The role of AhR in transcriptional regulation of immune cell development and function

Journal Pre-proof The role of AhR in transcriptional regulation of immune cell development and function

Prashant Trikha, Dean A. Lee PII:

S0304-419X(19)30107-6

DOI:

https://doi.org/10.1016/j.bbcan.2019.188335

Reference:

BBACAN 188335

To appear in:

BBA - Reviews on Cancer

Received date:

26 July 2019

Revised date:

2 December 2019

Accepted date:

2 December 2019

Please cite this article as: P. Trikha and D.A. Lee, The role of AhR in transcriptional regulation of immune cell development and function, BBA - Reviews on Cancer(2019), https://doi.org/10.1016/j.bbcan.2019.188335

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© 2019 Published by Elsevier.

Journal Pre-proof

The role of AhR in transcriptional regulation of immune cell development and function.

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Prashant Trikha*,1 and Dean A. Lee*

*Cellular Therapy & Cancer Immunotherapy Program Center for Childhood Cancer & Blood Diseases WA-4112 Abigail Wexner Research Institute Nationwide Children’s Hospital 700 Children's Drive Columbus, Ohio 43205 Email: [email protected] 1

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Address:

Phone: 614 3555 3601

Abstract The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor (TF) that is a member of the Per-Arnt-Sim family of proteins. AhR regulates diverse processes, including ma-

Journal Pre-proof lignant transformation, hematopoietic cell development, and fate determination of immune cell lineages. Moreover, AhR forms a crucial link between innate and adaptive arms of the immune system. Malignant cells frequently evolve multiple mechanisms for suppressing tumor-specific responses, including the induction of suppressive pathways involving AhR and its metabolic byproducts in the tumor microenvironment that promote immune evasion and tumor progression. Thus, interest is high in further defining the role of AhR in carcinogenesis and immune development and regulation, particularly regarding the therapeutic interventions that unleash immune responses to cancer cells. Here, we provide an overview of the role of AhR in the regulation of

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innate and adaptive immune response and discuss the implications of targeting this pathway to

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augment the immune response in cancer patients.

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Keywords: AhR, Natural killer cells, Immune evasion, Cancer immunity, Immunotherapy

Journal Pre-proof 1. Introduction Aryl hydrocarbon receptor (AhR), a ligand-activated transcriptional factor (TF), shares several structural similarities with other members of the Pern-Arnt-Sim (PAS) superfamily of TFs. This superfamily comprises an ancient signaling system that mediates communication between the host and the external environment. AhR was initially identified as a receptor that could bind to dioxins and was primarily involved in detoxification and metabolism of environmental carcinogens (1-3). However, the role of AhR extends beyond xenobiotic metabolism, and a concerted effort is underway to elucidate its role during the development of and in the regulation of im-

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mune cell functions. AhR and its other PAS family members (i.e., AhR nuclear translocator

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(ARNT), HIF-1, and Per proteins) influence a diverse range of biological functions ranging from cellular differentiation, malignant transformation, regulation of immune responses, control

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of circadian rhythms and cellular metabolism (Figure 1).

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Numerous studies over the last two decades have shown that AhR signaling has a central role in

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shaping the innate and adaptive immune responses. An elegant study from Mellor’s laboratory was the first to demonstrate the role of tryptophan (Trp) in mediating immune suppression by

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showing that the establishment of the conceptus requires Trp catabolism. They showed that mammalian conceptus cells found at the maternal-fetal interface express the Trp metabolizing enzyme, indoleamine 2,3-dioxygenase (IDO), which can suppress the proliferation of T cells by

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reducing the Trp concentration. Mellor’s study also showed that IDO protects the fetus from ma-

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ternal T-cell attack reducing the risk of rejection (4).

Deregulation of the AhR pathway and its metabolites in the tumor microenvironment (TME) promotes immune evasion that supports tumor progression. Although no significant AhR mutations have been reported in any cancers, high AhR activity in cancer is prevalent through the upregulation of endogenous ligands (5). Furthermore, cancer cells have developed a variety of mechanisms to suppress tumor-specific responses via generation of suppressive immune cells, including myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs), and through the production of immunosuppressive molecules and cytokines. Tryptophan metabolites, particularly L-kynurenine, drive tumor invasion through an AhR-dependent mechanism, which seems to be the most pertinent in the cancer setting (6).

Journal Pre-proof 2. AhR structure and function 2.1. Regulation of AhR expression AhR serves as a cytosolic signal sensor upon binding to its ligands, which include polycyclic aromatic hydrocarbons (PAHs). AhR undergoes a conformational change, and is transported from the cytoplasm to the nucleus.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one such high-

affinity ligand that mediates its toxic effect via activating AhR by binding to the receptor (7, 8). Analysis of the AhR protein structure reveals three regions: 1) an N-terminal DNA binding domain (DBD), which is composed of the basic helix-loop-helix (bHLH) region and nuclear local-

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ization signal (NLS); 2) a central PAS domain which consists of two degenerate repeats, and 3) a

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C-terminal transactivation domain (TAD) (Figure 2). Furthermore, phylogenetic analysis has revealed that AhR is a protein of ancient origin with functional orthologues present in reptile,

tural differences.

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amphibians, birds, and mammals. Nevertheless, human and murine AhR genes have many strucSequence analysis has revealed up to 85% structural similarities in the N-

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terminus of the receptor, whereas the C-terminus exhibits lower homology. The TAD or the N-

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terminus is the least conserved (9). The C-terminal half is highly variable. This largely unstructured region contains a transcriptionally active domain and is involved in receptor transformation

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(10, 11).

In the cytoplasm, AhR forms a multi-protein complex with the heat shock protein-90 and

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X-associated protein 2. However, in the presence of a ligand or an agonist, the AhR complex

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translocates from the cytoplasm to the nucleus and forms a heterodimer with ARNT. The AhR/ARNT complex binds to dioxin response elements (DRE) located on the proximal site of gene promoters with a core sequence of 5ʹ-GCGTG-3ʹ. Both AhR and ARNT can recruit other transcriptional co-activators to regulate the expression of genes, including cytochrome P450 (CYP) and AhR repressor (AhRR). Once in the nucleus, AhR is degraded by the proteasome pathway (12). Like other signaling pathways, the AhR signaling cascade has a negative feedback arm. The AhR functionsare regulated and can be attenuated by another PAS family member, AhRR; the levels of AhRR rapidly increase following AhR activation (13).

