AGONIST OF ARYL HYDROCARBON RECEPTOR FOR USE IN CANCER COMBINATION THERAPY

Abstract

The present invention relates to an AhR agonist for use in combination with at least one immune checkpoint modulator in the treatment of cancer. The present invention also encompasses product containing an AhR agonist and at least one immune checkpoint modulator as defined in any one of the preceding, claims, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

Claims

1. An AhR agonist for use in combination with at least one immune checkpoint modulator in the treatment of cancer.

2. An AhR agonist for use according to claim 1, wherein the AhR agonist is selected from the group comprising dietary indoles, dietary flavonoids, tryptophan metabolites and synthetic weak AhR agonists.

3. An AhR agonist for use according to claim 1, wherein the AhR agonist is a dietary indole or a derivative thereof, preferably the dietary indole or derivative thereof is an indole glucosinolate, preferably the dietary indole or derivative thereof is selected from the group comprising indole-3-carbinol, 3,3-diindoylmethane, and indolo[3,2b]carbazole.

4. An AhR agonist for use according to claim 1, wherein the AhR agonist is a dietary flavonoid or derivative thereof, preferably the dietary flavonoid or derivative thereof is selected from the group comprising quercetin, galangin, daidzein, naringenin, baicalein, diosmin and diametin.

5. An AhR agonist for use according to claim 1, wherein the AhR agonist is a dietary indole or a dietary flavonoid, preferably the dietary indole or the dietary flavonoid is in the form of a natural product extract, preferably the AhR agonist is a dietary indole in the form of a cruciferous vegetable extract.

6. An AhR agonist for use according to claim 1, wherein the AhR agonist is a synthetic weak AhR agonist, preferably the synthetic weak AhR agonist is selected from the group comprising benzimidazole derivatives, such as omeprazole and lansoprazole, primaquine, leflutamide, VAF347 ([4-(3-chloro-phenyl)-pyrimidin-2-yl]-(4trifluoromethyl-phenyl)-amine), TSU-16 ((Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2indolinone), synthetic flavonoids such as TMF (6,2,4-trimethoxyflavone) and MNF (3-methoxy-4nitroflavone), M50367 (ethyl 3-hydroxy-3-[2-(2-phenylethyl)benzoimidazol-4-yl]propanoate), M50354 (3-[2-(2-phenylethyl)benzoimidazole-4-yl]-3-hydroxypropanoic acid).

7. An AhR agonist for use according to claim 1, wherein the AhR agonist is a tryptophan metabolite, preferably the tryptophan metabolite is selected from the group comprising Kynurenic acid, Kynurenine, 6-formylindolo[3,2b]carbazole (FICZ) and Indoxyl sulfate.

8. An AhR agonist for use according to claim 1, wherein the AhR agonist is suitable for oral administration, preferably the AhR agonist is in the form of a medical food composition.

9. An AhR agonist for use in combination with at least one immune checkpoint regulator according to claim 1, wherein said at least one immune checkpoint modulator is an inhibitory immune checkpoint molecule and/or a stimulatory immune checkpoint agonist.

10. An AhR agonist for use in combination with at least one immune checkpoint regulator according to claim 9, wherein the inhibitory immune checkpoint molecule is selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 TIGIT, VISTA, CD96, CD112R, CD160, CD244 (or 2B4) DCIR (C-type lectin surface receptor), ILT3, ILT4 (Immunoglobulin-like transcript), CD31 (PECAM-1) (Ig-like R family), CD39, CD73, CD94/NKG2, GP49b (immunoglobulin superfamily), KLRG1, LAIR-1 (Leukocyte-associated immunoglobulin-like receptor 1) CD305 , PD-L1, PD-L2 and SIRP, preferably, the inhibitory checkpoint molecule is selected from CTLA-4, PD1, PDL1 or a combination thereof.

11. An AhR agonist for use in combination with at least one immune checkpoint regulator according to claim 9, wherein the stimulatory immune checkpoint agonist is selected from CD27, CD40, OX40, GITR, ICOS, TNFRSF25, 41BB, HVEM, CD28, TMIGD2, CD226, 2B4 (CD244) and agonist CD48, B7-H6 Brandt (NK agonist), LIGHT (CD258, TNFSF14) and CD28H.

12. An AhR agonist for use in combination with at least one immune checkpoint modulator according to claim 1, wherein the immune checkpoint modulator is an antibody or a fusion protein.

13. An AhR agonist for use in combination with at least one immune checkpoint modulator according to claim 1, wherein the immune checkpoint modulator is an anti-PD-1 or an anti-PD-L1 antibody.

14. A product containing an AhR agonist and at least one immune checkpoint modulator as defined in claim 1, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

15. Method for treating a patient suffering from cancer, wherein said method comprises the combined administration of an effective amount of one or more AHR agonist and an effective amount of at least one immune checkpoint modulator.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0145] FIG. 1: IRF4 and MafB are essential for mo-DC and mo-Mac differentiation. (A) Volcano plot showing the fold change and significance of transcription factor genes between ascites mo-DC and mo-Mac. Genes not expressed in in vitro-generated mo-DC or mo-Mac were filtered out. Adjusted p values determined by differential expression analysis. (B) Cell-sorted ascites mo-DC and mo-Mac, and blood CD14.sup.+ monocytes (Mono) were analyzed by Immuno Blot. Representative of 4 independent experiments. (C) CD14.sup.+ monocytes were cultured with MCSF, IL-4 and TNF- for 3 h, 6 h, 12 h or 24 h, or processed directly after isolation (0). IRF4 and MAFB expression were analyzed by RT-qPCR in total cells. Each color represents an individual donor (n=5 in 3 independent experiments). (D) CD14.sup.+ monocytes were cultured with MCSF, IL-4 and TNF- for 5 days. Total cells were lyzed at different days and analyzed by Immuno Blot. Representative of 5 independent experiments. (E-H) Monocytes were infected at day 0 with lentivirus containing sh RNA against IRF4 (E-F), or MAFB (G-H), or control sh RNA. After 5 days of culture, cells were analyzed by Immuno Blot (E and G), or by flow cytometry (F and H). (E and G) Silencing quantified based on Immuno Blot stainings. (F and H) Proportions of DC and macrophages in the culture at day 5. Each symbol represents an individual donor (F n=6 in 3 independent experiments, H n=8 in 4 independent experiments). *p<0.05, **p<0.01.

