Baan, C.C.

Tacrolimus Inhibits NF-κB Activation in Peripheral Human T Cells (Article)

2013-04-01T00:00:00Z

The calcineurin inhibitor, tacrolimus (TAC), inhibits the protein phosphatase activity of calcineurin, leading to suppression of the nuclear translocation of NFAT and inhibition of T cell activation. Apart from NFAT also the transcription factor NF-κB plays a key functional role in T cell activation. Therefore, blockade of the NF-κB activation cascade by immunosuppressive drugs prevents immune activation. Here we studied whether TAC blocks NF-κB activation in peripheral human T cells. After anti-CD3/CD28-activation of T cells from healthy volunteers, NF-κB (p65) phosphorylation was measured by flow cytometry in CD3+ T cells, CD4+ helper T cells and CD8+ cytotoxic T cells in the absence and presence of TAC 10 ng/mL, sotrastaurin 500 nM (positive control) and mycophenolic acid 10 μg/mL (negative control; n = 6). NF-κB transcriptional activity was measured by ELISA and intracellular TNFα protein, a downstream target, was measured by flow cytometry to assess the functional consequences of NF-κB blockade. Anti-CD3/28-activation induced NF-κB phosphorylation in CD3+ T cells, CD4+ T cells and CD8+ T cells by 34% (mean), 38% and 30% resp. (p<0.01). Sotrastaurin inhibited NF-κB activation in the respective T cell subsets by 93%, 95% and 86% (p<0.01 vs. no drug), while mycophenolic acid did not affect this activation pathway. Surprisingly, TAC also inhibited NF-κB phosphorylation, by 55% (p<0.01) in CD3+ T cells, by 56% (p<0.01) in CD4+ T cells and by 51% in CD8+ T cells (p<0.01). In addition, TAC suppressed NF-κB DNA binding capacity by 55% (p<0.05) in CD3+ T cells and TNFα protein expression was inhibited in CD3+ T cells, CD4+ T cells and CD8+ T cells by 76%, 71% and 93% resp. (p<0.01 vs. no drug), confirming impaired NF-κB signaling. This study shows the suppressive effect of TAC on NF-κB signaling in peripheral human T cell subsets, measured at three specific positions in the NF-κB activation cascade.

Genetic variants of FOXP3 influence graft survival in kidney transplant patients (Article)

2013-03-25T00:00:00Z

FOXP3+regulatory T cells (Treg) play a role in controlling alloreactivity. It has been shown that short (GT)ndinucleotide repeats (≤(GT)15; S) in the promoter region of the FOXP3 gene enhance the promoter activity when compared to long (GT)nrepeats (≥(GT)16; L). The present study retrospectively investigated the influence of this (GT)nFOXP3 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)nFOXP3 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.

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.

The functional polymorphism Ala258Ser in the innate receptor gene ficolin-2 in the donor predicts improved renal transplant outcome (Article)

2012-09-15T00:00:00Z

BACKGROUND: Innate immunity plays a role in controlling adaptive immune responses. METHODS: We investigated the clinical relevance of single nucleotide polymorphisms in 22 genes encoding innate, secreted, and signaling pattern recognition receptors in a total of 520 donor-recipient pairs of postmortem, human leukocyte antigen-DR-compatible kidney transplantations. Associations with rejection incidence were tested in an a priori randomized training set and validation set. RESULTS: Polymorphisms in TLR-3 (rs3775296) in the recipients and in Ficolin-2 (rs7851696; Ala258Ser) and C1qR1 (rs7492) in the donors showed the strongest association with severe rejection. In multivariate analysis, presence of the Ficolin-2 Ala258Ser variant in the donor predicted lower incidence of severe rejection (odds ratio=0.3; 95% confidence interval, 0.1-0.9; P=0.024) and of graft loss (hazard ratio=0.5; 95% confidence interval, 0.2-1.0; P=0.046) independently of clinical risk factors. Ficolin-2 messenger RNA expression was detected in pretransplantation biopsies from 69 donor grafts. Serum and tissue Ficolin-2 levels were unaffected by genotype. Ficolin-2 protein, which bound to dying cells, was detected in donor kidneys in a passenger leukocyte-like pattern. Indeed, monocytes, monocyte-derived macrophages, and peripheral blood mononuclear cells expressed Ficolin-2. Donor grafts with the Ficolin-2 Ala258Ser variant contained significantly elevated expression of interleukin 6, having ascribed cytoprotective effects. It has been described that Ala258Ser leads to increased binding capacity of Ficolin-2 to N-acetylglucosamine. CONCLUSIONS: Presence of the Ficolin-2 Ala258Ser polymorphism in the donor independently predicts improved graft outcome. Based on mechanistic data, we propose that this functional polymorphism leads to more efficient handling of injured cells by phagocytozing cells, resulting in decreased intragraft exposure to danger signals and dampened alloimmune responses. Copyright

Pharmacodynamic analysis of tofacitinib and basiliximab in kidney allograft recipients (Article)

2012-09-15T00:00:00Z

BACKGROUND: The common γ-chain (γc) cytokines signal through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and play pivotal roles in lymphocyte activation. We investigated the effect of immunosuppressive drugs targeting this pathway, the JAK inhibitor tofacitinib (CP-690,550) and the anti-interleukin (IL)-2R antibody basiliximab, as part of a phase 2 study. METHODS: After whole-blood activation with the γccytokines IL-2, IL-7, and IL-15, STAT5 phosphorylation was determined in T cells of de novo kidney transplantation patients treated with tofacitinib/basiliximab (n=5), calcineurin inhibitor (CNI) (cyclosporine A)/basiliximab (n=4) or CNI (tacrolimus)-based immunosuppression (n=6). The IC50 for phosphorylated STAT (P-STAT) 5 inhibition by tofacitinib was determined in cytokine-activated CD4+and CD8+T cells from healthy individuals (n=4). RESULTS: IC50 was 26, 72, and 37 ng/mL for IL-2, IL-7, and IL-15 activation, in CD4+T cells, respectively; and 35, 61, and 76 ng/mL for IL-2, IL-7, and IL-15 activation, in CD8+T cells, respectively. In kidney transplantation patients, 7 days after starting tofacitinib/basiliximab treatment, cytokine-induced P-STAT5 was inhibited in CD4+T cells (92% for IL-2 activation, 60% for IL-7, and 75% for IL-15), which persisted for the 2-month study period. In contrast, CNI/basiliximab treatment did not affect IL-7-activated or IL-15-activated P-STAT5; only IL-2-activated P-STAT5 was reduced by 77% on day 7 and recovered to pretreatment levels within 2 months. CD8+T cells showed a comparable profile to CD4+T cells. P-STAT5 was not inhibited in CNI-treated control patients. CONCLUSIONS: Tofacitinib therapy strongly inhibits γccytokine-induced JAK/STAT5 activation, whereas basiliximab suppresses IL-2-stimulated activation only. Pharmacodynamic monitoring offers a unique tool to evaluate the biologic effects of immunosuppressive drugs. Copyright