AhRR, which is

structurally related to AhR but unlike AhR, has a transcriptional repressor domain and dimerizes with ARNT even in the absence of an agonist to effectuate its functions (14).

Journal Pre-proof 2.2. AhR knockout mice The generation of AhR knockout (AhR -/-) mice has provided valuable insight into elucidating the physiological functions and role of AhR during developmental and cancer. The AhR knockout mice were generated by ablating the exon-2 that encodes for the bHLH-DNA binding domain (15, 16). Targeting this domain leads to a frameshift resulting in the premature termination of the AhR protein. The AhR-/- mice are viable and fertile but show defects in liver development and hepatic functions. These mice have low liver weight and exhibit fatty metamorphosis and increased residual extra-medullary hematopoiesis. Also, the AhR-/- mice have a suppressed im-

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mune system with up to 80% reduction in the number of splenic cells, which resolves with age (16). AhR-/- mice are susceptible to developing liver cancer after treatment with the carcinogen

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diethylnitrosamine (17). Similarly, AhR-/- mice have a higher incidence of cecal and prostate

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cancer (18). Overexpression of AhR suppresses tumorigenesis in mice, suggesting that AhR may

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function as a tumor suppressor (19).

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AhR expression varies widely in organs, the liver, spleen, lungs, and kidneys have the highest expression. Epithelial cells have the highest AhR expression in the tissues.

Immunohistochemi-

liver cancers (20).

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cal studies have reported a high AhR expression in breast, prostate, gastric, small cell lung, and

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Adenomatous polyposis coli (APC) gene mutations, which cause the activation of the Wnt sig-

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naling pathway, lead to an increase in the levels of beta-catenin, a contributing factor to the initiation and development of colon carcinogenesis. Kawajiri et al., showed that AhR−/− mice were prone to develop tumors in the cecum, but not in the small intestine. This finding was in contrast to ApcMin/+ mutant mice, which developed numerous polyps in the small intestine. The authors further observed an increased expression of AhR in Paneth cells of the small intestine and the cecum adjacent to the ileocecal junction. Since β-Catenin accumulates in the intestines of AhR−/− mice, the authors concluded that β-catenin accumulation, together with microbial interaction, could promote cecal carcinogenesis (21). The generation of mice that express human AhR under the regulatory control of the mouse AhR locus should further help in elucidating the function of human AhR.

Journal Pre-proof 3. AhR and Signal Transduction AhR is involved in the regulation of diverse cellular processes such as cell proliferation, metabolism, and immune regulation. AhR mediates these effects either by directly binding to a gene promoter or through its interaction and regulation of signaling pathways (Figure 3).

3.1. Cell cycle control The role of AhR in cell cycle regulation is complex; multiple mechanisms have been proposed to explain its proliferative and anti-proliferative in response to AhR ligands in vitro. Promoters of

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many genes contain AhR binding sites or DRE, which exhibit enhanced expression following

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exposure to AhR ligands. AhR can function as a transcriptional activator by inducing the expression of genes involved and signaling pathways that are involved in cell proliferation, such as In contrast,

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platelet-derived growth factor and vascular endothelial cell growth factor-A (22).

exogenous ligands, particularly TCDD, can inhibit cell proliferation and induce cell cycle arrest.

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AhR following activation can bind to gene promoters like cyclin-dependent kinase p27kip1 (p27),

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leading to cell cycle arrest. The presence of an LXCXE motif within the PAS domain of AhR suggests that protein-protein interactions might mediate the effects of TCDD in cell proliferation

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(23).

The Rb-E2F pathway, which helps control the cell cycle, is disrupted in cancer (24, 25). One of

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the mechanisms through which AhR ligands regulate the cell cycle is via interaction with reti-

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noblastoma (Rb). Activated AhR forms a complex with hypo-phosphorylated Rb that prevents E2F-dependent transcriptional activation of S-phase target genes leading to cell cycle arrest (23). Similarly, AhR activation severely compromises the ability of hepatoma cells to progress through G1 phase due to the induction of p27. Consistent with a primary role of p27 in the cell cycle arrest in response to TCDD, fetal thymus cultures from p27-deficient mice were more resistant to the toxic effects of TCDD compared to control mice (26).

AhR also interacts with epidermal growth factor receptor (EGFR) and regulates cellular proliferation. Treatment of human colon cancer cell lines (H508 and SNU-C4) with TCDD induces cell proliferation due to the activation of EGFR, ERK1/2, and Src kinases. AhR can regulate Src activity by phosphorylating tyrosine residues.

These results demonstrate that cross-talk between

Journal Pre-proof Src and AhR induces the rapid proliferation of human colon cancer cells that leads to EGFR activation (27). Together, these studies demonstrate that the role of AhR in cell cycle regulation is complex and cell-type specific. AhR mediates many of its functions through interactions with other signaling pathways. The interaction of AhR with some of the major signaling pathways are briefly reviewed.

3.2. Estrogen receptor signaling Estrogens have an essential role in the growth, differentiation, and function of a broad range of

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target tissues in the human body. The estrogen receptor-α/β (ER) mediates estrogen’s functions.

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ER act as ligand-activated transcription factors (28). Similar to AhR, estrogen binds to estrogen response elements (EREs) located on gene promoters, regulating gene expression. Kociba et al.

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(1978) first reported the crosstalk between the AhR and ER signaling by examining the longterm effects of TCDD treatment in Sprague Dawley rats (29). They observed that female rats fed a

diet

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AhR

agonist

TCDD

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dimethylbenz[a]anthracene and induced mammary and uterine tumors. Activated AhR inhibits ER activity through many different mechanisms, including the induction of CYP1A1/CYP1B1 that can increase oxidative metabolism of E2 or the activation of proteasomes by AhR leading to

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the degradation of ERα (30). Benzo[a]pyrene(B[a]P), another AhR ligand, inhibited BRCA-1 expression and reduced cell proliferation in a dose and time-dependent manner in MCF-7 breast

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cancer cells (31). Collectively, these studies indicate that constitutive activation of AhR appears

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to have anti-estrogenic effects.

