WO2020169472A2 - Methods of inducing phenotypic changes in macrophages - Google Patents

Abstract

Tumor-associated macrophages (TAM) have been shown to have important roles in the malignant progression of various cancers. However, macrophages also possess intrinsic tumoricidal activity and can promote the activity of cytotoxic lymphocytes, but they rapidly adopt an alternative phenotype within tumors, associated with immune-suppression and trophic functions that support tumor growth. The mechanisms that promote TAM polarization in the tumor-microenvironment remain poorly understood, these mechanisms may represent important therapeutic targets to block the tumor-promoting functions of TAM and restore their anti-tumor potential. Here the inventors have characterized TAM in a mouse model of metastatic ovarian cancer. They show that ovarian cancer cells promote membrane-cholesterol efflux and the depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4 mediated reprogramming while inhibiting IFN-induced gene expression. These studies reveal an unexpected role for tumor-induced membrane-cholesterol efflux in driving the IL-4 signaling and the tumor-promoting functions of TAM, while rendering them refractory to pro-inflammatory stimuli. Thus, preventing cholesterol efflux in TAM could represent a novel therapeutic strategy to block pro-tumor functions and restore anti-tumor immunity.

Description

METHODS OF INDUCING PHENOTYPIC CHANGES IN MACROPHAGES

FIELD OF THE INVENTION:

The field of the invention is immunology.

BACKGROUND OF THE INVENTION:

There is now a wealth of clinical and experimental evidence that strongly links tumor- associated macrophages (TAM) with tumor progression, invasion and metastasis (Noy and Pollard, 2014). In the vast majority of published studies, increased numbers of TAM correlate with poor prognosis, but in some cases, specific TAM subsets have been associated with beneficial outcomes (de Vos van Steenwijk et ah, 2013; Ino et al., 2013). Indeed, macrophages have been shown to posses intrinsic tumoricidal activity and promote the activation of cytotoxic lymphocytes (Bonnotte et al., 2001; Hagemann et al., 2008; Mytar et al., 1999), but they rapidly adopt an alternative phenotype within tumors, associated with immune-suppression and trophic functions that support tumor growth (Mantovani et al, 2008). However, the mechanisms that promote TAM reprogramming in the tumor-microenvironment remain poorly understood.

In mammals, macrophages are found in all tissues after birth and are endowed with trophic functions that contribute to organ development and remodelling (Pollard, 2009). Recent advances in genetic fate-mapping techniques have revealed that the majority of tissue-resident macrophages, at least in steady-state, develop from embryonic precursors and are maintained by local proliferation with little input from hematopoeitc stem cells (HSC) in the bone marrow (Schulz et al., 2012). Subsequent studies have shown that embryonic macrophages can be gradually replaced by HSC-derived blood monocytes, to varying degrees depending on the specific context (Ginhoux and Guilliams, 2016). But the functional implications of these distinct developmental origins and certainly their respective contributions to tumor progression remain unclear. In a recent study, both tissue-resident macrophages of embryonic origin and monocyte-derived TAM were shown to contribute towards tumor growth in a mouse model of pancreatic cancer (Zhu et al., 2017).

The phenotype of tissue-resident macrophages is dictated by the tissue-specific signals in their respective niche (Gosselin et al, 2014; Lavin et al., 2014). However, during inflammation or tissue stress, monocyte-derived macrophages can be recruited into tissues and their functional reprogramming is dictated by the pathological context. It is now widely appreciated that macrophages follow a multi-dimensional model of activation states with distinct phenotypic and functional properties in response to different stimuli in the tissue microenvironment and can maintain considerable plasticity (Murray et al, 2014; Xue et al.,

2014). Along these lines, TAM in various experimental models and human cancers have been shown to express uniques sets of gene patterns including the production of specific chemokines, cytokines and growth factors linked with tumor progression, such as CCL2, TNF, VEGF, basic fibroblast growth factor (bFGF) and matrix metalloproteinases (MMPs) (Kratochvill et al.,

2015). Nevertheless, TAM are invariably reprogrammed towards a functional state that supports tumor growth and immune-suppression and away from inflammatory phenotypes that could be associated with anti-tumor functions. The specific mechanisms that drive TAM accumulation and polarization in different tumors remain unclear. Several studies have shown that TAM are CSF-1 dependent, as are most tissue macrophages, and CSF-1 signaling has been suggested to be an important factor in their reprogramming towards pro-tumor functions (Martinez et al., 2006; Noy and Pollard, 2014). CSF-1 and IL-4 signaling in TAM was later shown to cooperatively promote growth of lung metastatses in the MMTV-pyMT mouse model of mammary carcinonogenesis (DeNardo et al, 2009). However, although primary tumor- development in this model was CSF-1 dependent (Lin et al., 2006), IL-4 signaling in TAM did not impact primary tumors (DeNardo et al, 2009). Subsequent studies showed that the development of lung metastases, but not primary tumors, in the same model critically requires the recruitment of CCR2-dependent monocytes (Qian et al., 2011). Suggesting that IL-4 signaling, specifically in monocyte-derived TAM, promotes metastatic disease in this model.

SUMMARY OF THE INVENTION:

As defined by the claims, the present relates to a method of inducing a phenotypic change in a population of macrophages in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that modulates cholesterol efflux in said population of macrophages.

DETAILED DESCRIPTION OF THE INVENTION:

Tumor-associated macrophages (TAM) have been shown to have important roles in the malignant progression of various cancers. However, macrophages also possess intrinsic tumoricidal activity and can promote the activity of cytotoxic lymphocytes, but they rapidly adopt an alternative phenotype within tumors, associated with immune-suppression and trophic functions that support tumor growth. The mechanisms that promote TAM polarization in the tumor-microenvironment remain poorly understood, these mechanisms may represent important therapeutic targets to block the tumor-promoting functions of TAM and restore their anti-tumor potential. Here the inventors have characterized TAM in a mouse model of metastatic ovarian cancer. They show that ovarian cancer cells promote membrane-cholesterol efflux and the depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4 mediated reprogramming while inhibiting IFNy-induced gene expression. These studies reveal an unexpected role for tumor-induced membrane-cholesterol efflux in driving the IL-4 signaling and the tumor-promoting functions of TAM, while rendering them refractory to pro- inflammatory stimuli. Thus, preventing cholesterol efflux in TAM could represent a novel therapeutic strategy to block pro-tumor functions and restore anti-tumor immunity.

Accordingly, the first object of the present invention relates to a method of inducing a phenotypic change in a population of macrophages in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that modulates cholesterol efflux in said population of macrophages.

As used herein, the term“macrophage” has its general meaning in the art and refers to a type of antigen-presenting cell of the mammalian immune system that have phagocytic activities. These cells are characterized by their distinctive morphology and high levels of surface MHC-class II expression. A macrophage is a monocyte-derived phagocyte which is not a dendritic cell or a cell that derives from tissue macrophages by local proliferation. In the body these cells are tissue specific and refer to e. g. Kupffer cells in the liver, alveolar macrophages in the lung, microglia cells in the brain, osteoclasts in the bone etc. The skilled person is aware how to identify macrophage cells, how to isolate macrophage cells from the body of a human or animal, and how to characterize macrophage cells with respect to their subclass and subpopulation.

Macrophages have historically been divided into two phenotypically diverse populations, i.e. a Ml -polarized or "classically activated" population, and a macrophage M2 - polarized or "alternatively activated" population. However, it is well appreciated in the art that a continuum of phenotypes exists between the macrophage Ml - polarized and macrophage M2- polarized populations, and in some cases macrophages assume a phenotype that does not fit well within any of these defined phenotypic groups.

Macrophages exhibiting a Ml phenotype are pro-inflammatory, and are capable of either direct (pathogen pattern recognition receptors) or indirect (Fc receptors, complement receptors) recognition of pathogens and tumor antigens (i.e. they exhibit anti -tumor activity). Ml macrophages produce reactive oxygen species and secrete pro-inflammatory cytokines and chemokines, such as, for example, but without limitation, TNFa, IL-1, IL-6, IL-15, IL-18, IL- 23, and iNOS. Ml macrophages also express high levels of MHC, costimulatory molecules, and FCyR. The Ml phenotype is triggered by GM-CSF and further stimulated by interferon-g (IFN-g), bacterial lipopolysaccharide (LPS), or tumor necrosis factor a (TNFa), and is mediated by several signal transduction pathways involving signal transducer and activator of transcription (STAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB), and mitogen-activated protein kinases (MAPK). These events enhance the production of agents such as the reactive oxygen species and nitric oxide (NO) and promote subsequent inflammatory immune responses by increasing antigen presentation capacity and inducing the Thl immunity through the production of cytokines such as IL-12.

In contrast, macrophages exhibiting a M2 phenotype are often characterized as being anti-inflammatory and immunosuppressive as they suppress T-cell responses and are involved in the Th2-type immune response. The M2 macrophage phenotype facilitates tissue repair, wound healing, and is profibrotic. M2 macrophages often undesirably infiltrate and surround tumors, where they provide an immunosuppressive microenvironment that promotes rather than suppresses tumor progression. M2 macrophages are characterized by high surface expression of I1-4R, FcsR, Dectin-1, CD 136, CD206, and CD209A. M2 macrophages include IL-4/IL- 13 -stimulated macrophages, IL-10-induced macrophages, and immune complex-triggered macrophages.

The method of the present invention is thus suitable for inducing one or more phenotypic changes in a population of macrophages to adjust a population of macrophages to have a desired phenotype, e.g. a pro-inflammatory phenotype (Ml) or an immunosuppressive phenotype (M2).

As used herein, a "phenotypic change" encompasses an observable or detectable change in a characteristic, property, attribute, or function of the population of macrophages. For example, phenotypic characteristics/properties/functions of macrophages that can be modified or modulated in accordance with the methods of the present invention include, without limitation, pro-inflammatory activity, anti-inflammatory activity, immunogenic activity, tolerogenic activity, tissue damaging activity, tissue healing activity, cytotoxic activity, migratory activity, bone-resorbing activity, angiogenic activity, anti- angiogenic activity, suppressor activity, antigen presenting activity, or phagocytic activity. A phenotypic change in the macrophages may be observed or detected in any of a number of ways. For example, a phenotypic change may be observed or detected either by performing a test, observation, or measurement on the macrophages themselves or by performing a test, observation, or measurement, on other cells, tissues, organs, etc., that may be affected by the monocytes/macrophages, or by performing a test, observation, or measurement on a subj ect that contains the phenotypically modified macrophages.

