Aryl Hydrocarbon Receptor-Dependent Pathways in Immune Regulation.

The idea of possible involvement of the aryl hydrocarbon receptor (AhR) in transplant tolerance can be traced back >30 years, when very low doses of dioxin-the most potent AhR ligand-were found to markedly reduce the generation of cytotoxic T lymphocytes in response to alloantigen challenge in vivo. AhR is a ligand-activated transcription factor that is activated by dioxins and other environmental pollutants. We now know that AhR can bind a broad variety of activating ligands that are disparate in nature, including endogenous molecules and those formed in the gut from food and bacterial products. Consequently, in addition to its classical role as a toxicological signal mediator, AhR is emerging as a transcription factor involved in the regulation of both innate and adaptive immune responses in various immune cell types, including lymphocytes and antigen-presenting cells (APCs). Allograft rejection is mostly a T cell-mediated alloimmune response initiated by the recognition of alloantigens presented by donor and recipient APCs to recipient CD4(+) and CD8(+) T cells. Based on those findings, AhR may function as a critical sensor of outside and inside environments, leading to changes in the immune system that may have relevance in transplantation.

Cytotoxic T lymphocyte antigen 4-immunoglobulin G is a potent adjuvant for experimental allergen immunotherapy.

Allergen-specific immunotherapy (SIT) is the only treatment for allergic diseases that targets allergen-specific T helper type 2 (Th2) cells, which are the cause of the disease. There is an unmet requirement for adjuvants that increase the clinical efficacy of SIT allowing application of lower doses of the allergen, thereby reducing the risk of anaphylactic reactions. Cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA-4-Ig) has been shown to induce immunological tolerance in autoimmunity and allograft transplantation by blocking T cell co-stimulation and induction of the immunoregulatory enzyme indoleamine 2,3 dioxygenase (IDO). Previously, we showed that CTLA-4-Ig treatment at the time of allergen inhalation induced tolerance to subsequent allergen exposure in a mouse model of asthma. In this study, we test the hypothesis that CTLA-4-Ig acts as an adjuvant for experimental SIT. We evaluated the adjuvant effects of CTLA-4-Ig on SIT in a mouse model of ovalbumin-driven asthma. We used both wild-type and IDO-deficient mice to assess the role of IDO in the adjuvant effects of CTLA-4-Ig. Co-administration of CTLA-4-Ig strongly increased SIT-induced suppression of airway hyperreactivity (AHR), specific IgE in serum, airway eosinophilia and Th2 cytokine levels. Moreover, we found that CTLA-4-Ig, as an adjuvant for SIT, is equally effective in IDO-deficient and wild-type mice, demonstrating that the effect of CTLA-4-Ig is independent of IDO expression. We show that CTLA-4-Ig acts as a potent adjuvant to augment the therapeutic effects of SIT. As the adjuvant activity of CTLA-4-Ig is independent of IDO, we conclude that it acts by blocking CD28-mediated T cell co-stimulation.

The signaling function of IDO1 incites the malignant progression of mouse B16 melanoma.

Indoleamine 2,3 dioxygenase 1 (IDO1), a leader tryptophan-degrading enzyme, represents a recognized immune checkpoint molecule. In neoplasia, IDO1 is often highly expressed in dendritic cells infiltrating the tumor and/or in tumor cells themselves, particularly in human melanoma. In dendritic cells, IDO1 does not merely metabolize tryptophan into kynurenine but, after phosphorylation of critical tyrosine residues in the non-catalytic small domain, it triggers a signaling pathway prolonging its immunoregulatory effects by a feed-forward mechanism. We here investigated whether the non-enzymatic function of IDO1 could also play a role in tumor cells by using B16-F10 mouse melanoma cells transfected with either the wild-type Ido1 gene (Ido1WT ) or a mutated variant lacking the catalytic, but not signaling activity (Ido1H350A ). As compared to the Ido1WT -transfected counterpart (B16WT), B16-F10 cells expressing Ido1H350A (B16H350A) were characterized by an in vitro accelerated growth mediated by increased Ras and Erk activities. Faster growth and malignant progression of B16H350A cells, also detectable in vivo, were found to be accompanied by a reduction in tumor-infiltrating CD8+ T cells and an increase in Foxp3+ regulatory T cells. Our data, therefore, suggest that the IDO1 signaling function can also occur in tumor cells and that alternative therapeutic approach strategies should be undertaken to effectively tackle this important immune checkpoint molecule.

B7-1 engagement of cytotoxic T lymphocyte antigen 4 inhibits T cell activation in the absence of CD28.

Ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) appears to inhibit T cell responses. Four mechanisms have been proposed to explain the inhibitory activity of CTLA4: competition for B7-1 and B7-2 binding by CD28; sequestration of signaling molecules away from CD28 via endocytosis; delivery of a signal that antagonizes a CD28 signal; and delivery of a signal that antagonizes a T cell receptor (TCR) signal. As three of these potential mechanisms involve functional antagonism of CD28, an experimental model was designed to determine whether CTLA4 could inhibit T cell function in the absence of CD28. TCR transgenic/recombinase activating gene 2-deficient/CD28-wild-type or CD28-deficient mice were generated and immunized with an antigen-expressing tumor. Primed T cells from both types of mice produced cytokines and proliferated in response to stimulator cells lacking B7 expression. However, whereas the response of CD28+/+ T cells was augmented by costimulation with B7-1, the response of the CD28-/- T cells was strongly inhibited. This inhibition was reversed by monoclonal antibody against B7-1 or CTLA4. Thus, CTLA4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA4.

Mechanisms of CTLA-4-Ig in tolerance induction.

The size of the peripheral T lymphocyte pool remains relatively constant throughout adult life, but individual populations undergo expansion and contraction upon antigen encounter due to signals delivered by members of the B7-CD28 family of costimulatory molecules. This family includes receptors on T cells that can provide either activating or inhibitory signals. In general, activation occurs in response to pathogens, when lymphocyte expansion and acquisition of effector functions is appropriate. Conversely inhibitory receptors provide down-modulating signals that help terminate immune responses and maintain self-tolerance. The activating receptor CD28 engages the same B7-1 and B7-2 molecules as the inhibitory receptor cytotoxic T lymphocyte antigen 4 (CTLA-4), although with reduced affinity than CTLA-4. In addition to this direct competitive mechanism, CTLA-4 can directly inhibit T cell receptor (TCR) signals independently of CD28 expression and recent findings indicate that CTLA-4 may also operate through reverse signaling on ligand-expressing cells. Fusion proteins between the extracellular domain of CTLA-4 and an immunoglobulin Fc portion have been created that have potent immunosuppressive properties in animal models of transplantation and autoimmunity and that show great promise in clinical trials. Like CTLA-4, CTLA-Ig, is thought to selectively prevent activation of CD28 by interacting with B7-1 and B7-2. In addition, CTLA-4-Ig can bind to B7 molecules expressed on dendritic cells and activate a pathway of tryptophan catabolism that can lead to indirect inhibition of lymphocyte activation and T cell death. In this review, we will focus on the current knowledge of the mechanisms of action of CTLA-4 and CTLA-4-Ig.

T cell apoptosis by tryptophan catabolism.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We show here that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and of Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions, and was associated with the activation of caspase-8 and the release of cytochrome c from mitochondria. When administered in vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data suggest that the selective deletion of T lymphocytes may be a major mechanism whereby tryptophan metabolism affects immunity under physiopathologic conditions.

Immunization of HLA-A2+ melanoma patients with MAGE-3 or MelanA peptide-pulsed autologous peripheral blood mononuclear cells plus recombinant human interleukin 12.

Vaccination with dendritic cells (DCs) pulsed with tumor antigen peptides has shown promise in the treatment of melanoma. Interleukin (IL)-12 production by DCs is a key component for their efficacy. Murine studies have shown that IL-12 promotes potent antitumor immunization when coadministered with peptides loaded onto other class I MHC+ cells, thus bypassing the need to use DCs. The easiest cell source to obtain in large quantity from human patients is peripheral blood mononuclear cells (PBMCs). A Phase I clinical trial was thus performed in patients with metastatic melanoma using immunization with autologous PBMCs pulsed with a MAGE-3 or a MelanA peptide, coadministered with various doses of recombinant human (rh)IL-12. Patients receiving low-to-moderate doses of rhIL-12 developed increased specific CD8+ T-cell responses. Of the eight patients showing increased immunity, six had evidence of clinical activity, with one complete, one partial, one minor, and three mixed responses observed. In two patients with mixed responses, growing tumors were found to lack expression of the antigen used to immunize. Thus, vaccination with peptide-pulsed PBMCs plus rhIL-12 induces specific immunity and has clinical activity, without the need to generate DCs. Outgrowth of antigen-negative tumors argues for the future development of polyepitope vaccines.

