IFN-γ and tumor gangliosides: Implications for the tumor microenvironment.

Gangliosides shed by tumors into their microenvironment (TME) are immunoinhibitory. Interferon-γ (IFN-γ) may boost antitumor immune responses. Thus we wondered whether IFN-γ would counteract tumor ganglioside-mediated immune suppression. To test this hypothesis, we exposed human monocyte-derived LPS-activated dendritic cells (DC) to IFN-γ and to a highly purified ganglioside, GD1a. DC ganglioside exposure decreased TLR-dependent p38 signaling, explaining the previously observed ganglioside-induced down-modulation of pro-inflammatory surface markers and cytokines. Strikingly, while increasing LPS-dependent DC responses, IFN-γ unexpectedly did not counteract the inhibitory effects of GD1a. Rather, induction of indoleamine 2,3-dioxygenase (IDO1), and expression of STAT1/IRF-1 and programmed cell death ligand (PD-L1), indicated that the immunoinhibitory, not an immune stimulatory, IFN-γ-signaling axis, was active. The combination, IFN-γ and DC ganglioside enrichment, markedly impaired DC stimulatory potential of CD8+ T-cells. We suggest that gangliosides and IFN-γ may act in concert as immunosuppressive mediators in the TME, possibly promoting tumor progression.

B-cell deficiency and severe autoimmunity caused by deficiency of protein kinase C δ.

Primary B-cell disorders comprise a heterogeneous group of inherited immunodeficiencies, often associated with autoimmunity causing significant morbidity. The underlying genetic etiology remains elusive in the majority of patients. In this study, we investigated a patient from a consanguineous family suffering from recurrent infections and severe lupuslike autoimmunity. Immunophenotyping revealed progressive decrease of CD19(+) B cells, a defective class switch indicated by low numbers of IgM- and IgG-memory B cells, as well as increased numbers of CD21(low) B cells. Combined homozygosity mapping and exome sequencing identified a biallelic splice-site mutation in protein C kinase δ (PRKCD), causing the absence of the corresponding protein product. Consequently, phosphorylation of myristoylated alanine-rich C kinase substrate was decreased, and mRNA levels of nuclear factor interleukin (IL)-6 and IL-6 were increased. Our study uncovers human PRKCD deficiency as a novel cause of common variable immunodeficiency-like B-cell deficiency with severe autoimmunity.

Ambivalent effects of dendritic cells displaying prostaglandin E2-induced indoleamine 2,3-dioxygenase.

Prostaglandin E2 (PGE(2)), an abundantly produced lipid messenger in mammalian organisms, has been attributed to possess potent albeit ambivalent immunological functions. Recently, PGE(2) has been reported to stimulate the commonly believed immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway in human dendritic cells (DCs), but without promoting DC immunosuppressive activity. Here, we report that PGE(2) used as a DC maturation agent apparently has more diverse functions. PGE(2)-matured DCs acquired powerful IDO activity, which was sustained even after removing PGE(2). These IDO-competent DCs were able to stimulate allogeneic T-cell proliferation, but achieved inhibitory activity as their content in DC/T-cell co-cultures increased. The DC inhibitory activity was reversed upon blockade of IDO activity, confirming that the suppressive effect was in fact mediated by IDO and occurred in a dose-dependent fashion. IDO-mediated T-cell suppression was restored upon re-stimulation of T cells in the absence of IDO activity, confirming its reversibility. T cells stimulated by PGE(2)-matured IDO-competent DCs were sensitized to produce multiple cytokines, comprising Th1, Th2, and Th17 phenotypes. Collectively, these data suggest that T cells stimulated by PGE(2)-matured DCs are not terminally differentiated and their ultimate type of response may be formed by microenvironmental conditions.

Mannan-binding lectin deficiency - Good news, bad news, doesn't matter?

Mannan-binding lectin (MBL) deficiency has been classified as a commonly occurring immune disorder, affecting approximately 30% of the human population. MBL, being part of the innate immune system, supports the recognition of infectious pathogens by binding to carbohydrate moieties expressed on microorganisms and activates the lectin pathway of the complement system. MBL2 gene polymorphisms are associated with quantitative and qualitative MBL abnormalities in the serum. The clinical impact of MBL deficiency and its association to a wide variety of diseases has been extensively studied. The picture is puzzling as the studies suggest a detrimental or beneficial or no impact of low or high MBL serum levels on disease susceptibility. In this review we attempt to extract what is relevant from the literature and address controversial issues. We finally suggest that a comprehensive understanding of the role of MBL in human diseases requires considering its context-dependency.

Interferon-gamma-triggered indoleamine 2,3-dioxygenase competence in human monocyte-derived dendritic cells induces regulatory activity in allogeneic T cells.

The role of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in down-regulating human alloresponses has recently been controversially debated. We here demonstrate that human monocyte-derived dendritic cells (mDCs) can be endowed with sustained IDO competence in vitro by 48-hour activation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). IFN-gamma also amplified proinflammatory cytokine secretion during activation. Yet, on reculture after activation cytokine production ceased, whereas IDO enzymatic activity continued. Manipulation of tryptophan metabolism did not affect proinflammatory cytokine release, suggesting that IFN-gamma triggers IDO activity and proinflammatory cytokine release as distinct cellular programs. IDO-competent DCs down-regulated allogeneic T-cell responses, but this IDO-mediated effect was overcome by slightly modifying cell culture conditions. Nevertheless, the CD4(+)CD25(+) T-cell fraction stimulated by IDO-competent DCs displayed substantial suppressor activity. This suppressive activity (1) required allogeneic stimulation for its induction, (2) affected third-party T cells, and (3) was reduced by the IDO inhibitor methyl-thiohydantoin-tryptophan. It became also manifest when DC/T-cell cocultures were initiated with naive (CD4(+)CD25(-)CD45RA(+)) T cells, indicating the differentiation of adaptive regulatory T cells. Together, these findings suggest that IFN-gamma triggered IDO competence in human mDCs constitutes a critical factor for endowing allogeneic T cells with regulatory activity.

Intact indoleamine 2,3-dioxygenase activity in human chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by a disability to produce reactive oxygen intermediates (ROI) caused by a defect of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. A hyperinflammatory response to immune activation has been reported to contribute to the pathology of CGD. The tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is considered critical for regulating immune responses and suppression of inflammation. IDO is generally believed to require ROI for enzymatic activity and was found to be inactive in murine CGD. Here, we report that, strikingly, in human CGD IDO metabolic activity is intact. Monocyte-derived dendritic cells generated from CGD patients, harbouring X-linked and autosomal recessive forms of CGD, and from healthy controls produced similar amounts of the tryptophan metabolite kynurenine upon activation with lipopolysaccharide and interferon-γ. Thus, in humans, ROI apparently are dispensable for IDO activity. Hyperinflammation in human CGD cannot be attributed to disabled IDO activation.

Indoleamine 2,3-dioxygenase in hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) is complicated by unwelcome side-effects that arise on the basis of an altered immune system. Infectious complications and alloreactive T-cell responses trigger a process of ongoing immune activation and inflammation. Negative-feedback mechanisms to counteract inflammation involve the induction of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO), which mediates anti-inflammatory activities and T-cell inhibition via tryptophan catabolism. However, persistent immune activation and generalized release of pro-inflammatory cytokines deviate immune regulation towards chronic suppression incapable to abrogate the inflammatory response. This review focuses on the unique role of tryptophan catabolism in modulating inflammatory processes and T-cell responses after HSCT.

Expression of the putatively regulatory T-cell marker FOXP3 by CD4(+)CD25+ T cells after pediatric hematopoietic stem cell transplantation.