Phenotypic change or modulation can be assessed by detecting or measuring, for example, (i) a change in the expression of one or more genes (e.g., cytokines, inflammatory mediators, etc.); (ii) the change in secretion of one or more molecules (e.g., cytokines, inflammatory mediators, etc.); (iii) an increase or decrease in migration to one or more sites in the body; (iv) a change in the ability to cause an alteration in one or more phenotypic characteristics or phenotypes of another macrophage-related cell or ability to cause an alteration in one or more phenotypic characteristics or phenotypes of a non-macrophage-related cell. Methods for observing, detecting, and measuring these phenotypic changes are known in the art and described herein. For example, gene expression profiles can be assessed at the RNA level using cDNA or oligonucleotide microarray analysis, Northern blots, RT-PCR, sequencing, etc. Protein expression can be measured using, for example, immunoblotting, immunohistochemistry, protein microarrays, etc. Various cell-based assays and animal models readily known and utilized in the art can also be used.

In some embodiments, the method of the present invention is particularly suitable for inducing a phenotypic change in a population of tumor-associated macrophages (TAM). More specifically, the method of the present invention is particularly suitable for blocking pro-tumor functions and restoring anti-tumor immunity of tumor-associated macrophages.

As used herein, the term’’tumor associated macrophage” or“TAM” has its general meaning in the art and is intended to describe a type of cell belonging to the macrophage lineage. They are found in close proximity or within tumor masses. TAMs are derived from circulating monocytes or resident tissue macrophages, which form the major leukocytic infiltrate found within the stroma of many tumor types. TAM are characterized by the expression of CD 163. As used herein, the term“cholesterol efflux” or“cholesterol efflux activity” refers to the efflux of cholesterol from population of macrophages as described in the EXAMPLE. Accordingly the term refers to the movement of cholesterol from the cell to the cell's exterior.

As used herein, the term "modulate" when applied to the term“cholesterol efflux” refers to an increase or a decrease in said cholesterol efflux. In some embodiments, thus the agent can be an agent that increases said cholesterol efflux or an agent that decreases said cholesterol efflux. In some embodiments, the increase can be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%. 250%. 300%, 400%, 500% or more. In some embodiments, the decrease can be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. As used herein, the term "decrease" thus includes the inhibition or blockage of said cholesterol efflux.

As used herein, the term“agent” refers to any chemical entity, including, without limitation, a glycomer, a protein, an antibody, a lectin, a nucleic acid, a small molecule, and any combination thereof. Examples of possible agents include, but are not limited to, a ribozyme, a DNAzyme and a siRNA molecule. In some embodiments, the agent is an antibody.

As used herein, the term“antibody” has its general meaning in the art and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-id) antibodies (including, e.g., anti-id antibodies to antibodies of the invention), intrabodies, and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI and IgA2) or subclass.

In some embodiments, the antibody of the present invention is a monoclonal antibody. The term“monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cells that are uncontaminated by other immunoglobulin producing cells. Alternative production methods are known to those trained in the art, for example, a monoclonal antibody may be produced by cells stably or transiently transfected with the heavy and light chain genes encoding the monoclonal antibody.

Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with the appropriate antigenic forms (i.e. polypeptides of the present invention). The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG- containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes. Briefly, the recombinant polypeptide of the present invention may be provided by expression with recombinant cell lines. Recombinant forms of the polypeptides may be provided using any previously described method. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods. Following culture of the hybridomas, cell supernatants are analysed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

In some embodiments, the monoclonal antibody of the invention is a chimeric antibody, in particular a chimeric mouse/human antibody. As used herein, the term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody. In some embodiments, the human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a human chimeric antibody, it may be any region which belongs to Ig, and those of kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).

In some embodiments, the monoclonal antibody of the invention is a humanized antibody. In particular, in said humanized antibody, the variable domain comprises human acceptor frameworks regions, and optionally human constant domain where present, and non human donor CDRs, such as mouse CDRs. According to the invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody. The humanized antibody of the present invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred. Examples of tandem type humanized antibody expression vector include pKANTEX93 (WO 97/10354), pEE18 and the like. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

In some embodiments the antibody of the invention is a human antibody. As used herein the term "human antibody is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, cur. Opin. Pharmacol. 5; 368-74 (2001) and lonberg, cur. Opin. Immunol. 20; 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech. 23;1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075, 181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application publication No. US 2007/0061900, describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 13 : 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86(1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc. Natl. Acad. Sci. USA, 103 :3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human igM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein,, Histology and Histopathology, 20(3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005). Fully human antibodies can also be derived from phage- display libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; and Marks et al, 1991, J. Mol. Biol. 222:581). Phage display techniques mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT publication No. WO 99/10494. Human antibodies described herein can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

In some embodiments, the agent that modulates the cholesterol efflux is an agent that modulates the activity or expression of a cholesterol efflux mediating protein.

As used herein, the term“cholesterol efflux-mediating protein” refers to any protein which, when properly situated in and/or on a macrophage, facilitates the efflux of cholesterol from the macrophage. Examples of a“cholesterol efflux-mediating protein” include, without limitation, ABCl, ABCGl and ABCG4.

In some embodiment, the agent that modulates the cholesterol efflux is an agent that modulates the activity or expression of ABCl, ABCGl and ABCG4.

As used herein, the term“ABCl” has its general meaning in the art and refers to the ATP -binding cassette sub-family A member 1. An exemplary human amino acid sequence is represented by SEQ ID NO: 1. The extracellular domain of ABCl corresponds to the amino acid sequence that ranges from amino acid residue at position 43 to the amino acid residue at position 639 in SEQ ID NO: 1.

SEQ ID NO: 1 >sp | 095477 | ABCA1 HUMAN ATP-binding cassette sub-family A member 1 OS=Homo sapiens OX=9606 GN=ABCA1 PE=1 SV=3

MACWPQLRLLLWKNLTFRRRQTCQLLLEVAWPLFIFLILISVRLSYPPYEQHECHFPNKA MPSAGTLPWVQGIICNANNPCFRYPTPGEAPGWGNFNKSIVARLFSDARRLLLYSQKDT SMKDMRKVLRTLQQIKKSSSNLKLQDFLVDNETFSGFLYHNLSLPKSTVDKMLRADVILH KVFLQGYQLHLTSLCNGSKSEEMIQLGDQEVSELCGLPREKLAAAERVLRSNMDILKPIL RTLNSTSPFPSKELAEATKTLLHSLGTLAQELFSMRSWSDMRQEVMFLTNWSSSSSTQI YQAVSRIVCGHPEGGGLKIKSLNWYEDNNYKALFGGNGTEEDAETFYDNSTTPYCNDLMK NLESSPLSRIIWKALKPLLVGKILYTPDTPATRQVMAEWKTFQELAVFHDLEGMWEELS PKIWTFMENSQEMDLVRMLLDSRDNDHFWEQQLDGLDWTAQDIVAFLAKHPEDVQSSNGS VYTWREAFNETNQAIRTISRFMECWLNKLEPIATEVWLINKSMELLDERKFWAGIVFTG ITPGSIELPHHVKYKIRMDIDNVERTNKIKDGYWDPGPRADPFEDMRYVWGGFAYLQDW EQAIIRVLTGTEKKTGVYMQQMPYPCYVDDIFLRVMSRSMPLFMTLAWIYSVAVIIKGIV YEKEARLKETMRIMGLDNSILWFSWFISSLIPLLVSAGLLWILKLGNLLPYSDPSWFV FLSVFAWTILQCFLISTLFSRA LAAACGGIIYFTLYLPYVLCVAWQDYVGFTLKIFAS LLSPVAFGFGCEYFALFEEQGIGVQWDNLFESPVEEDGFNLTTSVSMMLFDTFLYGVMTW YIEAVFPGQYGIPRPWYFPCTKSYWFGEESDEKSHPGSNQKRISEICMEEEPTHLKLGVS IQNLVKVYRDGMKVAVDGLALNFYEGQITSFLGHNGAGKTTTMSILTGLFPPTSGTAYIL GKDIRSEMSTIRQNLGVCPQHNVLFDMLTVEEHIWFYARLKGLSEKHVKAEMEQMALDVG LPSSKLKSKTSQLSGGMQRKLSVALAFVGGSKWILDEPTAGVDPYSRRGIWELLLKYRQ GRTIILSTHHMDEADVLGDRIAIISHGKLCCVGSSLFLKNQLGTGYYLTLVKKDVESSLS SCRNSSSTVSYLKKEDSVSQSSSDAGLGSDHESDTLTIDVSAISNLIRKHVSEARLVEDI GHELTYVLPYEAAKEGAFVELFHEIDDRLSDLGISSYGISETTLEEIFLKVAEESGVDAE TSDGTLPARRNRRAFGDKQSCLRPFTEDDAADPNDSDIDPESRETDLLSGMDGKGSYQVK GWKLTQQQFVALLWKRLLIARRSRKGFFAQIVLPAVFVCIALVFSLIVPPFGKYPSLELQ PWMYNEQYTFVSNDAPEDTGTLELLNALTKDPGFGTRCMEGNPIPDTPCQAGEEEWTTAP VPQTIMDLFQNGNWTMQNPSPACQCSSDKIKKMLPVCPPGAGGLPPPQRKQNTADILQDL TGRNISDYLVKTYVQIIAKSLKNKIWWEFRYGGFSLGVSNTQALPPSQEWDAIKQMKK HLKLAKDSSADRFLNSLGRFMTGLDTKNNVKVWFNNKGWHAI SSFLNVINNAILRANLQK GENPSHYGITAFNHPLNLTKQQLSEVALMTTSVDVLVSICVIFAMSFVPASFWFLIQER VSKAKHLQFI SGVKPVIYWLSNFVWDMCNYWPATLVI 11 FICFQQKSYVSSTNLPVLAL

LLLLYGWSITPLMYPASFVFKIPSTAYWLTSWLFIGINGSVATFVLELFTDNKLNNIN DILKSVFLIFPHFCLGRGLIDMVKNQAMADALERFGENRFVSPLSWDLVGRNLFAMAVEG WFFLITVLIQYRFFIRPRPWAKLSPLNDEDEDVRRERQRILDGGGQNDILEIKELTKI YRRKRKPAVDRICVGIPPGECFGLLGWGAGKSSTFKMLTGDTTVTRGDAFLNKNSILSN IHEVHQNMGYCPQFDAITELLTGREHVEFFALLRGVPEKEVGKVGEWAIRKLGLVKYGEK YAGNYSGGNKRKLSTAMALIGGPPWFLDEPTTGMDPKARRFLWNCALSWKEGRSWLT SHSMEECEALCTRMAIMVNGRFRCLGSVQHLKNRFGDGYTIWRIAGSNPDLKPVQDFFG LAFPGSVLKEKHRNMLQYQLPSSLSSLARIFSILSQSKKRLHIEDYSVSQTTLDQVFWF AKDQSDDDHLKDLSLHKNQTWDVAVLTSFLQDEKVKESYV

As used herein, the term“ABCG1” has its general meaning in the art and refers to the ATP -binding cassette sub-family G member 1. An exemplary human amino acid sequence is represented by SEQ ID NO: 2. The extracellular domain of ABC1 corresponds to the amino acid sequence that ranges from amino acid residue at position 446 to the amino acid residue at position 456 in SEQ ID NO:2.