[0146] FIG. 2: AHR is a molecular switch for mo-DC versus mo-Mac differentiation. (A-B) CD14.sup.+ monocytes were infected at day 0 with lentivirus containing sh RNA against AHR, or control sh RNA. After 5 days of culture, cells were analyzed by Immuno Blot (A) or by flow cytometry (B). (A) Silencing quantified based on Immuno Blot stainings. (B) Proportions of DC and macrophages in the culture at day 5. Each symbol represents an individual donor (n=6 in 3 independent experiments). *p<0.05. (C) Monocytes were cultured with MCSF, IL-4 and TNF- for 5 days, in the presence of various concentrations of FICZ (AHR agonist) or SR1 (AHR inhibitor). Proportions of DC and macrophages at day 5. Each symbol represents an individual donor (n=10 in 5 independent experiments for FICZ and n=8 for SR1 in 4 independent experiments). (D-E) CD14.sup.+ monocytes were analyzed directly after isolation, or were cultured for 3h in medium alone or with various combinations of MCSF, IL-4, TNF-, FICZ or SRI. (D) Relative expression of IRF4 and MAFB measured by RT-qPCR. Box plots representing the 5-95 percentile (n=6 in 3 independent experiments). (E) Expression of IRF4 and MAFB at the single cell level was measured by fluorescent in situ hybridization coupled to flow cytometry. Proportions of IRF4.sup.+ and MAFB.sup.+ cells. Each symbol represents an individual donor (n=6 in 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001.

[0147] FIG. 3: AHR acts on mo-DC differentiation through BLIMP-1. (A) CD14.sup.+ monocytes were cultured with MCSF, IL-4 and TNF- for 3 h, 6 h, 12 h or 24 h in the presence or absence of FICZ or SR1, or processed directly after isolation (0). PRDM1 expression measured by RT-qPCR. Each color represents an individual donor (n=5 in 3 independent experiments). (B) Monocytes were cultured with MCSF, IL-4 and TNF- for 5 days. Total cells were lyzed at different days and analyzed by Immuno Blot. Representative of 4 independent experiments. (C-E) Monocytes were infected at day 0 with lentivirus containing sh RNA against PRDM1, or control sh RNA. After 5 days of culture, cells were analyzed by Immuno Blot (C) or by flow cytometry (D-E). (C) Silencing quantified based on Immuno Blot stainings. (D) Proportions of DC and macrophages in the culture at day 5. Each symbol represents an individual donor (n=8 in 4 independent experiments). *p<0.05. **p<0.01. (E) Cells were cultured in presence or absence of 62 nM FICZ. Proportions of DC and macrophages in the culture at day 5. Each symbol represents an individual donor (n=6 in 3 independent experiments). *p<0.05.

[0148] FIG. 4: AhR is involved in mo-DC differentiation in vivo in the mouse. (A-C) Ear skin from individual AhR.sup./ mice or WT littermates (A-B), or C57BL/6 mice fed with an experimental diet supplemented or not with indole-3-carbinol (I3C) (C), was digested to prepare single-cell suspensions. After gating on live CD45.sup.+CD3.sup.NK1.1.sup.CD19.sup.Ly6G.sup.CD24.sup. CD11b.sup.+ cells, cells were separated into 5 subsets based on the expression of Ly6C, CD64, MHC II and CCR2. (A) One representative AhR.sup./ and WT mouse is shown. (B-C) Proportions of monocytes, early mo-DC, mo-DC, MHC II.sup.+ macrophages and resident MHC II.sup. macrophages among Ly6C.sup.+ or CD64.sup.+ cells. Each symbol represents an individual mouse (B n=9 in 2 independent experiments, C n=12 in 2 independent experiments).*p<0.05, **p<0.01. (D-E) Cells from the peritoneal lavage were analyzed by flow cytometry. After gating on live CD115.sup.+CD11b.sup.+ cells, cells were separated into MHCII.sup.+CD226.sup.+ cells and ICAM2.sup.+MHCII.sup. cells. (D) Cell-sorted cells were analyzed after cytospin and Giemsa/May-Grnwald staining. Bar=10 m. Representative of 2 independent experiments. (E) C57BL/6 mice fed with an experimental diet supplemented or not with I3C, and treated or not with a cocktail of antibiotics. Percentage of MHCII.sup.+CD226.sup.+ mo-DC among CD115.sup.+CD11b.sup.+ cells is shown. Each symbol represents an individual mouse (n=6 in 2 independent experiments). *p<0.05, **p<0.01, ****p<0.0001.

[0149] FIG. 5: AhR agonist improves the efficacy of anti-PD1 treatment in tumor-bearing mice. Mice were injected subcutaneously with B16-OVA (A-B) or MCA101-OVA (C-D) tumor cells. Tumor growth was monitored twice a week. Mice were fed with a control diet or supplemented with indole-3-carbinol (I3C). Mice were treated with PBS (vehicle control) or with anti-PD1 antibody. (A-B) B16-OVA tumor growth. Mean+/SEM (n=12 for A, n=8 for B). (C) MCA-OVA tumor growth. Mean+/SEM (n=16). (D) Number of mice showing complete tumor rejection, tumor regression or tumor progression, for each treatment.

