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Transplantation of allogeneic human cardiac/stem progenitor cells (hCSCs) is currently being tested in several phase I/II clinical trials as a novel and promising therapy for restoration of myocardial tissue function in acute myocardial infarction (AMI) patients. Previous findings demonstrate that these cells have an immune suppressive profile interacting with different populations from the immune system, resulting in overall attenuation of myocardial inflammation. However, transplanted hCSCs are still recognized and cleared from the injured site, impairing long retention times in the tissue that could translate into a higher clinical benefit.In this work, through modeling allogeneic hCSC/T lymphocyte interaction in vitro by direct contact, transwell inserts, and hCSC conditioned medium, our results demonstrate that hCSCs exert an immune-suppressive effect on T lymphocyte proliferation not only through the previously described cell contact-dependent programmed cell death-1 (PD1)/programmed death ligand-1 (PDL-1) axis but also through a paracrine mechanism associated with indoleamine 2,3-dioxygenase (IDO) enzyme-mediated tryptophan metabolism. Such findings constitute a step forward in better understanding the mechanisms of action of transplanted hCSCs in allogeneic settings.

After organ transplantation, recipient T cells contribute to graft rejection. Mesenchymal stromal cells from the bone marrow (BM-MSCs) are known to suppress allogeneic T-cell responses, suggesting a possible clinical application of MSCs in organ transplantation. Human liver grafts harbor resident populations of MSCs (L-MSCs). We aimed to determine the immunosuppressive effects of these graft-derived MSCs on allogeneic T-cell responses and to compare these with the effects of BM-MSCs. BM-MSCs were harvested from aspirates and L-MSCs from liver graft perfusates. We cultured them for 21 days and compared their suppressive effects with the effects of BM-MSCs on allogeneic T-cell responses. Proliferation, cytotoxic degranulation, and interferon-gamma production of alloreactive T cells were more potently suppressed by L-MSCs than BM-MSCs. Suppression was mediated by both cell-cell contact and secreted factors. In addition, L-MSCs showed ex vivo a higher expression of PD-L1 than BM-MSCs, which was associated with inhibition of T-cell proliferation and cytotoxic degranulation in vitro. Blocking PD-L1 partly abrogated the inhibition of cytotoxic degranulation by L-MSCs. In addition, blocking indoleamine 2,3-dioxygenase partly abrogated the inhibitive effects of L-MSCs, but not BM-MSCs, on T-cell proliferation. In conclusion, liver graft-derived MSC suppression of allogeneic T-cell responses is stronger than BM-MSCs, which may be related to in situ priming and mobilization from the graft. These graft-derived MSCs may therefore be relevant in transplantation by promoting allohyporesponsiveness.

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.

While most studies have suggested multipotential stromal cell or mesenchymal stem cell (MSC) therapies are useful for immune-mediated diseases, MSCs' immunomodulatory effects were not entirely reproduced in some studies, indicating the necessity to determine the underlying mechanism of MSCs' effects on immune response regulation to maximize their immunomodulatory effects. We have identified the transcription factor early growth response gene-2 (EGR2) as a novel molecular switch regulating known immunomodulatory molecules in human MSCs. EGR2 binds to the promoter regions of these genes, interleukin-6 (IL6), leukemia inhibitory factor (LIF), indoleamine dioxygenase-1 (IDO1), and cyclooxygenase-2/prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), and siRNA against EGR2 was shown to downregulate these genes and reduce the production of prostaglandin E2, an immunomodulatory mediator produced downstream of COX2/PTGS2. Moreover, EGR2 knockdown restores T-lymphocyte proliferation reduced by MSC coculture. Therefore, EGR2 is a potential target for the optimization of immunomodulatory properties of MSC-based therapies.

