25+ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4+CD25+FoxP3+CD127-/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25+ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-, perforin and granzyme B in contrast to natural CD25 + T cells (all P = 0·03), while FoxP3 was expressed at a lower level (P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4 +CD25+ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.

Characterization of rabbit antithymocyte globulins-induced CD25+ regulatory T cells from cells of patients with end-stage renal disease (Article)

2010-03-01T00:00:00Z

Background: Rabbit antithymocyte globulins (rATGs) are known to convert CD4CD25FoxP3 T cells from healthy individuals to CD4CD25FoxP3 T cells. In this study, we investigated the effect of rATG on the induction of regulatory T cells (Tregs) from blood cells of patients with end-stage renal disease who are candidates for transplantation and rATG-induction therapy. The induced Tregs were analyzed and compared with naturally occurring CD4CD25FoxP3T cells. Methods: The CD25 T cells of pretransplant patients (n=7) and healthy controls (n=4) were stimulated with rATG or control rabbit immunoglobulins for 24 hr. The phenotype of induced Tregs was examined by flow cytometry, and their function was studied in the conventional suppression assay. Further characterization was performed by mRNA analyses. Results: After 24 hr, the percentage of CD4CD25FoxP3CD127 T cells and CD8CD25FoxP3CD127 T cells became higher in the rATG-treated samples compared with the rabbit immunoglobulin-treated samples (P<0.01). The rATG-induced CD25T cells, whether CD4 or CD8 inhibited the allogeneic responses of CD25 effector T cells as vigorously as natural CD25T cells. However, the proportion of FoxP3 within the top 2% rATG-induced CD4CD25T-cells was lower than within the natural CD4CD25T-cells (11%±2% vs. 95%±5%, P<0.01). The mRNA-expression levels of interleukin-27, interleukin-10, interferon-γ, perforin, and granzyme B were markedly higher compared with natural CD25T-cells (all P=0.03), whereas CTLA4 (P=0.03), transforming growth factor-β (P=0.02), and RORγt (P=0.04) were lower. Conclusion: rATG allows the induction of Tregs from patient peripheral blood mononuclear cell in vitro. In comparison with natural Tregs, the rATG-induced Tregs are phenotypically distinct but have similar regulatory activities. rATG may beneficially contribute to the mechanisms that control alloreactivity.

Monitoring of the immunomodulatory effect of CP-690,550 by analysis of the JAK/STAT pathway in kidney transplant patients (Article)

2009-10-01T00:00:00Z

BACKGROUND.: The small molecule drug CP-690,550 inhibits Janus kinase 3 at nanomolar concentrations and has recently been shown to prevent allograft rejection in rodents and nonhuman primates. METHODS.: As part of a phase 1 clinical trial, we investigated the effect of CP-690,550 after 29 days of 30 mg twice daily treatment at the cellular level in eight kidney transplant patients by studying ex vivo phosphorylation of STAT5 (P-STAT5), the key substrate of JAK3. RESULTS.: As determined by quantitative fluorescent western blotting, interleukin-2-induced P-STAT5 in YT cells was reduced by a median of 73% (P<0.01) in the presence of serum collected on day 29 compared with pretreatment baseline. When evaluated by phosphospecific flow cytometry, CP-690,550 also reduced interleukin-2-induced P-STAT5 in CD3 (median 20%; P<0.05), CD3CD4 (median 37%; P<0.05), and CD3CD8 (median 34%; P<0.01) populations in patient-derived peripheral blood mononuclear cells. At the functional level, the inhibitory effect of CP-690,550 was confirmed by determining the expression of several STAT5 targets genes. CONCLUSION.: Analysis of P-STAT5 may, therefore, be used to determine the immunomodulatory effect of CP-690,550 at the cellular level in transplant patients.