EXAMPLES

Example 1: Aryl Hydrocarbon Receptor Controls Monocyte Differentiation into Dendritic Cells Versus Macrophage

Introduction

[0150] Mononuclear phagocytes are divided into three groups: macrophages, monocytes and dendritic cells (DC). Macrophages derive from embryonic precursors whose differentiation is strongly imprinted by the micro-environment (Gosselin et al., 2014; Haldar et al., 2014; Lavin et al., 2014; Okabe and Medzhitov, 2014). By contrast, classical DC derive from pre-committed precursors that follow a pre-determined developmental program primed at an early stage, independently of their tissue of residence (Breton et al., 2016; Schlitzer et al., 2015). When entering tissues, monocytes can differentiate into either macrophages or DC (Mildner et al., 2013; Segura and Amigorena, 2013). Whether mo-DC and mo-Mac represent variations of one highly plastic cell type or distinct bona fide lineages remains unclear (Guilliams et al., 2014). In addition, what environmental cues drive monocyte fate towards mo-Mac versus mo-DC and what molecular regulators orchestrate this process remains to be established.

[0151] In mouse models, mo-DC and mo-Mac appear during inflammation but are also found in the steady-state at mucosal sites such as intestine and skin (Bain et al., 2014; Mildner et al., 2013; Segura and Amigorena, 2013; Tamoutounour et al., 2013). There is strong experimental evidence that the same scheme applies to humans. Monocyte-derived cells are found in the steady-state in human skin (McGovern et al., 2014). In inflammatory conditions, monocyte recruitment is observed in the gut of inflammatory bowel disease patients (Grimm et al., 1995), in cantharidin-induced skin blisters (Jenner et al., 2014) and in the nasal mucosa of subjects with induced allergic rhinitis (Eguiluz-Gracia et al., 2016). Inflammatory macrophages and DC have been described in atopic dermatitis (Wollenberg et al., 1996), Crohn's disease (Bain et al., 2013; Kamada et al., 2008), psoriasis (Zaba et al., 2009), allergic rhinitis (Eguiluz-Gracia et al., 2016), rheumatoid arthritis and tumor ascites (Segura et al., 2013). Transcriptomic analysis shows that ascites DC share gene signatures with in vitro-generated monocyte-derived cells, supporting the idea that these DC represent tissue mo-DC (Segura et al., 2013).

[0152] We hypothesized that transcription factors differentially expressed between human mo-DC and mo-Mac may be involved in their differentiation from monocytes. In this study, we have identified candidate transcription factors by comparative transcriptomic analysis, and established a model of human monocyte differentation to test the role of these candidates. We found that IRF4 and MAFB were required for mo-DC and mo-Mac differentiation, respectively. We also have shown that AHR is essential for driving monocyte differentiation towards mo-DC. Finally, we validated the role of AHR in mo-DC differentiation in vivo in a mouse model and by analyzing clinical data from leprosy patients.

Results

Culturing Human Monocytes with M-CSF, IL-4 and TNF- Yields mo-DC and mo-Mac Resembling Those Found in Tumor Ascites

[0153] To address the ontogeny of human monocyte-derived cells, we first searched for transcription factors that are differentially expressed between monocytes, ascites mo-DC and ascites mo-Mac. Using our transcriptomic data (Segura et al., 2013), we established a list of candidates. To test their role, we turned to an in vitro model of monocyte differentiation. Current culture models yield either mo-DC or mo-Mac depending on cytokines used. By contrast, we needed a model that would mimic the differentiation of monocytes into mo-DC and mo-Mac in the same environment. We therefore established a new in vitro system enabling the differentiation of both mo-DC and mo-Mac in the same culture.

[0154] Macrophage colony-stimulating factor (M-CSF) and its receptor are essential for mo-DC and mo-Mac differentiation in vivo during inflammation in mice (Davies et al., 2013; Greter et al., 2012), and M-CSFR is highly expressed on human mo-DC and mo-Mac found in vivo (McGovern et al., 2014; Segura et al., 2013). Therefore, we designed a cytokine cocktail based on M-CSF or IL-34, the two ligands of M-CSFR. We also included IL-4, a cytokine known to induce the expression by cultured monocytes of CD1 molecules, which are highly expressed on ascites mo-DC (Segura et al., 2013). Finally, we added TNF-, a major mediator of inflammation. Culturing human blood CD14.sup.+ monocytes with M-CSF, IL-4 and TNF-or IL-34, IL-4 and TNF- yielded in the same culture two main populations expressing CD16 or CD1a, and displaying a typical macrophage or DC morphology, respectively. Only CD1a.sup.+ cells could efficiently induce allogeneic naive CD4.sup.+ T cell proliferation, confirming that they were bona fide DC. We then characterized the properties of cultured mo-DC and mo-Mac compared to that of ascites mo-DC and mo-Mac. Both mo-DC and mo-Mac secreted IL-6 after stimulation with CD40L, but only mo-DC secreted IL-23, similar to what we observed with ascites cells (Segura et al., 2013). Phenotypic comparison of mo-DC and mo-Mac differentiated with M-CSF, or IL-34, IL-4 and TNF- with ascites mo-DC and mo-Mac showed similar expression for various surface markers, except for CD14 which was down-regulated upon culture. CD14.sup.+ monocytes were routinely isolated by positive selection using magnetic beads with 90-95% purity, contaminating cells being CD14.sup.+CD16.sup.+ monocytes. To address whether the presence of CD14.sup.+CD16.sup.+ monocytes could influence the culture outcome, we isolated highly pure CD14.sup.+ monocytes by cell sorting. The absence of contaminating CD14.sup.+CD16.sup.+ monocytes did not impact monocyte differentiation into both mo-DC and mo-Mac. In addition, CD16.sup.+ monocytes (isolated using magnetic beads) had a low survival rate and did not maintain CD16 expression in culture, suggesting that potential contaminating CD16.sup.+ monocytes had a negligeable effect on the final proportions of mo-DC and mo-Mac. Of note, monocytes differentiated with GM-CSF and IL-4, a widely used culture system, yielded only CD1a.sup.+ mo-DC. The phenotype of mo-DC derived with GM-CSF and IL-4, with or without TNF-, was less similar to that of ascites mo-DC. When stimulated with a toll-like receptor (TLR)7 and TLR8 ligand (R848) and an endogenous danger signal (uric acid cristals), mo-DC differentiated with M-CSF, or IL-34, IL-4 and TNF- secreted high concentrations of inflammatory cytokines (IL-1 and IL-6), consistent with the secretory ability of ascites mo-DC (Segura et al., 2013). Of note, mo-DC differentiated with GM-CSF and IL-4 were less efficient for the secretion of IL-1 and IL-6, although they secreted TNF- and the chemokine CXCL10 at similar concentrations as mo-DC differentiated with M-CSF, or IL-34, IL-4 and TNF-.

