[Show abstract][Hide abstract] ABSTRACT: Graft survival in transplant recipients depends on pharmacokinetics and on individual susceptibility towards immunosuppressive drugs. Nevertheless, pharmacodynamic changes in T-cell functionality in response to drugs and in relation to pharmacokinetics are poorly characterized. We therefore investigated the immunosuppressive effect of calcineurin inhibitors and steroids on general T-cell functionality after polyclonal stimulation of whole blood samples.
General T-cell functionality in the absence or presence of immunosuppressive drugs was determined in vitro directly from whole blood based on cytokine induction after stimulation with the polyclonal stimulus Staphylococcus aureus enterotoxin B. In addition, diurnal changes in leukocyte and lymphocyte subsets, and on T-cell function after intake of immunosuppressive drugs were analyzed in 19 patients during one day and compared to respective kinetics in six immunocompetent controls. Statistical analysis was performed using non-parametric and parametric tests.
Susceptibility towards calcineurin inhibitors showed interindividual differences. When combined with steroids, tacrolimus led to more pronounced increase in the inhibitory activity as compared to cyclosporine A. While circadian alterations in leukocyte subpopulations and T-cell function in controls were related to endogenous cortisol levels, T-cell functionality in transplant recipients decreased after intake of the morning medication, which was more pronounced in patients with higher drug-dosages. Interestingly, calcineurin inhibitors differentially affected circadian rhythm of T-cell function, as patients on cyclosporine A showed a biphasic decrease in T-cell reactivity after drug-intake in the morning and evening, whereas T-cell reactivity in patients on tacrolimus remained rather stable.
The whole blood assay allows assessment of the inhibitory activity of immunosuppressive drugs in clinically relevant concentrations. Circadian alterations in T-cell function are determined by dose and type of immunosuppressive drugs and show distinct differences between cyclosporine A and tacrolimus. In future these findings may have practical implications to estimate the net immunosuppressive effect of a given drug-regimen that daily acts in an individual patient, and may contribute to individualize immunosuppression.
Full-text · Article · Dec 2015 · Journal of Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: The risk of rejection by cellular alloreactivity to the transplant donor is not routinely assessed. Here we analyzed alloreactive T cells in kidney transplant recipients and report how their detection may have helped to prevent rejection of a second kidney graft in a patient with a history of acute accelerated steroid-resistant nonhumoral rejection. Alloreactive CD4 and CD8 T cells were quantified using a flow-cytometric mixed lymphocyte reaction assay based on interferon-γ induction. A group of 16 nonrejecting transplant recipients did not show any alloreactive T-cell immunity to their respective donors, whereas alloreactivity to third-party controls was detectable. In the patient with rejection, HLA-specific antibodies were not detectable before and shortly after rejection, but after transplantation the patient showed exceptionally high frequencies of alloreactive T cells against 2 of 11 HLA-typed controls (0.604% and 0.791% alloreactive CD4 T cells and 0.792% and 0.978% alloreactive CD8 T cells) who shared HLA alleles (HLA-A*24, -B*44, -C*02, -DQB1*5) with the kidney donor. These HLA alleles were subsequently excluded for allocation of a second graft. No alloreactive T cells were observed toward the second kidney donor, and this transplantation was performed successfully. Thus, shared HLA alleles between the donor and third-party controls may suggest that alloreactive T cells had contributed to rejection of the first graft. The rejecting patient highlights that determination of cellular alloreactivity before transplantation may be applied to identify unacceptable mismatches and to reduce the risk for acute cellular rejection episodes.
No preview · Article · Oct 2015 · Transplantation Proceedings
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) play an important role in the induction of immune responses. Patients with end stage renal disease (ESRD) suffer from chronic inflammation, leading to a secondary, uremic immunodeficiency associated with alterations in monocyte subpopulations with increased proinflammatory capacities.
The aim of this study was to examine, under isolated conditions, whether alterations in monocyte subpopulations may affect in vitro maturation of dendritic cells (DC) in patients with ESRD, thus allowing us to draw conclusions for the situation in vivo.
Monocytes from 30 patients undergoing hemodialysis (HD) and 15 healthy volunteers were enriched from peripheral blood leukocytes, differentiated into immature DC (iDC) in medium containing IL-4 and GM-CSF, and were induced with LPS to differentiate into mature DC (mDC). Monocyte subpopulations and DC maturation stages were phenotypically characterized using flow-cytometry.