SEQ ID NO: 2 >sp | P45844 | ABCG1_HUMAN ATP-binding cassette sub-family G member 1 OS=Homo sapiens OX=9606 GN=ABCG1 PE=1 SV=3

MACLMAAFSVGTAMNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDNNL

TEAQRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMGPS

GAGKSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMV

SAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFF

DEPTSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGK

VCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGD

AEVNPFLWHRPSEEVKQTKRLKGLRKDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDS

VLTHLRITSHIGIGLLIGLLYLGIGNEAKKVLSNSGFLFFSMLFLMFAALMPTVLTFPLE

MGVFLREHLNYWYSLKAYYLAKTMADVPFQIMFPVAYCSIVYWMTSQPSDAVRFVLFAAL

GTMTSLVAQSLGLLIGAASTSLQVATFVGPVTAI PVLLFSGFFVSFDTIPTYLQWMSYIS

YVRYGFEGVILSIYGLDREDLHCDIDETCHFQKSEAILRELDVENAKLYLDFIVLGIFFI

SLRLIAYFVLRYKIRAER As used herein, the term“ABCG4” has its general meaning in the art and refers to the ATP -binding cassette sub-family G member 4. An exemplary human amino acid sequence is represented by SEQ ID NO: 3. The extracellular domain of ABC1 corresponds to the amino acid sequence that ranges from amino acid residue at position 415 to the amino acid residue at position 425 in SEQ ID NO:3.

SEQ ID NO: 3 >sp | Q9H172 | ABCG4_HUMAN ATP-binding cassette sub-family G member 4 OS=Homo sapiens OX=9606 GN=ABCG4 PE=1 SV=2

MAEKALEAVGCGLGPGAVAMAVTLEDGAEPPVLTTHLKKVENHITEAQRFSHLPKRSAVD

IEFVELSYSVREGPCWRKRGYKTLLKCLSGKFCRRELIGIMGPSGAGKSTFMNILAGYRE

SGMKGQILVNGRPRELRTFRKMSCYIMQDDMLLPHLTVLEAMMVSANLKLSEKQEVKKEL

VTEILTALGLMSCSHTRTALLSGGQRKRLAIALELVNNPPVMFFDEPTSGLDSASCFQW

SLMKSLAQGGRTIICTIHQPSAKLFEMFDKLYILSQGQCIFKGWTNLIPYLKGLGLHCP

TYHNPADFIIEVASGEYGDLNPMLFRAVQNGLCAMAEKKSSPEKNEVPAPCPPCPPEVDP

IESHTFATSTLTQFCILFKRTFLSILRDTVLTHLRFMSHWIGVLIGLLYLHIGDDASKV

FNNTGCLFFSMLFLMFAALMPTVLTFPLEMAVFMREHLNYWYSLKAYYLAKTMADVPFQV

VCPWYCSIVYWMTGQPAETSRFLLFSALATATALVAQSLGLLIGAASNSLQVATFVGPV

TAI PVLLFSGFFVSFKTIPTYLQWSSYLSYVRYGFEGVILTIYGMERGDLTCLEERCPFR

EPQSILRALDVEDAKLYMDFLVLGIFFLALRLLAYLVLRYRVKSER

In some embodiments, the agent that modulates the activity of ABC1, ABCG1 or ABCG4 is an antibody that binds to the extracellular domain of ABC1, ABCG1 or ABCG4 respectively.

In some embodiments, the agent that modulates the cholesterol efflux is an agent that modulates the activity or expression of a receptor for hyaluronic acid.

As used herein, the term“hyaluronic acid” or“HA” refers to the polymer having the formula:

where n is the number of repeating units. All sources of hyaluronic acid are useful in this invention, including bacterial and avian sources. Hyaluronic acids useful in this invention have a molecular weight superior to lOOkDa (“high molecular weight”).

In some embodiments, the agent that modulates the the cholesterol efflux modulates the activity or expression of CD44 or Lyve-1 that are receptors for HA. As used herein, the term“CD44” has its general meaning in the art and refers to a receptor for hyaluronic acid. The term is also known as CDw44, Epican, Extracellular matrix receptor III (ECMR-III), GP90 lymphocyte homing/adhesion receptor, HUTCH-I, Heparan sulfate proteoglycan, Hermes antigen, Hyaluronate receptor, Phagocytic glycoprotein 1 (PGP- 1), and Phagocytic glycoprotein I (PGP-I). An exemplary human amino acid sequence is represented by SEQ ID NO:4. The extracellular domain of CD44 corresponds to the amino acid sequence that ranges from amino acid residue at position 21 to the amino acid residue at position 649 in SEQ ID NO:4.

SEQ ID NO: 4 >sp | PI 6070 | CD44_HUMAN CD44 antigen OS=Homo sapiens

OX=9606 GN=CD44 PE=1 SV=3

MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSISRTEAADLCKAFNSTL PTMAQMEKALSIGFETCRYGFIEGHWIPRIHPNSICAANNTGVYILTSNTSQYDTYCFN ASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQKGEYRTNPEDIYPSNPTDDDVSS GSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTDRIPATTLMSTSATATETATKRQE TWDWFSWLFLPSESKNHLHTTTQMAGTSSNTISAGWEPNEENEDERDRHLSFSGSGIDDD

EDFISSTISTTPRAFDHTKQNQDWTQWNPSHSNPEVLLQTTTRMTDVDRNGTTAYEGNWN PEAHPPLIHHEHHEEEETPHSTSTIQATPSSTTEETATQKEQWFGNRWHEGYRQTPKEDS HSTTGTAAASAHTSHPMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRRMDMDSSHSIT LQPTANPNTGLVEDLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDV TGGRRDPNHSEGSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNR

SLSGDQDTFHPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPEWLIILASLLAL ALILAVCIAVNSRRRCGQKKKLVINSGNGAVEDRKPSGLNGEASKSQEMVHLVNKESSET PDQFMTADETRNLQNVDMKIGV As used herein, the term“Lyve-1” has its general meaning in the art and refers to the lymphatic vessel endothelial hyaluronic acid receptor 1. An exemplary human amino acid sequence is represented by SEQ ID NO: 5. The extracellular domain of CD44 corresponds to the amino acid sequence that ranges from amino acid residue at position 20 to the amino acid residue at position 238 in SEQ ID NO: 5.

SEQ ID NO; 5 >sp | Q9Y5Y7 | LYVE1 HUMAN Lymphatic vessel endothelial hyaluronic acid receptor 1 OS=Homo sapiens OX=9606 GN=LYVE1 PE=1 SV=2 MARCFSLVLLLTSIWTTRLLVQGSLRAEELSIQVSCRIMGITLVSKKANQQLNFTEAKEA CRLLGLSLAGKDQVETALKASFETCSYGWVGDGFWISRISPNPKCGKNGVGVLIWKVPV SRQFAAYCYNSSDTWTNSCIPEIITTKDPIFNTQTATQTTEFIVSDSTYSVASPYSTIPA PTTTPPAPASTSIPRRKKLICVTEVFMETSTMSTETEPFVENKAAFKNEAAGFGGVPTAL

LVLALLFFGAAAGLGFCYVKRYVKAFPFTNKNQQKEMIETKWKEEKANDSNPNEESKKT DKNPEESKSPSKTTVRCLEAEV

In some embodiments, the agent modulates the activity of CD44 or Lyve-1 is an antibody that binds to the extracellular domain of CD44 or Lyve-1.

In some embodiments, the agent is an inhibitor of expression; such as an inhibitor of. ABC1, ABCG1, ABCG4, CD44 or Lyve-1 expression.

In some embodiment, the agent that modulates the cholesterol efflux is an inhibitor of expression of ABC1, ABCG1, ABCG4 CD44 or Lyve-1. An“inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of Proprotein convertase (PC) mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Proprotein convertase (PC), and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding Proprotein convertase (PC) can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6, 107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Proprotein convertase (PC) gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Proprotein convertase (PC) gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing Proprotein convertase (PC). Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor of expression is an endonuclease. The term“endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, and cleave only at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term“CRISPR- cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797. In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

In some embodiments, the agent that modulates the cholesterol efflux is selected as depicted in Table A.

Table A: examples of agents capable of modulating cholesterol efflux in macrophages.

Accordingly, in some embodiments, the agent that modulates the cholesterol efflux is an agent that decreases the cholesterol efflux in macrophages. In one embodiment, the agent that decrease the cholesterol efflux in macrophages is an a antagonistic antibody that binds to the extracellular domain of ABC 1 , ABCG1 ABCG4, CD44 or Lyve-1 respectively

In one embodiment, the agent that decrease the cholesterol efflux in macrophages is an inhibitor of expression of ABC1, ABCG1, ABCG4 CD44 or Lyve-1

Accordingly, in some embodiments, the agent that modulates the cholesterol efflux is an agent that increases the cholesterol efflux in macrophages.

In one embodiment, the agent that increase the cholesterol efflux in macrophages is an a agonistic antibody that binds to the extracellular domain of ABC1, ABCGl ABCG4, CD44 or Lyve-1 respectively

In one embodiment, the agent that increase the cholesterol efflux in macrophages is a hyaluronic acid.

The method as described herein particularly suitable for the purpose of treating a disease or condition that is caused or exacerbated, at least in part, by macrophages exhibiting one or more undesirable phenotypes.