FOXP3 has been proposed to be critical for the regulatory function of CD4(+)CD25+ T cells and it has been reported that its expression correlates with protection from graft-versus-host-disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Here, by monitoring 28 pediatric HSCT recipients, we found that the levels of FOXP3-mRNA expression in highly enriched CD4(+)CD25+ cells were identical to those in healthy controls irrespective of GvHD status. Moreover, FOXP3-mRNA was abundant in recently in vitro stimulated CD4(+)CD25+ cells that lacked regulatory function. Together these findings suggest that FOXP3-mRNA expression primarily reflects CD4(+)CD25+ cell frequency rather than defining the regulatory potential of CD4(+)CD25+ T cells and GvHD risk after HSCT.

Selective IgA deficiency in children with recurrent parotitis of childhood.

Recurrent parotitis of childhood is defined as the relapsing form of juvenile (idiopathic) parotitis and represents a rare inflammatory disorder of the parotid gland with potentially significant morbidity. We reviewed the charts of patients who were diagnosed with inflammatory parotid diseases in our institution between 1992 and 2002. There were 91 patients presenting with juvenile parotitis (1 of 6117 of all clinical visits). Of these 91 cases, 23 patients (28%) had the relapsing form of juvenile parotitis, and the median number of episodes was 5 (range, 2-20). Laboratory investigations revealed that 5 patients had selective IgA deficiency. The prevalence (22%) is different from the cumulative prevalence of IgA deficiency in a healthy population (0.3%; P < 0.001).

Loss of intrahepatic bile ducts: an important feature of familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, fatal disorder of early infancy. We report two siblings with FHL whose symptoms were dominated by hepatic failure. Both presented with sudden-onset fever and hepatosplenomegaly with progressive abnormalities of clinical biochemistry indices of liver function. One died of hepatorenal failure. The other underwent liver transplantation. Autopsy and explant liver displayed portal and periportal infiltrates of T lymphocytes and histiocytes; an activation of the hepatic mononuclear phagocytic system with focal hemophagocytosis; and almost complete loss of interlobular bile ducts. Paucity of bile ducts dominated in a pre-transplant liver biopsy specimen (and transiently obscured the diagnosis of FHL). Disease recurred in the allograft, again with lymphohistiocytic infiltration and destruction of interlobular bile ducts. Consequently the patient underwent haploidentical peripheral stem cell transplantation. This patient is alive 5 years later. Loss of bile ducts may be an important feature of hepatic involvement by FHL.

CTLA4-Ig preserves thymus-derived T regulatory cells.

BACKGROUND: CTLA-4 immunoglobulin fusion proteins (CTLA4-Ig) suppress immune reactions by blocking the T-cell costimulatory CD28-CD80-86 pathway and are used in clinical trials for diseases featuring exaggerated T-cell reactivity including autoimmune diseases and allograft rejection. However, because CTLA4-Ig has been suspected to interfere with T regulatory (Treg) cell homeostasis and function, recently, substantial concerns on CTLA4-Ig's potentially antitolerogenic effects have been raised. METHODS: We tested immunoregulatory CTLA4-Ig explicitly for its effect on Treg cell numbers, frequencies and function in an in vitro murine major histocompatibility complex mismatched setting using C57BL/6 bone marrow-derived dendritic cells as stimulators of allogeneic Balb/c Foxp3 T cells, which allowed for tracing Treg cells in a straightforward fashion. RESULTS: The presence of CTLA4-Ig in mixed leukocyte reactions-while dampening the global proliferative response of allostimulated Balb/c T cells-resulted in a relative increase of the frequency of thymus-derived CD4CD25Foxp3 Treg cells with intact suppressive activity. This relative increase was caused by a selective inhibitory effect of CTLA4-Ig on proliferating conventional T cells, whereas the proliferative capacity of Treg cells in cell cultures remained unaffected. Additionally, in the presence of CTLA4-Ig, the frequency of apoptosis was decreased in these cells. CONCLUSION: Our findings unequivocally demonstrate that CTLA4-Ig does not negatively affect Treg cell frequencies and function in vitro.

CTLA4-Ig immunosuppressive activity at the level of dendritic cell/T cell crosstalk.