Phosphospecific flow cytometry for pharmacodynamic drug monitoring: Analysis of the JAK-STAT signaling pathway (Article)

2012-09-08T00:00:00Z

Cytokines of the IL-2 receptor family act via activation of the JAK-STAT (janus tyrosine kinase-signal transducer and activator of transcription) signaling pathway. These cytokines are pivotal for the development and function of lymphocyte subsets involved in the immune response after organ transplantation including T, B and natural killer cells. The new small drug molecule and JAK1/3 inhibitor, tofacitinib, is currently being tested in phase II and III clinical trials for rheumatoid arthritis, psoriasis and in organ transplantation. This agent specifically targets the JAK-STAT signaling pathway. Here we discuss phosphospecific flow cytometry as a novel tool to monitor the JAK-STAT signaling pathway in kidney transplant patients and speculate that through the use of this pharmacodynamic tool the efficacy of immunosuppressive drugs can be assessed enabling optimization of the immunosuppressive therapy for individual transplant patients.

Inhibitory effect of tacrolimus on p38 mitogen-activated protein kinase signaling in kidney transplant recipients measured by whole-blood phosphospecific flow cytometry. (Article)

2012-06-27T00:00:00Z

Tacrolimus (TAC), the cornerstone of immunosuppressive therapy after solid organ transplantation, inhibits calcineurin activation. Despite pharmacokinetic monitoring, patients frequently experience toxicity or lack of efficacy, which could be prevented by pharmacodynamic monitoring. In Jurkat T-cell lines, it has been shown that TAC, in addition to calcineurin, inhibits the p38 mitogen-activated protein kinase (MAPK) pathway, which is important in T-cell activation and is therefore a potential drug-specific biomarker. We studied whether TAC inhibits p38 MAPK signaling in primary human T cells and ex vivo in healthy volunteers and kidney transplant recipients. Phorbol-12-myristate-13-acetate/ionomycin-induced MAPK signaling was measured by whole-blood phosphospecific flow cytometry. In vitro, 10-ng/mL TAC inhibited p38 MAPK phosphorylation by a mean of 27% in CD3, 26% in CD4, and 34% in CD8 T cells (P<0.01 compared with baseline). In healthy adults (n=4), 2 hr after a single oral dose of 10-mg TAC, the p38 MAPK activation was inhibited by 35% in CD3, CD4, and CD8 T cells (P<0.05 compared with baseline). In kidney transplant recipients (n=24), TAC predose concentrations (range, 3.2-10.5 ng/mL) were inversely correlated with p38 MAPK activation in CD3, CD4, and CD8 T cells (r=0.51, 0.34, and 0.37, respectively; P<0.01). TAC inhibits activation of the MAPK pathway in a dose-dependent manner in kidney transplant patients and may be a potential marker for immune monitoring.

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.

How does auxiliary liver transplantation regulate alloreactivity in sensitized kidney transplant patients? (Article)

2011-04-27T00:00:00Z

Report of a joint ESOT and AST meeting: Highlights in biologic agents and transplantation (Article)

2011-04-01T00:00:00Z

A joint meeting organized by the European (ESOT) and American (AST) Societies of Transplantation occurred in Nice, France, October 1-3, 2010. Focused on emerging use of biologic agents in solid organ transplantation, it served as a venue for state-of-the-art updates in basic immunology and clinical science, with an emphasis on the interrelatedness of the two. This meeting summary is designed to highlight important insights communicated in Nice, offer an overview of novel therapeutics in development, and entice members of all societies to consider attending a second joint symposium, under consideration for 2012.

Discontinuation of calcineurin inhibitors treatment allows the development of FOXP3+ regulatory T-cells in patients after kidney transplantation (Article)

2011-01-01T00:00:00Z

This study investigated specific gene expression profiles in patients with donor-specific cytotoxic-hyporesponsiveness, reflected by cytotoxic T-lymphocyte precursor frequency (CTLpf). The effect of calcineurin inhibitor (CNI) withdrawal was studied on markers for cytotoxicity (perforin, granzyme B), apoptosis (Fas,FasL), Th1 and Th2 cytokines (IL-2, IL-10), Th1 and Th2 transcription factors (T-bet, GATA 3), Th17 transcription factor and cytokine (RORγt, IL-17), and for immune regulation/activation (CD25, FOXP3). Peripheral blood samples from renal allograft recipients (n=18) more than twoyr after transplantation with stable renal function were analyzed before and fourmonths after CNI withdrawal. Additionally, systolic and diastolic blood pressure, cholesterol, serum creatinine and proteinuria were evaluated, and no significant differences were measured before and after CNI withdrawal. However, CNIs' discontinuation influenced peripheral gene expression profiles. After CNI withdrawal, the mRNA expression of Granzyme B, Perforin, Fas, FasL, T-bet, GATA3 and CD25 were significantly lower than during CNI treatment. After CNI discontinuation, donor-specific CTLpf decreased, while FOXP3 expression discriminated between detectable and non-detectable donor-specific cytolysis reactivity; FOXP3 transcript values were highest in absence of donor-specific cytotoxicity (p<0.01). Our study shows CNI withdrawal in stable kidney transplant recipients twoyr after transplantation is safe. Moreover, discontinuation of CNIs' treatment allows FOXP3+ regulatory T-cells development, resulting in a significant decrease of anti-donor immune reactivity.

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.

ITregs by vitamins: Commentary on 'retinoic acid attenuates acute heart rejection by increasing regulatory t cell and repressing differentiation of th17 in the presence of TGF-β' (Article)

2010-10-01T00:00:00Z

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.

Advancement of mesenchymal stem cell therapy in solid organ transplantation (MISOT) (Article)

2010-07-27T00:00:00Z

There is evolving interest in the use of mesenchymal stem cells (MSC) in solid organ transplantation. Pre-clinical transplantation models show efficacy of MSC in prolonging graft survival and a number of clinical studies are planned or underway. At a recent meeting of the MISOT consortium (MSC In Solid Organ Transplantation) the advances of these studies were evaluated and mechanisms underlying the potential effects of MSC discussed. Continued discussion is required for definition of safety and eventually efficacy endpoints for MSC therapy in solid organ transplantation.

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.