Although phenotypically indistinguishable, the number of both iDC and mDC that were generated from uremic monocytes was significantly higher compared to those from healthy controls (p = 0.02 and p = 0.03, respectively). This was associated with an increased number of CD14+ CD16+ monocytes (p = 0.02) and by a higher maturation efficiency of mDC in patients (p = 0.04).
A high percentage of CD14+ CD16+ monocytes in patients with ESRD is associated with an increased propensity to differentiate into DC. This indicates that chronic inflammation may substantiate the biased consistence of monocyte subpopulations leading to profound alteration in DC generation and maturation in ESRD.
No preview · Article · Mar 2015 · Advances in Clinical and Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: Background:
Varicella zoster virus (VZV) establishes lifelong persistence and may reactivate in individuals with impaired immune function. To investigate immunologic correlates of protection and VZV reactivation, we characterized specific immunity in 207 nonsymptomatic immunocompetent and 132 immunocompromised individuals in comparison with patients with acute herpes zoster.
VZV-specific CD4 T cells were quantified flow cytometrically after stimulation and characterized for expression of interferon-γ, interleukin 2, and tumor necrosis factor α and surface markers for differentiation (CD127) and anergy (cytotoxic T lymphocyte antigen 4 [CTLA-4] and programmed death [PD]-1). Immunoglobulin G and A levels were quantified using an enzyme-linked immunosorbent assay.
In healthy individuals, VZV-specific antibody and T-cell levels were age dependent, with the highest median VZV-specific CD4 T-cell frequencies of 0.108% (interquartile range, 0.121%) during adolescence. VZV-specific T-cell profiles were multifunctional with predominant expression of all 3 cytokines, CD127 positivity, and low expression of CTLA-4 and PD-1. Nonsymptomatic immunocompromised patients had similar VZV-specific immunologic properties except for lower T-cell frequencies (P<.001) and restricted cytokine expression. In contrast, significantly elevated antibody- and VZV-specific CD4 T-cell levels were found in patients with zoster. Their specific T cells showed a shift in cytokine expression toward interferon γ single positivity, an increase in CTLA-4 and PD-1, and a decrease in CD127 expression (all P<.001). This phenotype normalized after resolution of symptoms.
VZV-specific CD4-T cells in patients with zoster bear typical features of anergy. This phenotype is reversible and may serve as adjunct tool for monitoring VZV reactivations in high-risk patients.
No preview · Article · Sep 2014 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Background
After kidney transplantation, immunosuppressive therapy causes impaired cellular immune defense leading to an increased risk of viral complications. Trough level monitoring of immunosuppressants is insufficient to estimate the individual intensity of immunosuppression. We have already shown that virus-specific T cells (Tvis) correlate with control of virus replication as well as with the intensity of immunosuppression. The multicentre IVIST01-trial should prove that additional steering of immunosuppressive and antiviral therapy by Tvis levels leads to better graft function by avoidance of over-immunosuppression (for example, viral infections) and drug toxicity (for example, nephrotoxicity).
The IVIST-trial starts 4 weeks after transplantation. Sixty-four pediatric kidney recipients are randomized either to a non-intervention group that is only treated conservatively or to an intervention group with additional monitoring by Tvis. The randomization is stratified by centre and cytomegalovirus (CMV) prophylaxis. In both groups the immunosuppressive medication (cyclosporine A and everolimus) is adopted in the same target range of trough levels. In the non-intervention group the immunosuppressive therapy (cyclosporine A and everolimus) is only steered by classical trough level monitoring and the antiviral therapy of a CMV infection is performed according to a standard protocol. In contrast, in the intervention group the dose of immunosuppressants is individually adopted according to Tvis levels as a direct measure of the intensity of immunosuppression in addition to classical trough level monitoring. In case of CMV infection or reactivation the antiviral management is based on the individual CMV-specific immune defense assessed by the CMV-Tvis level. Primary endpoint of the study is the glomerular filtration rate 2 years after transplantation; secondary endpoints are the number and severity of viral infections and the incidence of side effects of immunosuppressive and antiviral drugs.
