Five-year follow-up of a phase I trial of donor-derived modified immune cell infusion in kidney transplantation.

Background: The administration of modified immune cells (MIC) before kidney transplantation led to specific immunosuppression against the allogeneic donor and a significant increase in regulatory B lymphocytes. We wondered how this approach affected the continued clinical course of these patients. Methods: Ten patients from a phase I clinical trial who had received MIC infusions prior to kidney transplantation were retrospectively compared to 15 matched standard-risk recipients. Follow-up was until year five after surgery. Results: The 10 MIC patients had an excellent clinical course with stable kidney graft function, no donor-specific human leukocyte antigen antibodies (DSA) or acute rejections, and no opportunistic infections. In comparison, a retrospectively matched control group receiving standard immunosuppressive therapy had a higher frequency of DSA (log rank P = 0.046) and more opportunistic infections (log rank P = 0.033). Importantly, MIC patients, and in particular the four patients who had received the highest cell number 7 days before surgery and received low immunosuppression during follow-up, continued to show a lack of anti-donor T lymphocyte reactivity in vitro and high CD19+CD24hiCD38hi transitional and CD19+CD24hiCD27+ memory B lymphocytes until year five after surgery. Conclusions: MIC infusions together with reduced conventional immunosuppression were associated with good graft function during five years of follow-up, no de novo DSA development and no opportunistic infections. In the future, MIC infusions might contribute to graft protection while reducing the side effects of immunosuppressive therapy. However, this approach needs further validation in direct comparison with prospective controls. Trial registration: https://clinicaltrials.gov/, identifier NCT02560220 (for the TOL-1 Study). EudraCT Number: 2014-002086-30.

Correction: Oweira et al. Kynurenine Is the Main Metabolite of Tryptophan Degradation by Tryptophan 2,3-Dioxygenase in HepaRG Tumor Cells. J. Clin. Med. 2022, 11, 4794.

There was an error in the original publication [...].

Induction of Long-Lasting Regulatory B Lymphocytes by Modified Immune Cells in Kidney Transplant Recipients.

BACKGROUND: We recently demonstrated that donor-derived modified immune cells (MICs)-PBMCs that acquire immunosuppressive properties after a brief treatment-induced specific immunosuppression against the allogeneic donor when administered before kidney transplantation. We found up to a 68-fold increase in CD19 + CD24 hi CD38 hi transitional B lymphocytes compared with transplanted controls. METHODS: Ten patients from a phase 1 clinical trial who had received MIC infusions before kidney transplantation were followed to post-transplant day 1080. RESULTS: Patients treated with MICs had a favorable clinical course, showing no donor-specific human leukocyte antigen antibodies or acute rejections. The four patients who had received the highest dose of MICs 7 days before surgery and were on reduced immunosuppressive therapy showed an absence of in vitro lymphocyte reactivity against stimulatory donor blood cells, whereas reactivity against third party cells was preserved. In these patients, numbers of transitional B lymphocytes were 75-fold and seven-fold higher than in 12 long-term survivors on minimal immunosuppression and four operationally tolerant patients, respectively ( P <0.001 for both). In addition, we found significantly higher numbers of other regulatory B lymphocyte subsets and a gene expression signature suggestive of operational tolerance in three of four patients. In MIC-treated patients, in vitro lymphocyte reactivity against donor blood cells was restored after B lymphocyte depletion, suggesting a direct pathophysiologic role of regulatory B lymphocytes in donor-specific unresponsiveness. CONCLUSIONS: These results indicate that donor-specific immunosuppression after MIC infusion is long-lasting and associated with a striking increase in regulatory B lymphocytes. Donor-derived MICs appear to be an immunoregulatory cell population that when administered to recipients before transplantation, may exert a beneficial effect on kidney transplants. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: MIC Cell Therapy for Individualized Immunosuppression in Living Donor Kidney Transplant Recipients (TOL-1), NCT02560220.

Individualised immunosuppression with intravenously administered donor-derived modified immune cells compared with standard of care in living donor kidney transplantation (TOL-2 Study): protocol for a multicentre, open-label, phase II, randomised controlled trial.