Enhancement by IL-12 of the cytolytic T lymphocyte (CTL) response of mice immunized with tumor-specific peptides in an adjuvant containing QS21 and MPL.

Immunization of cancer patients with tumor-specific antigenic peptides is currently being tested in several clinical studies. We have examined the induction of CTL responses in mice after various modalities of peptide vaccination, to explore protocols that could be applied to humans. Our first model antigen was P198, which results from a point mutation in a normal gene. While two immunizations with peptide P198 in SBAS-1c adjuvant induced measurable CTL responses in less than 10% of DBA/2 mice, the addition of IL-12 to the peptide adjuvant mixture resulted in high CTL responses in nearly all mice. This strong enhancing effect of IL-12 was observed with 1,000 and 300 units and decreased gradually as the doses were reduced to 30 units. When IL-12 was replaced by other cytokines acting on T cells or antigen-presenting cells, such as IFN-gamma, IL-2, IL-6, IL-7, GM-CSF or MCP-3, no significant enhancing effect was observed. The same effect of IL-12 was obtained with peptide P1A, which is a major tumor-specific antigen of mastocytoma P815 and is encoded by a gene that is specifically activated in tumors.

Targeting metabotropic glutamate receptors in neuroimmune communication.

L-Glutamate (L-Glu) is the principal excitatory neurotransmitter in the Central Nervous System (CNS), where it regulates cellular and synaptic activity, neuronal plasticity, cell survival and other relevant functions. Glutamatergic neurotransmission is complex and involves both ionotropic (ligand-gated ion channels; iGluRs) and metabotropic receptors (G-protein coupled receptors). Recent evidence suggests that glutamatergic receptors are also expressed by immune cells, regulating the degree of cell activation. In this review we primarily focus on mGluRs and their role in the crosstalk between the central nervous and immune systems during neuroinflammation.

Using an ancient tool for igniting and propagating immune tolerance: IDO as an inducer and amplifier of regulatory T cell functions.

Although most CD4(+)CD25(+) regulatory T (T(reg)) cells develop in the thymus (i.e., natural T(reg) or nT(reg)), accumulating evidence suggests that they can also develop in the periphery (adaptive/induced T(reg) or iT(reg)). Both types of cells are functionally associated with the expression of Foxp3, a transcription factor that is constitutively expressed in nT(reg) cells and inducible during iT(reg) cell generation from CD4+CD25- T lymphocytes. Multiple factors are involved in the generation and function of T(reg) cells, but a major role seems to be played by indoleamine 2,3-dioxygenase (IDO). IDO can both deplete tryptophan in local tissue microenvironments and generate immunoregulatory catabolites, known as kynurenines. Tryptophan starvation and presence of kynurenines can induce the conversion of naïve CD4(+)CD25(-) T cells into highly suppressive CD4(+)CD25(+)Foxp3(+) T(reg) cells. In turn, T(reg) cells induce IDO in dendritic cells (DCs) and convert inflammatory into regulatory DCs, which can further expand the T(reg) cell compartment by tryptophan catabolism. Evidence suggests that the modulation of IDO activity favors the interconversion between T(reg) cells and T helper type 17 (T(H)17) inflammatory cells. Thus, in the periphery, tolerogenic immune responses mediated by T(reg) cells can be induced and amplified by IDO, a tryptophan catabolizing enzyme that also contributes to the plasticity of the T(reg) cell lineage.

Intranasally delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways protects from aspergillosis.

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Although some degree of inflammation is required for protection, progressive inflammation may worsen disease and ultimately prevents pathogen eradication. To define molecular pathways leading to or diverting from pathogenic inflammation in infection, we resorted to dendritic cells (DCs), known to activate distinct signaling pathways in response to pathogens. We found that distinct intracellular pathways mediated the sensing of conidia and hyphae by lung DCs in vitro, which translate in vivo in the activation of protective Th1/Treg responses by conidia or inflammatory Th2/Th17 responses by hyphae. In vivo targeting inflammatory (PI3K/Akt/mTOR) or anti-inflammatory (STAT3/IDO) DC pathways by intranasally delivered small interfering RNA (siRNA) accordingly modified inflammation and immunity to infection. Thus, the screening of signaling pathways in DCs through a systems biology approach may be exploited for the development of siRNA therapeutics to attenuate inflammation in respiratory fungal infections and diseases.

IL-22 defines a novel immune pathway of antifungal resistance.

The role of IL-17 and Th17 cells in immunity vs. pathology associated with the human commensal Candida albicans remains controversial. Both positive and negative effects on immune resistance have been attributed to IL-17/Th17 in experimental candidiasis. In this study, we provide evidence that IL-22, which is also produced by Th17 cells, has a critical, first-line defense in candidiasis by controlling the growth of infecting yeasts as well as by contributing to the host's epithelial integrity in the absence of acquired Th1-type immunity. The two pathways are reciprocally regulated, and IL-22 is upregulated under Th1 deficiency conditions and vice versa. Whereas both IL-17A and F are dispensable for antifungal resistance, IL-22 mediates protection in IL-17RA-deficient mice, in which IL-17A contributes to disease susceptibility. Thus, our findings suggest that protective immunity to candidiasis is made up of a staged response involving an early, IL-22-dominated response followed by Th1/Treg reactivity that will prevent fungal dissemination and supply memory.

Balancing inflammation and tolerance in vivo through dendritic cells by the commensal Candida albicans.

We analyzed the contribution of intracellular signaling to the functional plasticity of dendritic cells (DCs) presenting Candida albicans, a human commensal associated with severe diseases. Distinct intracellular pathways were activated by recognition of different fungal morphotypes in distinct DC subsets and in Peyer's patches DCs. Inflammatory DCs initiated Th17/Th2 responses to yeasts through the adaptor myeloid differentiation factor-88 (MyD88), whereas tolerogenic DCs activate Th1/T regulatory cell (Treg) differentiation programs to hyphae involving Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) as an intermediary of signaling. In addition, signal transducer and activator of transcription 3 (STAT3), affecting the balance between canonical and non-canonical activation of nuclear factor-kappaB (NF-kappaB) and 2,3 indoleamine dioxygenase (IDO), pivotally contributed to DC plasticity and functional specialization. As Candida-induced tolerogenic DCs ameliorated experimental colitis, our data qualify Candida as a commensal with immunoregulatory activity, resulting from the orchestrated usage of multiple, yet functionally distinct, receptor-signaling pathways in DCs. Ultimately, affecting the local Th17/Treg balance might likely be exploited by the fungus for either commensalism or pathogenicity.

Rational development of tumour antigen-specific immunization in melanoma.

The identification and molecular characterization of antigens expressed on tumour cells, but not on most normal host tissues, has opened the possibility of specific immunization in the therapy of cancer, particularly of melanoma. Most antigens defined are class I MHC-binding peptides recognized by CD8+ cytolytic T lymphocytes (CTL). Methodologies for active immunization to induce effective anti-tumour CTL are under development in a number of laboratories, and some of these approaches have entered clinical trials. Optimization of the anti-tumour immune response will depend on a thorough knowledge of the signals required for T cell activation, differentiation, and inactivation.

Cutting edge: differentiation of antitumor CTL in vivo requires host expression of Stat1.

Several lines of evidence suggest that an IFN-gamma-producing, Th1/Tc1 phenotype may be optimal for tumor rejection. Recent work has indicated that IFN signaling on tumor cells is important for protection against carcinogenesis. However, the potential involvement of IFN signaling among host immune cells has not been carefully examined. To this end, Stat1-deficient mice were employed as tumor recipients. In contrast to wild-type mice, Stat1-/- mice failed to reject immunogenic tumors and did not support regression of poorly immunogenic tumors when treated with an IL-12-based vaccine. T cells from immunized Stat1-/- mice produced 50% of the levels of IFN-gamma and lacked cytolytic activity compared with wild-type mice, and NK lytic activity also was not observed. Lack of cytolytic function correlated with a failure to up-regulate serine esterase activity. Thus, IFN-mediated signaling on host cells is required for the development of antitumor lytic effector cells.

Endogenous IL-12 is necessary for rejection of P815 tumor variants in vivo.