3.3. HIF-1 signaling

Hypoxia-inducible factor (HIF), another member of the PAS family of TFs, activates in response to reduced oxygen levels (32, 33). HIF has a vital role in sensing environmental cues and in coordinating the transcriptional control of metabolic pathways that drive glycolysis (34). Both AhR and HIF-1 are environmental sensors that can dimerize with ARNT (35). HIF-1 is frequently upregulated in glioblastoma (GBM) and can synergize with AhR to support GBM growth (36). HIF-1 also promotes the expression of hexokinase-2 and has a central role in the initiation of glycolysis (37). Studies using HIF-1R deficient T cells show that glycolysis in effector CD8+- T cells requires HIF-1/ARNT (38). Furthermore, the HIF-1/HIF-1 complex can trans-

Journal Pre-proof locate to the nucleus to regulate the expression of genes by binding to the hypoxiaresponse elements in genes involved in glycolysis, such as pyruvate dehydrogenase kinase 1 (39). Cross-talk between AhR and HIF signaling pathways occurs in other cell types, including macrophages. Lipopolysaccharide (LPS) stimulation leads to the activation of AhR and HIF in macrophages (40).

3.4. NFB pathway The NFB signaling pathway has a central role in regulating immune response, inflammation,

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and tumor development. In mammals, NFB has five family members (i.e., NFB1/B2, RelA/B,

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and c-Rel). NFB1 and NFB2 are synthesized as large precursors, which undergo posttranslational processing to generate the mature NFkB subunits p50 and p52, respectively. NFB

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signaling can regulate cellular processes through canonical (classic) or non-canonical pathways.

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In classic pathways, the pro-inflammatory cytokines such as IL-1 phosphorylate IB activate NFB. In the non-canonical cascade, the NFB2 p100/RelB complexes are inactive in the cyto-

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plasm (41).

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A link between the NF-κB and AhR signaling pathways was established while investigating the effects of IL-1β and TNF-α in primary hepatocytes (42). IL-1β could attenuate the TCDD mediA potential suppression

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ated induction of the AhR target gene cytochrome P450 isoforms.

mechanism involved NF-κB binding to the κB sites upstream of the AhR gene, which regulates

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its expression. Studies by Kimura et al., further helped elucidate the interaction between the AhR and NFB signaling pathways. The levels of IL-6 in Stat1−/− macrophages increased following LPS stimulation. AhR and Stat1 proteins can interact with the p50 subunit, which suggests the existence of a tripartite complex. Activation of AhR and Stat1 blocks the inflammatory response initiated by NF-κB (14, 43). AhR can also interact with RelB, a component of the non-canonical pathway. Studies using human macrophages U937 and hepatocytes HepG2 have revealed that transcriptional activation of the chemokine gene IL-8 requires a novel RelB/AhR responsive element (44). Moreover, AhR reduces inflammation in AhR−/− mice through the stabilization of the RelB protein (45).

Journal Pre-proof 3.5. STAT pathway The Stat signaling pathway has a fundamental role in mediating the inflammatory response. The Stat family consists of seven family members (Stat1-4, Stat5a-5b, and Stat6), which each have specific DNA-binding motifs for differentiation activation of gene transcription profiles. Gene knockout studies have helped elucidate various components of this pathway (46).

Analysis of the human IDO-1 promoter shows two STAT binding sites, and STAT3 can activate IDO expression in mouse dendritic cells (DCs) (47). Similarly, AhR promoter has Stat binding

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sites,. there was an increased expression in response to oncostatin M stimulation in HepG2,

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demonstrating that that AhR is a downstream target of IL6 (48). Inhibition of IL-6 or use of STAT3 inhibitors reduced IDO expression as well as the production of kynurenine (Kyn). Ulrike

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et al., demonstrate that Kyn, produced by IDO in tumor cells, activates the AhR, inducing IL-6 that drives IDO expression mediated via STAT3 in vitro. Inhibition of the AhR/IL-6/STAT3 axis

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was able to restore T cell proliferation in mixed leukocyte reaction. Moreover, increased expres-

lung adenocarcinoma (49).

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4. AhR and Immune Regulation

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sion of IDO and STAT3/CYP1B1 is associated with poor relapse-free survival in patients with

The role of AhR extends beyond the metabolism of xenobiotic and environmental carcinogens.

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Elucidating AhR’s role during development and in the regulation of innate and adaptive immune

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cell functions (Figure 4).

4.1. Hematopoietic stem cells Hematopoietic stem cells (HSCs) are multipotent, self-renewing progenitor cells that divide and differentiate to give rise to various immune cell lineages.

Under homeostatic conditions, HSCs

are in a quiescent state to prevent premature exhaustion and reduce their susceptibility to DNA damage. Under conditions of disease, tissue damage, or pathogen exposure, HSCs undergo selfrenewal and expansion to develop into mature immune cells to help fight infection and promote tissue regeneration and repair (50).

Journal Pre-proof Persistent AhR activation due to dioxin exposure affects the numbers and function of HSCs in mice. AhR usually acts as a negative regulator to curb excessive proliferation. Loss of AhR expression and/or function leads to a loss of quiescence. HSCs isolated from AhR-/- mice are hyperproliferative. Moreover, aging AhR-/- mice show features characteristic with premature bone marrow exhaustion (1).

The deletion of AhR leads to increased proliferation and self-renewal of

long term HSCs, in part, by influencing the microenvironment that regulates the equilibrium between quiescence and proliferation (51). AhR can affect the self-renewal capacity of HSC through the regulation of genes involved in hematopoiesis, such as PU.1, c-MYC, CXCR4, HES-

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1, and CEBP (1, 52).

TCDD, a classified human carcinogen,exposure to TCDD is associated with an increased inci-

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dence of leukemia and lymphoma (53). Several pathways are affected upon exposure to TCDD, but the precise mechanism(s) by which TCDD disrupts immune functions remains elusive. HSCs

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isolated from TCDD-treated mice exhibit a diminished capacity to reconstitute, home to marrow

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of irradiated recipients, and differentiate into mature immune cell lineages (54). Furthermore, TCDD treatment of donor mice diminished their capacity for LSK reconstitution (55). AhR promoter hypermethylation that leads to a reduction of AhR activity has been reported in acute lym-

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phocytic leukemia (ALL). Regulated AhR activity may provide a permissive environment for

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the selection and expansion of cancer cell clones in leukemias (56).

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Several strategies are being pursued to grow and expand HSC for clinical studies. StemRegenin 1 (SR1), a purine derivative, is a selective AhR antagonist that promotes the expansion of CD34+ progenitor cells. Culture of HSC with SR1 significantly enhanced the capability of HSC for long-term engraftment in immune-deficient mice. Studies show that SR1 promotes HSC expansion by antagonizing AhR function and is used for ex vivo expansion of HSC (57). In 2017 Kaufman’s laboratory corroborated these findings, demonstrating that AhR inhibition or deletion using CRISPR enhanced the differentiation of hematopoietic progenitor cells (58).