Cell contact interaction between adipose-derived stromal cells and allo-activated T lymphocytes (Article)

2009-12-01T00:00:00Z

Mesenchymal stromal cells regulate immune cell function via the secretion of soluble factors. Cell membrane interactions between these cell types may play an additional role. Here, we demonstrate that subpopulations of allo-activated T cells are capable of binding to human adipose-derived stromal cells (ASC). The bound T-cell population contained CD8+T cells and was enriched for CD4-CD8-T cells, whereas the proportion of CD4+T cells was decreased compared with the non-bound T-cell population. Bound CD4+T cells had high proliferative activity and increased CD25 and FoxP3 expression. However, they also expressed CD127, excluding regulatory T-cell function. In CD8+T cells, IL-2 sensitivity, as determined by the analysis of phosphorylated STAT5, was lower in the presence of ASC and even lower in bound cells. In contrast, IL-2-induced phosphorylated STAT5 levels were higher in bound CD4+T cells than in non-bound CD4+T cells. Additionally, pro-proliferative TGF-β signalling via endoglin and SMAD1/5/8 phosphorylation was detected in bound CD4+T cells. Even after prolonged co-culture with ASC, the activated phenotype of bound CD4+T cells persisted. In conclusion, these results demonstrate that the binding of lymphocytes to ASC represents an immunomodulatory mechanism in which CD8+T cells are inhibited in their responsiveness to pro-inflammatory stimuli and reactive CD4+T cells are depleted from the immune response.

Inosine monophosphate dehydrogenase messenger RNA expression is correlated to clinical outcomes in mycophenolate mofetil-treated kidney transplant patients, whereas inosine monophosphate dehydrogenase activity is not (Article)

2009-10-01T00:00:00Z

Measurement of the pharmacodynamic biomarker inosine monophosphate dehydrogenase (IMPDH) activity in renal transplant recipients has been proposed to reflect the biological effect better than using pharmacokinetic parameters to monitor mycophenolate mofetil therapy. The IMPDH assays are however labor intensive and this complicates implementation into patient care. Quantification of IMPDH messenger RNA (mRNA) could form an attractive alternative. This study was designed to correlate IMPDH mRNA levels with IMPDH activity and clinical outcome in renal transplant recipients. From a cohort of 101 renal transplant patients, blood samples were drawn pre transplantation and at 4 times after transplantation. IMPDH activity, IMPDH type 1 and type 2 mRNA levels, and mycophenolic acid concentrations were measured and correlated to clinical outcomes. No correlation was found between IMPDH type 1 and type 2 mRNA levels and IMPDH activity in pre- and posttransplant samples. A significant increase in IMPDH mRNA levels was found between day 6 and day 140 after transplantation. IMPDH type 1 and type 2 mRNA levels before transplant showed a trend toward statistically significant higher levels in patients with an acute rejection (P = 0.052 and P = 0.058). After transplant, the IMPDH type 1 and type 2 mRNA levels were significantly lower in patients with an acute rejection (P = 0.026 and P = 0.007). We conclude that IMPDH mRNA levels do not correlate with IMPDH activity but are nevertheless correlated with acute rejections. Furthermore, although the regulation of the expression of the 2 isoforms is presumed to be different, in this study, the changes in the expression of type 1 mRNA closely paralleled those of type 2.

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.

Regulatory T cells in alloreactivity after clinical heart transplantation (Article)

2009-10-01T00:00:00Z

Purpose of review As the knowledge of CD4+CD25bright+FoxP3+regulatory T cells in experimental transplant models grows, we need to understand how and to what extent these suppressor cells regulate donor-directed immune events in the transplantation clinic. This review focuses on the function of regulatory T cells in the peripheral blood and the transplanted organ of patients after heart transplantation during immunological quiescence and rejection. Recent findings Here, we present data that peripheral CD4+CD25bright+FoxP3+T cells of heart transplant patients who experience acute rejection have inadequate immune regulatory function in vitro compared with those of nonrejecting patients. During rejection, potent donor-specific T-cell suppressors are present in the transplanted organ. Summary The studies in transplant patients' show that the function of CD4+CD25bright+FoxP3+regulatory T cells in alloimmunity is to inhibit the activation of effector T cells, to prevent rejection, and to control the antidonor response at the graft itself at later stages of immune reactivity.

Toward MSC in solid organ transplantation: 2008 position paper of the MISOT study group (Article)

2009-09-15T00:00:00Z

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance-agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008. Copyright

Monotherapy rapamycin allows an increase of CD4+ CD25 bright+ FoxP3+ T cells in renal recipients (Article)

2009-09-01T00:00:00Z

CD4+ CD25bright+ FoxP3+ regulatory T cells (Tregs) may control donor-specific allogeneic responses in kidney transplant recipients. Recent evidence demonstrated that three phenotypical Treg-subsets, naive (CCR7+CD45RO-), central-memory (CCR7 +CD45RO+) and effector-memory (CCR7-CD45RO +), are essential for the development and function of antigen-specific suppression in the lymphoid and peripheral tissues. Also, it has been appreciated that Tregs are affected by immunosuppressive agents. In clinical practice, however, the effect of a single drug remains to be determined. Therefore, we analyzed the effect of several immunosuppressive agents on the number, phenotype and function of peripheral Tregs from 46 stable kidney transplant recipients. These patients were converted to monotherapy with tacrolimus (n = 15), rapamycin (n = 17) or mycophenolate mofetil (n = 14). Blood was obtained at inclusion and 6 months thereafter. The number of Tregs increased significantly in patients on monotherapy with rapamycin (P < 0.001), which was caused by increased numbers of Tregs with a central-memory and an effector-memory phenotype (both P < 0.05). At 6 months after conversion, however, the suppressive function of Tregs did not significantly change in co-cultures stimulated with donor-Ag. Therefore, monotherapy with rapamycin allows the signals that are needed to increase the number of functional Tregs with a memory phenotype, thereby enhancing the potential capacity to regulate donor-specific responses in the lymphoid and the peripheral tissues.