Inadequate immune regulatory function of CD4+CD25bright+FoxP3+ T cells in heart transplant patients who experience acute cellular rejection (Article)

2009-04-27T00:00:00Z

BACKGROUND.: CD4CD25FoxP3 regulatory T cells are suppressors of antigen-activated immune reactivity. Here, we assessed the clinically relevant role of these cells in the control of immune responses directed to a transplanted heart. METHODS.: We investigated the phenotype and function of peripheral CD4CD25FoxP3 T cells in heart transplant patients free from acute rejections (n=9) and in rejectors (n=12) before and during acute cellular rejection. RESULTS.: Between rejectors and nonrejectors, the proportion of CD4CD25 T cells and of FoxP3 cells within this population was comparable. Yet, CD4CD25FoxP3 T cells of rejectors had a higher CD127 expression than those of nonrejectors (P=0.0001). Depletion of CD4CD25 T cells from peripheral blood mononuclear cells increased the antidonor proliferative response of both nonrejectors (P≤0.0005) and rejectors (P≤0.03). In rejectors, however, only a 2-fold increase was measured, whereas the nonrejectors' response became 14 times higher (P=0.002). Reconstitution of CD4CD25 T cells only suppressed the overall antidonor proliferative response of CD25 responder cells of nonrejectors significantly (P=0.001). Moreover, the percentage inhibition of the response was higher in nonrejectors than in rejectors (P=0.02). Analyses of pretransplant samples revealed that CD4CD25 T cells of rejectors already had a lower suppressive capacity than those of nonrejectors before transplantation (P=0.04). CONCLUSION.: CD4CD25FoxP3 T cells of heart transplant patients who experience acute rejection had an up-regulated CD127 expression and an inadequate regulatory function compared with those of nonrejecting patients. Our observations suggest that the function of circulating CD4CD25FoxP3 regulatory T cells may be pivotal for the prevention of acute cellular rejection after clinical heart transplantation.

Donor-derived mesenchymal stem cells suppress alloreactivity of kidney transplant patients (Article)

2009-03-27T00:00:00Z

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.

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

2009-01-15T00:00:00Z

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), CD3CD16CD56 natural killer-cells (P<0.01), and CD4CD25 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 CD4CD25 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 CD4CD25 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.

Donor-derived mesenchymal stem cells remain present and functional in the transplanted human heart (Article)

2009-01-01T00:00:00Z

Mesenchymal stem cells (MSC) are characterized by their multilineage differentiation capacity and immunosuppressive properties. They are resident in virtually all tissues and we have recently characterized MSC from the human heart. Clinical heart transplantation offers a model to study the fate of transplanted human MSC. In this study, we isolated and expanded MSC from heart tissue taken before, and 1 week up to 6 years after heart transplantation. MSC from posttransplantation tissue were all of donor origin, demonstrating the longevity of endogenous MSC and suggesting an absence of immigration of recipient MSC into the heart. MSC isolated from transplanted tissue showed an immunophenotype that was characteristic for MSC and maintained cardiomyogenic and osteogenic differentiation capacity. They furthermore preserved their ability to inhibit the proliferative response of donor-stimulated recipient peripheral blood mononuclear cells. In conclusion, functional MSC of donor origin remain present in the heart for several years after transplantation.

Susceptibility of human mesenchymal stem cells to tacrolimus, mycophenolic acid, and rapamycin (Article)

2008-11-15T00:00:00Z

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.

FOXP3 mRNA expression analysis in the peripheral blood and allograft of heart transplant patients (Article)