In summary,

these studies demonstrate that AhR is involved in the regulation of HSC functions, including self-renewal, proliferation, and differentiation.

Deregulation of AhR’s function may have an es-

sential role in the etiology and/or development of hematopoietic diseases associated with aging.

Journal Pre-proof 4.2. T lymphocytes T lymphocytes are a significant component of the adaptive immune system and have a central role in fighting infection and the eradication of cancer cells. Regulatory T (Treg) cells are a subpopulation of T cells that help maintain tolerance to self-antigens and prevent the development of autoimmune diseases. T lymphocytes regulate the strength of the immune response through processes often, but not exclusively, involving specialized Treg cells. Kerkvliet et al., demonstrated that TCDD could suppress the function of alloantigen-specific cytotoxic T cells by promoting the expansion of CD4+CD25+ Tregs. Since then, several studies have shown that AhR is involved in

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Treg development (59). For example, TCDD treatment decreases the incidence of type-1 diabe-

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tes and colitis in non-obese diabetic mice. Administration of TCDD promotes Treg generation in mice (60) suggests that the presence of endogenous AhR ligands may promote Treg cell devel-

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opment in the TME (59). Further evidence comes from a recent study using a green-fluorescent protein knocked into the endogenous AhR gene locus, which shows that Tregs found in the gut

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had the highest expression of AhR. This study confirmed the role of AhR in the development of

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Tregs in mice (61).

In healthy individuals, different subsets of Treg cells regulate the immune response. The Tregs

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produced in the thymus, referred to as natural Treg, and those generated in the periphery in response to various tolerogenic stimuli are known as induced Treg cells or iT(reg). Gandhi et al.

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demonstrated that AhR promotes the development of CD4+Foxp3--T cells (62). These cells pro-

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duce IL-10 and control the function of responder T cells via the production of granzyme B. However, the activation of AhR in the presence of TGF- promote the generation of Foxp3+iT(reg) cells, thus, opening the possibility of generating Treg via AhR activation for clinical studies (62). T helper 17 (Th17) cells are a unique subset in the CD4+ T cells that produce Th17 cytokine and have a pivotal role in the pathogenesis of several inflammatory diseases. These cells differentiate in response to IL-6 and TGF-. The transcription factors ROR-and RORt define the Th17 lineage and along with Stat3 are essential for IL-17 production (63). AhR promotes the differentiation of Th17 cells through several mechanisms. AhR can regulate Th17 expression via binding to the DRE sites on the Th17 promoter. Also, AhR can cooperate with Stat3 to induce the expres-

Journal Pre-proof sion of Aiolos (IKZF3), a member of the Ikaros family to reduce IL-2 expression, promoting the generation of Th17 cells (64).

Type 1 regulatory T cells (Tr1 cells), a type of regulatory T cells, produce IL-10 and have a crucial role in suppressing tissue inflammation, autoimmunity, and graft-versus-host disease. Tr1 cells require c-Maf, a TF, to produce IL-10. AhR is an essential regulator of T-cell differentiation. After T cell activation under Tr1 skewing conditions, AhR binds to c-Maf, promoting transactivation of the IL-10 and IL-21 promoters, leading to the generation of Tr1 cells. Studies from

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opment of murine and human regulatory Tr1 cells (65).

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Kuchroo’s lab have shown that the interaction between AhR and c-Maf is essential for the devel-

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Interleukin 22 (IL-22), an IL-10 family member, is involved in inflammation and has been linked to the development of autoimmune diseases. Epithelial cells lining the respiratory and di-

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gestives tracts predominately express the IL-22 receptor, which is composed of two chains, IL-

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10R2 and IL-22R. IL-22 was assumed to be associated with T helper type 1 (TH1) cytokine but has since been linked to Th17 cells. T cells isolated from AhR--/- mice are not able to produce IL22, which suggests that this receptor is essential for IL-22 production. The IL-22-producing cells

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have an essential role in skin homeostasis. Natural killer (NK) and NKT cells also produce IL-

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22. AhR agonists significantly alter the balance between IL-22 and IL-17-producing cells (66).

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4.3. B Cells

B lymphocytes, which are central components of the humoral immunity, are endowed with a vast repertoire of specificities against a variety of pathogens. Activation of a naive B cell upon antigen receptor stimulation results in their clonal expansion, antibody isotype switching, and differentiation into antibody-secreting plasma cells to effect a robust immune response (67).

During

infection, mature B cells in lymphoid nodes and secondary lymphoid organs undergo somatic hypermutation and recombine to generate cells with higher antigen affinity and distinct effector functions (68). During this course, B cells undergo differentiation to generate antibodyproducing plasma cells.

Journal Pre-proof AhR’s involvement in the development of B lymphocytes was reported by Li et al., using cord blood CD34 cells and a feeder cell line that promotes B cell development. Activation of AhR suppressed the generation of early B cell and pro-B cells. The authors further demonstrated that AhR regulates B cell differentiation via transcriptional suppression of early B cell genes, EBF1, and PAX5 (69).

AhR is highly induced following B cell activation and is essential in regulating activationinduced cell fate outcomes. Vaidyanathan et al., showed that AhR negatively regulates class-

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switch recombination by altering the expression of activation-induced cytidine deaminase. They

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demonstrated that AhR suppresses the differentiation of B cells into plasmablasts and antibodysecreting plasma cells (70). Further, evidence regarding the role of AhR in B cell was provided

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by Villa et al., found that AhR expression increases following treatment with IL-4 and B cell receptor (BCR) engagement. However, AhR‐ deficient B cells exhibited a reduced ability to pro-

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liferate and were unable to enter S-phase. Moreover, B cells lacking AhR were not able to com-

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pete with AhR+/+ B cell’s diminished ability to reconstitute an empty host and were incapable of mounting an antigen-dependent proliferative response in mice. Gene-expression profiling re-

control (71).

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4.4. Innate lymphoid cells

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vealed that ablation of AhR reduced the expression of cyclin O, a gene involved in cell cycle

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Innate lymphoid cells (ILCs) are a heterogeneous population of immune cells that develop from the common lymphoid progenitors (CLPs) but lack antigen-specific receptors. ILCs have a pivotal role in protection against micro-organisms and are involved in tissue remodeling after injury (72). ILCs are broadly divided into three-categories ILC1, ILC2, and ILC3; however, this classification is a simplistic view.

ILC diversity and functional programs are

more complex , especially during an immune response. Studies show that ILC subsets are functionally plastic and capable of converting into other subsets in response to appropriate cytokine stimulation (73).