[0155] To determine whether these populations had a stable phenotype over time, we sorted CD1a.sup.CD16.sup. cells, mo-DC and mo-Mac after 5 days of culture and re-cultured them separately with M-CSF, IL-4 and TNF-. After 2 days of re-culture, the phenotype of CD1a.sup. CD16.sup. cells, mo-DC or mo-Mac remained stable based on the expression of CD16, CD163 and CD1a. After 4 days of re-culture, only CD1a.sup.CD16.sup. cells and mo-Mac were still viable, and their phenotype was largely unchanged. These results show that mo-DC and mo-Mac are maintained as stable populations over the course of the culture, and that mo-DC or mo-Mac do not emerge from CD1a.sup.CD16.sup. cells at later time points. To address whether a single monocyte could give rise to both mo-DC and mo-Mac in our culture model, we stained monocytes with a proliferation dye and analyzed cell proliferation after 5 days. As a positive control, we stimulated monocytes with the mitogen phytohaemagglutin-L (PHA-L), which induced the proliferation of a portion of monocytes. Monocytes did not proliferate in the culture, suggesting that there was a precursor-product relationship between a single monocyte and a single mo-DC, or mo-Mac, progeny.

[0156] Finally, to complete the characterization of our culture model, we compared the transcriptome of cell-sorted mo-DC and mo-Mac differentiated with M-CSF, or IL-34, IL-4 and TNF- with that of cell-sorted ascites mo-DC and mo-Mac, blood CD14.sup.+ monocytes, blood CD1c.sup.+ DC and mo-DC differentiated with GM-CSF and IL-4. Supervised analysis of the micro-array data showed differential expression for selected phenotypic markers as expected. Comparative transcriptomic analysis showed that mo-Mac and mo-DC differentiated with M-CSF, IL-4 and TNF- were highly similar to those differentiated with IL-34, IL-4 and TNF-. In addition, these in vitro-generated mo-Mac and mo-DC clustered close to ascites mo-Mac and mo-DC respectively, while the transcriptome of mo-DC differentiated with GM-CSF and IL-4 was closer to that of blood CD1c.sup.+ DC. These results show that our culture system yields mo-DC and mo-Mac populations that closely resemble mo-DC and mo-Mac present in human tumor ascites. For the rest of the study, we used monocytes cultured with M-CSF, IL-4 and TNF- as a model to analyze monocyte-derived cell differentiation.

IRF4 and MAFB are Essential for the Development of mo-DC and mo-Mac

[0157] To refine our list of candidates for the differentiation of monocyte-derived cells, we removed transcription factors that were not expressed or not differentially expressed in in vitro-derived mo-DC versus in vitro-derived mo-Mac (FIG. 1A). We selected for further validation two candidates, IRF4 and MAFB, previously proposed to be involved in the development of a subset of mouse classical DC (Murphy et al., 2015), and in macrophage differentiation (Kelly et al., 2000) respectively. IRF4 and MAFB were differentially expressed in mo-DC versus mo-Mac, both in vitro and in vivo. In addition, we confirmed the differential expression of IRF4 and MAFB in blood monocytes, ascites mo-DC and ascites mo-Mac at the protein level (FIG. 1B).

[0158] IRF4 and MAFB were expressed early during the culture both at the mRNA (FIG. 1C) and protein levels (FIG. 1D), consistent with their possible role as master regulator transcription factors. To address the role of IRF4, we silenced its expression by infecting monocytes at the start of the culture with lentiviral vectors containing shRNA against IRF4, or control shRNA (FIG. 1E). Inhibition of IRF4 expression induced a dramatic reduction of mo-DC while maintaining the mo-Mac population (FIG. 1F). We used a similar strategy to analyze the role of MAFB (FIG. 1G). Silencing of MAFB resulted in a strong decrease in mo-Mac and an increase in mo-DC differentiation (FIG. 1H). Analysis of additional phenotypic markers confirmed the disappearance of mo-DC or mo-Mac from the culture, rather than the mere down-regulation of CD1a or CD16 expression. These results show that IRF4 and MAFB are essential for mo-DC and mo-Mac development, respectively.