Clinical rejection and persistent immune regulation in kidney transplant patients (Article)

2009-07-01T00:00:00Z

We evaluated whether the regulatory function of CD4+CD25high+FoxP3+T-cells from patients on tacrolimus and mycophenolate mofetil (MMF) is affected by preceding steroid and anti-CD25 mAb induction therapy and whether this function is associated with rejection after kidney transplantation. Kidney recipients (N = 15) were randomized to receive either anti-CD25 mAb induction (i.e., daclizumab) or steroids for 4 months. We analyzed the presence and suppressive activity of CD4+CD25high+FoxP3+peripheral T-cells in samples obtained at pre and 4-6 months after transplantation. Anti-CD25 mAb therapy and treatment with steroids did not significantly affect protein expression of FoxP3. However, at the functional level, significant differences were found in the regulatory activities of CD4+CD25high+T-cells from the anti-CD25 group vs those from the steroid group. At 4-6 months after transplantation, the regulatory activities of CD4+CD25high+T-cells were comparable to those before anti-CD25 mAb therapy; 49 ± 13% (mean ± SEM) vs 40 ± 14% at a 1:20 ratio (CD25high+:CD25-/dim), respectively. In contrast, the regulatory capacities of CD+D25bright+T-cells from the steroid patient group became significantly impaired. The percentage inhibition of the anti-donor response decreased from 57 ± 12% before transplantation to 12 ± 7% after transplantation (p < 0.01). Five out of 15 patients experienced a rejection episode. At 4-6 months after transplantation, the CD25high+cells from these rejectors (who all received daclizumab induction therapy) had clear regulatory function, while suppression by CD25high+cells from non-rejectors (N = 10) was significantly lower. The percentage inhibition of the anti-donor response was 48 ± 14% (mean ± SEM) vs 10 ± 7%, respectively, p = 0.02. Anti-CD25 mAb induction therapy does not negatively influence the regulatory function of CD4+CD25high+FoxP3+T-cells from kidney transplant recipients on tacrolimus and MMF. The majority of these patients experienced an acute rejection episode, which suggests that immune activation is required for persistent immunoregulatory function.

The authors' reply: Mesenchymal stem cells and immunosuppressive drug interactions (Article)

2009-06-27T00:00:00Z

End-stage renal failure and regulatory activities of CD4 +CD25bright+FoxP3+ T-cells (Article)

2009-06-01T00:00:00Z

Background. The defensive immune system in patients with end-stage renal failure is impaired at multiple levels. This state of immune incompetence is associated with continuous activation of the immune system. An additional explanation for this state of activation may be the disturbed function of CD4+CD25bright+FoxP3+regulatory T-cells.Methods. The phenotype and function of peripheral regulatory T-cells from patients with end-stage renal failure (N = 80) and healthy controls (N = 17) was studied by flow cytometry, RT-PCR and mixed lymphocyte reaction. Patients were on haemodialysis (N = 40), peritoneal dialysis (N = 26) or not treated with dialysis yet (N = 14). The latter group had a glomerular filtration rate of <20 mlmin 1.73 m2.Results. The basal IL-2 mRNA level was high in patient-PBMC (P = 0.0002 versus healthy controls). The absolute number of CD4+CD25bright+T-cells was low in patients (P < 0.05 versus healthy controls). Furthermore, proliferation of patient-PBMC upon allogeneic stimulation was impaired (P < 0.0001 versus healthy controls). The regulatory function of CD4+CD25bright+T-cells was determined in the setting of direct allorecognition. First, the effect of depletion of CD25bright+cells from patient-PBMC on proliferation was low. Second, co-culture of CD25bright+cells with CD25negdimcells (1:10 ratio) showed impaired regulatory function (P < 0.001 versus healthy controls), which was especially pronounced in patients on dialysis. The FOXP3 mRNA level was also low upon stimulation (P = 0.0002 versus healthy controls).Conclusions. In line with previous studies, we observed an overactivated but functionally compromised immune system in patients with end-stage renal failure. It now appears that in this setting, regulation by CD4+CD25bright+FoxP3+T-cells is also impaired.

Deficient TNF-α and IFN-γ production correlates with nondetectable donor-specific cytotoxicity after clinical kidney transplantation (Article)

2009-05-27T00:00:00Z

BACKGROUND.: We previously reported that no cytotoxic T-lymphocyte precursor frequencies (CTLpf) were found in 60% of patients on azathioprine or mycophenolate mofetil+Pred long after kidney transplantation. We questioned whether the absence of donor-specific CTLpf was associated with low levels of stimulatory Th1 (tumor necrosis factor [TNF]-α, interferon [IFN]-α) or high levels of regulatory Th2 (interleukin [IL]-4, IL-6, IL-10) cytokines. METHODS.: In this study, peripheral blood monocyte cells (PBMC) were stimulated with irradiated donor cells. After 7 days, cytokine production was determined by cytokine bead array, and CTLpf by limiting dilution assay. RESULTS.: Patients with detectable CTLpf (ĝ‰¥10/10 PBMC) had significantly higher levels of TNF-α (P=0.04) and IFN-α (P=0.02) than patients with nondetectable CTLpf (<10/10 PBMC). Donor-reactive IL-4, IL-6, and IL-10 production was comparable in both patient groups. Additionally, CTLpf was positively correlated with TNF-α (rs=0.54, P=0.0003) and IFN-α (rs=0.64, P<0.0001) production. CONCLUSION.: The absence of donor-specific CTLp after transplantation correlates with low levels of stimulatory cytokines, not with elevated levels of regulatory cytokines.

The effect of rabbit anti-thymocyte globulin induction therapy on regulatory T cells in kidney transplant patients (Article)

2009-05-01T00:00:00Z

Background. Prevention of alloreactivity by rabbit anti-thymocyte globulins (rATG) may not only result from immunodepletion but also from the induction of T cells that control allogeneic immune responses. In the present prospective and controlled study, we investigated the effect of rATG on the frequency, function and phenotype of peripheral immunoregulatory CD4+ T cells in kidney transplant (KTx) patients.Methods. After transplantation, 16 patients received ATG-induction therapy and triple therapy consisting of tacrolimus, MMF and steroids. The control group (n = 18) received triple therapy only. By flow cytometry, T cells were analysed for CD25, FoxP3, CD127, CD45RO and CCR7. To study their suppressive capacities, CD25bright T cells were co-cultured with CD25-dim effector T cells (Teff) in mixed lymphocyte reactions (MLR), stimulated with donor and third party (3P) antigens.Results. Pre-transplant levels of FoxP3+CD127-low T cells were 6 of CD4+ T cells. One week post-ATG treatment, no measurable numbers of regulatory T cells were present (P < 0.01). After 4 weeks, the cell numbers of CD4+FoxP3+CD127-low T cells slowly reappeared and thereafter remained low (P < 0.01). At 14 weeks, a significant shift towards the CD45RO+CCR7+ (central memory) phenotype within CD4+FoxP3+ T cells was observed (P < 0.01). At 26 weeks, the proliferative alloresponses of the PBMC and CD25-dim Teff profoundly decreased compared to pre-transplant (P = 0.01 and P = 0.02 respectively), while the regulatory capacity of the CD25bright T cells, of which 90 consisted of FoxP3+CD127-low T cells, remained unaffected. The CD25bright T cells suppressed the anti-donor (94) and 3P responses (93).Conclusion. Our findings show that rATG therapy does not spare peripheral immunoregulatory T cells in vivo, but after regeneration preserves their suppressive activity.

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.