Distinct ILC subsets develop from hematopoietic precursors in a process orchestrated by transcription factors. The three major groups of ILCs are further classified based on their cytokine

Journal Pre-proof signature, expression of transcription factors, and phenotypic markers. ILC1 express T-bet; Eomes produce interferon-γ (IFN-γ). ILC2 secrete IL-5, IL-9, IL-13, amphiregulin, and GATA3. ILC3s express Rorγt+ and produce IL-22 and IL-17. ILC2 and ILC3 rely primarily on IL-7, whereas natural killer (NK) cells and ILC1s depend on IL-15 (73). Several ILC1 subsets overlap considerably with NK. In fact, NK cells are the first ILCs with the characteristic property effector functions that help define the prototypical ILC (74).

AhR is highly expressed in ILC3 promoting their development, and ablation of AhR leads to de-

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fects in IL-22-production (75, 76). Both human and murine ILC22 cells express AhR. The ge-

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netic ablation of AhR reduces the number of ILC22 cells in mice (77), although the precise mechanism of how AhR regulates IL-22 expression remains largely unknown. AhR interacts

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with RORγt, and AhR deficient ILCs have a higher rate of apoptosis (75). Decreased levels of IL-22, reduced anti-microbial peptide production, and expansion of Th17 cells in the gut epithe-

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lial cells of AhR-deficient mice increasing the susceptibility of mice on developing bacterial in-

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fection. This study demonstrates a regulatory role for ILCs in controlling Th17 cell responses in mice (78).

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IL-1β is involved in the maintenance of expression of IL-22 and AhR in vitro while blocking the differentiation of immature NK cells into conventional NK cells, suggesting a central role for IL-

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1β in the development of IL-22-producing cells (66, 73). Only a subset of immature NK cells

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expresses IL-1 receptor type 1 (IL-1R1). Inhibition of AhR using an antagonist, or its silencing, promotes the differentiation of human tonsillar IL-1R1high ILC3s into CD56bright CD94+ mature NK cells, demonstrating that AhR prevents the differentiation of ILC3s into CD56bright NK cells. The authors postulated that IL-1 and AhR work collectively to block IL-1R1high ILC3 from differentiation into stage-IV mature-NK cells. IL-1 activates AhR promoting the expansion of IL22-producing IL-1R1hi ILC3s in response to environmental stimuli (79).

The WASH regulatory complex, a Wiskott-Aldrich syndrome protein (WASP) and SCAR homologue, is an actin-nucleating factor. Deletion of WASP leads to defects in hematopoiesis and impairs T cell proliferation in mice. WASP activates AhR expression by recruiting AT-Rich

Journal Pre-proof Interaction Domain 1A (Arid1a) to the AhR promoter, demonstrating that WASH-mediated AhR activation is essential for the maintenance of ILC3s in the gut (80).

The AhR pathway has an essential role in the gut immune system. Genetic studies have identified AhR as a locus for inflammatory bowel disease (81). AhR activation induces CYP1 enzymes, which facilitate metabolic clearance and detoxification. Thus, CYP1 enzymes have an important feedback role that curtails the duration of AhR signaling. Schiering et al., demonstrated that constitutive expression of CYP1A1 is restricted to intestinal epithelial cells, which lead to

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loss of AhR-dependent type 3 innate lymphoid cells and Th17 cells, increasing the risk of devel-

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oping enteric infection. This study suggests that intestinal epithelial cells serve as a pool of AhR ligands and highlights the importance of the feedback control to regulate the AhR pathway (82). reported the expression of AhR in gut ILC2 in mice. The authors

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A recent study by Li et al.,

showed that AhR inhibits ILC2 function in a cell-intrinsic manner, and ablation of AhR enhances

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gut ILC2 function during anti-helminth immunity. Furthermore, cell-specific activation of AhR

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enhances gut ILC3 function and is involved in anti-bacterial immunity demonstrating that the AhR pathway is involved in maintaining the balance between ILC2/ILC3 in the gut (83). In summary, the cross-regulation between innate and adaptive immune systems is important for the

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4.5. Natural killer cells

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immune balance, and the AhR-IL-22 axis has a critical role in maintaining homeostasis.

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NK cells are large granular lymphocytes that participate in the innate immune response to virally infected and neoplastic cells via specialized receptors. The cytotoxic function of NK cells is regulated by the interplay between inhibitory and activating signals. Activated NK cells secrete cytokines such as interferon IFN- that inhibit tumor cell proliferation, enhance antigen presentation, and aid in the recruitment of T cells (84, 85). NK cells are defined as lineage Lin−CD56+ lymphocytes that lack CD3 but express other pan-NK cell markers, like NKp46 (86, 87). Human NK cells have two subsets based on CD56 expression: CD56bright and CD56dim. Traditionally the CD56dim NK cell subset is thought to mediate antitumor responses, whereas the CD56bright subset is involved in immunomodulation. The CD56 dim subset, which comprises 80%–95% of peripheral blood NK cells, represents the most mature NK

Journal Pre-proof cell population in humans (stage V) and expresses the inhibitory receptors killer cell immunoglobulin-like receptors (KIRs) and cytotoxic effector molecules (perforin and granzyme B), and highly expresses CD16 (FcγRIIIa) (88, 89). In contrast, the less mature CD56bright cells, representing ~10% of the circulating NK cells, are potent cytokine producers. CD56 bright cells with low CD16 expression have a higher capacity for ex vivo proliferation and a lower capacity for natural cytotoxicity compared to the CD56dim cells. CD56bright NK cells have lower expression of cytotoxic molecules and primarily rely on CD94/NKG2A receptors rather than KIRs for selftolerance (84). Furthermore, CD56bright NK cells are thought to act as precursors of CD56dim NK

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followed by an accumulation of CD56dim NK cells (85).

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cells, since CD56bright NK cells appear first in the blood following bone marrow transplantation

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The signaling pathways and genes involved in the differentiation of CD56 dim to CD56bright subset have remained an enigma. Although AhR modulates the function of NK cells in murine NK

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cells, AhR’s involvement in effecting the differentiation of human NK cell subsets has only been

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recently demonstrated (90). Studies from other labs and our lab have recently shown higher expression of AhR in CD56bright NK cells compared to CD56dim cells. Moreover, a reduction in AhR levels in NK cells is observed during development as NK cells acquire a more mature phe-

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notype. Interestingly, inhibition of AhR using the antagonist StemRegenin-1 (SRI) promotes the development of the mature CD56dim subset (unpublished data). We believe AhR functions as a

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molecular switch that is involved in the differentiation of CD56 bright to CD56dim cells. However,

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more studies are required to elucidate the molecular mechanism(s) through which AhR mediates the switch between the two NK cell subsets.