Monocytes are not Heterogeneous for the Expression of mo-DC Gene Signatures

[0159] These results could be explained either by the presence of two distinct precursor populations or by the existence among blood CD14.sup.+ monocytes of two transcriptionally primed populations that would be pre-committed to become mo-DC or mo-Mac (Schlitzer et al., 2015). To address the heterogeneity of CD14.sup.+ monocytes, we performed single-cell RNA-seq on CD14.sup.+ monocytes from 2 donors isolated using magnetic beads. Cell purity as assessed by flow cytometry was 93% and 95% respectively. We generated single-cell transcriptomes using a droplet-based method enabling 3 mRNA counting (Zheng et al., 2017). To evaluate the heterogeneity of CD14.sup.+ monocytes, we clustered cells using a graph-based approach with the Seurat package, which combines dimensionality reduction and graph-based partitioning algorithms for unsupervised clustering (Satija et al., 2015). For vizualisation of the cell clusters, we used t-Distributed Stochastic Neighbor Embedding (t-SNE). We found two clusters, one of which represents around 25% of total cells and corresponds to cells expressing FCGR3A (encoding CD16), high amounts of MHC class II molecules and several genes preferentially expressed in CD14.sup.+CD16.sup.+ and CD16.sup.+ monocytes including IFITM2 and IFITM3 (Villani et al., 2017; Wong et al., 2011). In addition to contaminating CD14.sup.+ CD16.sup.+ monocytes, some of these cells may correspond to CD14.sup.+ monocytes en route to differentiating into CD14.sup.+CD16.sup.+ monocytes (Patel et al., 2017). To address the potential heterogeneity of the CD16.sup. cluster, we performed a second analysis excluding cells from the CD16.sup.+ cluster. We did not detect subgroups of transcriptionally distinct cells within the CD16.sup. cluster, confirming that the CD14.sup.+ monocyte population is likely homogeneous, as previously reported (Villani et al., 2017). To confirm these results, we sought to address whether subpopulations of monocytes displayed transcriptional similarity with mo-DC or mo-Mac. Because of the limited number of genes detected per cell in our analysis (average of 1185), we interrogated published single-cell RNA-seq data obtained using the Smart-seq2 approach (Villani et al., 2017), characterized by a lower number of cells analyzed but a higher number of genes per cell (average of 5326). We defined gene signatures for mo-DC and mo-Mac using our transcriptomic data by identifying genes that were (i) more expressed in ascites mo-DC than in blood monocytes, (ii) more expressed in ascites mo-DC than in ascites mo-Mac, and (iii) more expressed in in vitro-derived mo-DC than in in vitro-derived mo-Mac (and vice-versa for mo-Mac). We then queried genes with at least a 2-fold change. Among these, 35 genes for mo-DC and 35 genes for mo-Mac were expressed in the single-cell RNA-seq data set. While none of the monocytes expressed the mo-DC signature, the mo-Mac signature was partially expressed by all monocyte subsets. We also assessed the expression of selected genes that could be involved in determining monocyte fate, including receptors for the cytokines used in our model (CSF1R, IL4R, TNFRSF1A), candidate transcription factors (IRF4, MAFB, AHR) or genes recently proposed to distinguish DC-biased monocytes in the mouse (FLT3, SPI1) (Menezes et al., 2016). While IRF4 was not expressed by monocytes, MAFB was detected in all monocyte subsets. We conclude that human CD14.sup.+ monocytes are not heterogeneous in their expression of mo-DC transcriptional signature. While monocytes do not contain a subpopulation that would be pre-committed towards mo-DC differentiation, they all express a partial mo-Mac gene signature, including MAFB, suggesting a default differentiation pathway towards mo-Mac if no other environmental triggers are encountered.

AHR is a Molecular Switch for Monocyte Fate

[0160] Given that monocytes do not seem to be transcriptionally primed, we then hypothesized that environmental signals play a major role in driving monocyte fate. Among candidate transcription factors (FIG. 1A), we identified AHR, a ligand-activated transcription factor sensing tryptophan catabolites and metabolites generated by dietary intake, UV exposure, or microbiota (Stockinger et al., 2014). AHR was differentially expressed by mo-DC and mo-Mac at the mRNA and protein levels (FIG. 1B).

[0161] To address the role of AHR, we first inhibited its expression by targeted silencing using lentiviral vectors (FIG. 2A). AHR silencing reduced mo-DC differentiation while slightly increasing mo-Mac (FIG. 4B). Because culture medium contains small amounts of AHR ligands (Veldhoen et al., 2009) and AHR silencing was incomplete (FIG. 2A), we sought to confirm these results using a different approach. We cultured monocytes in the presence of various doses of a natural AHR agonist (6-Formylindolo(3,2-b)carbazole, FICZ) or an AHR inhibitor (stemregenin-1, SR1) and assessed mo-DC and mo-Mac differentiation. AHR activation by FICZ increased mo-DC while decreasing mo-Mac development (FIG. 2C). Conversely, AHR inhibition by SR1 increased mo-Mac while decreasing mo-DC proportions (FIG. 2C). Of note, the phenotype of FICZ-treated mo-DC or SR1-treated mo-Mac was similar to that of untreated cells, while the phenotype of CD1a.sup.CD16.sup. cells remained unchanged. These results suggest that AHR is a molecular switch for mo-DC versus mo-Mac differentiation.

[0162] To decipher how AHR shapes monocyte fate, we assessed IRF4 and MAFB expression in monocytes after 3 h of culture with various combinations of M-CSF, IL-4, TNF-, SR1 and FICZ (FIG. 2D). We also analyzed the expression of CYP1A1, a known direct target of AHR (Stockinger et al., 2014), as a control for AHR activation. IL-4 induced IRF4 expression, as previously reported (Lehtonen et al., 2005). This induction was inhibited in the presence of SR1, indicating that IRF4 expression is dependent on AHR signaling, presumably in response to small amounts of AHR ligand from the culture medium. However, IRF4 was not induced in the presence of FICZ alone. The expression of IL-4-induced IRF4 was further increased in the presence of TNF- and with FICZ. By contrast, MAFB expression was induced by culture medium alone, and further increased by M-CSF (FIG. 2D). AHR signaling had no significant impact on MAFB expression at this time point. To address whether quantities of mRNA expressed per cell or proportions of expressing cells were regulated by these signals, we analyzed the expression of IRF4 and MAFB mRNA at the single-cell level using fluorescent in situ hybridization coupled to flow cytometry (FIG. 2E). After 3 h of culture, monocytes upregulated MAFB in response to M-CSF, but in the presence of IL-4 and TNF-, the proportion of MAFB-expressing monocytes was dramatically reduced while a distinct population of IRF4-expressing monocytes appeared, which was further increased in the presence of FICZ. These results show that external signals polarize monocytes towards mo-DC versus mo-Mac differentiation. In line with this, increasing concentrations in the culture of IL-4 or TNF- increased the proportion of mo-DC while reducing that of mo-Mac. In addition, the effect of FICZ on the culture outcome was strongly diminished in the absence of TNF-. These results indicate that IL-4, TNF- and AHR signaling synergize for IRF4 induction and mo-DC differentiation. Collectively, these results suggest the existence of a default differentiation pathway into mo-Mac, which can be switched to mo-DC differentiation in response to IL-4, TNF- and AHR ligands.