Conversion from calcineurin inhibitor to mycophenolate mofetil-based immunosuppression changes the frequency and phenotype of CD4+FOXP3+ regulatory T cells (Article)

2009-04-15T00:00:00Z

BACKGROUND.: CD4Foxp3 regulatory T cells (Treg) depend on interleukin (IL)-2 for their function and survival. By interfering with the IL-2 production, calcineurin inhibitors (CNI) may negatively affect Treg. Here, we describe the effects of conversion from CNI to mycophenolate mofetil (MMF) monotherapy on renal function, and on Treg frequency and phenotype in liver transplant recipients. METHODS.: Patients (=16) with renal impairment on CNI were converted to MMF and received a single dose of IL-2-receptor blocking antibody (Daclizumab). Control patients (=8) continued CNI treatment. RESULTS.: Renal function rapidly and significantly improved after conversion. Daclizumab treatment resulted in a 75% blocking of CD25 at 1 month causing a significant reduction in the percentage of CD4CD25 cells but not affecting the percentage of CD4CD25Foxp3 cells. Six months after conversion to MMF, the percentage of CD4CD25Foxp3 cells increased significantly by 125%. FOXP3 mRNA analysis of mononuclear cells confirmed the enrichment of Foxp3 in peripheral blood. Interestingly, the CD25 expression level on CD4Foxp3, but not CD4Foxp3, cells significantly increased compared with preconversion. CONCLUSION.: Conversion to MMF increases the percentage and CD25 expression of CD4Foxp3 cells indicating that MMF therapy can overturn the repressive effect of CNI on circulating Treg levels and therefore may promote Treg-mediated suppression of alloreactivity.

Potential of mesenchymal stem cells as immune therapy in solid-organ transplantation (Article)

2009-04-01T00:00:00Z

Over the last decade, there has been a rising interest in the use of mesenchymal stem cells (MSCs) for clinical applications. This interest stems from the beneficial properties of MSCs, which include multi-lineage differentiation and immunosuppressive ability, suggesting there is a role for MSC therapy for tissue regeneration and in immunologic disease. Despite recent clinical trials investigating the use of MSCs in treating immune-mediated disease, their applicability in solid-organ transplantation is still unknown. In this review, we identified topics that are important when considering MSC therapy in clinical organ transplantation. Whereas, from other clinical studies, it would appear that administration of MSCs is safe, issues like dosing, timing, route of administration, and in particular the use of autologous or donor-derived MSCs may be of crucial importance for the functional outcome of MSCs treatment in organ transplantation. We discuss these topics and assess the feasibility of MSCs therapy in organ 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.

Generation of donor-specific regulatory t-cell function in kidney transplant patients (Article)

2009-02-15T00:00:00Z

Background.: In the search for mechanisms that can induce and maintain transplant tolerance, donor-specific CD4CD25FoxP3 regulatory T cells have been frequently mentioned. However, it remains to be demonstrated, whether these cells are generated after clinical transplantation. Methods.: We prospectively analyzed the phenotype and function of peripheral regulatory CD4CD25 T cells of 79 patients before, 3, 6, and 12 months after kidney transplantation. The immune regulatory capacities of CD4CD25 T cells were assessed by their depletion from peripheral blood mononuclear cells and in co-culture with CD25 responder T-cells in the mixed lymphocyte reactions. Results.: In the first year after transplantation, the number and proportion of CD4CD25 T cells significantly decreased (P<0.05 and P<0.001, respectively). In the mixed lymphocyte reactions, we observed donor-specific hyporesponsiveness in the presence of significantly increased proliferation to third and fourth Party-Ag, (P<0.001 and P<0.05, respectively). Furthermore, functional analysis of CD25 cells showed that the effect of depletion of these cells from peripheral blood mononuclear cells, and their suppressive capacities in co-culture with donor-Ag stimulated CD25 responder T-cells (1:10 ratio) significantly improved (P<0.01 and P<0.001, respectively). Moreover, the difference between the stimulation with donor-Ag and third Party-Ag became apparent at 6 months after transplantation. Conclusions.: These findings demonstrate that donor-specific CD4CD25 regulatory T-cell function is generated in fully immunosuppressed renal recipients in the first year after transplantation.

Donor-specific immune regulation by CD8+ lymphocytes expanded from rejecting human cardiac allografts (Article)

2009-02-01T00:00:00Z

To assess whether regulatory T cells are present in rejecting human cardiac allografts, we performed functional analyses of graft lymphocytes (GLs) expanded from endomyocardial biopsies (EMB; n = 5) with histological signs of acute cellular rejection. The GL cultures were tested for their proliferative capacity and regulatory activity on allogeneic-stimulated peripheral blood mononuclear cells (PBMC) of the patient (ratio PBMC:GLs = 5:1). Three of these GL cultures were hyporesponsive to donor antigens and suppressed the antidonor proliferative T-cell response of PBMC, but not the anti-third-party response. Interestingly, it was the CD8+GL subset of these cultures that inhibited the antidonor response (65-91% inhibition of the proportion of proliferating cells); the CD4+GLs of the expanded GL cultures were not suppressive. In conclusion, CD8+GLs expanded from rejecting human cardiac allografts can exhibit donor-specific immune regulatory activities in vitro. We suggest that during acute cellular rejection, GLs may not only consist of graft-destructing effector T cells, but also of cells of the CD8+type with the potential to specifically inhibit antidonor immune reactivity.

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.

Functional analysis of CD4+ CD25 bright T cells in kidney transplant patients: Improving suppression of donor-directed responses after transplantation (Article)

2008-10-01T00:00:00Z

Background: The role of CD4+ CD25bright regulatory T cells (Treg) in controlling alloreactivity is established, but little is known whether antigen-specific Treg are induced in fully immunosuppressed kidney transplant patients. Methods: The frequency and function of CD25bright T cells of nine stable kidney transplant patients before and 0.5-2yr after transplantation were measured. Patients received triple therapy consisting of cyclosporine, mycophenolate mofetil and prednisone. To investigate the influence of transplantation and immunosuppression on Treg function, we compared their suppressive capacities pre- and post-transplantation using mixed lymphocyte reactions and kept the CD25-/dim effector T-cell (Teff) population constant. Results: After transplantation, the percentage of CD4+ CD25bright T cells significantly decreased from 8.5% pre-transplant to 6.9% post-transplant (median, p=0.05). However, the lower percentage of post-transplant CD4+ CD25bright T cells was not associated with reduced, but rather improved suppressor function of these cells. The proliferative response of pre-transplant Teff to donor-antigens was more profoundly suppressed by post-transplant Treg than by pre-transplant Treg (pre-transplant 18% vs. post-transplant 55% median, p=0.03) and was comparable against third party antigens at a CD25bright:CD25-/dim ratio of 1:20. Conclusions: In immunosuppressed kidney transplant patients, the donor-directed suppressive capacity of CD4+ CD25bright regulatory T cells improved, which may contribute to the development of donor-specific hyporesponsiveness against the graft.