This IVIST01-trial will answer the question whether the new concept of steering immunosuppressive and antiviral therapy by Tvis levels leads to better future graft function. In terms of an effect-related drug monitoring, the study design aims to realize a personalization of immunosuppressive and antiviral management after transplantation. Based on the IVIST01-trial, immunomonitoring by Tvis might be incorporated into routine care after kidney transplantation.
EudraCT No: 2009-012436-32, ISRCTN89806912 (17 June 2009).
[Show abstract][Hide abstract] ABSTRACT: Cell-mediated immunity assays could be valuable for risk assessment of organ donors, but no data exist on their feasibility in deceased donors. In this study, 105 deceased donors (52.3 ± 16.9 years) were screened at the time of organ procurement. Pathogen-specific stimulation was performed using a cytomegalovirus (CMV) lysate, tuberculin (purified protein derivative [PPD]) and soluble Mycobacterium tuberculosis-specific ESAT-6/CFP-10 proteins in combination with an in-house fluorescence-activated cell sorting (FACS) assay or commercial assay formats (QuantiFERON-CMV/TB for ELISA, T-SPOT.TB for ELISPOT). CMV-IgG antibody titers were determined as gold standard for CMV infection; 51.4% of samples were CMV seropositive. Indeterminate results were observed in 47.6% of ELISA, 12.5% of FACS and 0% of ELISPOT assays. Agreement with serology was highest for FACS (95.6%, κ = 0.91), followed by ELISPOT (84.0%, κ = 0.68) and ELISA (80.0%, κ = 0.60). Agreement between ELISA and serology increased if the CMV lysate was used as stimulus (96.7%, κ = 0.92). Among the T cell assays, agreement between ELISPOT and FACS was highest (κ = 0.70). PPD-positive results among valid samples differed between assays (26.5% for ELISA, 23.1% for FACS and 50.5% for ELISPOT); 2.0% were QuantiFERON-TB positive, 3.3% were ESAT-6/CFP-10-positive in FACS and 13.4% were positive in the T-SPOT.TB assay. In conclusion, cellular immunity may be analyzed from samples of deceased donors, although the assays differ in the rate of positivity and indeterminate results.
Preview · Article · Jul 2014 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: BK polyomavirus (BKPyV) infection is widespread and typically asymptomatic during childhood, but may cause nephropathy in kidney transplant recipients. However, there is only limited knowledge on BKPyV-specific immunity in children and adults, and its role in BKPyV-replication and disease posttransplant. We therefore characterized BKPyV-specific immunity from 122 immunocompetent individuals (1–84 years), 38 adult kidney recipients with (n = 14) and without BKPyV-associated complications (n = 24), and 25 hemodialysis (HD) patients. Blood samples were stimulated with overlapping peptides of BKPyV large-T antigen and VP1 followed by flow-cytometric analysis of activated CD4 T cells expressing interferon-γ, IL-2 and tumor necrosis factor-α. Antibody-levels were determined using enzyme-linked immunosorbent assay. Both BKPyV-IgG levels and BKPyV-specific CD4 T cell frequencies were age-dependent (p = 0.0059) with maximum levels between 20 and 30 years (0.042%, interquartile range 0.05%). Transplant recipients showed a significantly higher BKPyV-specific T cell prevalence (57.9%) compared to age-matched controls (21.7%) or HD patients (28%, p = 0.017). Clinically relevant BKPyV-replication was associated with elevated frequencies of BKPyV-specific T cells (p = 0.0002), but decreased percentage of cells expressing multiple cytokines (p = 0.009). In conclusion, BKPyV-specific cellular immunity reflects phases of active BKPyV-replication either after primary infection in childhood or during reactivation after transplantation. Combined analysis of BKPyV-specific T cell functionality and viral loads may improve individual risk assessment.
Full-text · Article · Apr 2014 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: Wir sahen eine 41-jährige hämodialysepflichtige Patientin, bei der ein vorbekannter langjähriger Diabetes mellitus Typ 1 mit diabetischer Nephropathie bestand, mit dysurischen Beschwerden, linksseitigen Flankenschmerzen, Übelkeit und Erbrechen. Wegen gleicher Symptome war sie zuvor stationär mit Ciprofloxacin behandelt, nach Beschwerdebesserung auf eigenen Wunsch allerdings wieder entlassen worden. Dieser Fall unterstreicht, dass auch junge dialysepflichtige Patienten durch die urämische Immundysfunktion infektgefährdet sind.