Accordingly, a further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that modulates cholesterol efflux in a population of macrophages.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In some embodiments, an agent that increases cholesterol efflux in a population of macrophages would be suitable for the treatment of autoimmune inflammatory disease. Autoimmune inflammatory diseases typically involve the undesired actions of pro- inflammatory macrophages (Ml). Using the method of the present invention to induce a macrophage M2 phenotypic change in the Ml pro-inflammatory macrophages that are involved in these diseases would be suitable for alleviating one or more symptoms of the diseases.

In some embodiments, the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal garnmopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channel opathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channel opathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia- reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman- Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Lesihmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott- Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

In some embodiments, an agent that supresses cholesterol efflux in a population of tumor-associated macrophages would be suitable for the treatment of cancer.

Many studies indicate that tumor- associated macrophages (TAMs) exhibit a macrophage M2-like phenotype. These M2 macrophages are important tumor-infiltrating cells and play pivotal roles in tumor growth and metastasis. In most solid tumors, the existence of TAMs is advantageous for tumor growth and metastasis. These TAMs produce interleukin IL- 10 and transforming growth factor (TGF) b to suppress general antitumor immune responses. Meanwhile, TAMs promote tumor neo- angiogenesis by the secretion of pro-angiogenic factors and define the invasive microenvironment to facilitate tumor metastasis and dissemination. Therefore, employing the method of the present invention to induce a Ml phenotypic change in the TAMs to enhance anti-tumor immunity will significantly alter the progression of the cancer.

As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In some embodiments, by inhibiting the immunosuppressive phenotype of TAM, the agent that decreases the cholesterol efflux would thus be suitable for enhancing the proliferation, migration, persistence and/or activity of cytotoxic T lymphocytes (CTLs).

Thus a further of the present invention relates to a method of enhancing the proliferation, migration, persistence and/or activity of cytotoxic T lymphocytes (CTLs) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that suppresses cholesterol efflux in TAM. As used herein, the term“cytotoxic T lymphocyte” or“CTL” has its general meaning in the art and refers to a subset of T cells which express CD8 on their surface. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions. They are MHC class I- restricted, and function as cytotoxic T cells. Cytotoxic T lymphocytes are also called, CD8+ T cells, T-killer cells, cytolytic T cells, or killer T cells. The ability of the proprotein convertase (PC) inhibitor to enhance proliferation, migration, persistence and/or cytotoxic activity of cytotoxic T lymphocytes may be determined by any assay well known in the art. Typically said assay is an in vitro assay wherein cytotoxic T lymphocytes are brought into contact with target cells (e.g. target cells that are recognized and/or lysed by cytotoxic T lymphocytes). For example, the proprotein convertase (PC) inhibitor can be selected for the ability to increase specific lysis by cytotoxic T lymphocytes by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%, or more of the specific lysis obtained at the same effector: target cell ratio with cytotoxic T lymphocytes that are contacted by the proprotein convertase (PC) inhibitor of the present invention. Examples of protocols for classical cytotoxicity assays are conventional.

In some embodiments, the administration of the agent that decreases the cholesterol efflux in TAM is combined with an immune checkpoint inhibitor.

As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM-3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist.

In some embodiments, PD-1 (Programmed Death-1) axis antagonists include PD-1 antagonist (for example anti-PD-1 antibody), PD-L1 (Programmed Death Ligand-1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg). In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in W02007/005874. Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010/077634 Al . MEDI4736 is an anti-PD- Ll antibody described in WO2011/066389 and US2013/034559. MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and W02006/121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009/114335. CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009/101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342. Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-Ll antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in W02015033301 & WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.

In some embodiments, CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA- 4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA- 4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No. WO 2001/014424, the antibodies disclosed in PCT Publication No. WO 2004/035607, the antibodies disclosed in U.S. Publication No. 2005/0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 B. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014. Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98/42752; U.S. Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al, J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP- 675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7, 132,281. A preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00- 9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01/14424. With regard to CTLA-4 antagonist (antibodies), these are known and include Tremelimumab (CP- 675,206) and Ipilimumab.

Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al, 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al, 2010, J. Exp. Med. 207:2187-94). As used herein, the term“TIM-3” has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galectin 9 (Gal9). Accordingly, the term“TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and/or block the binding of TIM-3 to galectin-9. Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO2011155607, WQ2013006490 and WO2010117057. In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1 -methyl-tryptophan (IMT), b- (3-benzofuranyl)-alanine, b-(3- benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 - methyl tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3- diacetate, 9- vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a b-carboline derivative or a brassilexin derivative. Preferably the IDO inhibitor is selected from 1 -methyl-tryptophan, b-(3- benzofuranyl)-alanine, 6-nitro-L- tryptophan, 3- Amino-nap htoic acid and b-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

In some embodiments, the administration of the agent that decreases the cholesterol efflux in TAM is combined with a T-cell immunotherapy to boost the patient's anti-tumor immune response. Suitable T-cell immunotherapies include, without limitation, adoptive T-cell therapy, tumor-infiltrating lymphocyte therapy, chimeric antigen receptor (CAR) T-cell therapy, and antigen-specific T-cell receptor transduced T-cell therapy. As used herein the term "CAR-T cell" refers to a T lymphocyte that has been genetically engineered to express a CAR. The definition of CAR T-cells encompasses all classes and subclasses of T-lymphocytes including CD4+ , CD8+ T cells, gamma delta T cells as well as effector T cells, memory T cells, regulatory T cells, and the like. The T lymphocytes that are genetically modified may be "derived" or "obtained" from the subject who will receive the treatment using the genetically modified T cells or they may "derived" or "obtained" from a different subject. As used herein, the term“Chimeric Antigen Receptor” or alternatively a“CAR” refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as“an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27 and/or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. In some embodiments, CARs comprise domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAPIO, and/or 0X40. In some embodiments, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. As used herein, the terms“combined treatment”, “combined therapy” or“therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

Such administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate.

As used herein, the term“administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term“administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different

As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the agent of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the agent of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of agent of the present invention employed in the pharmaceutical composition at levels lower than that required achieving the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above.

For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a patient. One of ordinary skill in the art would be able to determine such amounts based on such factors as the patient's size, the severity of the patient's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1- 20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered overtime or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the human monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of a inhibitor of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of a inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1,

1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

Typically, the agent of the present invention is administered to the patient in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m² and 500 mg/m². However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Tumor cell-derived hyaluronic acid (HA) oligomers deplete lipid rafts in macrophages. (A) BMDM were co-cultured with live or paraformaldehyde-treated (fixed) ID8 cells, prior to CTB staining, quantification of CTCF is shown. (B) High and low molecular weight fractions of ID8-CM were prepared using Centricon filters with 100 kDa pores, each fraction was compared with unfractionated ID8-CM for effects on CTB staining. (C) BMDM were treated with ID8-CM with and without hyaluronidase (HAse) treatement prior to CTB staining and quantification. (D) BMDM were incubated with HA oligomers of increasing molecular weights before CTB staining and quantification, ID8-CM was used as a positive control.

Figure 2. Cholesterol efflux promotes IL-4 mediated macrophage reprogramming.

(A,B) BMDM were treated with or without ID8-CM before stimulation with increasing concentrations of IL-4 or IFNy for 8 hours. (A) Quantitative PCR (qPCR) analysis of IL-4 induced gene expression; Argl, Chi3l3, Mrcl, Retnla and (B) IFNy-induced expression of Nos2 and 1112b. (C) BMDM were treated with different cholesterol depleting agents; 9-cis-retenoic acid (9cRA), high-density lipoprotein (HDL) or apolipoprotein A1 (ApoAl), as well as ID8- CM; lipid raft density was subsequently measured by CTB staining, quantification of CTCF is shown. (D) IL-4 (20 ng/ml) induced Argl and IFNy (20 ng/ml) induced Nos2 expression in BMDM treated with 9cRA, HDL or ApoAl, compared to ID8-CM. (E) BMDM from Abcal/g and Abcal/gl^ALyz2 mice were incubated with ID8-CM, 9cRA, HDL or ApoAl and lipid raft density was measure by CTB staining, quantification of CTCF is shown. (F) IL-4 induced Argl and IFNy induced Nos 2 expression in BMDM from A bcaJ gjd and A heal gU^{l vz2} mice with and without ID8-CM treatment.

EXAMPLE:

Material & Methods

Materials

The following materials were employed throughout the study: LY294002, Torin, Rapamycin (Merck); hyaluronic acid, hyaluronidase, methyl-P-cyclodextrin, apolipoprotein Al, high-density lipoprotein, 9-cis-retenoic acid and cholesterol-methyl- -cyclodextrin (Sigma); recombinant mouse IL-4, IFNy and M-CSF (Peprotech); IL4Ra neutralizing antibody (eBioscience).

Mice

C57B1/6 mice were obtained from Charles River. All transgenic mouse strains were backcrossed to a C57B1/6 background. Abcal^tmlJp Abcgl^tmlTal1 (A heal gl^ff) and Lyz2^tml(cre)I^° ( Lyz2^Cre ) mice were obtained from Jackson Laboratories. Slal6 mice were kindly donated by Dr. Bernard Malissen (CIML, Marseille, FR), A heal mice by Prof. Miranda VanEck (Leiden University, NL) and Pik3cct mice by Prof. Martin Turner (Babraham Institute, Cambridge, UK). Generation of shielded chimeras was performed as previously described (Scott et al., 2016); briefly, CD45.1 congenic mice mice were anaesthetised with Ketamine (150 mg/kg) and Xylazine (10 mg/kg) and placed in 6 mm thick lead cylinders, exposing only the hind legs. With the peritoneal cavity protected, mice were irradiated with 9 Gy and reconstituted with 10⁷ bone marrow cells from Ccr2^~/~ (CD45.2) and CD45.1/2 mice, at a ratio of 4: 1. After 5 weeks, chimerism of blood leukocytes was assessed by flow cytometry. All mice were housed under specific pathogen-free conditions and animal experimentation was conducted in strict accordance with good animal practice as defined by the French animal welfare bodies relative to European Convention (EEC Directive 86/609) and approved by the Direction Departmental des Services Veterinaires des Bouches du Rhones.