INTRODUCTION: Donor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in a cellular phenotype that was directly and indirectly suppressive to the recipient's immune system allowing for reduction of conventional immunosuppressive therapy. Here, we describe a protocol for a randomised controlled, multicentre phase-IIb clinical trial of individualised immunosuppression with intravenously administered donor MIC compared with standard-of-care (SoC) in living donor kidney transplantation (TOL-2 Study). METHODS AND ANALYSIS: Sixty-three living donor kidney transplant recipients from six German transplant centres are randomised 2:1 to treatment with MIC (MIC group, N=42) or no treatment with MIC (control arm, N=21). MIC are manufactured from donor peripheral blood mononuclear cells under Good Manufacturing Practice conditions. The primary objective of this trial is to determine the efficacy of MIC treatment together with reduced conventional immunosuppressive therapy in terms of achieving an operational tolerance-like phenotype compared with SoC 12 months after MIC administration. Key secondary endpoints are the number of patient-relevant infections as well as a composite of biopsy-proven acute rejection, graft loss, graft dysfunction or death. Immunosuppressive therapy of MIC-treated patients is reduced during follow-up under an extended immunological monitoring including human leucocyte antigen-antibody testing, and determination of lymphocyte subsets, for example, regulatory B lymphocytes (Breg) and antidonor T cell response. A Data Safety Monitoring Board has been established to allow an independent assessment of safety and efficacy. ETHICS AND DISSEMINATION: Ethical approval has been provided by the Ethics Committee of the Medical Faculty of the University of Heidelberg, Heidelberg, Germany (AFmu-580/2021, 17 March 2022) and from the Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institute, Langen, Germany (Vorlage-Nr. 4586/02, 21 March 2022). Written informed consent will be obtained from all patients and respective donors prior to enrolment in the study. The results from the TOL-2 Study will be published in peer-reviewed medical journals and will be presented at symposia and scientific meetings. TRIAL REGISTRATION NUMBER: NCT05365672.

Kynurenine Is the Main Metabolite of Tryptophan Degradation by Tryptophan 2,3-Dioxygenase in HepG2 Tumor Cells.

There are two main enzymes that convert tryptophan (Trp) to kynurenine (Kyn): tryptophan-2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Kyn accumulation can promote immunosuppression in certain cancers. In this study, we investigated Trp degradation to Kyn by IDO and TDO in primary human hepatocytes (PHH) and tumoral HepG2 cells. To quantify Trp-degradation and Kyn-accumulation, using reversed-phase high-pressure liquid chromatography, the levels of Trp and Kyn were determined in the culture media of PHH and HepG2 cells. The role of IDO in Trp metabolism was investigated by activating IDO with IFN-γ and inhibiting IDO with 1-methyl-tryptophan (1-DL-MT). The role of TDO was investigated using one of two TDO inhibitors: 680C91 or LM10. Real-time PCR was used to measure TDO and IDO expression. Trp was degraded in both PHH and HepG2 cells, but degradation was higher in PHH cells. However, Kyn accumulation was higher in the supernatants of HepG2 cells. Stimulating IDO with IFN-γ did not significantly affect Trp degradation and Kyn accumulation, even though it strongly upregulated IDO expression. Inhibiting IDO with 1-DL-MT also had no effect on Trp degradation. In contrast, inhibiting TDO with 680C91 or LM10 significantly reduced Trp degradation. The expression of TDO but not of IDO correlated positively with Kyn accumulation in the HepG2 cell culture media. Furthermore, TDO degraded L-Trp but not D-Trp in HepG2 cells. Kyn is the main metabolite of Trp degradation by TDO in HepG2 cells. The accumulation of Kyn in HepG2 cells could be a key mechanism for tumor immune resistance. Two TDO inhibitors, 680C91 and LM10, could be useful in immunotherapy for liver cancers.

The combination of mitomycin-induced blood cells with a temporary treatment of ciclosporin A prolongs allograft survival in vascularized composite allotransplantation.