Prostaglandin E2 has been shown to enhance the maturation, migration, and antigen-presenting capacity of DCs. It is therefore included in many maturation cocktails for the generation of monocyte-derived DCs. Paradoxically, PGE2 is also an important tumor-derived immunosuppressive factor and has inhibitory effects on DC differentiation and function. To further investigate these seemingly contradictory results we studied whether the DC:T cell ratio has an impact on the outcome of the interaction between PGE2-treated DCs and T cells. Surprisingly, at high DC:T cell ratios T cell proliferation was inhibited while at low ratios PGE2-treated DCs displayed enhanced T cell-stimulatory properties. The inhibitory function of PGE2-treated DCs depended primarily on the PGE2-induced induction of indoleamine 2,3-dioxygenase competence. In summary, we show that PGE2-treated DCs can have either an immunogenic or tolerogenic function depending on the DC:T cell ratio. This finding could explain the conflicting results regarding the influence of PGE2 on DC function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan into kynurenine metabolites that suppress effector T-cell function. In this study, we investigated IDO and its metabolite, 3-hydroxyanthranilic acid (3HAA), in regulating lung allograft rejection, using a murine orthotopic lung transplant model with a major mismatch (BALB/c donor and C57BL6 recipient). IDO was overexpressed in murine donor lungs, using an established nonviral (polyethylenimine carrier)-based gene transfer approach, whereas 3HAA was delivered daily via intraperitoneal injection. Increased IDO expression or its metabolite, 3HAA, resulted in a remarkable therapeutic effect with near normal lung function and little acute rejection, approximately A1, compared with A3 in untreated allografts (grading based on International Society for Heart and Lung Transplantation guidelines). We found that a high IDO environment for 7 days in lung allografts resulted in impaired T-cell activation, the production of multiple effector cytokines (IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, IL-12, and IL-13), and the generation of effector memory T cells (CD62L(lo)CD44(hi) phenotype). In isolated murine splenocytes, we observed that IDO/3HAA impaired T-cell receptor (TCR)-mediated T-cell activation, and more importantly, a decrease of intracellular calcium, phospholipase C-γ1 phosphorylation, and mitochondrial mass was evident. This work further illustrates the potential role of a high IDO environment in lung transplantation, and that the high IDO environment directly impairs TCR activation via the disruption of calcium signaling.

A delicate balance between immunostimulatory and immunosuppressive signals mediated by dendritic cells (DCs) and other antigen-presenting cells (APCs) regulates the strength and efficacy of antiviral T-cell responses. HIV is a potent activator of plasmacytoid DCs (pDCs), and chronic pDC activation by HIV promotes the pathogenesis of AIDS. Cholesterol is pivotal in maintaining HIV envelope integrity and allowing HIV-cell interaction. By depleting envelope-associated cholesterol to different degrees, we generated virions with reduced ability to activate pDCs. We found that APC activation was dissociated from the induction of type I IFN-α/β and indoleamine-2,3-dioxygenase (IDO)-mediated immunosuppression in vitro. Extensive cholesterol withdrawal, resulting in partial protein and RNA loss from the virions, rendered HIV a more powerful recall immunogen for stimulating memory CD8 T-cell responses in HIV-exposed, uninfected individuals. These enhanced responses were dependent on the inability of cholesterol-depleted HIV to induce IFN-α/β.

Background: Increasing evidence suggests that, by the production of indoleamine 2,3-dioxygenase (IDO), dendritic cells (DC) may reduce the activity of T lymphocytes and inhibit T lymphocyte proliferation-induced immune tolerance. One promising way is inspired by increasing IDO expression in DC cells for immune tolerance after transplantation. The aim of this work was to examine the effect of interferon-γ (IFN-γ) on the expression of IDO by DC., Methods: Spleen-derived rat DCs were cultured and induced by cytokines, and the expression of OX62 and surface molecules CD80 and CD86 were measured with flow cytometry. After the DCs were induced by IFN-γ at different concentrations (0, 100, 300, 500 U/ml), the expression levels of IDO mRNA were measured with real-time PCR, and the expression levels of IDO protein in DCs were measured with Western blotting. The allogeneic mixed lymphocyte reaction (MLR) was used to test the effects of DCs incubated with different concentrations of IFN-γ on allogeneic T lymphocyte proliferation., Results: Under the microscope, the DCs induced by IFN-γ showed a typical dendritic morphology. The expression rate of OX62 was above 80% and the positive expression rates of CD80 and CD86 were both about 80%. The expressions of IDO mRNA and IDO protein increased gradually with the increase of IFN-γ concentration, showing statistical significance in the differences between the groups (P < 0.05). Compared with the control DC, the DC incubated with IFN-γ had a notable decrease in allostimulatory activity (P < 0.05). With the increasing IFN-γ concentration, the T lymphocyte proliferation decreased, and the difference between the groups was also statistically significant (P < 0.05)., Conclusions: The highly purified spleen derived rat DCs can be successfully acquired through the improved adhesion in-vitro method. IFN-γ can induce increased expression of IDO in spleen-derived rat DCs and reduce the spleen DCs' capacity to stimulate the proliferation of allogeneic T cells.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.

Ligation of CD40 on dendritic cells (DCs) induces early production of inflammatory mediators via canonical NF-kappaB signaling, as well as late expression of the anti-inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) via unknown signal transduction. By selective blocking of either the canonical NF-kappaB pathway using the NEMO-binding domain peptide or the noncanonical NF-kappaB pathway by small interfering RNA, we demonstrate that IDO expression requires noncanonical NF-kappaB signaling. Also, noncanonical NF-kappaB signaling down-regulates proinflammatory cytokine production in DCs. In addition, selective activation of the noncanonical NF-kappaB pathway results in noninflammatory DCs that suppress T-cell activation and promote the development of T cells with regulatory properties. These findings reveal an important role of the noncanonical NF-kappaB pathway in the regulation of immunity.

