Discovery and Characterization of a Novel Aryl Hydrocarbon Receptor Inhibitor, IK-175, and Its Inhibitory Activity on Tumor Immune Suppression.

Aryl hydrocarbon receptor (AHR) is a transcription factor that regulates the activity of multiple innate and adaptive immune cells subsequent to binding to numerous endogenous and exogenous ligands. For example, AHR is activated by the metabolite kynurenine, which is secreted into the tumor microenvironment by cancer cells leading to broad immunosuppression. Therefore, AHR inhibition provides a novel and ideal approach to stimulate immune-mediated recognition and subsequent eradication of tumor cells. We report here the discovery and characterization of IK-175, a novel, potent and selective AHR antagonist with favorable ADME and pharmacokinetic profiles in preclinical species. IK-175 inhibits AHR activity in experimental systems derived from multiple species including mouse, rat, monkey, and humans. In human primary immune cells, IK-175 decreased AHR target gene expression and anti-inflammatory cytokine release and increased proinflammatory cytokine release. Moreover, IK-175 led to a decrease in suppressive IL17A-, IL-22+ expressing T cells in a Th17 differentiation assay. IK-175 dose dependently blocks ligand-stimulated AHR activation of Cyp1a1 transcription in mouse liver and spleen, demonstrating on-target in vivo activity. IK-175 increases proinflammatory phenotype of the tumor microenvironment in mouse syngeneic tumors and in adjacent tumor-draining lymph nodes. As a monotherapy and combined with an anti-PD-1 antibody, IK-175 demonstrates antitumor activity in syngeneic mouse models of colorectal cancer and melanoma. IK-175 also demonstrates antitumor activity combined with liposomal doxorubicin in syngeneic mouse tumors. These studies provide rationale for targeting AHR in patients with cancer. IK-175 is being evaluated in a phase I clinical trial in patients with advanced solid tumors.

Transfused platelets enhance alloimmune responses to transfused KEL-expressing red blood cells in a murine model.

BACKGROUND: Factors influencing the development of alloantibodies against blood group antigens on transfused red blood cells are poorly defined. We hypothesised that transfused platelets may act as a danger signal to recipients and affect humoral immune responses to transfused red blood cells. MATERIALS AND METHODS: Platelet-rich plasma prepared from wild-type C57BL/6 or CD40L knock-out donors was transfused into wild-type or CD40L knock-out recipients. Leucoreduced red blood cells from transgenic donors expressing high levels of the human KEL glycoprotein in an erythrocyte-specific manner (KELhi donors) were transfused after the platelets, and anti-KEL responses were measured longitudinally. In some experiments, recipients were treated with poly (I:C), monoclonal CD40L-blocking antibody, or CD4-depleting antibody prior to transfusion. RESULTS: Transfusion of wild-type C57BL/6 platelets or treatment with poly (I:C) prior to KELhi red blood cell transfusion led to an anti-KEL alloimmune response in wild-type recipients. Transfusion of platelets from wild-type but not CD40L knock-out donors prior to KELhi red blood cell transfusion led to an IgG anti-KEL alloimmune response in CD40L knock-out recipients; unexpectedly, transfusion of platelets from CD40L knock-out donors prior to KELhi red blood cell transfusion led to a robust anti-KEL alloimmune response in wild-type recipients. Recipient treatment with MR1 CD40L-blocking antibody or CD4-depleting antibody prevented KEL alloimmunisation altogether. DISCUSSION: Transfused platelets serve as an adjuvant in this T-dependent murine model of anti-KEL red blood cell alloimmunisation, with CD40/CD40L interactions being involved to some degree but with additional mechanisms also playing a role. These findings raise questions about the role that transfused or endogenous platelets may play in other innate/adaptive immune responses.

B cells require Type 1 interferon to produce alloantibodies to transfused KEL-expressing red blood cells in mice.