Regulatory T cells after organ transplantation: Where does their action take place? (Article)

2008-07-01T00:00:00Z

Regulatory T cells are considered to be pivotal for the induction of tolerance to donor antigens. In the past decades, several regulatory T-cell subsets have been identified, such as CD4+CD25+ regulatory T cells and the CD8+CD28- suppressor T cells. Although many studies have investigated the role of these regulators in transplant tolerance, relatively little attention has focused on the exact place where these cells suppress immune responses directed to donor antigens. The localization of regulatory T cells may influence their effect on allogeneic immune responses. More insight into the localization and migration of regulatory T cells in transplant recipients is therefore important, especially when these cells are to be used for monitoring purposes and for cellular immune therapy. In the present review we summarize current knowledge about the presence of functional donor-directed regulatory T cells in the secondary lymphoid organs, peripheral blood, and the transplanted organ itself. In addition, we discuss the importance of the appropriate localization for the control of anti-donor immune reactivity.

The Control of Anti-Donor Immune Responses by Regulatory T Cells in Organ Transplant Patients (Article)

2008-06-01T00:00:00Z

The role of regulatory T cells in the induction and maintenance of transplant tolerance has been widely investigated in experimental animal models. Their involvement in the regulation of allogeneic immune reactivity in immunosuppressed organ transplant patients, however, remains unclear. Measurements of regulatory T cells after clinical organ transplantation may contribute to understanding their role in anti-donor immune responses. Several studies have investigated the frequency and/or immune regulatory function of peripheral regulatory T-cell populations, such as, the CD4+CD25+regulatory T cells or the CD8+CD28-suppressor T cells, in clinically stable organ transplant patients and patients with acute or chronic rejection. This review summarizes these studies and discusses the correlations found between the number and function of regulatory T cells and the immunological state of the patient. This knowledge is warranted for a better understanding of the control of allogeneic immune responses by regulatory T cells. Subsequently, monitoring regulatory T cells may help us to identify patients in whom the anti-donor reactivity is actively suppressed.

Ficoll-separated mononuclear cells from sepsis patients are contaminated with granulocytes (Article)

2008-05-01T00:00:00Z

Objective: To determine the cell content and purity of Ficoll-separated peripheral blood mononuclear cells and granulocyte isolates in sepsis patients compared to healthy controls. Design and setting: Prospective study in the adult and pediatric intensive care departments of the Erasmus University Medical Center in the Netherlands. Patients: Three sepsis patients (two adults, one child) and four healthy controls. Measurements and results: Blood leukocytes were separated by Ficoll into an interface and a bottom fraction. The cell content and purity was analyzed by cytospin and flow-cytometric immunofluorescence. In sepsis patients, the interface consisted of 11-52% mononuclear cells only, due to high contamination with granulocytes (48-89%). This was in contrast to a high proportion of mononuclear cells (88-100%) in healthy controls. The bottom fraction showed a cell purity of ≥92% polymorphonuclear granulocytes in sepsis patients as well as in healthy controls. Conclusions: Ficoll-separated leukocytes of sepsis patients are not suitable for studying mononuclear cells but can be used for studying granulocytes with high purity. The mononuclear cell fraction is highly contaminated with granulocytes. Additional separation techniques are necessary to obtain a pure cell fraction.

After discontinuation of calcineurin inhibitors, tapering of mycophenolate mofetil further impairs donor-directed cytotoxicity (Article)

2008-03-01T00:00:00Z

Background: Recently, we described a significant decrease in donor-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) after discontinuation of calcineurin inhibitors (CNI), while the proliferative capacity in mixed lymphocyte culture (MLC), and the number of interferon-γ (IFN-γ) producing cells (pc) in Elispot remained unchanged. Methods: We tested T-cell reactivity in CNI free patients with stable renal graft function, on mycophenolate mofetil (MMF) or azathioprine (AZA) plus prednisone, who were tapered to 50% of their MMF or AZA dose. Results: Furthermore, tapering of the MMF or AZA dose resulted in a decrease of donor-reactive CTLpf in all patients with detectable CTLpf. Detectable numbers decreased from a median of 32 to 8 CTLp/106 peripheral blood mononuclear cell (PBMC). No effect on third-party reactive CTLpf was found, while the T-cell reactivity to donor and third-party cells as tested in MLC and in IFN-γ Elispot was not affected either by tapering of immunosuppression. Third-party reactivity was significantly higher than donor-specific reactivity in all tests. A control group showed no changes in any of the in vitro assays. Conclusion: Both withdrawal of CNI and tapering of MMF or AZA dose decreases the donor-specific CTLpf. Our data suggest that reduction of immunosuppression results in a specific decrease of donor-directed cytotoxic capacity of immunocompetent cells, while their proliferation and cytokine production capacity remained unchanged. Immunosuppression hinders development of cytotoxic non-responsiveness.

Impact of immunosuppressive drugs on CD4+CD25+FOXP3+ regulatory T cells: Does in vitro evidence translate to the clinical setting? (Article)

2008-03-01T00:00:00Z

Success of solid-organ transplantation requires the continuous administration of immunosuppressive drugs to prevent graft rejection. The currently prescribed immunosuppressive medication targets the immune system in a nonspecific fashion, leading to debilitating side effects that diminish patient survival and quality of life. Therefore, it is important to minimize immunosuppression, but this requires the development of alternative therapeutic strategies to induce and maintain transplant tolerance. One such strategy would be to allow and facilitate the induction of alloantigen-specific immune regulation by regulatory T cells (Treg). Recent experimental studies indicate that several commonly used immunosuppressive drugs have detrimental effects on the induction and function of Treg, whereas other drugs appear to spare these cells or may even be beneficial. These differential effects may be explained by differences in signaling pathways between Treg and effector T cells. In this review, we provide a comprehensive overview of the current literature on the effects of immunosuppressive drugs on CD4CD25FOXP3 Treg and discuss whether these in vitro data are substantiated by in vivo evidence from the clinic. A greater understanding of the impact of immunosuppression on Treg may help to create future opportunities to manipulate the host allo-immune response and achieve operational tolerance in transplantation.

Hepatitis B vaccine-specific CD4+ T cells can be detected and characterised at the single cell level: Limited usefulness of dendritic cells as signal enhancers (Article)

2008-01-31T00:00:00Z

Hepatitis B surface antigen (HBsAg)-specific T cells were analysed in 16 healthy individuals vaccinated against hepatitis B, using optimised protocols. HBsAg-specific IFN-γ producing CD4+T cells were found at a similar low frequency (0.01%) within the naive T cell subset, the central and the effector memory T cell subset. Overall, HBsAg-specific total CD4+T cells were detected in approximately 81% of the HBV vaccinees. The use of dendritic cells substantially increased the otherwise low proliferative responses but not the percentage of IFN-γ producing cells. The analysis of readily detectable CMV-specific CD4+T cell responses was used as a positive control and confirmed the adequacy of our assays. T cell responses associated with (in-) adequate hepatitis B vaccination can now be analysed in more detail.