BACKGROUND: Vascularized composite allotransplantation (VCA) is a rapidly expanding field of transplantation and provides a potential treatment for complex tissue defects. Peripheral blood mononuclear cells (PBMCs) shortly incubated with the antibiotic and chemotherapeutic agent mitomycin C (MMC) can suppress allogeneic T cell response and control allograft rejection in various organ transplantation models. MMC-incubated PBMCs (MICs) are currently being tested in a phase I clinical trial in kidney transplant patients. Previous studies with MICs in a complex VCA model showed the immunomodulatory potential of these cells. The aim of this study is to optimize and evaluate the use of MICs in combination with a standard immunosuppressive drug in VCA. METHODS: Fully mismatched rats were used as hind limb donors [Lewis (RT11)] and recipients [Brown-Norway (RT1n)]. Sixty allogeneic hind limb transplantations were performed in six groups. Group A received donor-derived MICs combined with a temporary ciclosporin A (CsA) treatment. Group B received MICs in combination with a temporarily administered reduced dose of CsA. Group C served as a control and received a standard CsA dose temporarily without an additional administration of MICs, whereas Group D was solely medicated with a reduced CsA dose. Group E received no immunosuppressive therapy, neither CsA nor MICs. Group F was given a continuous standard immunosuppressive regimen consisting of CsA and prednisolone. The endpoint of the study was the onset of allograft rejection which was assessed clinically and histologically. RESULTS: In group A and B, the rejection-free interval of the allograft was significantly prolonged to an average of 23.1 ± 1.7 and 24.7 ± 1.8 days compared to the corresponding control groups (p < 0.01). Rejection in groups C, D, and E was noted after 14.3 ± 1.1, 7.8 ± 0.7, and 6.9 ± 0.6 days. No rejection occurred in control group F during the follow-up period of 100 days. No adverse events have been noted. CONCLUSION: The findings of this study show that the combination of MICs with a temporary CsA treatment significantly prolongs the rejection-free interval in a complex VCA model. The combination of MICs with CsA showed no adverse events such as graft-versus-host disease. MICs, which are generated by a simple and reliable in vitro technique, represent a potential therapeutic tool for prolonging allograft survival through immunomodulation.

Autoantigen-specific immunosuppression with tolerogenic peripheral blood cells prevents relapses in a mouse model of relapsing-remitting multiple sclerosis.

BACKGROUND: Dendritic cells (DCs) rendered suppressive by treatment with mitomycin C and loaded with the autoantigen myelin basic protein demonstrated earlier their ability to prevent experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis (MS). This provides an approach for prophylactic vaccination against autoimmune diseases. For clinical application such DCs are difficult to generate and autoantigens hold the risk of exacerbating the disease. METHODS: We replaced DCs by peripheral mononuclear cells and myelin autoantigens by glatiramer acetate (Copaxone(®)), a drug approved for the treatment of MS. Spleen cells were loaded with Copaxone(®), incubated with mitomycin C (MICCop) and injected into mice after the first bout of relapsing-remitting EAE. Immunosuppression mediated by MICCop was investigated in vivo by daily assessment of clinical signs of paralysis and in in vitro restimulation assays of peripheral immune cells. Cytokine profiling was performed by enzyme-linked immunosorbent assay (ELISA). Migration of MICCop cells after injection was examined by biodistribution analysis of (111)Indium-labelled MICCop. The number and inhibitory activity of CD4(+)CD25(+)FoxP3(+) regulatory T cells were analysed by histology, flow cytometry and in vitro mixed lymphocyte cultures. In order to assess the specificity of MICCop-induced suppression, treated EAE mice were challenged with the control protein ovalbumin. Humoral and cellular immune responses were then determined by ELISA and in vitro antigen restimulation assay. RESULTS: MICCop cells were able to inhibit the harmful autoreactive T-cell response and prevented mice from further relapses without affecting general immune responses. Administered MICCop migrated to various organs leading to an increased infiltration of the spleen and the central nervous system with CD4(+)CD25(+)FoxP3(+) cells displaying a suppressive cytokine profile and inhibiting T-cell responses. CONCLUSION: We describe a clinically applicable cell therapeutic approach for controlling relapses in autoimmune encephalomyelitis by specifically silencing the deleterious autoimmune response.

Cell therapy for immunosuppression after kidney transplantation.