Background: Suppressive dendritic cells (DCs) are a promising tool for tolerance induction in transplantation. A human DCs subpopulation, which constitutively expresses indoleamine-2,3-dioxygenase (IDO), a molecule shown to prevent the rejection of fetus during pregnancy, has recently been described. This subset, characterized by nonadherence and CD123/CCR6 expression, exhibited sustained IDO production if exposed to interleukin (IL)-10. In the present work, we generated human nonadherent, CD123/CCR6 DCs secreting IL-10., Methods: Monocytes were separated by plastic adherence and differentiated to DCs in the presecence of IL-3 and IL-4. Expression of IDO was determined by reverse-transcriptase polymerase chain reaction and enzyme activity by reverse-phased high-performance liquid chromatography. Mixed lymphocyte cultures were performed with allogeneic nylon wool-purified T-cells., Results: Contradicting previous findings, CD123+/CCR6+ DCs did not express IDO. Maturation of these cells with inducer-cytokines up-regulated IDO, but the allogeneic T-cell stimulatory capacity of these DCs was even stronger than that of immature IDO DCs, and chemical abrogation of IDO activity did not increase T-cell proliferation. In parallel, we generated mature IDO DCs, but these cells also did not induce stronger T-cell stimulation than their IDO counterpart., Conclusions: In conclusion, CD123/CCR6 DCs do not constitutively express IDO and "induced" IDO DCs, even coexpressing anti-inflammatory IL-10, do not suppress allogeneic T-cell responses.

A hallmark of T cell-mediated autoimmunity is the persistence of autoreactive T cells. However, it remains to elucidate the manner in which synovial T cells are sustained in patients with rheumatoid arthritis (RA). We found that dendritic cells (DC) and tissues from the synovial joints of RA patients expressed higher levels of IDO than DC from healthy donors. Interestingly, T cells derived from the joint synovial fluid (SF) of RA patients proliferated in response to either autologous or allogeneic IDO-positive DC, an outcome that was not affected by the addition of IDO inhibitor 1-methyl-D-tryptophan (1-MT). In contrast, addition of 1-MT to the culture stimulated with allogeneic or autologous IDO-positive DC significantly enhanced the proliferation of T cells derived from peripheral blood of healthy donors or from peripheral blood of RA patients. Furthermore, we found that functionally active tryptophanyl-tRNA-synthetase (TTS) was significantly elevated in T cells derived from the SF of RA patients, leading to enhanced storage of tryptophan in T cells and to subsequent resistance to IDO-mediated deprivation of tryptophan. The RA SF enhancement of TTS expression in T cells was blocked by mAb to IFN-gamma and TNF-alpha. These results suggest that the resistance of T cells to IDO-mediated deprivation of tryptophan represents a mechanism by which autoreactive T cells are sustained in vivo in RA patients. Specifically, blocking of the up-regulation of TTS expression in T cells presents an avenue for development of a novel therapeutic approach to treatment of RA.

Although dendritic cells (DCs) strongly stimulate the immune response, they can also induce unresponsiveness. Recently, a human monocyte-derived DC subpopulation was described that constitutively expresses indoleamine 2,3-dioxygenase (IDO). These DCs were defined as nonadherent CD123+/CC chemokine receptor 6+ (CCR6+) cells that suppress the allogeneic T-cell response. In the present study, we generated nonadherent, mature DCs from human blood monocytes. As expected, in addition to the classic markers, these cells expressed CD123 and CCR6. Reverse transcription-polymerase chain reaction (RT-PCR), however, did not show IDO gene transcription, nor did we detect enzymatic IDO activity. Treating the cells with interferon-gamma (IFN-gamma) resulted in significant IDO production. Subsequently, we studied the regulatory properties of IDO-producing DCs on autologous and allogeneic T-cell responses. Neither OKT3-stimulated T cells of healthy donors nor myelin basic protein (MBP)-specific T cells of patients with multiple sclerosis (MS) were suppressed by autologous IDO DCs. However, whereas IDO(neg) DCs supported further stimulation of preactivated MBP-specific T cells of an MS patient, IDO(pos) DCs had lost this capacity. The allogeneic T-cell response was only marginally suppressed by IDO DCs. Our findings show that nonadherent CD123+/CCR6+ human DCs do not constitutively express IDO, and, even if they express the enzyme after IFN-gamma treatment, they possess only limited T-cell regulatory function.