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The aim of the present study was to investigate association of serum amino (AA) acids and metabolites of AAs with post-transplant outcome in liver transplant recipients. Eighty-nine patients with end-stage liver diseases and available pre- and early post-transplant serum were characterised as patients with (GI) and without one-year mortality (GII) and patients with and without early graft dysfunction (EAD). A panel of pre- and early post-transplant serum levels of AAs and early and metabolites of tryptophan were measured using tandem mass spectrometry. Patient groups had significantly higher pre-transplant serum levels of phenylalanine, tryptophan, and tryptophan metabolites than healthy controls (for all p<0.001). Pre-transplant serum levels of all these parameters were significantly higher in GI than in GII (for all p<0.001). GI had a higher MELD score and re-transplantation number than GII (p≤0.005 for both investigations). Serum bilirubin on day 5 and serum phenylalanine on day 10 post-transplant were associated parameters of mortality, whereas day 1post-transplant phenylalanine and kynurenine and female gender were associated parameters of EAD. Our results indicate that pre- and early post-transplant levels of phenylalanine, tryptophan and metabolites of tryptophan are increased in patients and are associated with EAD and one-year mortality in liver transplant recipients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Our previous studies in rats showed that incubation of monocytic dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders the cells immunosuppressive. Donor-derived MMC-DCs injected into the recipient prior to transplantation prolonged heart allograft survival. Although the generation of DCs is labour-intensive and time-consuming, peripheral blood mononuclear cells (PBMCs) can be easily harvested. In the present study, we analyse under which conditions DCs can be replaced by PBMCs and examine their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs completely abrogated their suppressive effect, indicating that monocytes are the active cell population. Suppression of rejection was donor-specific. The injected MICs migrated into peripheral lymphoid organs and led to an increased number of regulatory T-cells (Tregs) expressing cluster of differentiation (CD) markers CD4 and CD25 and forkhead box protein 3 (FoxP3). Tolerance could be transferred to syngeneic recipients with blood or spleen cells. Depletion of Tregs from tolerogenic cells abrogated their suppressive effect, arguing for mediation of immunosuppression by CD4⁺CD25⁺FoxP3⁺ Tregs. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs generated from donor monocytes were applied for the first time in humans in a patient suffering from therapy-resistant rejection of a haploidentical stem cell transplant. We describe, in the present paper, a simple method for in vitro generation of suppressor blood cells for potential use in clinical organ transplantation. Although the case report does not allow us to draw any conclusion about their therapeutic effectiveness, it shows that MICs can be easily generated and applied in humans.

Purpose: Human corneal endothelial cells (HCEC) are a potential target of immune attack after corneal transplantation. The aim of this in vitro study was to investigate the role of HCEC during the alloimmune response of T-cells by examining cytokine profiles, function of the immunosuppressive enzyme indoleamine 2,3-dioxigenase (IDO), major histocompatibility complex (MHC-I/-II), T-cell proliferation, and the induction of cell death., Methods: Real-time PCR and RP-HPLC were used to determine IDO expression and activity. Multiplex assay was performed for quantification of cytokine levels. T-cell proliferation was assessed by thymidine incorporation, and HCEC cell death was measured by flow cytometry., Results: Human corneal endothelial cells induce strong proliferation of allogeneic T-cells and an increase of proinflammatory cytokines such as interleukin-1α (IL-1α), IL-1β, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Tumor necrosis factor-alpha (and to a lesser extent IFN-γ) induces apoptosis. Moreover, IFN-γ strongly upregulates MHC-II molecules and IDO activity in HCEC as reflected by high kynurenine (Kyn) concentrations. Interestingly, the T-cell response was not affected by increased IDO activity, since blocking of IDO did not affect the proliferation rate. Indoleamine 2,3-dioxigenase-induced Kyn levels did not exceed concentrations of 175 ± 20 μM. Concentrations of ≥400 μM Kyn were required to suppress T-cell proliferation., Conclusions: Our data show that T-cell attack on HCEC leads to increased concentrations of proinflammatory cytokines. Inflammatory cytokines induce apoptosis and upregulate MHC-II molecules and IDO in HCEC. Although increased IDO activity does not influence the T-cell response, it constitutes an inflammatory marker of the alloimmune response toward HCEC.