PURPOSE: To give an overview over cell therapeutic approaches to immunosuppression in clinical kidney transplantation. A focus is on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). METHODS: Literature review with an emphasis on already existing therapies. RESULTS: Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness are now being tested in early phase I and phase II trials in clinical kidney transplantation. Cell products such as regulatory T cells or regulatory macrophages, or other myeloid suppressor cell therapies, may either consist of donor-specific, third-party, or autologous cell preparations. Major problems are the identification of the suppressive cell populations and their expansion to have sufficient amount of cells to achieve donor unresponsiveness (e.g., with regulatory T cells). We show a simple and safe way to establish donor unresponsiveness in living-donor kidney transplantation by MIC therapy. A phase I clinical trial is now under way to test the safety and efficacy of this cell therapeutic approach. CONCLUSIONS: Cell therapeutic approaches to immunosuppression after kidney transplantation may revolutionize clinical transplantation in the future.

Generation of suppressive blood cells for control of allograft rejection.

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.

Heat shock proteins purified from autologous tumors using antibody-based affinity chromatography.

Heat shock proteins (HSP) isolated from autologous tumors have become a promising tool for active-specific anticancer immunotherapy due to their properties as carriers of antigenic peptides on one hand and as immunostimulatory adjuvants on the other. Rapid and efficient isolation of HSP-peptide complexes from a patient's tumor is fundamental for their clinical application. Herein, we describe the purification of the HSP Gp96 and Hsc70/Hsp70 from human autologous tumor sources by one-step antibody-based affinity chromatography. Recombinant anti-Gp96 and anti-Hsp70 single-chain Fv antibodies are covalently coupled to a chromatographic bead resin to obtain highly specific affinity matrices. Chromatographic columns are assembled and then used to simultaneously isolate various HSP from the supernatant of lysates of human tumor samples of different origin in a single chromatographic step.

Role of human corneal endothelial cells in T-cell-mediated alloimmune attack in vitro.

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-1ß, 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.

Increased serum levels of quinolinic acid indicate enhanced severity of hepatic dysfunction in patients with liver cirrhosis.

BACKGROUND: The Model for End-Stage Liver Disease (MELD) score is a tool for assessment of the degree of hepatic insufficiency/failure. Quinolinic acid (QuinA) is a tryptophan metabolite produced by activated macrophages. Here we investigate whether the degree of systemic inflammation (QuinA, neopterin, CRP and IL-6) correlates with clinical liver dysfunction according to the MELD Score. METHOD: Ninety-four patients with liver cirrhosis were categorized into 2 groups according to baseline MELD score (group I, MELD <20, n = 61, and group II, MELD ≥20, n = 33). RESULTS: Serum levels of QuinA, neopterin, CRP, and IL-6 significantly correlated with MELD score (r = 0.77, 0.75, 0.57, and 0.50; p < 0.0001, respectively). Patients of group II had significantly higher serum levels of QuinA, neopterin, CRP, and IL-6 than group I (p0.0001). ROC curve analysis showed that QuinA and neopterin are more sensitive markers for severity of liver disease than established markers of inflammation such as CRP and IL-6 (sensitivity = 86% and 79%, respectively) (AUC=0.89 and 0.89, respectively). QuinA provided the most sensitive index with regard to the identification of patients with hepatic encephalopathy. CONCLUSION: Serum levels of QuinA reflect the degree of liver dysfunction. Moreover, high levels of QuinA may serve as a sensitive indicator of hepatic encephalopathy.

HIV-Specific CD8(+) T Lymphocytes in Blood of Long-Term HIV-Infected Hemophilia Patients.

Hemophilia patients infected with human immunodeficiency virus (HIV) 30 years ago show increased proportions of activated CD8(+)DR(+) blood lymphocytes. We hypothesized that this might indicate a cellular immune response directed against HIV and might be the reason for long-term clinical stability of these patients. CD8(+) peripheral blood lymphocytes (PBL) reactive with six HIV and two cytomegalovirus (CMV) pentamers were determined in heparinized whole blood. Additional lymphocyte subsets as well as plasma cytokines and HIV-1 load were studied. Long-term HIV-infected hemophilia patients with (n=15) or without (n=33) currently detectable HIV-1 load in the plasma showed higher proportions of CD8(+) lymphocytes reactive with HIV (p<0.001) and CMV pentamers (p=0.010) than healthy individuals. The cellular anti-HIV response tended to be stronger and more polyclonal in patients during periods of viral replication than in patients with retroviral quiescence (p=0.077). Anti-HIV CD8(+) lymphocyte responses were strongest in patients with high counts of activated CD8(+)DR(+) T (r=0.353; p=0.014) and low CD19(+) B lymphocyte counts (r=-0.472; p=0.001). Patients with or without HIV-1 viral load showed normal Th1 and Th2 plasma cytokine levels and high plasma interleukin-6 (versus healthy controls, p=0.001) and tumor necrosis factor-α (p=0.020). Hemophilia patients who have been living with HIV for more than 30 years showed a polyclonal CD8(+) T-cell response against HIV and CMV. This cellular antiviral immune response was strongest during periods of HIV-1 replication and remained detectable during periods of HIV-1 quiescence. We hypothesize that the consistent cellular anti-HIV-1 response in combination with highly active antiretroviral therapy ensures stability and survival of these chronically HIV-1-infected hemophilia patients.

Mitomycin-C-treated peripheral blood mononuclear cells (PBMCs) prolong allograft survival in composite tissue allotransplantation.

BACKGROUND: Composite tissue allotransplantation (CTA) was introduced as a potential treatment for complex reconstructive procedures and has become a clinical reality. Hand and face transplantation, the most widely recognized forms of CTA, have intensified immunological research in this emerging field of transplantation. Mitomycin C (MMC) is an alkylating agent that suppresses allogeneic T-cell responses. MMC-treated dendritic cells/PBMCs have been shown to induce donor-specific tolerance in solid organ allograft transplantations. METHODS: Fully mismatched rats were used as hind limb donors [Lewis (RT1(1))] and recipients [Brown-Norway (RT1(n))]. Fifty-five allogeneic hind limb transplantations were accomplished in six groups. Group A (n = 10) received donor-derived MMC-treated PBMCs on transplantation day. Group B (n = 10) rats received no immunosuppression, group C (n = 10) received FK506 and prednisolon, group D consisted in isograft transplantation without immunosuppression, group E (n = 10) received non-treated PBMCs, and group F (n = 5) received PBS without any donor-derived cells. Rejection was assessed clinically and histologically. RESULTS: In group A, the survival times of the allografts were prolonged to an average of 8.0 d. Rejection was significantly delayed compared with the averages of the corresponding control groups B, E, and F (5.5, 5.9, and 5.8 d). No rejection was seen in control groups C and D. CONCLUSION: These results demonstrate that MMC-treated donor PBMCs significantly prolong allograft survival when administered systemically on the day of transplantation. However, the immunomodulatory effect is relatively modest with further research being required to clarify dose-effect relations, cell characteristics, and an optimized mechanism and timing for cell application.

Active-specific immunotherapy of human cancers with the heat shock protein Gp96-revisited.

The passive administration of specific antibodies that selectively target tumors is a well-known strategy in cancer treatment. Active immunotherapy using peptide vaccines, in contrast, is expected to induce specific, cytolytic T cells in the patient, which react against tumor antigens and destroy malignant cells. Although several concepts exist, the identification and low immunogenicity of tumor-specific peptides remain a serious problem. Heat shock proteins (HSPs), notably glycoprotein (Gp) 96, are of special interest, because they are able to take molecular peptide-fingerprints of the protein array characteristic for a particular cell. Association of Gp96 with peptides has been shown to be essential for crosspresentation and activation of T cells. Consequently, Gp96-peptide complexes extracted from cancer cells harbor the tumor-specific peptides and are immunogenic, thus offering a tool for active immunization against the tumor. Already, several immunotherapy studies of human cancers have been carried out, showing no severe adverse effects but unfortunately only limited improvement in the clinical outcome. Vitespen, a commercial HSP-peptide complex vaccine based on tumor-derived Gp96, seems to induce an improved overall survival for subsets of early stage melanoma and kidney cancer patients. The limited access to vaccine material derived from the autologous tumor requires the development of alternative protocols. Moreover, counteracting immunosuppressive mechanisms induced by the malignancy might further improve the efficacy of vaccinations. This review critically analyzes the current state of clinical immunotherapy with Gp96, with special attention to Vitespen.