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.

Kinetic analysis reveals potency of CD4+ CD25bright+ regulatory T-cells in kidney transplant patients (Article)

2007-11-01T00:00:00Z

Donor-specific hyporesponsiveness as occurs after allogeneic kidney transplantation may be mediated by repression of effector cells by a specific subset of T-cells: the CD4+CD25bright+FoxP3+regulatory T-cells (Tregs). Here, we examined the suppressive capacity of Tregs isolated from the leukafereses product of 6 kidney transplant recipients, by reconstituting Tregs to responder T-cells at several time-points after initiation of proliferation. We show that Tregs derived from kidney transplant patients potently restrain proliferation to donor-antigens and 3rd party-antigens in classic reconstitution assays (i.e. addition of Tregs at the start of the co-incubation). However, when Tregs were added 5 days after initiation of proliferation, they were still capable of suppressing proliferation to donor-antigens (by 38%) but no longer to 3rd party-antigens. Thus, we conclude that the potency of Tregs to suppress reactivity to specific antigens should be determined by reconstitution to ongoing reactions.

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.

Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities (Article)

2007-08-01T00:00:00Z

Mesenchymal stem cells (MSCs) have important tissue repair functions and show potent immunosuppressive capacities in vitro. Although usually isolated from the bone marrow, MSCs have been identified in other tissues, including the skin and liver. In the present study, we isolated and characterized MSCs from human heart, spleen, and perirenal adipose tissue. MSCs from these different tissue sites were similar to those derived from bone marrow in that they expressed comparable levels of the cell-surface markers CD90, CD105, CD166, and HLA class I, were negative for CD34, CD45, HLA class II, CD80, and CD86 expression, and were capable of osteogenic and adipogenic differentiation. Like bone marrow-derived MSCs, MSCs from these different tissue sources inhibited the proliferation of alloactivated peripheral blood mononuclear cells (PBMCs), giving 85%, 79%, 79%, and 81% inhibition, respectively. Also in line with bone marrow-derived MSCs they inhibited proliferative responses of PBMCs to phytohemagglutinin, a nonspecific stimulator of lymphocyte proliferation, and reduced-memory T lymphocyte responses to tetanus toxoid. The results of this study demonstrate that MSCs from various tissues have similar immunophenotypes, in vitro immunosuppressive properties, and differentiation potential.

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.

Intrahepatic detection of FOXP3 gene expression after liver transplantation using minimally invasive aspiration biopsy (Article)

2007-03-01T00:00:00Z

Intragraft accumulation of Forkhead box P3 (FOXP3)-positive regulatory T cells (Treg) is associated with local suppression of alloresponses in transplantation models. In the current study, the utility of the minimally invasive fine needle aspiration biopsy for the intragraft detection of FOXP3 and interferon (IFN)-γ mRNA expression was investigated in clinical liver transplantation (LTx). Intragraft FOXP3 increased within the first year after LTx, but not in blood. Elevated FOXP3, but not IFN-γ expression, in the liver was observed after hepatitis C virus (HCV) reinfection and after a previous episode of acute rejection. These data show the feasibility of aspiration biopsy for intragraft monitoring of gene expression. Intrahepatic FOXP3 levels are associated with HCV reinfection, a history of acute rejection, and increased within the first year after LTx. Differences in gene expression between the graft and blood underline the importance of local immune monitoring.

Functional CD25bright+ alloresponsive T cells in fully immunosuppressed renal allograft recipients (Article)

2007-01-01T00:00:00Z

Background: Evidence from animal studies indicate a crucial role for CD25bright+regulatory T cells in transplantation tolerance. Methods: To assess whether peripheral CD25bright+T cells control immune responses in immunosuppressed kidney transplant patients, we analyzed the suppressive capacities of these cells using mixed lymphocytes reactions. Results: Allogeneic stimulation of patients peripheral blood mononuclear cells was associated with IL-2 production and T-cell proliferation. Depletion of CD25bright+T cells resulted in a 35% (median) higher IL-2 production and a 38% higher proliferative response against third party cells, showing that functional regulatory CD25bright+T cells were present (p = 0.03 and 0.02 respectively). In eight out of 11 patients, we also demonstrated regulation activity against donor-activated T cells (p = 0.03). These data were confirmed in coculture experiments with isolated CD25-/dimT cells plus CD25bright+T cells. At a 1:2 ratio, the CD25bright+T cells suppressed the proliferation of CD25-/dimdonor- and third party-stimulated responder T cells. Conclusions: CD25bright+T cells with immune regulatory activities against anti-donor-responsive T cells are readily detectable in renal allograft recipients during treatment with full dosage immunosuppression. Whether CD25bright+T cells indeed play a role in graft acceptance after organ transplantation in patients remains to be elucidated.

Tapering immunosuppression in recipients of living donor kidney transplants. (Article)

2004-07-01T00:00:00Z

We have previously suggested that the in vitro donor-specific cytotoxic T-lymphocyte precursor (CTLp) assay can guide us to identify patients in which the immunosuppressive load can be tapered. In a clinical trial we had observed that a low (<10/10(6) PBMC) frequency of these CTLp was predictive for an uneventful rejection-free clinical course in patients that were converted from calcineurin inhibitors to mycophenolate mofetil or azathiopine. In the present prospective study in 81 stable kidney transplant recipients, already converted from calcineurin inhibitors, we measured CTLp frequencies and reduced the immunosuppressive load on a routine basis when CTLp were <10/10(6) PBMC. Donor-specific cytotoxicity could not be measured in 50/81 patients, while their reactivity against third-party lymphocytes was not impaired. These 50 patients were tapered in their immunosuppression. Only in one patient, who had stopped all his medication, was a rejection episode diagnosed. We conclude that in patients with a low donor-specific CTLp frequency it is safe to reduce the immunosuppression.