Although murine tumor cells have been transfected to express a multitude of different cytokines and shown to be rejected in vivo, it is unclear which of these factors might be useful to facilitate tumor Ag immunization schemes. A study of the normal immune mechanisms involved in tumor rejection when it naturally occurs should reveal critical signals for generation of antitumor CTL in vivo. The highly transfectable variant of P815, P1.HTR, was found to be rejected in the hind footpads by approximately one-third of syngeneic DBA/2 mice. Analysis of draining popliteal lymph nodes revealed a large influx of CD4+ and CD8+ T lymphocytes in all mice, indicating that a failure to reject was not due to the complete absence of an inflammatory response. However, although IL-2 and IL-3 were produced by lymph node cells from all mice, only approximately one-third generated a high IFN-gamma response. IL-4 was not detected. To explore a role for IL-12 in the induction of the IFN-gamma-producing phenotype, a histidine-tagged IL-12 fusion protein was expressed in mammalian cells and purified by nickel-chelate chromatography, and a rabbit antiserum was produced. Neutralization of IL-12 in vivo eliminated the high IFN-gamma response and prevented rejection of P1.HTR tumors and also of a more immunogenic tum- variant of P815, P198. Conversely, exogenous IL-12 delivered early during challenge with P1.HTR cells induced high IFN-gamma production and resulted in tumor rejection in most mice. Therefore, endogenous IL-12 is vital for the rejection of these tumors when it naturally occurs, supporting a role for exogenous administration of this cytokine to favor a Th1-like phenotype in the immunotherapy of cancer.

Antigen-specific regression of established tumors induced by active immunization with irradiated IL-12- but not B7-1-transfected tumor cells.

Transfection of modestly immunogenic tumors to express B7 family co-stimulator molecules results in their rejection by syngeneic mice, suggesting a possible clinical application in cancer patients. Immunization of naive mice with irradiated B7-1-transfected P1.HTR cells is sufficient to induce specific cytolytic T lymphocytes (CTL) and to protect against tumor challenge. However, patients to be treated will have an existing tumor burden; thus, preclinical models should examine therapeutic efficacy in an established tumor setting. Contrary to expectations, immunization of mice with irradiated B7-1-transfected P1.HTR cells had no impact on the growth of pre-established control-transfected tumors. Mice bearing control-transfected P1.HTR tumors successfully rejected living B7-1 transfectants on the contralateral flank, demonstrating the ability of tumor-bearing mice to respond to B7 co-stimulation. Inasmuch as IL-12 is another important factor for CTL maturation, P1.HTR transfectants expressing B7-1 and/or IL-12 were then constructed. Remarkably, regression of pre-established tumors was achieved following immunization with irradiated IL-12 transfectants, even without co-expression of B7-1. Rejection required a shared antigen with the tumor used for immunization, could not be reproduced with rIL-12 alone, depended on host T lymphocytes and correlated with a high IFN-gamma-producing T cell phenotype. In addition, IL-12-facilitated tumor rejection required co-operation with a CTLA-4 ligand provided by the host, and correlated with up-regulation of B7-1 and B7-2 on host antigen-presenting cells. Thus, active immunization in the established tumor setting is benefitted greatly by the provision of IL-12, which may recruit participation of sufficient B7 co-stimulation from the host that it need not be provided exogenously.

Improved efficacy of dendritic cell vaccines and successful immunization with tumor antigen peptide-pulsed peripheral blood mononuclear cells by coadministration of recombinant murine interleukin-12.

The well-characterized P815 tumor model was used to optimize anti-tumor immunization approaches in mice. Tumor peptides derived from antigens P198 or P1A were targeted to antigen-presenting cells (APC) by ex vivo pulsing. Initial experiments with irradiated pulsed splenic dendritic cells (sDC) injected weekly in the hind footpads for 3 weeks demonstrated cytolytic T lymphocyte (CTL) generation in 10-20% of mice. Because of the importance of interleukin-12 (IL-12) in tumor rejection responses, pulsed sDCs also were given together with recombinant murine IL-12 (rmIL-12). This strategy induced peptide-specific CTL in 100% of the mice. The IL-12 had to be injected in the footpads on days 0, 1 and 2 of each immunization week to achieve an optimal effect. The improvement seen with the addition of IL-12 prompted examination of other sources of APC. Purified resting B cells, lipopolysaccharide (LPS) blasts and nonfractionated splenocytes or peripheral blood mononuclear cells (PBMC) were pulsed with peptide and administered with the same schedule of rmIL-12. Because these cell types appeared to bind peptides less avidly than did DC, increasing peptide doses were used during pulsing. Interestingly, immunization with each of these APC also induced specific CTL in 100% of mice, provided rmIL-12 was coadministered. CTLs were detected both in the spleen and in the peripheral blood. Immunization with irradiated, P1A-pulsed PBMC plus rmIL-12 resulted in protection against challenge with tumors expressing the specific antigen in all mice. The ease by which human patient PBMCs can be prepared provides a straightforward vaccination approach to be used in clinical trials of peptide-based immunization in melanoma.