The mouse NK cells are similar to their human counterparts in many aspects, such as cytotoxic function and cytokine production; however, they lack the human homolog of CD56. Therefore, the assessment of analogous murine NK populations remained elusive for a long time. Murine NK cells are sub-divided into four majors subsets based on the surface expression of CD27 and CD11b (91). Gene signatures generated using mouse ILCs (CD127+) are enriched for genes expressed by CD56bright NK cells. Various cell-intrinsic factors are involved in the differentiation of CD56bright to CD56dim cells. Transcriptome analysis has revealed a higher expression of GATA3, TCF7 (TCF-1), AhR, SOX4, and RUNX2 in CD56bright cells. Whereas higher transcription of

Journal Pre-proof ZEB1, AIOLOS, T-bet, NFIL3, ZEB2, and PRDM1 is observed in CD56dim cells. This study suggests an ontological relationship between the mouse CD127 + ILC and CD56bright human NK cells (92, 93).

While the role of AhR in regulating the function of murine NK cells has been extensively studied, transcriptional regulation of AhR remains mostly unknown. In drosophila, the invertebrate Dlx ortholog Distal-less (Dll) regulates Spineless, which is involved in the development of distal antenna segments. The vertebrate ortholog of Spineless is AhR; Dlx3 is constitutively co-

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expressed with AhR in murine and human CD127 + NK cells. NK-92, a human NK cell line, transfected with human Dlx3 vector enhanced AhR expression and luciferase reporter activity in

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vitro, which demonstrated Dlx3 is a positive regulator of AhR expression in CD127 + NK cells

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(94).

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AhR expression is enhanced in NK cells following cytokine stimulation and has an essential role in controlling the anti-tumor response of NK cells. AhR-/- mice showed no change in the number

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of NK cells (NKp46+NK1.1+CD3−CD19−) compared to their littermate controls. AhR-/- mice inoculated with RMA lymphoma cells had a higher incidence of tumor growth, which was attribut-

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ed to a defect in the anti-tumor function of AhR deficient NK cells. In contrast, treatment with an AhR agonist, FIZC, resulted in a significant upregulation in NK cell cytotoxicity, and IFN-γ pro-

NK

cell

activity

(95).

AhR

is

also

required

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maintenance

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rine

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duction inhibited tumor growth demonstrating the involvement of AhR in the regulation of muCD49a+TRAIL+CXCR6+DX5− of resident NK cells, and AhR-/- mice show a significant reduction in number in the liver. Moreover, in the absence of memory NK cells, AhR-/- mice are unable to mount an effective response to hapten re-challenge. The authors concluded that longevity is the hallmark of memory cells and AhR promotes the generation of memory NK cells by reducing proliferation induced exhaustion (96).

Subversion of the anti-tumor function of NK cells by cancer cells is the principal mechanism involved in tumor progression and metastasis (97). Altered tryptophan (Trp) metabolism and increased secretion of tryptophan metabolites like kynurenine (Kyn) have been implicate immune evasion. Scoville et al., reported that AML blast suppression of NK cell function involved acti-

Journal Pre-proof vating the AhR signaling pathways, which lead to increased expression of miR-29b (98). Acute lymphoblastic leukemia (ALL) are resistant to NK cell-mediated killing. However, NK cell functions can be augmented using plasmacytoid dendritic cells (pDC). Díaz-Rodríguez et al., indicated that an AhR antagonist favors dendritic differentiation from CD34 + human progenitors. pDC obtained by in vitro differentiation of CD34 + progenitors in the presence of AhR antagonists were more efficient than PB-pDC to stimulate NK cell lytic function despite lower production of IFN-α. Moreover, these pDC augmented the cytotoxic function of NK cells, thereby en-

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hancing NK cells ability to destroy ALL tumor cells (99).

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In addition, to their anti-tumor functions, NK cells have a pivotal role in the control of infections. The secretion of potential AhR ligands by the parasite T. gondii suggests that AhR can function

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as a sensor that can detect metabolic products during an infection. AhR signaling also influences NK cell production of IL-10, which has a vital role in limiting inflammation during toxoplasmo-

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sis infection. Wagage et al., using IL-10 reporter mice, showed that IL-12 signaling alone was

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not sufficient to promote the secretion of IL-10 by NK cells, demonstrating that AhR’s role is a critical co-factor involved in production of IL-10. Furthermore, AhR-/- mice infected with T.

4.6. Dendritic cells

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(100).

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gondii had reduced levels of IL-10, which was associated with increased resistance to infection

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Dendritic cells (DCs) are professional antigen-presenting cells that help bridge between innate and adaptive immunity. Any defects in their development and/or function can lead to significant immune-deficiencies (101). Nguyen et al., observed increased expression of AhR in bone marrow-derived dendritic cell (BMDC) treated with LPS and CpG, and that AhR is involved in the regulation of IDO.

They further established that the levels of Kyn and IL-10 decreased in

AhR-deficient BMDC in response to LPS/CpG stimulation.

In contrast to wild type BMDC,

LPS/CpG-stimulation of AhR-/- BMDC inhibited the differentiation of naive T-cell into Treg cells, demonstrating the role of AhR in the regulation of immunogenic activities of DC (102).

Activated plasmacytoid DCs (pDCs) express IDO, which is postulated to be central to Treg generation from T cell precursors, although the precise mechanism of how IDO promotes the gener-

Journal Pre-proof ation of Tregs is not entirely understood. Both tryptophan starvation and its metabolite, Kyn, can promote the generation of Tregs. Kyn binds to the AhR on T cells, leading to their differentiation into Foxp3+-Treg cells. The absence of the AhR on T cells prevents this effect, suggesting that ligand-receptor interaction may be required for the generation of Treg (103).

AhR activation can affect BMDC differentiation; however, it does not affect the BMDC

ability of

to suppress antigen-specific activation of CD4+-T cells. AhR activation alters inflamma-

tory DC differentiation, generating DCs that are hyper-responsive to TLR stimulation and exhibit

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a regulatory phenotype in vitro (104). Similarly, in another study Vorderstrasse et al., revealed

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that DCs are sensitive targets of TCDD, and AhR activation results in a loss of splenic DCs (105). TCDD-treatment of BMDCs enhanced TNF-α-induced maturation and augmented CD95-

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mediated BMDC apoptosis (106). In summary, AhR activation can alter the differentiation and

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innate functions of inflammatory DCs but does not alter their ability to activate T cells.