Strong association of phenylalanine and tryptophan metabolites with activated cytomegalovirus infection in kidney transplant recipients.

Infection-induced inflammation triggers catabolism of proteins and amino acids. Phenylalanine and tryptophan are 2 amino acids related to infections that regulate immune responses. Polyomavirus BK (BKV) and cytomegalovirus (CMV) are important pathogens after kidney transplantation. We investigated the clinical relevance of phenylalanine, tryptophan, and tryptophan metabolites (kynurenine and quinolinic acid) plasma levels in kidney transplant recipients with active CMV (BKV(-)CMV(+), n = 12) or BK virus infection (BKV(+)CMV(-), n = 37). Recipients without active viral infections (CMV(-)BKV(-), n = 28) and CMV(-)BKV(-) healthy individuals (HCs, n = 50) served as controls. In contrast to BKV infection, activated CMV infection is tightly linked to increased phenylalanine and tryptophan metabolite plasma levels (p ≤ 0.002). The association of phenylalanine (cutoff 50 µmol/L) with CMV infection demonstrates high sensitivity (100%) and specificity (94%). By contrast, kynurenine (p = 0.029) and quinolinic acid (p = 0.003) values reflect the severity of CMV infection. In this early proof-of-concept trial, evidence indicates that activated CMV infection is strongly associated with increased phenylalanine as well as kynurenine and quinolinic acid plasma levels. Moreover, tryptophan metabolite levels correlate with disease severity. Measurement of these amino acids is an inexpensive and fast method expected to complete conventional diagnostic assays.

Increased pretransplantation plasma kynurenine levels do not protect from but predict acute kidney allograft rejection.

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.

Melan-A/MART1 analog peptide triggers anti-myeloma T-cells through crossreactivity with HM1.24.

The Melan-A(aa26-35) (EAAGIGILTV) peptide is a human leukocyte antigen (HLA)-A2-restricted T-cell epitope within the Melan-A/MART-1 tumor antigen expressed on malignant melanoma cells. Melan-A and Melan-A analog (ELAGIGILTV, Melan-A(aa26-35*A27L)) specific T-cells can be expanded reliably for immunotherapeutic approaches in vitro. We studied the ability of Melan-A analog (ELAGIGILTV, Melan-A(aa26-35*A27L)) specific T-cells to recognize the HM1.24(aa22-30) (LLLGIGILV) peptide within the HM1.24 antigen presented by normal and malignant plasma cells. Peripheral blood mononuclear cells from HLA-A2+ healthy donors and HLA-A2+ multiple myeloma (MM) patients were stimulated with Melan-A analog peptide-loaded autologous dendritic cells, and expanded in vitro. T-cell activation was assessed by interferon-gamma specific enzyme-linked immunosorbent spot and cytotoxicity by (51)Chromium-release-assays. The frequency of Melan-A analog specific CD8+ T-cells was detected by using tetramers. Melan-A analog specific T-cells from HLA-A2+ healthy donors and HLA-A2+ MM patients showed a interferon-gamma secretion mediated by HM1.24(aa22-30) peptide-pulsed T2 cells and lysed the HLA-A2+ HM1.24+ U266 and XG-1 human myeloma derived cell-lines as well as the B-lymphoblastoid cell-line IM-9. Melan-A analog specific T-cells from MM patients specifically lysed autologous MM cells. The current data demonstrate that Melan-A analog specific T-cells crossreact with HM1.24(aa22-30). They might be a tool for the future use in immunotherapy against MM.

Inhibitors of indoleamine-2,3-dioxygenase for cancer therapy: can we see the wood for the trees?

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme capable of inhibiting a destructive maternal T cell response against allogeneic fetuses. Expression of IDO is evident in tumours and is thought to enable escape from immunologically mediated rejection. Consequently, clinical trials using an inhibitor of IDO, 1-methyltryptophan (1MT), have been initiated. However, a review of the current literature indicates that we are far from understanding the biological relevance of IDO expression during tumorigenesis. A better understanding of IDO biology is needed to comprehend the effect of IDO inhibitors and to provide a rationale for their therapeutic application in cancer.

Mitomycin C-treated antigen-presenting cells as a tool for control of allograft rejection and autoimmunity: from bench to bedside.

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.