AHR Acts Through BLIMP-1

[0163] AHR activation triggers an autoregulatory feedback loop that restricts AHR signaling to a short timeframe (Stockinger et al., 2014). Therefore, we hypothesized that the effect of AHR activation on monocyte differentiation may be mediated by additional molecular regulators. In our differential transcriptomic analysis, we looked for transcription factors that could be induced by AHR activation based on literature. We identified PRDM1 (encoding BLIMP-1) as a candidate. Studies on cell lines have suggested that PRDM1 is a target of AHR (De Abrew et al., 2010; Ikuta et al., 2010). To analyze whether PRDM1 was induced by AHR signaling in monocytes, we measured PRDM1 expression during the early stages of monocyte culture in the presence or absence of FICZ or SR1 (FIG. 3A). We found that PRDM1 was rapidly induced upon AHR activation within 3 hours, suggesting that PRDM1 is a target of AHR. At the protein level, BLIMP-1 expression peaked during the first 24 h of the culture then decreased (FIG. 3B). To address whether PRDM1 was involved in monocyte differentiation, we silenced PRDM1 expression using shRNA (FIG. 3C). PRDM1 silencing significantly decreased mo-DC differentiation, while increasing the proportion of mo-Mac (FIG. 3D). To confirm that the effect of AHR was mediated by PRDM1, we silenced its expression in monocytes cultured in the presence or absence of FICZ (FIG. 3E). PRDM1 silencing abolished the promotion of mo-DC differentiation by FICZ, while the proportion of mo-Mac was not fully restored to that observed in control cells in the absence of FICZ. These results suggest that BLIMP-1 is essential for AHR-induced mo-DC differentiation.

AHR is Involved in mo-DC Differentiation In Vivo in the Mouse

[0164] To address the physiological relevance of our findings, we analyzed the role of AhR in mo-DC and mo-Mac differentiation in vivo in the mouse. In the steady-state dermis, mo-DC and mo-Mac continuously differentiate in situ from monocytes recruited to the skin (Tamoutounour et al., 2013). Five populations of macrophages and monocyte-related cells have been described in mouse skin: dermal monocytes, mo-DC at an early stage of differentiation, fully differentiated mo-DC, MHC class II.sup.+ macrophages (which contain a majority of mo-Mac) and resident MHC class II.sup. macrophages (Tamoutounour et al., 2013). Transcriptomic analysis showed that AhR and Irf4 were more expressed in mo-DC than in macrophages, while MafB was more expressed in macrophages (GEO accession code GSE49358), consistent with our findings in human monocyte-derived cells. In AhR-deficient mice, the proportion of skin mo-DC was decreased as compared to wild type (WT) littermates, while the proportion of MHC class II.sup.+ macrophages were increased (FIG. 4A-B). Proportions of other skin DC subsets and of monocyte subsets in the spleen were similar between AhR-deficient and WT mice. To confirm these findings, we increased AhR ligand availability in vivo by feeding mice with an experimental diet enriched for indole-3-carbinole (I3C), which is converted into an AhR ligand by stomach acids (Bjeldanes et al., 1991). In mice fed with IC-supplemented diet, mo-DC were significantly increased and MHC class II.sup.+ macrophages were decreased in the skin compared to mice fed with a control diet (FIG. 4C). These results show that AhR is involved in the in vivo differentiation of dermal mo-DC.

[0165] Recently, Irf4 has been shown in the mouse to be involved in the differentiation of a population of peritoneal and pleural MHC II.sup.+CD226.sup.+ monocyte-derived cells, proposed to be mo-Mac (Kim et al., 2016). However, our results identified CD226 as a marker of human mo-DC, both in vitro and in ascites (FIG. 1D). To determine the identity of mouse MHC II.sup.+CD226.sup.+ monocyte-derived cells, we isolated them from the peritoneal lavage of C57BL/6 mice, and compared their morphology to that of ICAM2.sup.+ peritoneal macrophages (FIG. 4D). MHC II.sup.+CD226.sup.+ cells displayed a typical DC morphology, distinct from that of bona fide ICAM2.sup.+ macrophages. Consistent with this, MHC II.sup.+CD226.sup.+ cells did not express the macrophage marker MerTK and CD226 was highly expressed by dermal mo-DC, but not by dermal macrophages. These results identify Irf4-dependent MHC II.sup.+CD226.sup.+ cells as mo-DC. As previously reported (Kim et al., 2016), this population of peritoneal mo-DC is decreased upon antibiotics treatment (FIG. 4E). Antibiotics induce the loss of intestinal bacteria species that are a major source of endogenous AhR ligand (Zelante et al., 2013). To address whether the decrease of peritoneal mo-DC upon antibiotics treatment was AhR-dependent, we fed antibiotics-treated mice with a I3C-supplemented or control diet, and analyzed cells from the peritoneal lavage (FIG. 3E). Supplementation in AhR ligand restored mo-DC differentiation in antibiotics-treated mice almost up to normal proportions.

[0166] Collectively, these results show that AhR activation in response to environmental stimuli has a key role in driving monocyte fate towards mo-DC in vivo.