[Show abstract][Hide abstract] ABSTRACT: Expression of the inhibitory receptor programmed death 1 (PD-1) on cytomegalovirus (CMV)-specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28(-) CD27(-) double negativity is known as a typical phenotype of CMV-specific CD4 T cells. Therefore, the co-expression of inhibitory receptors on CD28(-) CD27(-) CD4 T cells was assessed as a rapid, stimulation-independent parameter for monitoring CMV complications after transplantation. Ninety-three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross-sectional and longitudinal manner. CMV-specific CD4 T cell levels quantified after stimulation were compared to levels of CD28(-) CD27(-) CD4 T cells. PD-1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression on CD28(-) CD27(-) CD4 T cells were related to viremia. A percentage of ≥0.44% CD28(-) CD27(-) CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV-specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD-1 expression levels on CD28(-) CD27(-) CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA-4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD-1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD-1 expression on CD28(-) CD27(-) CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation-independent parameter to guide duration of antiviral therapy.
No preview · Article · Oct 2013 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: Specific T cell immunity in patients with active tuberculosis is associated with a decrease in multifunctionality. However, it is unknown whether cytokine profiles differ in patients with primary infection and those with prior contact. We therefore used intravesical immunotherapy with attenuated live Bacille Calmette-Guérin (BCG) in patients with urothelial carcinoma as a model to characterise the induction of systemic immunity towards purified protein derivate (PPD) and to study whether cytokine profiles differ depending on pre-existing immunity. Eighteen patients with non-muscle invasive bladder cancer were recruited during the BCG-induction course. Fifty-four healthy individuals served as controls. Interferon (IFN)-γ and interleukin (IL)-2 producing PPD-specific CD4 T cells were analysed longitudinally before each instillation using a rapid flow-cytometric whole blood immunoassay. Baseline levels of IFN-γ producing PPD-specific T cells were comparable to controls. T cells showed a 5-fold increase to 0.23% by week 2/3, and further increased 8-fold by week 4/5 (to 0.42%, p=0.0007). Systemic immunity was induced in all patients, although the increase was less pronounced in patients with pre-existing immunity. As in active TB, cytokine profiling during therapy revealed a lower percentage of multifunctional IFN-γ/IL-2 double-positive T cells compared to controls (60.2% vs. 71.9%, p=0.0003). Of note, when comparing patients with and without pre-existing immunity, cytokine profiles in patients with primary immunity were shifted towards IL-2 single producing T cells (p=0.02), whereas those in patients with pre-existing immunity were shifted towards IFN-γ single-positivity (p=0.01). In conclusion, systemic T cell responses were induced after BCG-therapy, and their kinetics and cytokine profile depended on pre-existing immunity. Decreased functionality is a typical feature of specific immunity in both patients with active tuberculosis and BCG-therapy. Among patients with active infection, a shift towards IL-2 or IFN-γ single-positive cells may allow distinction between patients with primary infection and cases with boosted immunity after prior contact, respectively.
[Show abstract][Hide abstract] ABSTRACT: To investigate correlates for the well-known impaired response of haemodialysis-patients to a variety of recommended vaccinations, the induction of antigen-specific cellular and humoral immunity was characterised after influenza-vaccination in two following seasons where the identical vaccine-composition was used. Influenza-specific T-cells were flow-cytometrically characterised from whole blood of 24 healthy controls and 26 haemodialysis-patients by proliferation-assays, induction of IFN-γ and TNF-α, and maturation markers. Antibody-titres were quantified using ELISA and hemagglutination-inhibition test. Influenza-specific CD4 T-cells were recently activated CD45RO+/CD27+ Th1-cells. Specific T-cell frequencies significantly increased 1-2 weeks after the first vaccination in both controls (mean increase by 0.50±0.64%, max: 3.01%) and haemodialysis-patients (by 0.55±0.71%, max: 3.44%). Thereafter, T-cell levels continuously decreased to pre-vaccination levels within approximately 7 weeks, whereas antibody-titres were more stable over time. By 6 months, haemodialysis-patients had significantly lower precursor-frequencies of proliferating influenza-specific memory T-cells (p=0.006). In the following season, memory-maintainance in immunocompetent individuals led to a significantly less pronounced increase in cellular immunity after re-vaccination (by only 0.12±0.09%, p=0.003), whereas the vaccine induced a strong increase in a second group of vaccination-naïve controls. Of note, haemodialysis-patients responded like vaccination-naïve individuals, as they showed a strong increase in cellular immunity after re-vaccination that was as pronounced as in the year before. In conclusion, the less pronounced T-cell increase after re-vaccination in controls may indicate maintainance of sufficient immunological memory. In contrast, the more rapid loss of proliferating cells in haemodialysis-patients may represent a sign of relative immunodeficiency and contribute to an increased incidence of recurrent infectious complications.