2008-01-01T00:00:00Z

Previously, we demonstrated in heart transplant patients that FOXP3, a gene required for the development and function of regulatory T cells, was highly expressed in the graft during an acute cellular rejection. In this study, we analyzed whether the FOXP3 gene expression in the peripheral blood also reflects anti-donor immune responses, and therefore may provide clues for non-invasive detection of non-responsiveness or acute rejection. We examined the FOXP3 expression patterns of peripheral blood mononuclear cells (PBMC; n = 69) of 19 heart transplant patients during quiescence and rejection in comparison with those of endomyocardial biopsies (EMB; n = 75) of 24 heart transplant patients. While the FOXP3 mRNA levels were abundantly expressed in rejecting EMB (ISHLT rejection grade > 1R) compared with EMB without histological evidence of myocardial damage (ISHLT rejection grade 0R-1R; p = 0.003), no association with rejection or non-responsiveness was found for the FOXP3 mRNA levels in the peripheral blood. Thus, in contrast to intragraft FOXP3 gene expression, the peripheral FOXP3 mRNA levels lack correlation with anti-donor immune responses in the graft, and, consequently, FOXP3 does not appear to be a potential candidate gene for non-invasive diagnosis of non-responsiveness or rejection.

Expression patterns of regulatory T-cell markers in accepted and rejected nonhuman primate kidney allografts (Article)

2007-10-01T00:00:00Z

The identification of FOXP3 expressing cells in recipients of an allograft, in particular inside the graft itself, may help to define criteria for immunosuppressive drug withdrawal. We therefore examined expression patterns of several regulatory T-cell (Treg) markers in kidney biopsies and kidney tissues taken at the time of graft rejection from monkeys treated with αCD40, αCD86, CsA, a combination of these or after drug withdrawal. In advanced stages of rejection, organized multifocal nodular infiltrates, with mature dendritic cells, T cells and B cells could be found. In contrast, interstitial infiltrates contain more macrophages, less T cells and few B cells. Cells expressing FOXP3, CD25 and CTLA-4 were mainly found in nodular infiltrates of rejected tissue samples. A significant correlation was found between the percentage FOXP3+cells and markers for rejection, i.e. creatinine levels and Banff interstitial and tubular infiltrate scores. The type of immunosuppression did not influence the percentage of cells expressing Treg markers. Three animals with prolonged drug-free survival showed low numbers of FOXP3+cells. We conclude that the presence of intragraft FOXP3+cells is not confined to tolerated grafts, but should be considered as part of the normal immune response during rejection.

Interleukin-21: An interleukin-2 dependent player in rejection processes (Article)

2007-06-01T00:00:00Z

BACKGROUND. Interleukin (IL)-21 is the most recently described cytokine that signals via the common cytokine receptor (γc), is produced by activated CD4+ T-cells, and regulates expansion and effector function of CD8+ T-cells. MATERIALS. To explore the actions of IL-21 with other γc-dependent cytokines in alloreactivity, mRNA expression of IL-21, IL-21R α-chain, and IL-2 proliferation and cytotoxicity was measured after stimulation in mixed lymphocyte reactions. Additionally, IL-21 and IL-21R α-chain expression was studied in biopsies of heart transplant patients. RESULTS. Analysis of mRNA expression levels of allostimulated T-cells showed a 10-fold induction of IL-21 and IL-21R α-chain. Interestingly, induction of IL-21 was highly dependent on IL-2 (as in the presence of anti-IL-2, anti-IL-2R α-chain, and the immunosuppressive drugs cyclosporine A, tacrolimus, and rapamycin) the transcription of IL-21 was almost completely inhibited, whereas in the presence of exogenous IL-2 the mRNA expression of IL-21 was even more upregulated. IL-21 functioned as a costimulator for IL-2 to augment proliferation and cytotoxic responses, while blockade of the IL-2 route abrogated these functions of IL-21. Blockade of the IL-21 route by anti-IL-21R α-chain monoclonal antibodies inhibited the proliferation of alloactivated T-cells. Also, in vivo alloreactivity was associated with IL-21/IL-21R α-chain expression. After heart transplantation, the highest intragraft IL-21, IL-21R α-chain, and IL-2 mRNA expression levels were measured during acute rejection (P<0.001, P=0.01, P=0.03). CONCLUSION. IL-21 is a critical cytokine for IL-2 dependent immune processes. Blockade of the IL-21 pathway may provide a new perspective for the treatment of allogeneic responses in patients after transplantation.

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

2007-06-01T00:00:00Z

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.