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The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ(+)CD8(+) T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα(+)IFNγ(+)CD8(+) T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

Notch activation, which is associated with basal-like breast cancer (BLBC), normally directs tissue patterning, suggesting that it may shape the tumor microenvironment. Here, we show that Notch in tumor cells regulates the expression of two powerful proinflammatory cytokines, IL1β and CCL2, and the recruitment of tumor-associated macrophages (TAM). Notch also regulates TGFβ-mediated activation of tumor cells by TAMs, closing a Notch-dependent paracrine signaling loop between these two cell types. We use a mouse model in which Notch can be regulated in spontaneous mammary carcinoma to confirm that IL1β and CCL2 production, and macrophage recruitment are Notch-dependent. In human disease, expression array analyses demonstrate a striking association between Notch activation, IL1β and CCL2 production, macrophage infiltration, and BLBC. These findings place Notch at the nexus of a vicious cycle of macrophage infiltration and amplified cytokine secretion and provide immunotherapeutic opportunities in BLBC. Significance: BLBC is aggressive and has an unmet need for effective targeted treatment. Our data highlight immunotherapeutic opportunities in Notch-activated BLBC. Effective IL1β and CCL2 antagonists are currently in clinical review to treat benign inflammatory disease, and their transition to the cancer clinic could have a rapid impact. Cancer Discov; 7(11); 1320–35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

Nuclear receptors (NRs) are key regulators of innate immune responses and tissue homeostasis. Evidence indicates that NRs significantly impact steady-state immune regulation, uptake and processing of apoptotic cells, tolerance induction, and control of inflammatory immunity. In this review, we describe our current understanding of the NR activity for balancing inflammation and tolerance, the signaling cascade inducing the NR activation and functional responses, and different mechanisms of the NR-driven immune effects in the context of autoimmune diseases. We further describe the ligand-activated transcription factor the aryl hydrocarbon receptor (AhR) that exhibits analogous functionality. Moreover, we will discuss the putative role of NRs and AhR in immune regulation and disease pathogenesis providing a rationale for therapeutic targeting as a unique opportunities in the clinical management of autoimmune diseases.

The Aryl hydrocarbon receptor (AhR) can potently modulate immunity at multiple levels, but its mechanistic role in immune regulation is still being elucidated. Here we show phagocytes exposed to apoptotic cells exhibit rapid activation of AhR driving IL-10 that antagonizes inflammatory cytokine production. AhR activation was dependent on apoptotic cell DNA-TLR9 interactions and reactive oxygen species (ROS) production in phagocytes that promoted nuclear accumulation and transcriptional activity. In vivo, apoptotic cell-induced AhR signals in myeloid cells were required for prevention of inflammatory innate and adaptive immunity against DNA and histones. Moreover, disease progression in lupus correlated with AhR signal strength, and disease course could be reduced or exacerbated by modulation of AhR activity. Finally, we observed myeloid specific deletion of AhR in mice resulted in systemic autoimmunity, and in SLE patients an increased AhR transcriptional signature correlated with disease. Thus, our findings suggest AhR activity influenced by apoptotic cell death is a key mechanism in maintenance of peripheral tolerance reducing inflammation and thereby retarding systemic autoimmune disease progression.

Tissue-resident macrophages (MΦ) are crucial in driving tolerance and preventing systemic autoimmunity. We have previously shown that exposure to apoptotic cells triggers a regulatory circuit dependent on IL-10 production in resident MΦ. However, key molecular mechanisms driving the regulatory response to apoptosis are not clear. RNA transcriptome analysis of MΦs after exposure to apoptotic cells identified strong transcript association with the aryl hydrocarbon receptor (AhR) signaling pathway, an association that was confirmed by phenotypic and biochemical analysis. When AhR activity was blocked, apoptotic cells induced an alteration in the mRNA signature enhancing proinflammatory effector expression. Functional analysis revealed that the DNA from apoptotic cells activated AhR in a reactive oxygen species (ROS) dependent mechanism and AhR is required for IL-10 production. Consequently, inhibition or deletion of AhR signals fundamentally altered immune responses to apoptotic cells in vivo resulting in proinflammatory cytokine production, increased effector T cell responses, and failure of long-term tolerance to apoptotic cell-associated antigens. Surprisingly, mice lacking AhR developed progressive systemic autoimmunity characterized by excessive MΦ and lymphocyte activation and renal pathology. Similarly, SLE-prone mice treated with AhR antagonist exhibited poor survival, while agonist treatment ablated disease pathology. Finally, an AhR transcriptional signature was significantly associated with active SLE flare in SLE patients. Thus, the data demonstrates the AhR pathway is a key molecular circuit responsible for apoptotic cell driven tolerance and suppression of inflammatory autoimmunity.

Abstract 2800 Poster Board II-776 Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic stem cell (HSC) chemotaxis, adhesion, proliferation, quiescence and differentiation are regulated by interactions with bone marrow (BM) niches. Two niches have been identified in the adult BM: the endosteal (close to the bone) and the perivascular niche (close to blood vessels). A vast body of literature has revealed the molecular basis for the interaction of HSCs with the two niches. However, the signals that regulate the communication between the two niches have not been well defined. Taking in consideration several clinical and experimental arguments this review highlights the molecular cues, involved in the communication between the BM niches, which regulate the basic properties of HSCs in physiological and malignant conditions. As such, it aims at clarifying the most important advances in basic and clinical research focusing on the role of different factors in the regulation of the BM microenvironment.