[Show abstract][Hide abstract] ABSTRACT: Serological identification of the cytomegalovirus (CMV) status in children <18 months of age is complicated by the variable persistence of maternal antibodies. As T cells are not passively transferred, we attempted to assess whether CMV-specific cellular immunity may be superior to determine the actual CMV-status; we also performed a functional characterisation of T-cell immunity in childhood. Antibodies from 59 mothers and 168 children were determined, and specific CD4(+) T cells were identified by induction of IFN-γ, IL-2, TNF-α, IL-4 and IL-17 after CMV-specific and polyclonal stimulation. Agreement between both tests was perfect for mothers and children >18 months. Among infants <18 months, 17/30 were concordantly negative. Interestingly, 8/13 seropositive children had detectable CMV-specific T cells, whereas only 5/13 were T-cell negative, indicating passive immunity. CMV-specific T cells from young infants differed in cytokine profiles from that of older age groups, and polyclonal effector T-cell frequencies were higher in young infants with detectable CMV-specific T cells compared with those without. In conclusion, the majority of young infants with CMV-specific antibodies show evidence of true infection, which indicates that passive immunity is overestimated. Our data may have important implications for improved risk stratification and CMV management in infants in the setting of transplantation.
No preview · Article · Apr 2013 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDLCKD), a population with high cardiovascular risk, we have demonstrated that HDLCKD in contrast to HDLHealthy promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDLCKD that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDLCKD reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension.
[Show abstract][Hide abstract] ABSTRACT: Background:
Programmed death receptor-1 (PD-1) compromises cytomegalovirus (CMV)-specific T-cell responses and has been linked to CMV viremia after transplantation. An impaired functional and proliferative capacity of PD-1-positive CMV-specific T cells may be reversed by the antibody-mediated blockade of PD-1 signaling. However, knowledge is limited on changes in "cytokinome" expression profiles associated with reversal of functional exhaustion.
The "cytokinome" was analyzed by 27-plex Luminex technology comparing renal transplant recipients with low (n = 5) and high (n = 5) PD-1 expression on CMV-specific T cells. The effect of blocking PD-1 by PD-ligand (PD-L) antibodies on restoration of cytokine expression was examined.
CMV-specific cytokine release and proliferation was lower in patients with high PD-1 expression on CMV-specific T cells. Antibody-mediated blockade of PD-L in CMV-stimulated samples restored expression levels of interleukin (IL)-1β, IL-2, IL-6, IL-9, IL-10, granulocyte colony-stimulating factor, interferon-γ, macrophage inflammatory protein-1α, and tumor necrosis factor-α. By contrast, no profound effect was observed for controls or patients with low PD-1 expression, or in staphylococcal enterotoxin B-stimulated cells.
Taken together, this pilot study provides evidence that a high PD-1 expression on CMV-specific T cells actively impairs proliferation and "cytokinome" responses in an antigen-specific manner. Importantly, blockade of PD-L restores CMV-specific T-cell proliferation and expression of a panel of different proinflammatory and/or type 1 cytokines, suggesting a common but as yet unknown regulatory principle. We conclude that PD-1 exhaustion is reversible and potentially amenable to therapeutic ex vivo and possibly in vivo manipulation. However, detailed knowledge of the differential effects on the "cytokinome" will be necessary to increase the safety and the efficacy of such manipulations.
No preview · Article · Nov 2012 · Transplant Infectious Disease