BACKGROUND: Alloantibodies to red blood cell (RBC) antigens can cause significant hemolytic events. Prior studies have demonstrated that inflammatory stimuli in animal models and inflammatory states in humans, including autoimmunity and viremia, promote alloimmunization. However, molecular mechanisms underlying these findings are poorly understood. Given that Type 1 interferons (IFN-α/ß) regulate antiviral immunity and autoimmune pathology, the hypothesis that IFN-α/ß regulates RBC alloimmunization was tested in a murine model. STUDY DESIGN AND METHODS: Leukoreduced murine RBCs expressing the human KEL glycoprotein were transfused into control mice (WT), mice lacking the unique IFN-α/ß receptor (IFNAR1-/- ), or bone marrow chimeric mice lacking IFNAR1 on specific cell populations. Anti-KEL IgG production, expressed as mean fluorescence intensity (MFI), and B-cell differentiation were examined. RESULTS: Transfused WT mice produced anti-KEL IgG alloantibodies (peak response MFI, 50.4). However, the alloimmune response of IFNAR1-/- mice was almost completely abrogated (MFI, 4.2; p < 0.05). The response of bone marrow chimeric mice lacking IFNAR1 expression in all hematopoietic cells or specifically in B cells was also diminished (MFI, 3.8 and 5.4, respectively, compared to control chimeras, MFI, 65.6; p < 0.01). Accordingly, transfusion-induced differentiation of IFNAR1-/- B cells into germinal center B cells and plasma cells was significantly reduced, compared to WT B cells. CONCLUSIONS: This study demonstrates that B cells require signaling from IFN-α/ß to produce alloantibodies to the human KEL glycoprotein in mice. These findings provide a potential mechanistic basis for inflammation-induced alloimmunization. If these findings extend to human studies, patients with IFN-α/ß-associated conditions may have an elevated risk of alloimmunization and benefit from personalized transfusion protocols.

CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model.

Approximately 3-10% of human red blood cell (RBC) transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that "non-responders" may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA) antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA) specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb)) demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken together, our data suggest that recipients may indeed become tolerized to antigens expressed on RBCs, with the recipient's immune status upon initial RBC exposure dictating future responses. Although questions surrounding mechanism(s) and sustainability of tolerance remain, these data lay the groundwork for future work investigating RBC immunity versus tolerance in reductionist models and in humans.

Type I IFN Is Necessary and Sufficient for Inflammation-Induced Red Blood Cell Alloimmunization in Mice.

During RBC transfusion, production of alloantibodies against RBC non-ABO Ags can cause hemolytic transfusion reactions and limit availability of compatible blood products, resulting in anemia-associated morbidity and mortality. Multiple studies have established that certain inflammatory disorders and inflammatory stimuli promote alloimmune responses to RBC Ags. However, the molecular mechanisms underlying these findings are poorly understood. Type I IFNs (IFN-α/ß) are induced in inflammatory conditions associated with increased alloimmunization. By developing a new transgenic murine model, we demonstrate that signaling through the IFN-α/ß receptor is required for inflammation-induced alloimmunization. Additionally, mitochondrial antiviral signaling protein-mediated signaling through cytosolic pattern recognition receptors was required for polyinosinic-polycytidylic acid-induced IFN-α/ß production and alloimmunization. We further report that IFN-α, in the absence of an adjuvant, is sufficient to induce RBC alloimmunization. These findings raise the possibility that patients with IFN-α/ß-mediated conditions, including autoimmunity and viral infections, may have an increased risk of RBC alloimmunization and may benefit from personalized transfusion protocols and/or targeted therapies.

Bortezomib decreases the magnitude of a primary humoral immune response to transfused red blood cells in a murine model.