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4.7. Myeloid cells

HSC in the bone marrow (BM) gives rise to two distinct progenitors: CLP and CMP. These progenitors lose their self-renewal ability and acquire lineage makers due to the expression of dis-

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tinct transcriptional activities. Circulating monocytes emigrate from the BM into the blood and give rise to tissue macrophages or dendritic cells. How monocyte fate is directed toward mono-

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cyte-derived macrophages or monocyte-derived DCs, and the role of transcription factors in con-

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trolling differentiation pathways remains unexplored. MafB is highly expressed by all mouse macrophage populations except for lung macrophages (107, 108). Using an in vitro culture model Goudot et al., demonstrated that IRF4 and MAFB are involved in differentiation of monocytes into DCs and macrophages, respectively. Activation of AhR promoted monocytes to DC differentiation via the induction of BLIMP-1, while interfering with the ability to differentiate into macrophages. The authors further demonstrated that AhR deletion reduces the differentiation of mouse DCs, thus identifying AhR as one of the TFs involved in determining the fate of monocyte/macrophages (109). LPS induces AhR expression in macrophages. The production of IL-6 and TNF-α was significantly elevated in AhR-deficient macrophages following LPS stimulation. In response to LPS stimulation, AhR forms a complex with Stat1 to regulate NF-κB-dependent pro-inflammatory responses and cytokine production in macrophages (43).

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Macrophages are also implicated in the pathogenesis of rheumatoid arthritis (RA). AhR activation is linked to RA pathogenesis, and AhR agonists can upregulate the expression of IL-1β in human synoviocytes. AhR can also facilitate the development of RA by promoting the development of Th17 cells (110). Levels of ARNT and CYP1B1 were significantly reduced in RA compared to osteoarthritis synovial tissue. Notch activation represses miR-223 expression in RA macrophages, thus, identifying miR-223 as a negative regulator of the AhR/ARNT pathway in

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RA derived macrophages (111).

NLRP3, an inflammasome, is a multi-protein complex required for the activation of inflammato-

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ry responses; NLRP3’s expression must be regulated to maintain homeostasis. AhR activation

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reduces NLRP3’s expression, caspase-1 activation, and subsequent IL-1β secretion by peritoneal macrophages. AhR can bind to a promoter of NLRP3 and prevent inflammasome activation.

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Thus, AhR is considered a potential therapeutic target for inflammatory diseases like atheroscle-

5. Targeting the AhR pathway

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rosis, obesity, and diabetes (112).

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Alteration in kynurenine pathway (KP) is associated with a number of pathological conditions, including cancers. Elevated levels of kynurenic acid (KA) has been reported in solid cancers

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such as colon, prostate, cervical, non-small cell lung cancer (NSCLC), liver and breast cancers

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(113-116). Tumor cell lines derived from patients with colon cancer have increased KA levels in the supernatants compared to the healthy controls (113). Similarly, serum samples obtained from NSCLC patients showed elevated levels of KA (114). In contrast, the levels of KA in serum samples of patients with GBM were reduced when compared to healthy donors, due to an increased ratio of Kyn to Trp (115). In another study, Optiz et al., reported that activation of the AhR pathway is associated with tumor progression and poor survival in patients with GBM. Furthermore, gene expression profiling of samples revealed that patients with high expression of TDO and AhR target gene CYP had lower overall survival compared to patients with intermediate expression of these genes, demonstrating that tumor-derived Kyn plays a pivotal role in the pathogenesis of GBM (6).

Journal Pre-proof The KP is involved in Trp catabolism and a prospective way that links the immune system and cancer. Kyn is a key intermediate and required for the synthesis of many downstream metabolites. The KP is also involved in the de novo synthesis of NAD+ and is a key cofactor involved in energy metabolism. Therefore, depletion of Kyn may alter the cellular energy supply that can affect immunological and neurological homeostasis (117). Degradation of the extracellular pool of Kyn by systemic or local delivery of recombinant kynureninase (KYNU) is an innovative approach of targeting the IDO1/TDO2/AhR axis (118). The depletion of Kyn prevents AhR activation and promotes anti-tumor immunity (119). It will be interesting to determine whether dual

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IDO1/TDO2 inhibitors, in combination with KYNU that depletes Kyn, can achieve a tolerable

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therapeutic effect in cancer patients.

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Several strategies targeting the Trp/IDO/Kyn axis, including the development of small-molecule AhR antagonist and IDO/TDO inhibitors, are being pursued to inhibit immune suppression. Ac-

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tivation of AhR can induce immune tolerance and promote immune evasion through the genera-

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tion of suppressive immune cells (MDSC and Tregs) and the production of molecules like Kyn (3). Therefore, modulating AhR function by targeting the AhR and related signaling pathways may provide a novel therapeutic option for cancer treatment and other inflammatory diseases (6).

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IDO1 inhibitors such as Epacadostat (INCB24360) and Navoximod (NLG-919) are at various phases of pre-clinical development. Clinical data indicates that these inhibitors are well tolerated

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with manageable toxicity (120, 121).

Like IDO, tryptophan 2,3-dioxygenase (TDO-2) is freinvolved Trp metabo-

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quently overexpressed in several cancers and represents another enzyme

lism (122). Since none of the IDO inhibitors target TDO, this provides a strong rationale and makes TDO an attractive target for inhibition in cancer. Early pre-clinical studies using TDO inhibitors show no signs of toxicity in mice. A possible consequence of systemic TDO inhibition is an increase in levels of Trp which can be metabolized into Kyn by the tumor cells (123).

The cells in the immune system are continuously being exposed to endogenous and exogenous AhR ligands that can interfere with physiological function altering immune homeostasis and develop into inflammatory diseases, autoimmune disorders, and cancer. are currently in development.

Several AhR antagonists

The inhibition of AhR can make immune-based therapies more

effective by relieving immune-suppression (124). These molecules compete for ligand binding

Journal Pre-proof sites, thereby preventing the translocation of AhR to the nucleus abrogating its function (125). Compared to IDO inhibitors, the development of anti-cancer therapy via direct AhR inhibition is still in its infancy and requires a more comprehensive understanding regarding the AhR’s role in cancer biology and tumor immunology. Some IDO inhibitors can bind and activate AhR, potentially counteracting the effect of inhibitors, which should open new research avenues and accelerate the development of AhR inhibitors since AhR is involved in the development of immune cells and modulates immune response/functions (126).

As with any small molecule inhibitors,

one challenge of targeting TF, like AhR, is to reduce off-target effects and to develop inhibi-

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tors/antagonists that can selectively inhibit AhR in target cells. Thus, new approaches are re-

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quired to enhance anti-tumor immunity and overcome AhR/IDO/Kyn-mediated immune suppression. In summary, these studies demonstrate the drugs that target the AHR/KP pathway can

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ameliorate tumor growth by activating immune responses and can be used as an immune checkpoint inhibitor alone or in combination with other immune/cell-based therapies for cancer treat-

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ment.

6. Conclusions and future directions

Now is an exciting time for AhR research! Investigating the role of AhR in immune cell develregulation of immune response, and development of immune tolerance has generated

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opment,

tremendous interest. AhR was initially identified as a sensor of environmental chemicals (diox-

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ins) and a regulator of drug-metabolizing enzymes. In the last decade, an appreciation of AhR’s

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role in the regulation of normal physiological processes has increased. As the number of AhRmediated processes grows, interest in determining the source of endogenous AhR ligands and their role in pathologic processes such as cancer is mounting.

However, many questions remain unanswered regarding the mechanism(s) via which AhR elicits these diversified effects. Global analysis of AhR-directed transcriptome, cistrome, interactome, and metabolome using chromatin immunoprecipitation coupled with next-generation sequencing of immune cells, will help in unraveling some of these questions. The emerging concept of AhR function in a cell-type-specific manner combined with differences between AhR activation in vitro and in vivo, present challenges for targeting the AhR pathway pharmacologically. Also, indepth investigations of AhR function in the TME (tumor cells and immune cells) will help in un-

Journal Pre-proof derstanding the crosstalk between the two components. Recent advances in immunotherapy, which include the use of immune checkpoint inhibitors (CKI), have opened new therapeutic options leading to improved patient survival (127, 128). However, not all cancer patients respond to CKI. To be effective, these therapies must be able to overcome barriers imposed by the suppressive TME. The TME is inherently suppressive due to the presence of high levels of immune suppressive cells (e.g., MDSC) and molecules like Kyn that can dampen the function of NK and T cells rendering immune-based therapies ineffective. A combination of CKI with agents targeting the AhR/IDO/Kyn axis can potentially augment the response of cell and immune-based thera-

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pies.

Journal Pre-proof Acknowledgments: We would like to thank Melody Davis for her help with editing and proofreading.

Disclosures: PT has no conflict of interest or financial disclosures. DAL reports stock ownership

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in CytoSen Therapeutics.

Journal Pre-proof Figure Legends: Figure 1. AhR signaling Aryl hydrocarbon receptor (AhR), a ligand-activated transcriptional factor is involved in the regulation of diverse cellular processes; cell proliferation, metabolism, and immune regulation. Genes and/or molecules associated with the various AhR regulated pathways are shown. STAT, signal transducer and activator of transcription; HIF-1 , hypoxia-inducible factor-1 alpha; HSC, hematopoietic stem cell; IL-6, interleukin-6; IL-10, interleukin-10; NFkB,_nuclear factor kappa B; NLRP3, NLR family pyrin domain containing 3; CYP, cytochrome P450; RB,

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retinoblastoma.

Figure 2. Structure of the human AhR gene.

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Schematic showing the organization of the human AhR gene by functional domains with its interacting proteins. The N-terminus consists of DNA binding domain (DBD), which is composed

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of a nuclear localization signal (NLS) and a basic helix-loop-helix (HLH) motif.

The N-

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terminus is involved in binding of DNA interaction and protein binding. AhR is composed of two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B). The PAS domains are involved in

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ligand binding and protein-protein interaction. The C-terminus contains a transactivation domain (TAD) composed of a proline-rich (Q) domain, acidic (AC), and (P/ST) regions. The Q and

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P/ST domains are involved in ligand-induced transport and nucleocytoplasmic shuttling. Genes and/or proteins associated with the different AhR domains are shown. HSP90, heat shock protein

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90; AIP, AhR interacting protein; ARNT, AhR nuclear translocator; HIF, hypoxia inducible factor: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; SRC-1. Steroid receptor coactivator-1; NcoA2, nuclear coactivator-2; CBP, CREB binding protein.

Figure 3. Interaction of AhR with other signaling pathways. Binding of the ligand to its cognate receptor AhR leads to activation of downstream signaling pathways that are involved in cell proliferation, migration, immune regulation, and inflammation. MAPK, mitogen activated kinase; ERK, extracellular signal regulated kinase.p27kip , cyclin-dependent kinase inhibitor-1B ; CDK4, cyclin dependent kinase 4; RB, retinoblastoma; PKC, protein kinase C; PLA2, phospholipase A2; PGE2, prostaglandin E2; SOCS, suppressor of cytokine signaling; JAK, janus kinase.

Journal Pre-proof Figure 4. Role of AhR ligands in immune cell development AhR interacts with exogenous and endogenous molecules originating from diverse sources. AhR ligands such as dioxins (e.g., TCDD), which industrial processes generate as a by-product, are present in the environment. Endogenous ligands like prostaglandin G2 (PGG2) are produced during inflammation from arachidonic acid.

Cruciferous vegetables also are a significant source

of AhR agonists. They are rich in sulfur-containing compounds, e.g., glucosinolates, that on hydrolysis produce indole-3-carbinol. Kynurenine, an immune-suppressive molecule, is produced during the tryptophan metabolism by cancer cells and myeloid in the tumor stroma. Upon bind-

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ing to the ligand, AhR translocates to the nucleus. AhR forms a hetrodimer with its partner,

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ARNT. The AhR/ARNT complex binds to xenobiotic response elements (XRE) that are located at the proximal site of gene promoter regulating gene expression. AhR is involved in regulating

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signaling pathways involved in differentiation and development of diverse immune cells. Green

opment and/or function of immune cells.

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arrows indicate that AHR activation promotes and red arrow shows AhR suppresses the devel-

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MDSC, myeloid-derived suppressor cells; IDO, indoleamine 2,3-dioxygenase; TDO, tryptophan 2,3-dioxygenase; Trp, tryptophan; Kyn, L-kynurenine; Th17, T helper cell 17; DC, dendritic

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lymphoid cells.

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cell; Treg, T regulatory cell; Tr1, type 1 regulatory T cells; NK, natural killer cells; ILCs, innate

Journal Pre-proof REFERENCES:

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