Cell Culture

Bone marrow derived macrophages (BMDM) were obtained as previously described (Hagemann et al., 2008); briefly, femurs and tibiae from mice aged 8 to 10 weeks were flushed and cells collected by centrifugation at 450 g for 5 min at 4°C. Cells were resuspended in DMEM supplemented with L-glutamine (2 mM), penicillin (100 U/ml)/streptomycin (100 pg/ml) (Gibco), 10 % heat-inactivated FBS and 10 ng/ml recombinant mouse M-CSF (Peprotech) and cultured at a density of 10⁶ cells/ml in non-tissue culture treated plastic dishes (BD Pharmingen) at 37°C and 5 % CO2. After 7 days, adherent cells were collected and resuspended in complete DMEM containing 10 ng/ml M-CSF. The ID8-Luc ovarian surface epithelial cell line was kindly provided by Prof. Frances Balkwill (Barts Cancer Institute, London, UK). To obtain ID8 cell-conditioned conditioned medium (ID8-CM); 13.75 xlO⁶ cells in 25 ml were incubated for 72 hours in a 175 cm² flasks in DMEM containing 4 % of FCS. Medium was filtered through a 22 mM filter, aliquoted and stored at -80°C.

Immunofluorescence and lipid raft staining

BMDM were grown in Lab-Tek chambered slides (ThermoFisher Scientific) and fixed with 4 % PFA, permeabilised (0.1 % Triton-X100) and blocked in 5 % BSA with 10 mM glycine. The following primary antibodies were used for incubation during 90 minutes at 4°C; anti-pSTATl, anti-pSTAT6, anti-pSer473-Akt, anti-pThr308-Akt, anti-PHL (Cell Signaling). After washing, anti-rabbit-Alexa488 (Invitrogen) and TO-PRO-3 (ThermoFisher Scientific) were added for 60 minutes. Lipid rafts were stained using the Vybrant Alexa Fluor 488 Lipid Raft Labelling Kit, following manufacturer’s instructions. Briefly, BMDM were grown and stimulated in Lab-Tek chambered slides. After washing with serum- free DMEM, they were incubated for 10 minutes with Alexa488-conjugated cholera toxin subunit B (CTB) at 4°C, followed by cross-linking with an anti-CTB antibody for 15 minutes at 4°C. Subsequently, the cells were fixed with 4 % Antigenfix (DiaPath) for 10 minutes on ice and nuclei were stained with TO-PRO-3. The di-4-ANEPPDHQ lipid raft staining protocol was adapted from the one described by Owen et al. (Owen et ak, 2011). Briefly, culture medium was replaced with fresh, serum- free DMEM containing 2 mΐ of di-4-ANEPPDHQ (5 mM). Dishes were shaken to ensure good mixing. After 30 min incubation at 37 °C in a humidified 5 % CO2 atmosphere, cells were fixed with 4 % PFA. Fluorescence was measured by confocal microscopy (Zeiss LSM780 or Leica SP5X) and analyzed with FIJI software. The corrected total cell fluorescence (CTCF) was measured for each cell in at least 6 different fields of view per well.

Cholesterol measurement

Total cell cholesterol content was measured in BMDM using the Amplex Red Cholesterol Assay kit (ThermoFisher Scientific), according to the manufacturer’s instructions.

Immunoblotting

BMDM were lysed on ice in lysis buffer supplemented with proteinase inhibitor cocktail, PNPP, b-glycerophosphate and DTT. Separation by SDS-PAGE was followed by blotting on PVDF membrane. Blots were blocked with 5 % skimmed milk in TBS- 0.05 %Tween20. The following primary antibodies were used; anti-Nos2, anti-Argl (Santa Cruz), anti-P-Actin (Sigma), anti-Akt and anti-pSer473-Akt (Cell Signaling). Primary antibodies were incubated overnight at 4°C and appropriate HRP-conjugated secondary antibodies (DAKO) for 1 hour at room temperature. Chemoluminescence was detected by Pierce ECL Western Blotting Substrate (Thermo Scientific).

Gene expression analysis

Total cellular RNA was extracted from BMDM using TRIzol and cDNA was synthesised with cDNA Synthesis Kit (Thermo Fisher Sientific) according to the manufacturer’s protocol. Gene expression was quantified using sequence specific primers in the presence of SYBR Green PCR Master Mix using an ABI 7900HT thermocycler (Applied Biosystems). Reactions were performed in duplicate or triplicate and Ct-values were normalised to the mean Ct-values of cyclophillin. Relative quantification of gene expression was calculated as 2AACt to controls.

Ovarian cancer model

One million ID8 cells were injected intraperitoneally in the different mouse strains using a 27G syringe. Mice were euthanised at the indicated times and peritoneal lavages were collected for cytometric analysis, ex vivo bioluminescence measurement and/or lipid raft staining. Briefly, 9 ml of ice cold PBS was injected intraperitoneally and after a careful massage to detach all the cells in the cavity, peritoneal fluid was collected through a 23 G syringe. Tubes were weighed to determine the recovered lavage volume and the cell density was assessed using a Casy cell counter (Innovatis). Cells were centrifugated and resuspended in 1 ml cold PBS. One million cells from each peritoneal lavage were stained for flow cytometry. 50 mΐ of the 1 ml cell suspension obtained from peritoneal lavage was used for luciferase activity measurements. Cells were plated in a white 96-well plate and 50 mΐ luciferin was added to each well, Luminescence [photons/s] was measured for each well using the Mithras Microplate Reader (Berthold Technologies).

Flow Cytometry

Peritoneal lavage cells underwent a short NFLCl red blood cell lysis and were incubated at 4°C for 10 min with the 2.4.G2 antibody to block Fc receptors. The cells were stained with the indicated antibodies for 30 min at 4°C. Dead cells were gated out using SYTOX Blue dead cell stain (Life Technlogies). After cell-surface staining, cells were fixed. Analysis was performed using an LSR-II flow cytometer or sorted using an Aria III cell sorter (both BD Biosciences) and data analysis was conducted with the FlowJo cytometric analytical software (Tree Star). Anti-CD l ib (Ml/70), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), NK1.1, Ly6G, anti-CD5 (53-7.3), anti-CD19 (1D3), anti-CD64, anti-Ly6C (AL-21), anti-F4/80 and anti-MHCII (M5/114) were purchased from BD Biosciences, eBioscience, BioLegend, and Life Technologies.

Microarray Analysis

RNA samples were hybridised on Affymetrix Mouse 430 2.0 or MoGene 1.0 st chips. Samples were processed as follows: The biotinylated cRNAs were prepared according to a double amplification protocol using MessageAmp™ II aRNA Amplification Kit (Ambion). The images of the chips were generated with Affymetrix software AGCC version 3.2. The expression data was then extracted with the Affymetrix Expression Console version 1.1 software using the RMA (log2 scale) and MAS5 (linear scale) algorithms. Gene Set Enrichment Analysis (GSEA, Broad Institute) (Subramanian et al, 2005) was used to examine differentially expressed genes (DEGs). The output of GSEA is an enrichment plot (ES), a normalised enrichment score (NES) which accounts for the size of the gene set being tested, a p-value, and an estimated False Discovery rate (FDR). We computed P values using 1,000 permutations for each gene set and corrected them with the false-discovery rate (FDR) method. When several probe sets were present for a gene, the mean of the probe set was used. Cell-specific gene sets were generated by performing pairwise comparisons between DEGs from different populations, applying a 1.5 FC threshold and a p value of 0.05, using the Minimal (pairwise[Mean(test)/Mean(ref)]) method. Sample correlation analysis was performed based on Pearson’s correlation coefficients using BioLayout Express3D (Theocharidis et al, 2009). GO enrichment analysis was applied using the Cytoscape plug-in BiNGO (v2.44) (Maere et al., 2005) with FDR q-value threshold of 0.05 as default. The Cytoscape plugins Enrichment Map (vl . l) (Merico et al., 2010) and Word Cloud (Oesper et al, 2011) were used to visualize the GO networks.

Statistical Analysis

Graphs were made and statistical analysis was performed using Prism software (Graphpad). All quantitative data are presented as mean ± SEM. Statistical significance was calculated using Student^'s t-test, Wilcoxon-Mann- Whitney test, Chi-square test for contingency tables or One-Way ANfOVA. P values <0.05 were considered as significant.

Results

Origins of TAM during ID8 tumor development. High grade serous ovarian cancer (HGSC) is frequently associated with colonisation of the peritoneal cavity by cancer cells (George et al., 2016). ID8 cells are spontaneously transformed mouse ovarian surface epithelial cells (Urzua et al., 2016), when adoptively transferred by intra-peritoneal (i.p.) injection in syngeneic mice, these cells progressively develop a malignant ascites with tumor nodules throughout the peritoneal cavity (Hagemann et al, 2008), which is characteristic of HGSC. The peritoneal cavity is populated by two major subsets of serosal macrophages; large peritoneal macrophages (LPM), which are most abundant, and a minor population of small peritoneal macrophages (SPM) (Ghosn et al., 2010). Previous studies have shown that SPM and LPM have distinct developmental origins; SPM develop from blood monocytes which are derived from bone marrow progenitors, whereas LPM are derived from embryonic progenitors and are maintained independently of blood monocytes, retaining proliferative capacity for self-renewal (Yona et al., 2013). More recent studies have shown that LPM can be progressively replaced by long-lived bone marrow-derived macrophages that maintain self-renewal potential (Bain et al, 2016). To monitor the dynamics of peritoneal macrophages (PM) during ID8 tumor growth, we first characterized macrophage subsets by flow cytometry. SPM and LPM can be distinguished by F4/80 and MHCII expression; SPM are MHCII¹¹¹ F4/80¹⁰ whereas LPM are F4/80¹" MHCII¹⁰ (data not shown). LPM represent approximately 80 % of PM in naive mice, however, after seeding of ID8 cells in the peritoneal cavity, a significant population of F4/80^mt MHCIT^nt PM rapidly accumulates (intPM; data not shown). Kinetic analysis of total cell numbers revealed that LPM numbers remain relatively constant throughout tumor progression, while intPM progressively accumulate and eventually become the dominant TAM population (data not shown).