BACKGROUND: Few therapeutic options currently exist to prevent or to mitigate transfusion-associated red blood cell (RBC) alloimmunization. We hypothesized that bortezomib, a proteasome inhibitor currently being utilized for HLA alloantibody and ADAMTS13 autoantibody reduction, may be beneficial in a transfusion setting. Herein, we utilized a reductionist murine model to test our hypothesis that bortezomib would decrease RBC alloimmune responses. STUDY DESIGN AND METHODS: Wild-type mice were treated with bortezomib or saline and transfused with murine RBCs expressing the human KEL glycoprotein. Levels of anti-KEL immunoglobulins in transfusion recipients were measured by flow cytometry. The impact of bortezomib treatment on recipient plasma cells (PCs) and other immune cells was also assessed by flow cytometry and immunofluorescence. RESULTS: After bortezomib treatment, mice had a 50% reduction in splenic white blood cells and a targeted reduction in marrow PCs. Mice treated with bortezomib before the transfusion of KEL RBCs became alloimmunized in three of three experiments, although their serum anti-KEL IgG levels were 2.6-fold lower than those in untreated mice. Once a primary antibody response was established, bortezomib treatment did not prevent an anamnestic response from occurring. CONCLUSION: To the extent that these findings are generalizable to other RBC antigens and to humans, bortezomib monotherapy is unlikely to be of significant clinical benefit in a transfusion setting where complete prevention of alloimmunization is desirable. Given the impact on PCs, however, it remains plausible that bortezomib therapy may be beneficial for RBC alloimmunization prevention or mitigation if used in combination with other immunomodulatory therapies.

Antigen modulation as a potential mechanism of anti-KEL immunoprophylaxis in mice.

Red blood cell (RBC) alloimmunization is a serious complication of transfusion or pregnancy. Despite the widespread use of Rh immune globulin to prevent pregnancy associated anti-D alloimmunization, its mechanism of action remains elusive. We have previously described a murine model in which immunoprophylaxis with polyclonal anti-KEL sera prevents alloimmunization in wild-type recipients transfused with transgenic murine RBCs expressing the human KEL glycoprotein. To investigate the mechanism of action, we have now evaluated the outcome of immunoprophylaxis treatment in mice lacking Fcγ receptors (FcγRs), complement (C3), both, or none. Whereas polyclonal anti-KEL sera completely prevented alloimmunization in wild-type and single-knockout (KO) mice lacking FcγRs or C3, double-KO mice lacking both FcγRs and C3 became alloimmunized despite immunoprophylaxis. Rapid clearance of essentially all transfused RBCs with detectable KEL glycoprotein antigen occurred within 24 hours in wild-type and single-KO recipients treated with immunoprophylaxis, with the transfused RBCs remaining in circulation having minimal KEL glycoprotein antigen detectable by flow cytometry or western blot. In contrast, transfused RBCs with the KEL glycoprotein antigen fully intact continued to circulate for days in double-KO mice despite treatment with immunoprophylaxis. Further, in vitro phagocytosis assays showed no consumption of opsonized murine RBCs by double-KO splenocytes. Taken in combination, our data suggest that modulation of the KEL antigen (and potentially RBC clearance) by redundant recipient pathways involving both FcγRs and C3 may be critical to the mechanism of action of polyclonal anti-KEL immunoprophylaxis. These findings could have implications for the development of immunoprophylaxis programs in humans.

Long-Term Exposure to House Dust Mite Leads to the Suppression of Allergic Airway Disease Despite Persistent Lung Inflammation.

BACKGROUND: Allergic asthma is a major cause of worldwide morbidity and results from inadequate immune regulation in response to innocuous, environmental antigens. The need exists to understand the mechanisms that promote nonreactivity to human-relevant allergens such as house dust mite (HDM) in order to develop curative therapies for asthma. The aim of our study was to compare the effects of short-, intermediate- and long-term HDM administration in a murine asthma model and determine the ability of long-term HDM exposure to suppress allergic inflammation. METHODS: C57BL/6 mice were intranasally instilled with HDM for short-term (2 weeks), intermediate-term (5 weeks) and long-term (11 weeks) periods to induce allergic airway disease (AAD). The severity of AAD was compared across all stages of the model via both immunological and pulmonary parameters. RESULTS: Short- and intermediate-term HDM exposure stimulated the development of AAD that included eosinophilia in the bronchoalveolar lavage fluid (BALF), pronounced airway hyperreactivity (AHR) and evidence of lung inflammation. Long-term HDM exposure promoted the suppression of AAD, with a loss of BALF eosinophilia and AHR despite persistent mononuclear inflammation in the lungs. Suppression of AAD with long-term HDM exposure was associated with an increase in both Foxp3+ regulatory T cells and IL-10-positive alveolar macrophages at the site of inflammation. CONCLUSIONS: This model recapitulates the key features of human asthma and may facilitate investigation into the mechanisms that promote immunological tolerance against clinically relevant aeroallergens.

Regulatory B cells in allergic airways disease and asthma.

B cells have long been known to participate in both innate and adaptive immune responses by contributing to antigen presentation and by producing antigen-specific antibodies. Recent evidence shows that certain B-cell subsets can also inhibit T-cell immune responses. Like regulatory T cells (Treg), these regulatory B cells (Breg) appear to comprise several subpopulations. How Breg cells are generated and how they control immune responses in vivo are just beginning to be elucidated. Here, we provide detailed instructions for the identification, isolation, and functional characterization of Breg cells in a murine model of allergic airway disease.

Bromelain Inhibits Allergic Sensitization and Murine Asthma via Modulation of Dendritic Cells.

The incidence of atopic conditions has increased in industrialized countries. Persisting symptoms and concern for drug side-effects lead patients toward adjunctive treatments such as phytotherapy. Previously, we have shown that Bromelain (sBr), a mixture of cysteine proteases from pineapple, Ananas comosus, inhibits ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). However, sBr's effect on development of AAD when treatment is administered throughout OVA-alum sensitization was unknown and is the aim of the present study. C57BL/6J mice were sensitized with OVA/alum and challenged with 7 days OVA aerosol. sBr 6 mg/kg/0.5 ml or PBS vehicle were administered throughout sensitization. Lung, bronchoalveolar lavage (BAL), spleen, and lymph nodes were processed for flow cytometry and OVA-specific IgE was determined via ELISA. sBr treatment throughout OVA-alum sensitization significantly reduced the development of AAD (BAL eosinophils and lymphocytes). OVA-specific IgE and OVA TET(+) cells were decreased. sBr reduced CD11c(+) dendritic cell subsets, and in vitro treatment of DCs significantly reduced CD44, a key receptor in both cell trafficking and activation. sBr was shown to reduce allergic sensitization and the generation of AAD upon antigen challenge. These results provide additional insight into sBr's anti-inflammatory and antiallergic properties and rationale for translation into the clinical arena.

LC-MS/MS Identification of a Bromelain Peptide Biomarker from Ananas comosus Merr.

Bromelain (Br) is a cysteine peptidase (GenBank AEH26024.1) from pineapple, with over 40 years of clinical use. The constituents mediating its anti-inflammatory activity are not thoroughly characterized and no peptide biomarker exists. Our objective is to characterize Br raw material and identify peptides in the plasma of Br treated mice. After SDS-PAGE in-gel digestion, Br (VN#3507; Middletown, CT, USA) peptides were analyzed via LC/MS/MS using 95% protein probability, 95% peptide probability, and a minimum peptide number = 5. Br spiked mouse plasma (1 ug/ul) and plasma from i.p. treated mice (12 mg/kg) were assessed using SRM. In Br raw material, we identified seven proteins: four proteases, one jacalin-like lectin, and two protease inhibitors. In Br spiked mouse plasma, six proteins (ananain, bromelain inhibitor, cysteine proteinase AN11, FB1035 precursor, FBSB precursor, and jacalin-like lectin) were identified. Using LC/MS/MS, we identified the unique peptide, DYGAVNEVK, derived from FB1035, in the plasma of i.p. Br treated mice. The spectral count of this peptide peaked at 6 hrs and was undetectable by 24 hrs. In this study, a novel Br peptide was identified in the plasma of treated mice for the first time. This Br peptide could serve as a biomarker to standardize the therapeutic dose and maximize clinical utility.

Splenic morphological changes are accompanied by altered baseline immunity in a mouse model of sickle-cell disease.

Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.