NK cell activity in response to K562 target cells as well as intracellular NFAT2 expression is retained in KTx patients compared to healthy individuals.

<p>(A) PBMC of healthy donors (n = 4, grey bar) or of KTx patients (n = 4, white bar) were incubated for 18h with K562 target cells and activation was quantified by IFN-γ ELISpot and multiplex analyses of supernatants for perforin and granzyme A/B. For statistical analyses, spots were normalized to 10.000 NK cells per well and mean values ± standard deviations are depicted, compared by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. (B) PBMC of healthy donors (n = 6) were pre-incubated with immunosuppressive drugs (5 μM) or DMSO solvent for 20 min and either left unstimulated (shaded bars) or P/I stimulated for additional 6h or 24h, respectively (grey bars, left and middle graph). KTx recipient-derived PBMC (n = 4) were stimulated identically, cells were stained intracellular for total NFAT2 and analyzed by flow cytometry. The right plot shows total NFAT2 in healthy donors compared to KTx patients after 24h stimulation. Mean values and standard deviations are displayed compared by two-sided One-way-ANOVA test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).</p

Cytokine response of PBMC derived from KTx patients is partially impaired compared to healthy individuals.

<p>PBMC of KTx patients (n = 4, white bars) were stimulated for 24h with P/I or left untreated as described, supernatants were collected, analyzed for cytokine production and compared to P/I stimulated PBMCs of healthy donors (n = 6, grey bars). Data are represented as mean values compared by two-sided One-way ANOVA test with Tukey’s post test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).</p

CNI but not mTORi suppress cytokine production of PBMC and isolated NK cells of healthy donors.

<p>PBMC of healthy donors (n = 6) were pre-incubated for 20 min with 5 μM inhibitor or DMSO solvent, stimulated with P/I for 24h, supernatants were collected and analyzed for cytokine secretion. Mean values ± standard deviation are shown. To determine statistical significance, Kruskal-Wallis test with Dunn’s post test comparing the different inhibitor treatments to DMSO control was performed. NK cells were negatively MACS-isolated from healthy donor PBMC and stimulated as described. To determine statistical significance, One-Way-ANOVA with Dunnett’s multiple comparison test was performed (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).</p

Surface expression of CD16, CD226 and CD161 is significantly reduced in KTx patients, while CD25, CD69 and HLA-DR surface expression is increased.

<p>Phenotypic characterization of peripheral NK cells from healthy individuals (n = 11, circles) and KTx patients (n = 29, triangles) was performed by flow cytometry. (A) CD16, CD226 (DNAM-1), CD161, HLA-DR, CD25 and CD69 expression was determined on CD56^dim NK cells, and compared between healthy donors (HD) and KTx patients (left plots). Displayed are mean values using unpaired Student’s t test (* = p≤0.05, ** = p≤0.01 and *** = p≤0.001, only significant values are shown). The impact of immunosuppression (right plots) was determined by grouping patients according to their immunosuppressive regimen: CsA, Tac or combination of Tac and Sir (T/S). Displayed are mean values, D'Agostino & Pearson omnibus normality test was performed to determine Gaussian distribution, subsequently either One-way-ANOVA or Kruskal-Wallis test were used to determine statistical significance. (B) Patients were grouped according to the histopathology of their biopsies (Banff classification): unsuspicious, borderline, T cell-mediated (TCMR) or antibody-mediated (AMR) rejection. (C) The impact of time after Tx was determined by grouping patients according to the time interval after Tx: ≤3, 6 or ≥ 9 months. Data are shown as scatter plots and display mean values. Asterisks indicate p-values * = p≤0.05, ** = p≤0.01 and *** = p≤0.001, only significant values are shown.</p

CD16 down-regulation is associated with IFN-γ induction following stimulation of NK cells from healthy individuals and KTx patients.

<p>PBMC of healthy donors (n = 6) or KTx-patients (n = 4) were pre-incubated with 5 μM inhibitor or equal concentrations of DMSO solvent, stimulated with P/I for 6 or 24 h, respectively and stained for surface CD3, CD56, CD16 and intracellular IFN-γ. (A) FACS dot plot analysis of gated CD3^-CD56⁺ NK cells of one representative healthy donor is shown. CD16^-IFN- γ⁺ subset used for statistical evaluation was labeled as 1. (B) Corresponding statistics of NK cells of 6 healthy individuals regarding IFN-γ-positive subsets in combination with CD16 after 6h stimulation are shown as mean values ± standard deviation compared by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test (* = p<0.05, ** = p<0.01, *** = p<0.001, only significant values are shown). (C) Corresponding statistics of NK cell subsets of healthy donors (n = 6) in comparison with KTx patients (n = 4) after 6h stimulation are displayed.</p

Immune monitoring after pediatric liver transplantation – the prospective ChilSFree cohort study

Abstract Background Although trough levels of immunosuppressive drugs are largely used to monitor immunosuppressive therapy after solid organ transplantation, there is still no established tool that allows for a validated assessment of functional degree of immunosuppression or the identification of clinically relevant over- or under-immunosuppression, depending on graft homeostasis. Reliable non-invasive markers to predict biopsy proven acute rejection (BPAR) do not exist. Literature data suggest that longitudinal measurements of immune markers might be predictive of BPAR, but data in children are scarce. We therefore propose an observational prospective cohort study focusing on immune monitoring in children after liver transplantation. We aim to describe immune function in a cohort of children before and during the first year after liver transplantation and plan to investigate how the immune function profile is associated with clinical and laboratory findings. Methods In an international multicenter prospective approach, children with end-stage liver disease who undergo liver transplantation are enrolled to the study and receive extensive immune monitoring before and at 1, 2, 3, 4 weeks and 3, 6, 12 months after transplantation, and whenever a clinically indicated liver biopsy is scheduled. Blood samples are analyzed for immune cell numbers and circulating levels of cytokines, chemokines and factors of angiogenesis reflecting immune cell activation. Statistical analysis will focus on the identification of trajectorial patterns of immune reactivity predictive for systemic non-inflammatory states, infectious complications or BPAR using joint modelling approaches. Discussion The ChilSFree study will help to understand the immune response after pLTx in different states of infection or rejection. It may provide insight into response mechanisms eventually facilitating immune tolerance towards the graft. Our analysis may yield an applicable immune panel for non-invasive early detection of acute cellular rejection, with the prospect of individually tailoring immunosuppressive therapy. The international collaborative set-up of this study allows for an appropriate sample size which is otherwise difficult to achieve in the field of pediatric liver transplantation

NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging.

To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56dim NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-γ, TNF-α, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-γ production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients