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.

Cell therapeutic approaches to immunosuppression after clinical kidney transplantation.

Refinement of immunosuppressive strategies has led to further improvement of kidney graft survival in recent years. Currently, the main limitations to long-term graft survival are life-threatening side effects of immunosuppression and chronic allograft injury, emphasizing the need for innovative immunosuppressive regimens that resolve this therapeutic dilemma. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness have been tested in early phase I and phase II clinical trials in kidney transplantation. The aim of this overview is to summarize current cell therapeutic approaches to immunosuppression in clinical kidney transplantation with a focus on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). MICs show great promise as a therapeutic agent to achieve the rapid and durable establishment of donor-unresponsiveness in living-donor kidney transplantation. Cell-based therapeutic approaches may eventually revolutionize immunosuppression in kidney transplantation in the near future.

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.

Early pregnancy serum neopterin concentrations predict spontaneous preterm birth in asymptomatic pregnant women.

OBJECTIVE: To investigate if early pregnancy serum neopterin concentrations (EPSN) could predict spontaneous preterm birth (SPB). METHODS: EPSN was measured in 92 sera collected from 46 pregnant women with birth at term and 40 sera from 20 pregnant women with preterm birth. Two sera were collected for each case: in the first and early second trimester. RESULTS: EPSN concentrations correlate with gestational age (ρ=0.275, P=0.001), a correlation which was present in both groups: term and preterm birth. EPSN were higher in pregnancies with SPB compared with normal pregnancies (6.27±1.03 vs. 6.04±0.15, P=0.039). Patients with SPB showed a considerable increase of EPSN in the second trimester compared with patients with birth at term (7.30±1.53 vs. 6.16±0.23, P=0.043). A sharper increase was found in the group with SPB before 32 weeks of pregnancy (wp) (9.83±4.36 vs. 6.16±0.23, P=0.016). Pregnant women with an early second trimester serum neopterin value of above 8 nmol/L are associated with a risk of SPB before 32 wp (odds ratio=14.4, P=0.01) and of SPB before 34 wp (odds ratio=3.6, P=0.05), respectively. CONCLUSIONS: EPSN increases with the gestational age and predicts SPB in asymptomatic pregnant women.

Local administration of Mitomycin-C-Treated peripheral blood mononuclear cells (PBMCs) prolongs allograft survival in vascularized composite allotransplantation.

BACKGROUND: VCA offers a potential treatment for extensive tissue defects. First results of systemic administration of Mitomycin C-treated PBMCs in VCA demonstrated a significant prolongation of allograft survival. The aim of this study is to evaluate if local administration of MMC-PBMCs prolongs allograft survival in allogeneic hind limb transplantations of the rat. METHODS: Sixty allogeneic hind limb transplantations in the rat were performed in six groups. Lewis rats (LEW) were used as hind limb donors and Brown-Norway rats (BN) as recipients. Animals in group A received donor-derived MMC-treated PBMCs locally (i.m.). Group B received no immunosuppressive therapy, group C received a standard immunosuppressive regime consisting of FK506 and Prednisolon, group D (BN to BN) comprised isograft transplantations without immunosuppressive treatment, group E received non-treated PBMCs (i.m.) and group F received phosphate buffered saline (PBS) without cells. The transplanted hind limbs were assessed for color, edema, skin, hair condition, and consistency of the thigh every 8 hours. RESULTS: Rejection in group A was delayed to an average of 7.2 ± 0.6 days. Survival times were significantly prolonged (P < 0.01) compared to control groups B, E, and F (5.5 ± 0.7, 5.8 ± 0.7, and 5.7 ± 0.5 days). Control groups C and D showed no signs of rejection. CONCLUSION: The findings of this study show that local administration of MMC-PBMCs has no side effects and significantly extends allograft survival. Further experiments with MMC-PBMCs treatments repeated at different time-points and being added to low dose immunosuppressive protocols need to be performed to improve experimental and eventually clinical outcome after VCA. © 2015 Wiley Periodicals, Inc. Microsurgery 36:417-425, 2016.

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.

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.

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 [...].

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.

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.

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.

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.

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.

Mitomycin C-treated dendritic cells inactivate autoreactive T cells: toward the development of a tolerogenic vaccine in autoimmune diseases.

Treatment of autoimmune diseases remains a challenge for immunological research. An ideal therapy should inhibit the immune reaction against the diseased organ and leave the rest of the immune response intact. Our previous studies showed that donor-derived dendritic cells (DCs) treated in vitro with mitomycin C (MMC) suppress rat heart allograft rejection if injected into recipients before transplantation. Here we analyze their efficacy in controlling autoimmunity. MMC-DCs loaded with myelin-basic-protein (MBP) inhibited specific T cells derived from multiple sclerosis patients in vitro. If coincubated with MMC-DCs, T cells were arrested in the G(0)/G(1) cell cycle phase. Microarray gene scan showed that MMC influences the expression of 116 genes in DCs, one main cluster comprising apoptotic and the second cluster immunosuppressive genes. Apparently, the combination of apoptosis with expression of tolerogenic molecules renders MMC-DCs suppressive. MBP-loaded MMC-DCs also inhibited mouse T cells in vitro and, in contrast to MBP-loaded naïve DCs, did not induce experimental autoimmune encephalitis. Most importantly, mice vaccinated with inhibitory DCs became resistant to the disease. Whereas this is not the first report on generation of suppressive DCs, it delineates a method using a clinically approved drug at nontoxic concentrations, which yields irreversibly changed DCs, effective across species in vitro and in vivo.

[Immunosuppressive effect of mitomycin C-treated peripheral mononuclear blood cells (MICs) in vascularised composite allotransplantation]. / Immunsuppressive Wirkung von Mitomycin-C-behandelten mononukleären Zellen des peripheren Blutes (MICs) in der Vaskularisierten Composite Allotransplantation.

BACKGROUND: Vascularized Composite Allotransplantation (VCA) enables the restoration of complex tissue defects. Since the first successful hand and face transplants were performed, clinical and experimental research has consistently improved immunosuppressive therapies. The incubation of peripheral blood mononuclear cells (PBMCs) with mitomycin C (MMC) results in immunomodulatory cells (MICs). In previous studies, the systemic application of MICs on the day of allogeneic hind limb transplantation led to a significant immunosuppression in rats. The aim of this study is to further investigate the optimal point in time of MIC application in a complex VCA model. MATERIAL AND METHODS: In six groups, 60 allogeneic hind limb transplantations were performed. Fully mismatched rats were used as hind limb donors [Lewis (LEW)] and recipients [Brown-Norway (BN)]. Group A received donor-derived MICs seven days preoperatively. Group B received no immunosuppression; group C received untreated PBMCs seven days prior to transplantation. Animals in group D received cell culture media, whereas group E was treated with a standard immunosuppression consisting of tacrolimus and prednisolone. In group F, syngeneic hind limb transplantations (BN→BN) were performed. Transplant rejection was assessed clinically and histologically. RESULTS: Group A showed a significantly earlier onset of allograft rejection after 3.5 ± 0.2 days (p < 0.01) when compared with control groups B, C and D (5.5 ± 0.7, 5.3 ± 0.7 und 5.7 ± 0.5). Groups E and F showedno allograft rejection. CONCLUSION: This study shows that the time of application determines the immunomodulatory effects of MICs. Whereas the systemic application of MICs on the day of transplantation led to a significant immunosuppression in previous studies, this study demonstrates that preoperative injections of MICs lead to an acceleration of allotransplant rejection. Follow-up studies are necessary to investigate further modifications of application time as well as dose-effect relations and cell characteristics of these potential immunosuppressive cells.

Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites.

Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the catabolism of tryptophan, is expressed in certain cells and tissues, particularly in antigen-presenting cells of lymphoid organs and in the placenta. It was shown that IDO prevents rejection of the fetus during pregnancy, probably by inhibiting alloreactive T cells, and it was suggested that IDO-expression in antigen-presenting cells may control autoreactive immune responses. Degradation of tryptophan, an essential amino acid required for cell proliferation, was reported to be the mechanism of IDO-induced T cell suppression. Because we wanted to study the action of IDO-expressing dendritic cells (DCs) on allogeneic T cells, the human IDO gene was inserted into an adenoviral vector and expressed in DCs. Transgenic DCs decreased the concentration of tryptophan, increased the concentration of kynurenine, the main tryptophan metabolite, and suppressed allogeneic T cell proliferation in vitro. Kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, but no other IDO-induced tryptophan metabolites, suppressed the T cell response, the suppressive effects being additive. T cells, once stopped in their proliferation, could not be restimulated. Inhibition of proliferation was likely due to T cell death because suppressive tryptophan catabolites exerted a cytotoxic action on CD3(+) cells. This action preferentially affected activated T cells and increased gradually with exposure time. In addition to T cells, B and natural killer (NK) cells were also killed, whereas DCs were not affected. Our findings shed light on suppressive mechanisms mediated by DCs and provide an explanation for important biological processes in which IDO activity apparently is increased, such as protection of the fetus from rejection during pregnancy and possibly T cell death in HIV-infected patients.

Comparison of early pregnancy serum concentration of neopterin, neopterin/creatinine ratio, C-reactive protein, and chitotriosidase, in pregnant women with birth at term and spontaneous preterm birth.

Inflammatory mechanisms are involved in achieving a normal pregnancy and in the development of certain pregnancy complications. These changes are more intense in pregnant women that suffer of pregnancy complications, such as spontaneous preterm birth (SPB). This study compared the course of inflammatory markers (IM) [neopterin (Neo), neopterin/creatinine ratio (Neo/Cre), C-reactive protein (CRP), and chitotriosidase (Chito)] serum concentration in the early pregnancy of women with birth at term (BT) and preterm birth (PB). IM concentration was measured in 90 sera sampled from 45 pregnancies with BT and 30 sera from 15 pregnancies with PB. Two sera were sampled from each pregnant woman: one in the first trimester and another one in the second trimester. Early pregnancy IM concentration showed a direct correlation with gestational age: Neo (rho=0.262, P=0.004), Neo/Cre (rho=0.372, P<0.001), CRP (rho=0.187, P=0.041), and Chito (rho=0.039, P=0.66). The correlation was present in both categories of patients with BT and PB. Patients with PB before 34 week of pregnancy (wp) and 32 wp showed higher Neo and Neo/Cre concentration than BT patients. A significant association was found between the risk of PB before 34 wp, PB before 32 wp, and Neo concentration (PB <34 wp: odds ratio (OR) =5.13, P=0.035) (PB <32 wp: OR=8.2, P=0.020) and, respectively, Neo/Cre concentration (PB <34 wp: OR=5.29, P=0.015) (PB <32 wp: OR=9.25, P=0.006). No association between CRP or Chito and PB age was found. IM concentration correlates with the gestational age at the time of blood sampling. Increased Neo and Neo/Cre concentration are associated with PB. Further studies are needed to evaluate the usefulness of these markers in clinical practice.

Phase I trial of donor-derived modified immune cell infusion in kidney transplantation.

BACKGROUNDPreclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor.METHODSIn this phase I trial, patients received either 1.5 × 106 MICs per kg BW on day -2 (n = 3, group A), or 1.5 × 108 MICs per kg BW on day -2 (n = 3, group B) or day -7 (n = 4, group C) before living donor kidney transplantation in addition to post-transplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360.RESULTSMIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 1010 donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19+CD24hiCD38hi transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature.CONCLUSIONMIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs.TRIAL REGISTRATIONEudraCT number: 2014-002086-30; ClinicalTrials.gov identifier: NCT02560220.FUNDINGFederal Ministry for Economic Affairs and Technology, Berlin, Germany, and TolerogenixX GmbH, Heidelberg, Germany.

IDO1 and IDO2 are expressed in human tumors: levo- but not dextro-1-methyl tryptophan inhibits tryptophan catabolism.

OBJECTIVES: Indoleamine-2,3-Dioxygenase (IDO) is an immunosuppressive molecule inducible in various cells. In addition to classic IDO (IDO1), a new variant, IDO2, has recently been described. When expressed in dendritic cells (DCs) or cancer cells, IDO was thought to suppress the immune response to tumors. A novel therapeutic approach in cancer envisages inhibition of IDO with 1-methyl-tryptophan (1MT). The levo-isoform (L-1MT) blocks IDO1, whereas dextro-1MT (D-1MT), which is used in clinical trials, inhibits IDO2. Here we analyze IDO2 expression in human cancer cells and the impact of both 1-MT isoforms on IDO activity. METHODS: Surgically extirpated human primary tumors as well as human cancer cell lines were tested for IDO1 and IDO2 expression by RT-PCR. IDO1 activity of Hela cells was blocked by transfection with IDO1-specific siRNA and analysed for tryptophan degradation by RP-HPLC. The impact of D-1MT and L-1MT on IDO activity of Hela cells and protein isolates of human colon cancer were studied. RESULTS: Human primary gastric, colon and renal cell carcinomas constitutively expressed both, IDO1 and IDO2 mRNA, whereas cancer cells lines had to be induced to by Interferon-gamma (IFN-gamma). Treatment of Hela cells with IDO1-specific siRNA resulted in complete abrogation of tryptophan degradation. Only L-1MT, and not D-1MT, was able to block IDO activity in IFN-gamma-treated Hela cells as well as in protein isolates of primary human colon cancer. CONCLUSIONS: Although IDO2 is expressed in human tumors, tryptophan degradation is entirely provided by IDO1. Importantly, D-1MT does not inhibit the IDO activity of malignant cells. If ongoing clinical studies show a therapeutic effect of D-1MT, this cannot be attributed to inhibition of IDO in tumor cells.