Indoleamine 2,3-dioxygenase (IDO), an enzyme expressed in many cell types, catalyses degradation of tryptophan (Trp) to kynurenine (Kyn) and may exert immunosuppressive functions, mediated mainly by kynurenines. Therefore, increased Kyn concentrations would be expected to protect allografts from rejection. We conducted this study to examine whether Kyn has predictive value for kidney graft outcome. End-stage renal disease patients (n = 210) demonstrated an increased Kyn/Trp ratio compared with healthy controls (n = 30). Both Kyn and Trp levels were significantly higher in patients who subsequently developed acute rejection than in patients who did not (p < 0.001 and p < 0.001, respectively). Furthermore, pretransplantation Kyn and Trp plasma concentrations were significantly different in patients who went on to develop acute rejection (high values) or acute tubular necrosis (low values) (p = 0.007 and p = 0.021, respectively). After transplantation Kyn levels decreased. Approximately 3 days before biopsy-confirmed rejection, Kyn was significantly increased in patients with rejection compared with those without rejection (p < 0.001). Contrary to expectation, high Kyn plasma levels before transplantation were not predictive of low rejection risk. Although informative in overall terms, at the present stage, Kyn levels do not allow the concise risk differentiation of individual patients., (Copyright © 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Cells have been previously used in experimental models for tolerance induction in organ transplantation and autoimmune diseases. One problem with the therapeutic use of cells is standardization of their preparation. We discuss an immunosuppressive strategy relying on cells irreversibly transformed by a chemotherapeutic drug. Dendritic cells (DCs) of transplant donors pretreated with mitomycin C (MMC) strongly prolonged rat heart allograft survival when injected into recipients before transplantation. Likewise, MMC-DCs loaded with myelin basic protein suppressed autoreactive T cells of MS patients in vitro and prevented experimental autoimmune encephalitis in mice. Comprehensive gene microarray analysis identified genes that possibly make up the suppressive phenotype, comprising glucocorticoid leucine zipper, immunoglobulin-like transcript 3, CD80, CD83, CD86, and apoptotic genes. Based on these findings, a hypothetical model of tolerance induction by MMC-treated DCs is delineated. Finally, we describe the first clinical application of MMC-treated monocyte-enriched donor cells in an attempt to control the rejection of a haploidentical stem cell transplant in a sensitized recipient and discuss the pros and cons of using MMC-treated antigen-presenting cells for tolerance induction. Although many questions remain, MMC-treated cells are a promising clinical tool for controlling allograft rejection and deleterious immune responses in autoimmune diseases.

The most important antigen-presenting cells are dendritic cells (DCs), which play a central role in the initiation of immunity and tolerance. Their immunoregulatory properties offer the potential of donor-specific control of graft rejection after organ transplantation. It has not been clarified which DC subpopulations mediate tolerance, and the use of natural DCs for therapeutic applications is therefore problematic. Suppressive DCs can be generated in vitro by treating the cells with biologic, pharmacologic, or genetic agents. Here we discuss approaches for generating inhibitory DCs and present DC-based animal models for control of allograft rejection. A prerequisite of suppressive DCs for therapeutic application in clinical transplantation is a reproducible method for their generation as well as the induction of irreversible suppressive function. Based on lessons learned from the use of DCs as tools in clinical vaccine trials in cancer, we discuss the unknown aspects and risks of DC therapy in transplantation.

We showed previously that dendritic cells (DCs) treated with mitomycin C (MMC) tolerize allogeneic T cells in vitro and this might be mediated by downregulation of CD80, CD86, and ICAM-1. Here we analyze the suppression of the T-cell response induced by MMC-DCs in vivo. Rats injected with allogeneic DCs developed a strong lymph node reaction, whereas MMC-DCs induced no reaction. The same effect was obtained when CD80, CD86, and ICAM-1 expressed by DCs were blocked with antibodies. One injection of donor MMC-DCs strongly prolonged heart allograft survival in a donor-specific manner. Suppression of rejection was also achieved when donor DCs were pretreated with a combination of anti-CD80, anti-CD86, and anti-ICAM-1 antibodies, showing that downregulation of these molecules confers the DCs inhibitory properties. We conclude that allogeneic MMC-DCs specifically inhibit the T-cell response in vivo and that downregulation of CD80, CD86, and ICAM-1 is a potential mechanism of this effect.

We have defined factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model and analyzed the influence of CTLA4Ig treatment on indirect alloactivation induced by donor MHC I peptides in a DA-->LEW heart allograft model. Indirect allorecognition of MHC I led to accelerated graft rejection and was accompanied by the induction of anti-peptide antibodies and donor peptide-activated T cells. In an attempt to block the B7-induced costimulatory signal of T cell activation, CTLA4Ig was administered to graft recipients in addition to MHC I peptide treatment. CTLA4Ig therapy, however, was not effective in preventing the humoral or cellular anti-donor immune response, nor did it prevent accelerated graft rejection.

The current series of experiments addressed the question of whether indirect priming with donor MHC antigens affects heart allograft survival. LEW (RT-1(1)) rats were immunized with a mixture of two peptides corresponding to the variable region of MHC class I locus Aa antigen (alpha1 and alpha2 domain). The recipients were transplanted with a DA (RT1-1a) heart 1 month after immunization, and graft survival was closely monitored by ECG. All peptide-treated recipients presented with anti-peptide antibodies at the time of transplantation and developed a strongly accelerated graft rejection. These findings indicated that indirect recognition of MHC I donor antigens promotes heart allograft rejection.