Differential expression of heme oxygenase-1 and vascular endothelial growth factor in cadaveric and living donor kidneys after ischemia-reperfusion (Article)

2003-01-01T00:00:00Z

The extent of graft damage after ischemia-reperfusion reflects the balance between deleterious events and protective factors. Heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) may contribute to cytoprotection by their anti-inflammatory and antiapoptotic properties. For investigating whether HO-1 and VEGF play a role in the adaptive response to ischemia-reperfusion injury after renal transplantation, kidney biopsies were analyzed from living (n = 45) and cadaveric (n = 16) donors, obtained at three time points: at the end of cold storage T(-1), after warm ischemia T(0), and after reperfusion T(+1). The mRNA expression levels of HO-1, VEGF(165), Bcl-2, Bax, and hypoxia inducible factor-1alpha were quantified by real-time reverse transcriptase-PCR, and the HO-1 and VEGF proteins were analyzed by immunohistochemistry. Cadaveric donor kidneys presented higher mRNA expression levels of hypoxia inducible factor-1alpha. In contrast, mRNA expression levels of HO-1, VEGF(165), and Bcl-2 were significantly lower in kidneys from cadaveric donors. Overall, a significant correlation was observed between mRNA expression of Bcl-2 and VEGF(165), between Bcl-2 and HO-1, and between HO-1 and VEGF(165). Moreover, protein expression of HO-1 and VEGF was detected in the same anatomical kidney compartments (glomerulus, arteries, and distal tubules). Renal function at the first week posttransplantation (analyzed by serum creatinine levels) showed a significant correlation with both HO-1 and VEGF mRNA expression, reinforcing the protective role of both genes in the early events of transplantation. It is concluded that the lower expression of HO-1, VEGF(165), and Bcl-2 in cadaveric donor kidneys can reflect a defective adaptation against ischemia-reperfusion injury that may affect their function in the short term.

Intragraft interleukin 2 mRNA expression during acute cellular rejection and left ventricular total wall thickness after heart transplantation (Article)

2002-01-01T00:00:00Z

OBJECTIVE: To assess whether diastolic graft function is influenced by intragraft interleukin 2 (IL-2) messenger RNA (mRNA) expression in rejecting cardiac allografts. DESIGN: 16 recipients of cardiac allografts were monitored during the first three months after transplantation. The presence of IL-2 mRNA in endomyocardial biopsies (n = 123) was measured by reverse transcriptase polymerase chain reaction. To determine heart function, concurrent M mode and two dimensional Doppler echocardiograms were analysed. RESULTS: Histological signs of acute rejection (International Society for Heart and Lung Transplantation (ISHLT) rejection grade > 2) were strongly associated with IL-2 mRNA expression (IL-2 mRNA was present in 12 of 20 endomyocardial biopsies (60%) with acute rejection and in 24 of 103 endomyocardial biopsies (23%) without acute rejection, p = 0.002). No significant relation was found between either histology or IL-2 mRNA expression alone and the studied echocardiographic parameters. However, stratification of the echocardiographic data into those of patients with and those without acute rejection showed that during acute rejection IL-2 mRNA expression was significantly associated with increased left ventricular total wall thickness (mean change in total wall thickness was +0.22 cm in patients with IL-2 mRNA expression versus -0.18 cm in patients without IL-2 mRNA expression, p = 0.048). CONCLUSIONS: An increase in left ventricular total wall thickness precedes IL-2 positive acute rejection after heart transplantation. Thus, cardiac allograft rejection accompanied by intragraft IL-2 mRNA expression may be indicative of more severe rejection episodes.

Quantitative flow cytometry shows activation of the TNF-alpha system but not of the IL-2 system at the single cell level in renal replacement therapy (Article)

2001-01-01T00:00:00Z

BACKGROUND: Immunological dysfunction in patients on haemodialysis may be related to imbalanced cytokine systems, such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-2. Despite activation of these systems, haemodialysis patients show high susceptibility for infections and malignancies, and have a poor immunological reaction to T-cell-dependent antigens, like hepatitis B vaccination. In this study we have determined the activation status of the two different cytokine systems, at the single cell level, using quantitative flow cytometry. METHODS: Using fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies directed against TNF-R2 (CD120b), IL-2Ralpha (CD25) and IL-2Rbeta (CD122), we measured the expression of these receptors at the single cell level in order to determine the level of activation of monocytes and T-lymphocytes. RESULTS: Significantly higher expression of the TNF-alpha receptor, TNF-R2, was present on both monocytes and T-lymphocytes in patients on renal replacement therapy (RRT) compared with pre-dialysis chronic renal failure (CRF) patients and controls, indicating activation of the TNF-alpha system. In contrast, IL-2R expression was comparable in all groups studied, which may reflect a non-activated state of the IL-2 system. CONCLUSIONS: The present study illustrates an activated state of the TNF-alpha system in patients on RRT, at the single cell level, while the IL-2 system seems to be unaffected. These findings support the hypothesis that the interaction between the TNF-alpha and IL-2 cytokine systems is disturbed.

The TNF-alpha system in heart failure and after heart transplantation: plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity (Article)

1999-01-01T00:00:00Z

BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To assess the activity of the TNF-alpha system in patients with heart failure and after heart transplantation. METHODS: We measured TNF-alpha mRNA expression of peripheral blood mononuclear cells, plasma levels of TNF-alpha and sTNF reverse transcriptase receptors, using polymerase chain reaction and ELISA and performed a TNF-alpha binding capacity analysis, quantitating the buffer capacity of patients' plasma. RESULTS: In 11 patients with heart failure and in 15 cardiac allograft recipients, the TNF-alpha mRNA expression was comparable to controls. This level of mRNA was not accompanied by detectable TNF-alpha plasma levels. Significantly higher sTNF receptors levels were found in patients: ( P <0.001; ANOVA). The TNF-alpha binding capacity of patients' plasma was significantly increased, which led to decreased TNF-alpha recovery ( P<0.05). Both sTNF receptors showed a linear correlation with serum creatinine (sTNF-RI: r=0.92; sTNF-R2: r=0.82, P<0.001). CONCLUSIONS:The TNF-alpha mRNA expression and plasma levels show that the 'peripheral' TNF-alpha system is not activated. The high sTNF-receptors levels and their elevated TNF-alpha binding capacity, resulting in decreased TNF-alpha bioavailability, may contribute to an immunosuppressed state in these patients.

Intragraft Cytokine mRNA Expression After Clinical Organ Transplantation (Doctoral Thesis)

1998-05-20T00:00:00Z

As the knowledge of the cytokine network in experimental transplant models grows, we need to understand how and to what extent cytokines mediate the various donordirected immune events in clinical situations. This overview on clinical cytokine measurements shows that specific intragraft cytokine messengerRNA (mRNA) expression profiles can be associated with acute rejection, may reflect the efficacy of immunosuppression, and can trace patients at risk for the development of early chronic rejection. Moreover, the literature also showed that acute rejection and immunological quiescence in man are not restricted to the cytokine patterns according to the type 1/type 2 paradigm. This apparent lack of association may be caused by the immunosuppression used in the clinic but may also be the result of the infinite diversity of donor and recipient factors of which polymorphisms in cytokines and cytokine receptor genes may playa central role.