Immunosuppressive cytotoxic T lymphocyte associated antigen-4 immunoglobulin fusion proteins (CTLA4-Ig) block the CD28:CD80/86 costimulatory pathway. On a cellular level, CTLA4-Ig is understood to dampen T cell responses. As a mechanism, CTLA4-Ig has been reported to affect dendritic cell (DC) function via inducing the immunosuppressive indoleamine 2,3 dioxygenase (IDO) pathway and promoting a DC regulatory phenotype. We here probed cellular mechanisms of CTLA4-Ig immunoregulation in an allogeneic setting using C57BL/6 splenic or bone marrow derived DCs (BMDCs) as stimulators of allogeneic Balb/c derived T cells. To address whether CTLA4-Ig immunosuppression affected DCs, we pre-exposed C57BL/6 splenic or BMDCs to CTLA4-Ig and removed unbound CTLA4-Ig before co-culture with allogeneic T cells. CTLA4-Ig disappeared rapidly (within 4 h) from the cell membrane by combined internalization and dissociation. These CTLA4-Ig pre-exposed DCs were fully capable of stimulating allogeneic T cell proliferation, suggesting that CTLA4-Ig does not impair the DC stimulatory capacity. Only the presence of CTLA4-Ig during DC/T cell co-culture resulted in the expected inhibition of proliferation. C57BL/6 splenic or BMDCs exposed to CTLA4-Ig did not display IDO activity. We conclude that CTLA4-Ig immunosuppressive activity does not depend on a DC regulatory phenotype but on its presence during DC/T cell interaction.

Indoleamine 2,3-dioxygenase in human hematopoietic stem cell transplantation.

In recent years tryptophan metabolism and its rate limiting enzyme indoleamine 2,3-dioxygenase (IDO) have attracted increasing attention for their potential to modulate immune responses including the regulation of transplantation tolerance. The focus of this review is to discuss some features of IDO activity which particularly relate to hematopoietic stem cell transplantation (HSCT). HSCT invariably involves the establishment of some degree of a donor-derived immune system in the recipient. Thus, the outstanding feature of tolerance in HSCT is that in this type of transplantation it is not rejection, which causes the most severe problems to HSCT recipients, but the reverse, graft-versus-host (GvH) directed immune responses. We will discuss the peculiar role of IDO activity and accelerated tryptophan metabolism at the interface between immune activation and immune suppression and delineate from theoretical and experimental evidence the potential significance of IDO in mediating tolerance in HSCT. Finally, we will examine therapeutic options for exploitation of IDO activity in the generation of allo-antigen-specific tolerance, i.e. avoiding allo-reactivity while maintaining immunocompetence, in HSCT.

Vaccination against tick-borne encephalitis virus tests specific IgG production ability in patients under immunoglobulin substitution therapy.

To assess B-cell function in patients under immunoglobulin (IgG)-replacement therapy, the non-licensed artificial bacteriophage (ΦX174)-neo-antigen may be used despite limited availability and experience. Active immunization against tick-borne encephalitis (TBE) is performed in few European countries. To test the feasibility of using licensed TBE vaccination as (neo-)antigen to determine residual or restored B-cell function in patients under regular IgG substitution, TBE-IgG levels were analyzed in 18 patients with ≥ 1-2 years of regular intravenous or subcutaneous IgG substitution and in pharmaceutical IgG-preparations (n=21 batches, 10 products). Six individuals were boosted against TBE. Although TBE-specific IgG was detectable in concentrates (281-57,100 VieU/0.5 µL), levels were only borderline in patient sera (n=31, 18 individuals; median 132 VieU; positive >155). Thus, TBE vaccination may be used to test B-cell function under IgG replacement therapy because IgG substitution appears insufficient to yield protective TBE-specific antibody levels in children.

A quadrivalent HPV vaccine induces humoral and cellular immune responses in WHIM immunodeficiency syndrome.