To determine the dynamics of PM subsets during ID8 tumor growth, we performed fate mapping studies with shielded radiation chimera mice. Radiation chimeras can be used to determine the contribution of bone marrow-derived progenitors towards cells in a given tissue. However, irradiation kills tissue-resident macrophages that then become replaced by monocyte- derived cells, thus to distinguish tissue-resident cells from monocyte-derived macrophages from the bone marrow, it is necessary to protect the tissue from the effects of radiation using lead shielding. To study the origins of PM subsets during ID8 tumor growth, we shielded the abdomen of host C57BL6 CD45.1 congenic mice during irradiation and then adoptively transferred a mixture of bone marrow cells from mice expressing both CD45.1 and CD45.2 (CD45.1/2) and Ccr2^_/ mice, expressing only CD45.2. This allowed the distinction between host (CD45.1) and donor cells (CD45.1/2 or CD45.2), as well as their CCR2-dependency, CD45.2 single-positive cells being CCR2-dependent. Due to the low engraftment efficiency of Ccr2 ^/ bone marrow cells, Ccr2^~ donor cells were mixed at a ratio of 4: 1 with competitor B6.CD45.1/2 cells. Five weeks after bone marrow engraftment, chimeric mice were injected with ID8 cells to track the contribution of bone marrow-derived cells to PM subsets (data not shown). CD45.1 and CD45.2 expression in TAM subsets was measured by flow cytometry a further 8 weeks after injection of ID8 cells, a total of 13 weeks after adoptive transfer of bone marrow cells, chimerism was normalised to blood monocytes. These experiments revealed that SPM and intPM were derived from CCR2-dependent bone marrow progenitors, with almost 100 % chimerism after 8 weeks of tumor growth (data not shown). However, at this time point, LPM only showed approximately 30 % chimerism, implying that LPM are more gradually replaced by bone marrow-derived cells during tumor development. To confirm the CCR2- dependency of intPM, we analyzed the accumulation of PM subsets in full CCR2 deficient mice (Ccr2^~/~), as expected, both SPM and intPM were drastically reduced in Ccr2^~ mice bearing ID8 tumors, whereas CCR2 deficiency had little impact on LPM numbers (data not shown).

To confirm the continuous contribution of blood monocytes to SPM and intPM populations throughout tumor growth, we used a fluorescent fate-mapping approach. The chemokine receptor Cx3crl is expressed by blood monocytes (Geissmann et ah, 2003) and previous studies have demonstrated the fate-mapping of monocyte-derived cells using knock- in mice that express a tamoxifen-inducible Cre-recombinase from the Cx3crl locus (Cx3crl^CreER ), crossed to mice expressing a ubiquitous lox-STOP-lox reporter cassette (Yona et al, 2013). As expected, we did not detect Cx3crl expression in steady-state LPM using the Cx3crl^egf^p/+ reporter mice, however, high levels of Cx3crl expression were observed in SPM and intermediated levels in intPM (data not shown), reflecting the likely monocyte origins of these cells. We crossed Cx3crl^CreER mice with Rosa26-lsl-tdRFP reporter mice (Cx3cr7^CreER:R26-tdRFP) and injected these mice with ID8 cells to track monocyte-derived cells during tumor growth. Six weeks after injection of ID8 cells, mice were given a single dose of 4-OHT by oral gavage (p.o.) and RFP expression in TAM subsets was measured by flow cytometry ten days later. These experiments showed strong RFP labelling in SPM and intPM within 10 days of 4-OHT administration, with very little labelling of LPM (data not shown). These data clearly demonstrated the contribution of blood monocytes to SPM and intPM during tumor growth, even within this short time frame.

Transcriptional profiling of TAM.

To evaluate the impact of the tumor-microenvironment on PM phenotype, we performed global gene expression analysis using microarrays on bulk PM from naive mice and at different time points during tumor progression. We isolated naive F4/80^hl PM and TAM at 5, 12 and 21 days during ID8 tumor development by flow cytometry (data not shown). Total RNA was extracted and samples were analyzed using MoGene l .Ost microarrays. RMA normalised data were filtered and analyzed for variations in gene expression. The generated heatmap shows the 1000 most variable genes in the dataset. To extract differentially expressed genes (DEGs) between naive PM and TAM at the different time points, we used Anova with an adjusted p value and a threshold of 1.5 fold change (FC). We then used Gene Ontology (GO) enrichment analysis to identify pathways affected in TAM at different time points (data not shown). DEGs are represented by edges and individual GO terms are represented by nodes. GO terms that are similar, as indicated by the intersection of DEGs in a given GO term, are closer to each other. This generates clusters of similar GO terms indicating common biological processes within the cluster. This analysis revealed a major cluster of upregulated genes related to immunity in TAM after 5 days (data not shown), possibly reflecting a tumoricidal response triggered by resident PM in response to ID8 cells. However, after 21 days, when tumors had become more established, the gene expression profile of TAM more closely resembled the phenotype of naive PM (data not shown). At this later time point, there was an upregulation of distinct gene clusters, including a large cluster of genes related to the innate immune response and tumor necrosis factor (TNF) signaling, in keeping with previous data showing an important role for TNF in this model (Charles et al, 2009; Hagemann et al, 2006), and also a distinctive cluster of genes associated with cholesterol metabolism and efflux (data not shown). To confirm the enrichment of genes related to cholesterol homeostasis, we merged several published genesets (Rayner et al, 2011) and known hallmarks to generate an extended gene list representing cholesterol homeostasis. This compiled geneset also showed a significant enrichment in TAM and among the up-regulated genes were known actors in cholesterol metabolism and efflux, including; Abcgl, Ldlr, Pparg, Hmgcsl, Hmgcr, Srebf2 (data not shown).

Increased membrane cholesterol efflux in TAM.

Changes in membrane cholesterol content have been shown to dramatically affect macrophage activation in response to pro-inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) (Fessler and Parks, 2011). This is thought, at least in part, to be due to the depletion of cholesterol rich membrane micro-domains, also called lipid rafts, which act as signaling platforms for certain receptors. But membrane cholesterol influences multiple facets of membrane structure and dynamics that can also affect receptor signaling. To confirm the finding that cholesterol efflux pathways were upregulated in TAM, we sought to measure effects on cholesterol membrane content in TAM from ID8 tumor bearing mice. Cholesterol rich membrane micro-domains are commonly measured using cholera toxin B (CTB) staining, which binds to ganglioside GM1, the accumulation of which is linked with membrane cholesterol content. We isolated naive PM and TAM at 5 and 21 days after injection of ID8 cells, stained the cells with Alexa Fluor 488-conjugated CTB and analyzed them by confocal microscopy. We observed that CTB staining was similar in naive PM and TAM isolated at 5 days, but was significantly decreased in TAM after 21 days of tumor growth (data not shown), indicating that the tumor-microenvironment may promote the depletion of cholesterol rich membrane micro-domains in TAM, in accordance with the upregulation of genes regulating cholesterol efflux in these cells (data not shown). To test if ID8 tumor cells had a direct effect on macrophage cholesterol efflux, we co-cultured ID8 cells with bone marrow-derived macrophages (BMDM) in vitro. After just one hour of co-culture, there was a significant decrease of CTB staining in BMDM (data not shown), indicating tumor cells actively promoted the depletion of membrane cholesterol in macrophages. To test if factors secreted by tumor cells were responsible for this effect, we incubated BMDM with conditioned medium obtained from ID8 cell cultures (ID8-CM). This also resulted in a rapid reduction in CTB staining, that was almost equivalent to the effects of methyl- -cyclodextrin (MCD), which extracts cholesterol from cell membranes (Ostrom and Liu, 2007) (data not shown). Although CTB is commonly used to measure cholesterol rich membrane micro-domains, this is a rather indirect measure of membrane cholesterol. Another method to assess membrane cholesterol content exploits the highly ordered structure of cholesterol-rich membrane microdomains by the use of phase-sensitive fluorescent probes such as Laurdan and di-4-ANEPPDHQ (Owen et al, 2011; Sonnino and Prinetti, 2013). These molecules adapt their emission wavelength based on local membrane order, which is a direct reflection of cholesterol content, independently of membrane-associated proteins. To confirm our findings, we labelled macrophages with di-4- ANEPPDHQ and found a significant decrease of membrane order in the presence of ID8-CM (data not shown). These assays confirmed that the reduction in CTB staining observed after ID8-CM treatment, correlated with alterations in membrane order that reflect reduced levels of membrane cholesterol. Furthermore, we measured total cholesterol levels in macrophages cultured in the presence or absence of tumor cell-conditioned medium and observed a significant decrease of total cellular cholesterol (data not shown). Finally, to directly measure cholesterol efflux from macrophages, we loaded BMDM with thymidine (³H)-labelled cholesterol and measured its efflux into the culture media after addition of the apolipoprotein Al (ApoAl). Membrane cholesterol efflux is mediated by the transfer of cholesterol to lipoproteins through ABC transporters (Zhao et al., 2010), in the case of ApoAl this occurs through the transporter ABCAl . These assays showed that addition of ID8-CM significantly increased cholesterol efflux from macrophages, which was reversed in BMDM from ABCAl- deficient mice ( Abcafl ), demonstrating that this was due to an increased efflux of membrane cholesterol (data not shown).

Tumor cell-derived hyaluronic acid (HA) drives cholesterol efflux in macrophages.

The studies described above showed that ID8 cells increased cholesterol efflux from macrophages. This effect could be recapitulated with conditioned medium but not with fixed cells (Fig.1 AT indicating that cholesterol efflux is promoted by a secreted factor. In order to further characterize this factor, we exposed ID8-CM to a series of treatments, including ultra centrifugation, boiling (95°C for 5 min), repeated freeze/thaw cycles, DNAse and proteinase K, none of which had any impact on the ability of ID8-CM to deplete CTB staining in macrophages (data not shown). However, size fractionation of ID8-CM with cut-offs at 3, 10, 30 or 100 kDa, revealed that this activity was present in a fraction with a molecular weight above 100 kDa (Fig.lBT Several previous studies have shown that the extracellular matrix (ECM) component hyaluronic acid (HA) can be produced by tumor cells and has been linked with increased tumor progression (Chanmee et al., 2016). HA also forms high molecular weight oligomers (>100 kDa) with distinct biological activity (Gomez-Aristizabal et al, 2016; Kolapalli et al., 2016; Rayahin et al., 2015). Furthermore, receptors for HA are expressed by TAM, namely CD44 and Lyve-1 (Chanmee et al., 2016; Turley et al, 2002). To test the hypothesis that HA in ID8-CM contributed to the effects on membrane cholesterol content, we treated ID8-CM with hyaluronidase (HAse) to degrade HA. Indeed, ID8-CM treated with HAse was no longer able to deplete CTB staining in macrophages (Fig.lCT Conversely, when HA of different molecular weights was added to macrophages in normal culture medium, we observed a reduction of CTB staining with increasing molecular weight (Fig.lDT These experiments suggested that high molecular weight HA produced by ID8 cells promotes membrane cholesterol depletion in macrophages. Given that HA is an important component of ECM in many cancers, including EOC (Kolapalli et al., 2016), this suggests HA could affect the phenotype of TAM through membrane cholesterol depletion.