AHR Activation Correlates With the Presence of mo-DC in Leprosy Lesions

[0167] Finally, to put these findings in the context of human disease, we assessed AHR signaling and monocyte-derived cells presence in leprosy lesions, that contain granulomas in which monocytes are constantly recruited (Russell et al., 2009; Schreiber and Sandor, 2010). We analyzed published micro-array data from lepromatous (L-lep) and tuberculoid (T-lep) leprosy lesions ((Montoya et al., 2009), GEO accession code GSE17763). Patients with L-lep lesions display poor immunological responses against Mycobacterium leprae, the causative agent of leprosy, while patients with T-lep lesions have strong anti-M. leprae T cell responses (Montoya et al., 2009). We first defined AHR agonist and AHR antagonist gene signatures based on publicly available transcriptomic data (Di Meglio et al., 2014). To assess whether AHR signaling was active in leprosy lesions, we performed Gene Set Enrichment Analysis (GSEA). We found that the AHR agonist signature was enriched in T-lep lesions, while the AHR antagonist signature was enriched in L-lep lesions. Consistent with this, AHRR and CYP1A1, which are upregulated upon AHR activation (Stockinger et al., 2014), were highly expressed in T-lep lesions while IFIT1, which is down-regulated by AHR (Di Meglio et al., 2014), was more expressed in L-lep lesions. These results suggest that the AHR pathway was preferentially activated in T-lep lesions. To address the presence of mo-Mac and mo-DC, we performed GSEA using our gene signatures. The mo-DC signature was enriched in T-lep lesions, while the mo-Mac signature was enriched in L-lep lesions. By contrast, gene signatures of human Langerhans cells, skin CD1c.sup.+ DC or dermal macrophages (from (Carpentier et al., 2016)) were not enriched in either dataset. Consistent with these results, MERTK, CD163 and FCGR3A (encoding CD16) were more expressed in L-lep lesions and CD1A, CD1B and FCER1A were more expressed in T-lep lesions. These results suggest that mo-DC are more abundant in T-lep lesions while mo-Mac are more numerous in L-lep lesions. This is in line with previous work showing the absence of CD1a.sup.+CD1b.sup.+CD1c.sup.+DC in L-lep lesions (Sieling et al., 1999) and the increased presence of CD163.sup.+ macrophages in L-lep lesions (Montoya et al., 2009). Collectively, these results show that AHR activation correlates with the selective presence of mo-DC in L-lep lesions.

Discussion

[0168] In this work, we have identified transcription factors involved in the differentiation of monocytes either into mo-Mac (MAFB) or into mo-DC (IRF4, AHR, BLIMP-1). These results show that mo-DC and mo-Mac do not represent different states of polarized monocytes, but rather are distinct lineages controlled by two different sets of molecular regulators. Several studies have evidenced that monocyte differentiation into mo-DC or mo-Mac is context-dependent (Bain et al., 2013; Zigmond et al., 2012). Here we identified micro-environmental cues that shape monocyte fate. Our results suggest that, in the presence of M-CSF, monocytes differentiate into mo-Mac by default, unless they are exposed to certain cytokines (IL-4 and TNF-) in conjunction with AHR ligands, promoting mo-DC differentiation.

[0169] Our conclusions are primarily based on the use of an in vitro human monocyte differentiation model, but are reinforced by the validation of our main finding in vivo in a mouse model and the correlation between AHR activation and mo-DC presence in leprosy lesions. In addition, BLIMP-1 and IRF4 were identified in both mouse and human as preferentially expressed in intestinal CD103.sup.+CD11b.sup.+ DC (a proposed mo-DC population), and mice deficient for Blimp-1 in DC displayed a strongly reduced population of CD103.sup.+CD11b.sup.+ DC in the intestine (Watchmaker et al., 2014), further supporting the physiological relevance of our findings.

[0170] It has been suggested that mouse monocytes can be separated into two subpopulations that are pre-committed to become mo-Mac in response to pathogens or mo-DC in response to GM-CSF (Menezes et al., 2016). Using two different datasets of single-cell RNA-seq, we could not identify distinct subpopulations of mo-DC and mo-Mac precursors within human CD14.sup.+ monocytes. This is consistent with a recent single-cell RNA-seq analysis showing that mouse Ly6C.sup.+ and Ly6C.sup. monocytes are not heterogeneous at the transcriptomic level (Mildner et al., 2017). Our results show that all monocyte subsets express a partial mo-Mac transcriptomic signature, but cues from the micro-environment can drive monocyte fate towards mo-DC or mo-Mac. One hypothesis to explain how the same signals can induce different outcomes within a transcriptionally homogeneous population could be the stochastic heterogeneity in chromatin accessibility (Buenrostro et al., 2015).

[0171] MafB is highly expressed by all mouse macrophage populations except for lung macrophages (Gautier et al., 2012). Based on in vitro over-expression in myeloid progenitor cells, MafB has been proposed to induce macrophage differentiation (Bakri et al., 2005; Kelly et al., 2000). However, subsequent work showed that MafB is dispensable both in vivo and in vitro for murine macrophage differentiation from fetal progenitors (Aziz et al., 2006), suggesting that MafB is not essential for the initial stages of differentiation of embryonic-derived macrophages. MafB is rather involved in their terminal differentiation by repressing self-renewal genes (Aziz et al., 2009). Whether MafB is important for the differentiation of mouse macrophages in an inflammatory setting remains to be addressed.

[0172] Irf4 is preferentially expressed by mouse CD11b.sup.+ DC. Whether it is required for their development, or rather their migration and survival, remains unclear (Murphy et al., 2015). We show that IRF4 was essential for human mo-DC differentiation, and its expression in human monocytes was induced by IL-4 in an AHR-dependent way. This is consistent with previous work showing IRF4 expression upon culture with IL-4 in human and mouse monocytes (Briseno et al., 2016; Lehtonen et al., 2005). In addition, Irf4.sup./ mouse monocytes cultured with GM-CSF and IL-4 fail to differentiate into mo-DC, but rather become mo-Mac (Briseno et al., 2016), supporting the idea of a default differentiation pathway into mo-Mac. We also showed that mouse Irf4-dependent peritoneal monocyte-derived cells, initially described as mo-Mac (Kim et al., 2016), actually correspond to mo-DC, based on their morphology and phenotype.

[0173] Previous evidence suggests a major role for AhR in the control of inflammatory responses, in particular through its action on T helper-17 (Th17) cell development and innate lymphoid cells homeostasis (Stockinger et al., 2014). Our work highlights an additional level of control by AhR ligands, by switching monocyte differentiation towards mo-DC, which are major producers of IL-23 and inducers of Th17 cells (Segura et al., 2013). Natural AhR ligands are derived from dietary intake (Bjeldanes et al., 1991) or produced through tryptophan catabolism at mucosal barriers (Fritsche et al., 2007; Zelante et al., 2013). AhR ligands can circulate throughout the body as evidenced by the regulation of astrocyte activity by microbiota-derived AhR ligands (Zelante et al., 2013), or the presence in milk of AhR ligands derived from the maternal microbiota (Gomez de Aguero et al., 2016).

[0174] Several studies have evidenced a deleterious role of monocyte-derived cells in pathological situations. mo-DC induce pathogenic T cells that mediate tissue damage in mice models of autoimmune or inflammatory diseases such as experimental autoimmune encephalomyelitis (Croxford et al., 2015) and colitis (Zigmond et al., 2012). Human inflammatory mo-DC likely contribute to the pathogenesis in Crohn's disease, rheumatoid arthritis and psoriasis through the secretion of high amounts of IL-23 and the induction of Th17 cells (Kamada et al., 2008; Segura et al., 2013; Zaba et al., 2009), two major players in the pathogenesis of these diseases. In tumors, mo-Mac play a central role in the suppression of anti-tumoral immune responses (Noy and Pollard, 2014). Monocyte-derived cells have therefore emerged in the past few years as attractive therapeutic targets (Getts et al., 2014; Leuschner et al., 2011). By enabling a better understanding of the molecular ontogeny of human monocyte-derived cells, our results should provide new opportunities for the therapeutic manipulation of monocyte differentiation.

[0175] The publication Goudot C, Coillard A, Villani A C, Gueguen P, Cros A, Sarkizova S, Tang-Huau T L, Bohec M, Baulande S, Hacohen N, Amigorena S, Segura E (2017). Aryl hydrocarbon receptor controls monocyte differentiation into dendritic cells versus macrophages. Immunity. 47(3): 582-596, is incorporated by reference to the present application. This publication includes the results of example 1. Using an in vitro culture model yielding human mo-DC and mo-Mac closely resembling those found in vivo in ascites, the inventors showed that IRF4 and MAFB were critical regulators of monocyte differentiation into mo-DC and mo-Mac, respectively. Activation of the aryl hydrocarbon receptor (AHR) promoted mo-DC differentiation through the induction of BLIMP-1, while impairing differentiation into mo-Mac. AhR deficiency also impaired the in vivo differentiation of mouse mo-DC. Finally, AHR activation correlated with mo-DC infiltration in leprosy lesions. These results establish that mo-DC and mo-Mac are controlled by distinct transcription factors, and show that AHR acts as a molecular switch for monocyte fate specification in response to micro-environmental factors.

Example 2: AhR Agonist Improves the Efficacy of Anti-PD1 Treatment in Tumor-Bearing Mice

Material and Methods

Mice

[0176] C57BL/6 female mice were obtained from Charles River Janvier and maintained under specific pathogen-free conditions at the animal facility of Institut Curie in accordance with institutional guidelines. C57BL/6 mice were maintained on a purified diet (AIN-93M, Safe diets) supplemented or not with 200 p.p.m. indole-3-carbinol (Sigma) for 3 weeks, starting when the mice were 3 weeks-old. 6 week-old mice used for tumor experiments.

Cells

[0177] B16.F10 OVA-expressing cells or MCA.101 OVA-expressing cells (Zeelenberg et al., 2008) were grown in RPMI-1640 containing 10% heat-inactivated FBS (Biowest), 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mM GlutaMAX, and 50 M -mercaptoethanol (all from Thermo Fisher Scientific).

Tumor Growth Experiments

[0178] Mice were injected subcutaneously in the flank with 0.5 10.sup.6 B16.F10-OVA melanoma cells or 0.5 10.sup.6 MCA.101-OVA cells. Tumor growth was measured twice a week and was followed until the tumor became necrotic or until the size reached 1,500 mm.sup.3. Mice were treated, or not, with anti-PD1 (Bio X cell) starting when the tumor was palpable for B16.F10-OVA or starting when the tumor was 100-200 mm.sup.3 for MCA.101-OVA. Treatment consisted of intra-peritoneal injections of 200 g of each antibody, delivered at day 7, day 10 and day 13 for B16.F10-OVA, or day 7 and day 14 for MCA.101-OVA. Control treatment consisted of intra-peritoneal injections of the same volume of PBS.

Results

[0179] To address whether increasing the availability of Aryl Hydrocarbon Receptor (AhR) ligands improves spontaneous anti-tumoral responses, we inoculated B16.F10-OVA melanoma cells to mice fed with a control diet or supplemented with indole-3-carbinol (I3C), which is cleaved into AhR ligands by stomach acids (Bjeldanes et al., 1991). Tumor growth in mice fed with I3C-supplemented was similar to that of the control group (n=12 mice) (FIG. 5A).

[0180] To address whether increasing the availability of AhR ligands improves the efficacy of anti-checkpoint therapy, we treated mice with anti-PD1 3 times (at day 7, day 10 and day 13 post-inoculation of tumor cells). Tumor growth in mice fed with I3C-supplemented and treated with anti-PD1 therapy was significantly delayed compared to that of untreated mice and mice treated with anti-PD1 therapy and fed with the control diel (n=8 mice) (FIG. 5B).

[0181] To confirm these results, we repeated this experiment with a second tumor model, MCA.101-OVA cells. We inoculated MCA.101-OVA cells to mice fed with a control diet or supplemented with I3C. Tumor growth in mice fed with I3C-supplemented diet was similar to the control group (n=16 mice) (FIG. 5C).

[0182] To address whether increasing the availability of AhR ligands improves the efficacy of anti-PD1 therapy, we treated mice with anti-PD1 twice (at day 7 and day 14 post-inoculation of tumor cells). Tumor volume at end point was significantly reduced in mice fed with I3C-supplemented diet and treated with anti-PD1 therapy compared to that of mice treated with anti-PD1 therapy and fed with the control diet (n=16 mice) (FIG. 5D). In addition, response to treatment in mice fed with I3C-supplemented diet was significantly improved compared to that of mice fed with the control diet, with 50% of mice responding to anti-PD1 treatment (regression or complete rejection) in the I3C diet group, versus 18.75% of mice responding in the control group (n=16 mice) (FIG. 5D).

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