WHIM-syndrome is an inherited immunodeficiency disorder with abnormal susceptibility to human papillomavirus (HPV) infection and diseases. We determined safety and immunogenicity to a quadrivalent HPV vaccine in WHIM-syndrome by detection of HPV-specific antibodies and lymphoproliferation. In virus-like-particle (VLP)-ELISA, a WHIM patient showed antibody titers up to 400 for HPV-6/11/16/18, whereas immuno-competent controls developed titers of 6400-25,600. In pseudovirion assays, the patient's neutralization titers ranged from 20 to 400 to the four HPV vaccine types, while titers of 1600-25,600 were detected in healthy vaccinees. Specific proliferation of PBMC of the WHIM patient to the HPV vaccine was demonstrated. This first report on response to HPV vaccination in WHIM-immunodeficiency highlights that patients with WHIM-syndrome, and probably other immunodeficiencies, may benefit from HPV immunoprophylaxis.

The role of indoleamine 2,3-dioxygenase in transplantation.

Indoleamine 2,3-dioxygenase (IDO), by enzymatic tryptophan degradation, has recently been proposed to have profound immunoregulatory activity. By most recent findings IDO induction follows reverse signaling of cytotoxic T-lymphocyte antigen-4 (CTLA-4) to its ligands CD80/86 and acts as a counter-regulatory mechanism to T-cell stimulation. With regard to transplantation, experimental evidence suggests that IDO has the potential to down-regulate allo-responses of T cells in vitro and to promote tolerance in murine models of pancreatic islet transplantation and of allogeneic T-cell transfer in vivo. However, the physiologic role of IDO in human organ transplantation still is to be elucidated. Experiments that clearly identify a significance of IDO in tolerance induction to vascularized organ allografts or in effecting costimulation blockade are required. In this review we provide a conceptual view of the current knowledge of IDO in the context of transplantation and, in light of its particular biological features, speculate about its potential application in novel therapeutic approaches for tolerance induction.

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells.

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN-gamma for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN-gamma for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.

Identical phenotype in patients with somatic and germline CD95 mutations requires a new diagnostic approach to autoimmune lymphoproliferative syndrome.

In a patient with a somatic mutation in the CD95 gene, the long-term evolution of the clinical phenotype was indistinguishable from that of patients with autoimmune lymphoproliferative syndrome caused by germline CD95 mutations. A new diagnostic algorithm for autoimmune lymphoproliferative syndrome is suggested incorporating studies on sorted TCRalpha/beta+CD3+CD8-CD4- T cells.

Monocyte-mediated T-cell suppression and augmented monocyte tryptophan catabolism after human hematopoietic stem-cell transplantation.

T-cell dysfunction after human hematopoietic stem-cell transplantation (HSCT) is generally attributed to intrinsic T-cell defects. Here we show that the characteristic impaired proliferative responses to polyclonal stimulation of post-HSCT peripheral blood mononuclear cells (PB-MCs) were markedly (4-fold) improved by T-cell enrichment. Conversely, addback of post-HSCT monocytes to these enriched T cells dampened their proliferative responses, suggesting that post-HSCT monocytes effectively mediate T-cell suppression. As a mechanism possibly contributing to monocyte-mediated T-cell suppression, we investigated monocyte tryptophan catabolism by indoleamine 2,3-dioxygenase into kynurenine, which has been implicated in regulating T-cell responses. Compared with controls, all post-HSCT monocyte-containing cell cultures (total PBMCs, monocytes, and monocyte/T-cell cocultures), but not monocyte-depleted populations, secreted elevated amounts of kynurenine. Blockade of tryptophan catabolism improved the proliferative responses. The slightly increased kynurenine release and substantial release of neopterin by unstimulated post-HSCT monocytes suggests that they were in a state of continuous activation. Superimposed on this state, stimulation of these cells caused a striking, additional increase (10-fold) in kynurenine release, and they triggered marked apoptosis of autologous post-HSCT T cells. We conclude that the amplified kynurenine release by post-HSCT monocytes, particularly induced upon stimulation, may underlie their suppressor activity, which in turn may contribute to the depressed T-cell immune responses after HSCT.