Cholesterol efflux promotes IL-4 mediated macrophage reprogramming.

Depletion of membrane cholesterol has been shown to profoundly affect macrophage activation in response to pro-inflammatory stimuli (Fessler and Parks, 2011; Pradel et al., 2009), which suggests that cholesterol efflux in TAM could affect their programming by signals in the tumor-microenvironment. In some instances, TAM have been shown to exhibit a tumor- promoting phenotype that can be driven by Th2 cytokines such as IL-4 or IL-13, and are skewed away from the pro-inflammatory and immunostimulatory activation state, for example induced by Thl cytokines such as IFNy (DeNardo et al, 2009; Murray et al, 2014). To test the effects of ID8 cells on macrophage reprogramming, we stimulated BMDM with IL-4 or IFNy in the presence or absence of ID8-CM and measured the induction of IL-4 and IFNy gene expression, respectively. ID8-CM pre-treatment profoundly increased the expression of IL-4 induced genes; Argl , Retnla , CM313 and Mrcl (Fig.2A). In contrast, ID8-CM inhibited the IFNy induced expression of Nos2 and III 2b (Fig.2B). as well as other IFNy-regulated genes including Cxc/9, CxcllO and Ciita (data not shown), demonstrating that ID8-CM promoted macrophage programming towards an IL-4 induced pro-tumor phenotype. Similar results were obtained after co-culture of ID8 cells with BMDM or with IL-13 treatment, which also signals through the IL4 receptor alpha chain (IL4RA) (data not shown). These effects were restricted to the high molecular weight (>100 kDa) fraction of ID8-CM (data not shown) and were not sensitive to freeze/thaw cycles, boiling, ultracentrufugation, DNAse or proteinase K treatment (data not shown).

To evaluate the effects of membrane cholesterol depletion on macrophage reprogramming we used several mechanistically distinct treatments to induce cholesterol efflux, in comparison with ID8-CM; 9-cis-retenoic acid (9cRA) upregulates expression of ABC transporters and thereby induces cholesterol efflux (Ricote et al, 2004), whereas high-density lipoprotein (HDL) and ApoAl strip cholesterol directly from the cell membrane (Zhao et al., 2010). Treatment of BMDM with 9cRA, HDL or ApoAl resulted in similar levels of reduction in CTB staining as seen upon ID8-CM treatment (Fig.2C). as well as increased IL-4 induced gene expression, while inhibiting IFNy induced genes (Fig.2D) In contrast, the addition of exogenous cholesterol to BMDM, reduced the effects of ID8-CM on IL-4 induced Argl and IFNy induced Nos2 expression (data not shown).

These data suggested that membrane cholesterol depletion promotes IL-4 mediated macrophage activation and abrogates IFNy signaling. To directly test the role of cholesterol efflux in macrophage reprogramming by ID8-CM, we used BMDM from mice with a combined myeloid deficiency in the ABCA1 and ABCG1 reverse cholesterol efflux transporters (Abcal/gl^²). Treatment with ID8-CM or other membrane cholesterol-depleting agents failed to reduce CTB staining in BMDM from Abcal/gl^² mice (Fig.2E). which indeed reversed the increase in IL-4 induced gene expression by ID8-CM and the inhibition of IFNy induced genes (Fig.2F). indicating that membrane cholesterol efflux through ABCA1 and/or ABCG1 promoted IL-4 mediated macrophage activation in the presence of ID8-CM.

Tumor-induced macrophage reprogramming is STAT6 and PI3K dependent. To further characterize the mechanisms behind increased IL-4 induced gene expression in the presence of ID8-CM and how this may relate to cholesterol efflux, we analyzed IL-4 receptor signaling pathways. First, we observed no increase in the expression levels of the IL- 4 receptor (IL4RA) on macrophages treated with ID8-CM (data not shown). Thus, we measured activation of signaling pathways downstream of the IL-4 receptor. IL-4 induced gene expression is regulated by JAK-mediated phosphorylation of the STAT6 transcription factor. Treatment of BMDM with ID8-CM increased levels of activated STAT6 (pY-STAT6) in response to IL-4 (data not shown) while reducing the accumulation of phosphorylated STAT1 (pY-STATl), upon IFNy activation (data not shown). As expected, IL-4 induced gene expression in the presence of ID8-CM was abolished in BMDM derived from STAT6 deficient mice (Stat6^/) (data not shown). IL-4 signaling also activates PI3K, which was recently shown to be an important pathway for the tumor-promoting functions of TAM (Kaneda et al, 2016a; Kaneda et al, 2016b), furthermore, increased PI3K signaling has been shown to promote IL-4 induced gene expression in macrophages (Rauh et al., 2005). To assess PI3K activation we measured phosphorylation of the downstream kinase Akt/PKB. We observed a marked increase in serine 473 phosphorylation of Akt (pS-Akt) in the presence of ID8-CM (data not shown), this correlated with increased accumulation of phosphatidylinositol (3,4,5)-triphosphate (PIP3), the product of PI3K activity, as measured by confocal microscopy (data not shown). To determine the contribution of PI3K to IL-4 mediated reprogramming in the presence of ID8- CM, we treated cells with the PI3K inhibitor LY294002. As expected, LY294002 treatment blocked the increase in pS-Akt by ID8-CM (data not shown) and also abrogated the increase in IL-4 induced Argl and CM313 expression (data not shown), indicating that PI3K activity was critical for ID8-CM induced reprogramming. To determine the role of cholesterol efflux in STAT6 and PI3K activation, we again treated macrophages with 9cRA and ApoAl to deplete membrane cholesterol, both treatments resulted in similar increases in the accumulation of pY- STAT6 and pS-Akt (data not shown). Furthermore, macrophages lacking the ABCAl and ABCGl cholesterol efflux transporters failed to increase pY-STAT6 and pS-Akt upon treatement with ID8-CM (data not shown). In addition, increased pY-STAT6 and pS-Akt accumulation was restricted to the high molecular weight (>100 kDa) fraction of ID8-CM and could be reversed by HAse treatment (data not shown), indicating that HA-mediated cholesterol efflux promoted increased STAT6 and Akt activation.

The specific accumulation of pS-Akt in the presence of ID8-CM was intriguing, serine 473 phosphorylation of Akt is mediated by mammalian target for rapamycin complex 2 (mTORC2) (Jacinto et al., 2006), which is activated by PI3K through PIP3 accumulation (Liu et al., 2015). Interestingly, mT0RC2 was also recently shown to promote IL-4 induced macrophage activation in response to metabolic stress (Huang et al, 2016). Thus, we hypothesized that ID8-CM induced PIP3 accumulation could activate mTORC2-mediated pS- AKT phosphorylation and increase IL-4 induced gene expression. In the absence of any specific mTORC2 inhibitors, to test the role of the mTORC complex we used rapamycin, which blocks mTORCl, and Torin which blocks both mTORCl and mTORC2. Rapamycin treatment only partially inhibited ID8-CM induced pS-Akt accumulation in macrophages, however, Torin treatment completely inhibited ID8-CM induced pS-Akt phosphorylation in a dose-dependent manner (data not shown), suggesting that mTORC2 activity is required for ID8-CM induced pS-Akt accumulation in macrophages.

In summary, IL-4 induced macrophage activation or reprogramming in response to ID8- CM requires PI3K-mTORC2-Akt activity and is driven by STAT6.

IL-4 induced STAT6 and PI3K signaling in TAM drives tumor progression in EOC.

To test the relevance of these pathways for TAM and tumor progression in vivo , we revisited our characterization of TAM in ID8 tumors. Our conclusions from the data presented in the first experimental section part of the study, was that monocyte-derived TAM gradually replaced resident macrophages during tumor progression and that TAM showed an enrichment for cholesterol efflux pathways (data not shown). However, these analyzes were performed on bulk TAM populations, including resident PM and monocyte-derived TAM. To refine our analysis and determine the specific gene expression signature of monocyte-derived cells, we isolated F4/80¹⁰ CCR2⁺ monocytes (MN), alongside F4/80¹" LPM, which were further divided into Tim4⁺ and Tim4 subsets (data not shown). Tim4 was previously shown to be a marker for proliferative, self-renewing LPM (Rosas et al., 2014), whereas Tim4 F4/80^hl cells represent monocyte-derived LPM, which are CCR2-dependent (data not shown). First, we collected these 3 populations from naive mice and at different time points during tumor progression for microarray analysis. We performed a pairwise comparison between the 3 populations in naive mice and extracted a specific gene signature for each subset, applying a 1.5 FC threshold and a p-value of 0.05. Using the Minimal method (pairwise[Mean(test)/Mean(ref)]), we identified sets of 553 genes specific for MN, 131 for Tim4⁺ PM and 84 for Tim4 PM (data not shown). Given that CCR2 was the highest DEG between Tim4 and Tim4⁺ populations, this strongly supported the monocytic origin of Tim4 cells, in keeping with our previous analysis (data not shown). We then used these gene sets to perform enrichment analysis (GSEA) with DEGs from the equivalent 3 subsets in ID8 tumor-bearing mice. This analysis showed a significant down- regulation of the naive Tim4⁺ PM gene signature and a strong enrichment of the MN and Tim4 gene signatures in Tim4⁺ TAM (data not shown), supporting our conclusion that the tumor- microenvironment promotes the replacement of resident PM with MN-derived cells that acquire a resident-like phenotype, including expression of Tim4 (data not shown). To determine the specific genes associated with this phenotype, we extracted the leading edges (LEs) for this enrichment, that is the genes most strongly associated with the enrichment of the MN gene signature in Tim4⁺ TAM. We identified 173 LEs that were enriched at all time points in Tim4⁺ TAM (data not shown), which we then used for Ingenuity Pathway Analysis (IP A). The most significant pathway associated with these genes was the IL-4 pathway (data not shown), suggesting that IL-4 in the tumor-microenvironment could be an important upstream regulator for the development of the monocyte-derived TAM phenotype.

To confirm the role of IL-4 signaling in tumor progression in vivo , we treated ID8 tumor bearing mice with an IL-4 receptor blocking monoclonal antibody (aIL4ra) and monitored tumor progression. Treatment with aIL4ra significantly reduced ID8 tumor growth in vivo (data not shown), suggesting that IL-4 signaling is an important factor for tumor progression in this model. Furthermore, chimeric mice with hematopoietic deficiency in STAT6 ( Stat6^{~ ~} ) or PI3K ( Pik3cd ^~ ), also showed significantly reduced tumor growth (data not shown), indicating that both signaling pathways in tumor stromal cells are important factors for tumor progression. To evaluate the impact of these pathways on TAM phenotype in vivo , we sorted bulk TAM from Pik3cd-/- chimeric mice by flow cytometry and isolated RNA for microarray analysis. Expression of Argl, 1110 , Ccl2 and Stabl , which have previsouly been shown to upregulated in TAM, were significantly downregulated in macrophages from Pik3cd chimeric mice compared to controls (data not shown), suggesting that PI3K activation contributes to the TAM phenotype. To further analyze the impact of PI3K activation on TAM, we generated a gene set from all DEGs between naive PM and TAM from wild-type mice. Subsequent GSEA showed a significant enrichment for genes expressed by naive PM in Pik3cd ^~ cells (data not shown), confirming that PI3K activity contributes to the promotion of the TAM phenotype. In addition, there was no enrichment of genes associated with the IL-4 dependent TAM phenotype (data not shown), described above (data not shown). These data indicates that PI3K is an important regulator of this gene set in TAM. Interestingly, using an established gene set for tumoricidal phenotype (GSE269I2), which was enriched in naive PM compared to TAM, we also observed an enrichment in Pik3cd TAM compared to wild-type cells, suggesting that these cells retained a more tumoricidal phenotype in the absence of PI3K activation (data not shown). Finally, to evaluate the role of cholesterol efflux in TAM in vivo , we established ID8 tumors in mice with a myeloid-specific deletion of both ABCA1 and ABCG1 (Abcal gl^{Al i2}). ID8 tumor progression was significantly impaired in Abcal/gl^² mice compared to littermate controls (data not shown). Furthermore, microarray analysis of TAM sorted from these mice showed a significant downregulation of genes associated with the IL-4 dependent TAM phenotype and a positive enrichment for tumoricidal genes (data not shown), reflecting the phenotype of PI3K deficient TAM.

Collectively, these data showed that IL-4 signaling in TAM plays an important role in tumor progression in this model. Furthermore, the PI3K pathway and increased cholesterol efflux, contribute significantly to the functional polarization of TAM and tumor progression in vivo.

Discussion:

It is now well appreciated that tumor-associated macrophages (TAM) can play an important role in cancer progression. TAM can contribute to tumor progression by various mechanisms, including immune-suppression and trophic functions, supporting angiogenesis, cell proliferation, invasion and metastasis. For example, increased expression of arginase I (Argl) in TAM, depletes arginine which is required by activated T cells and consequently increases polyamine synthesis, which supports cancer cell proliferation. However, macrophages also possess intrinsic anti-tumor potential, through direct tumoricidal functions and orchestrating anti-tumor immunity, which may be particularly relevant in response to therapy (Bonnotte et al., 2001; Hagemann et al., 2008; Mytar et al., 1999). But the mechanisms by which TAM become polarized towards pro-tumor functions remain poorly understood.

Here we have studied TAM in a mouse model of epithelial ovarian cancer (EOC), that reflects the peritoneal spread of high-grade serous ovarian cancer (HGSC). In mice, the peritoneal cavity contains a major resident macrophage population of embryonic origin (large peritoneal macrophages; LPM), as well as a minor population of monocyte (MN)-derived macrophages (small peritoneal macrophages; SPM). During tumor progression we showed that MN-derived TAM accumulate and gradually replace resident macrophages in the peritoneal cavity. We then analyzed the global changes in gene expression in TAM over time using microarrays and used pathway analysis to reveal changes in gene expression linked with different pathways and biological functions. At early time points, TAM displayed a more pro- inflammatory gene signature, which strongly distinguished them from naive resident PM. However, in established tumors, TAM acquired a phenotype more closely resembling resident PM, which suggested a dynamic reprogramming of TAM phenotype during tumor progression. Among the pathways upregulated in TAM from established tumors compared to naive PM was a cluster of genes related to cholesterol metabolism and reverse cholesterol efflux. Reverse cholesterol efflux in macrophages is regulated by membrane cholesterol efflux transporters, such as ABCA1 and ABCG1. These transporters regulate the levels of cholesterol in the plasma membrane, which has a profound influence on macrophage responses to extracellular stimuli. For example, ABCA1 deficient macrophages accumulate cholesterol in the membrane and are hyperresponsive to pro-inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) (Fessler and Parks, 2011; Pradel et al, 2009). This is thought to be due to the increase in cholesterol-rich membrane microdomains, also called lipid rafts, which are required to promote TLR4-signaling. However, previous studies have also shown that ABCA1 -deficient macrophages are hyporesponsive to other stimuli, including IL-4 and IL-13 (Pradel et al, 2009). Interestingly, ABCG1 deficiency in macrophages was shown to increase their pro-inflammatory phenotype and reduce growth of subcutaneous tumors in mice fed on a high-fat diet (Sag et al, 2015), suggesting that cholesterol accumulation in tumor-associated macrophages can abrogate their pro-tumor functions.

Here, we showed that ovarian cancer cells actively promoted membrane cholesterol efflux in macrophages, which was associated with increased IL-4 signaling and inhibition of IFNy-induced gene expression, resulting in transcriptional and functional reprogramming of TAM. Depletion of membrane cholesterol in macrophages increased PI3K activity and mTORC2-mediated Akt phosphorylation. Both PI3K and mTORC2 have previously been linked with IL-4 mediated macrophage activation in different contexts (Huang et al., 2016; Rauh et al, 2005). Furthermore, PI3K was recently shown to be a critical pathway to maintain the pro-tumor functions of TAM (Kaneda et al, 2016a; Kaneda et al., 2016b). The exact mechanism by which membrane cholesterol regulates PI3K/mTORC2 activation remains to be elucidated. Perhaps cholesterol-rich membrane microdomains are required to recruit negative regulators of PI3K activity, such as the lipid phosphatase SHIP-1. Previous studies have suggested that SHIP may reside in detergent-resistant membrane fractions (Galandrini et al., 2002) and SHIP-1 is known to inhibit IL-4 signaling in macrophages (Rauh et al., 2005).

The distinct metabolic environment of tumors has long been suggested to influence the phenotype of tumor-infiltrating immune cells, rendering them hyporesponsive and contributing to immune-suppression. Cancer cells rely heavily on cholesterol, which they can scavenge from the tumor-microenvironement through upregulation of apolipoproteins and their receptors (Guillaumond et al., 2015; Podzielinski et al., 2013; Villa et al., 2016). This may lead to cholesterol depletion in tumor-stromal cells, and particularly TAM which express high levels of the ABCA1 and ABCG1 efflux transporters. Our in vitro studies suggest hyaluronic acid (HA) could be an important factor produced by cancer cells that promotes this process. HA is a major component of the extracellular matrix (ECM) in many human cancers, including ovarian cancer and in many cases the degree of HA accumulation strongly correlates with poor prognosis (Kolapalli et al., 2016; Sironen et al., 2011). Macrophages express at least two distinct receptors for HA; CD44 and Lyvel . Interestingly, CD44 signaling has previously been associated with PI3K activation in TAM (Lenart et al., 2017). Co-incidentally, PI3K also upregulates ABCA1 expression in macrophages (Chen et al., 2012; Okoro et al., 2016), potentially creating a feed-forward loop for enhanced cholesterol efflux and IL-4 mediated reprogramming.

In summary, we describe an important role for membrane cholesterol efflux in the regulation of macrophage activation state in the tumor-microenvironment. Depletion of membrane cholesterol renders macrophages hyperresponsive to pro-tumor signals, such as IL- 4, but refractory to activation by the anti-tumor cytokine IFNy. We propose that cholesterol efflux pathways may represent novel targets to abrogate the pro-tumor functions of TAM while retaining potentially beneficial anti-tumor effects in response to therapy.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of inducing a phenotypic change in a population of macrophages in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that modulates cholesterol efflux in said population of macrophages.

2. The method of claim 1 for inducing pro-inflammatory phenotype (Ml) or an immunosuppressive phenotype (M2)

3. The method of claim 1 for inducing a phenotypic change in a population of tumor- associated macrophages (TAM).

4. The method of claim 1 for blocking pro-tumor functions and restoring anti-tumor immunity of tumor-associated macrophages (TAM).

5. The method of claim 1 wherein the agent that modulates the cholesterol efflux is an agent that modulates the activity or expression of a cholesterol efflux mediating protein, such as ABC1, ABCG1 or ABCG4.

6. The method of claim 1 wherein the agent that modulates the cholesterol efflux is an agent that modulates the activity or expression of a receptor for hyaluronic acid such as CD44 or Lyves-1.

7. The method of claim 1 wherein the agent is an antibody.

8. The method of claim 1 wherein the agent that decreases the cholesterol efflux in macrophages is an antibody that binds to the extracellular domain of CD44 or Lyve-1.

9. The method of claim 1 wherein the agent that increases the cholesterol efflux in macrophages is hyaluronic acid having a mass superior to lOOkDa.

10. A method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that modulates cholesterol efflux in a population of macrophages.

11. A method of treating an autoimmune inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that increases cholesterol efflux in a population of macrophages.

12. The method of claim 11 wherein the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE- mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin- dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen- antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal garnmopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channel opathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post- cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Lesihmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil- related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia- myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott- Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumo nocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

13. A method of treating an autoimmune inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that decreases cholesterol efflux in a population of macrophages.

14. The method of claim 13 wherein the cancer is selected from the group consisting of Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangio sarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant, Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplasia, Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, non-small cell lung cancer (NSCLC) which coexists with chronic obstructive pulmonary disease (COPD), Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath, meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastema, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute, lymphoblastic leukemia, T- cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Vemer Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

15. The method of claim 13 wherein the agent that decreases the cholesterol efflux in TAM is administrated in combination with an immune checkpoint inhibitor such as those selected from the group consisting of PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists.