Cellular Signalling 26 (2014) 2969–2978

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Cellular Signalling

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Review

Stromal-dependent tumor promotion by MIF family members☆

Robert A. Mitchell ⁎, Kavitha YaddanapudiJG Brown Cancer Center, Department of Medicine, University of Louisville, Louisville, KY 40202, United States

☆ Support: This work was supported in part by NIH CA1⁎ Corresponding author at: University of Louisville, Cl

Building, Suite 404, 505 S. Hancock St., Louisville, KY, U7698; fax: +1 502 852 5679.

E-mail address: robert.mitchell@louisville.edu (R.A. M

http://dx.doi.org/10.1016/j.cellsig.2014.09.0120898-6568/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 September 2014Accepted 23 September 2014Available online 30 September 2014

Keywords:MIFD-DTHypoxiaStromaCancerTumor microenvironment

Solid tumors are composed of a heterogeneous population of cells that interact with each other and with solubleand insoluble factors that, when combined, strongly influence the relative proliferation, differentiation, motility,matrix remodeling,metabolism andmicrovessel density ofmalignant lesions. One family of soluble factors that isbecoming increasingly associated with pro-tumoral phenotypes within tumor microenvironments is that ofthe migration inhibitory factor family which includes its namesake, MIF, and its only known family member,D-dopachrome tautomerase (D-DT). This review seeks to highlight our current understanding of the relativecontributions of a variety of immune and non-immune tumor stromal cell populations and, within thosecontexts, will summarize the literature associated with MIF and/or D-DT.

© 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29692. D-DT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29703. Cancer-associated fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29704. Mesenchymal stem cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29715. Myeloid-derived suppressor cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29716. Tumor-associated macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29737. NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29748. Cytotoxic T lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29749. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2976References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2976

1. Introduction

Intercellular communication between malignant cells and stromalcells in tumor microenvironments is essential for maintaining neo-vascularization, stromal remodeling, immune evasion and metabolicadaptation. Despite significant advances in our understanding of stro-mal cell contributions – and the factors involved – to solid tumor pro-gression, there is still much to be learned regarding individual stromal

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cell phenotypes, effectors and, perhaps more importantly, how to ther-apeutically target each one. Potentially complicating these efforts is thefact that each stromal cell type may be represented by severalindependent subtypes due to differences in activation, differentiationand/or reversible polarization. For example, late stage tumors maycontain up to five different monocytic lineage subpopulations: M2tumor-associated macrophages (TAMs), M1 TAMs, Tie-2-expressingTAMs, myeloid-derived suppressor cells (MDSCs) and bone marrow-derived monocytic cells (BMDCs).

Both tumor cell-derived and stromal cell-derived factors dictatestromal cell mobilization, recruitment, differentiation and polarization.One of these effectors, macrophage migration inhibitory factor (MIF),has been centrally implicated as a tumor cell- and stromal cell-derivedmediator of stromal cell recruitment, polarization and differentiation.Unlike prototypical cytokines and chemokines, MIF does not containa secretion signal peptide sequence and is non-classically secreted

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through anABCA1 transportermechanism [1]. Three-dimensional X-raycrystallographic studies reveal that human MIF exists as a homotrimerand is structurally related to the bacterial enzymes 4-oxalocrotonatetautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase [2,3]. MIF possesses the unusual ability to catalyze the tautomerization ofthe non-physiological substrates D-dopachrome and L-dopachromemethyl ester into their corresponding indole derivatives [4]. More re-cently, phenylpyruvic acid, p-hydroxyphenylpyruvic acid (HPP), 3,4-dihydroxyphenylaminechrome, and norepinephrinechrome also havebeen found to be MIF substrates [5]. However, high Michaelis constant(Km) values suggest that these also are unlikely natural substrates forMIF [5,6]. The N-terminal proline of MIF (Pro-1) appears to be a criticalresidue for tautomerase enzymatic activity, as site-directed mutagenesisthat substitutes a serine for this proline (P1S) is devoid of D-dopachrometautomerase activity [7]. Similarly, a proline to glycine (P1G)MIFmutantis also catalytically null for both D-dopachrome andHPP tautomerase ac-tivities [8,9]. Interestingly, a knock-inmouse expressing this catalyticallyinactive MIF exhibits a phenotype that is intermediate between micewith MIF wild-type alleles and those that are genetically deficient inMIF [10].

Despite being oneof the oldest cytokine activities ever identified [11,12], a primary cell surface receptor forMIFwas not identified until morethan 35 years after MIF's initial characterization [13]. Using expressioncloning and functional analyses, CD74 was identified by the Bucalagroup as a high affinity cell surface binding protein for MIF and wasfound to be responsible for extracellular MIF-dependent activation ofthe Erk1/2 MAP kinase cascade, cell proliferation and prostaglandin E2(PGE2) production. CD74 is a type II integral transmembrane proteinthat is expressed on monocytes/macrophages, B cells and mesenchy-mal, epithelial and endothelial cells. In antigen presenting cells, CD74functions as the invariant chain of the MHC class II receptor and servesto ferry class II proteins from the endoplasmic reticulum to theGolgi [14]. Extracellular MIF binds CD74's extracellular, C-terminaldomain which initiates CD74 signaling by intramembrane cleavage,co-activating CD44 [13,15,16] or co-activating chemokine receptorsCXCR2, CXCR4 and/or CXCR7 [17–20].

The purpose of this review is to discuss in detail the array of stromalphenotypic contributions made by MIF and MIF's only other knownfamily member, D-dopachrome tautomerase (D-DT), within the tumormicroenvironment which broadly serves to facilitate solid tumorprogression.

2. D-DT

Several studies indicate that gene targeting, immunoneutralizationor small molecule antagonism of MIF generally phenocopies loss orinhibition of CD74 but the effect is ~2-fold more pronounced in CD74targeted cells [17,21,22]. These seemingly incongruous observationshave now been rectified by studies that identified D-dopachrometautomerase (D-DT) – the only knownhomolog ofMIF – as cooperative-ly signaling with MIF in a CD74-dependent manner [21,23–25].Although human D-DT shares only 34% amino acid identify withhuman MIF, X-ray crystallography reveals that D-DT also exists as ahomotrimer and retains significant structural conservation with MIF,especially in their substrate binding pockets [2,21]. As its name implies,D-DT also catalyzes a tautomerization of D-dopachrome but, unlikeMIF,D-DT also decarboxylates D-dopachrome to give a final product of 5,6-dihydroxyindole [26].

Like MIF and CD74 [27,28], D-DT is highly expressed in a number ofhuman cancers [23–25]. MIF and D-DT overexpression allows for co-operative, additive and compensatory signaling in a CD74-dependentmanner. The first example of MIF and D-DT cooperative signalingcame from an investigation of MIF family members in human non-small cell lung carcinoma (NSCLC) [23]. Both MIF and D-DT werefound to be necessary for maximal expression and activity of pro-angiogenic growth factors in human lung adenocarcinoma cell

lines. Importantly, MIF and D-DT were able to fully compensate foreach other in providing maximal signaling – in a CD74-dependentmanner – to CXCL8 and VEGF expression [23]. More recently, MIF andD-DTwere found to cooperatively antagonize the lung adenocarcinomatumor suppressor pathways associated with activated AMP-activatedprotein kinase (AMPK) and tumor suppressor p53 [29,30]. In the caseof AMPK maintenance, MIF and D-DT additively signal through CD74to maintain glycolytic flux resulting in efficient steady state ATP gener-ation and NADPH reduction, ensuing control of oxidative stress thatcumulatively serves to reduce steady state AMPKactivation [29]. Impor-tantly, D-DT, like MIF, is a well-documented regulator of AMPK activityin a variety of cell types [31–33]. The central importance of AMPK indictating balances between energy homeostasis and inflammation[34] may provide an important clue as to potential effectors of MIFand/or D-DT-dependent monocyte/macrophage stromal contributionsto tumorigenesis [35] (to be discussed below).

3. Cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) within the tumor stroma havelong been known to promote several different aspects of tumorigenesis.CAFs are distinguished from normal tissue mesenchymal cells by anactivated phenotype that is acquired due to tumor-derived solublefactors such as TGF-β and/or PDGF [36]. These activated CAFs, whichexhibit traits similar to those of myofibroblasts [37], are responsiblefor producing chemokines that can directly promote tumor cell prolifer-ation as well as recruit endothelial cell progenitors into the tumorstroma which then leads to enhanced neovascularization processes[37]. In addition to chemokines, activated stromal fibroblasts producecopious amounts of both extracellular matrices as well as matrixmetalloproteases that, combined, broadly serve to remodel tumor/matrix interactions and allow for metastatic cell egress from primarylesions [38,39]. Toward this, several reports indicate a significantpotential for both autocrine and paracrine-derivedMIF inmesenchymalcell activation and upregulation ofmatrixmetalloproteases (MMPs) 1, 2and 3 from synovial fibroblasts [40,41]. Additionally, both extracellularand autocrine-derived MIF promote ERK MAP kinase, Rho GTPase,arachidonic acid metabolism, cell cycle progression and oncogene-induced malignant transformation in fibroblasts [42–45].

Hypoxia is a critically important byproduct and determinant oftumor stromal microenvironments generally [46] and CAFs specifically[47,48]. Arising as consequences of rapid tumor growth in the absenceof accompanying increased blood supply and low oxygen tension areas follows: 1) promotion of incomplete vascular formation leading tointermittent hypoxia that further exacerbates the cycle, 2) a decreasein the effectiveness of redox-requiring chemotherapeutics [49], 3) stabi-lization of hypoxia-inducible factor-1 alpha (HIF-1α) and/or HIF-2α(HIF-2α) transcription factors in malignant tumor cells that, in turn, in-creases the expression of gene products that promote stemness [50,51],anaerobic metabolism [52] and anti-oxidant defense [53] and, 4) initia-tion of differentiation/polarization of both circulating and intratumoralpro-tumorigenic monocytic cell types [54,55].

Arguably, one of the most compelling functional roles for MIFin fibroblast-associated phenotypes is its unique ability to counter-regulate hypoxia-induced cell senescence [56]. Welford and colleaguesidentified that MIF is a direct hypoxia-inducible factor-1α (HIF-1α)transcriptional target and MIF up-regulation by HIF-1α in fibroblastsserves to prevent hypoxia-induced cell senescence. MIF-deficiencywas found to phenocopy, almost exactly, HIF-1α-deficiency in the aber-rant induction of cell senescence induced by low oxygen tensions. It istempting to speculate, as the authors of this study do, that HIF-dependent MIF transcription prevents hypoxia-induced senescence byinhibiting tumor-suppressor p53 [56]. This potential mechanism forMIF-dependent senescence evasion is both feasible and likely as p53is a necessary determinant of hypoxia-induced senescence and/orapoptosis [57,58]. This, coupled with the fact that MIF functionally

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inhibits p53 in a variety of cell types, includingmesenchymal, monocyt-ic and lung adenocarcinoma cells [30,59,60], provides compellingevidence that MIF provides important survival/activation signals tocancer-associated fibroblasts in hypoxic tumor microenvironments.

Recent studies demonstrate that likeMIF, D-DT is also transcription-ally regulated by hypoxia-induced HIF-1α [25] and, intriguingly, MIFfamily members promote HIF-1α stability and/or mTOR-dependenttranslation [29,61–63]. These findings suggest an intriguing andpotentially very important paradigm for MIF family members inamplifying CAF-associated hypoxic signaling nodes in solid tumormicroenvironments. Given the importance of hypoxia-induced HIF inCAF-dependent neovascularization [48], ECM deposition [64] and,intriguingly, lactate generation and secretion that provide aerobicrespiration substrates to surrounding tumor cells [65], this MIF/HIFamplification loop likely represents a centrally important determinantof CAF-dependent tumor progression.

All things combined, it is becoming increasingly evident that bothtumor cell-derived and CAF-derived MIF family members serve topromote stromal fibroblast activation, survival and associated tumorprogression within solid tumor microenvironments. That being said, arecent study identified an unexpected anti-tumorigenic function fortumor-derived MIF in rhabdomyosarcoma (RMS)-mediated CAFrecruitment [20]. Tarnowski and colleagues discovered that MIF caninitiate signal transduction through the chemokine receptor CXCR7and that MIF-deficient RMS cells transplanted into immune-deficientmice develop larger tumors than MIF-competent RMS cells. MIF-deficient RMS tumors paradoxically displayed reduced neovasculariza-tion but had dramatically higher numbers of CAFs within the tumorstroma [20]. While there was no determination as to how much, ifany, direct tumor support the increased numbers of CAFs provided tothe MIF-deficient RMS tumors, it is likely that these CAFs were at leastpartially responsible for the observed increased tumor burden. Howev-er, several questions remain unresolved — not the least of which arewhether the CAFs present in MIF-deficient tumors are as functionallyactive as those found in MIF-competent tumors and what is the mecha-nism by which tumor-derived MIF acts to impede CAF intratumoralaccumulation. An even more pressing question is whether loss orinhibition of MIF in CAFs versus loss or inhibition in tumor cells wouldprovide an alternative phenotype. This will be especially important inany rigorous examination of relative MIF contributions to CAFs goingforward as loss of stromal MIF has been observed to play a dominantphenotypic role in promoting stromal-dependent tumor progressionwhen compared directly to loss of tumor-derived MIF [35,66].

4. Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are pluripotent cells that can beinduced to differentiate into a variety of cell types. These cell typescan be of either mesenchymal or non-mesenchymal lineages andinclude: adipocytes, osteoblasts, chondrocytes, tenocytes, myocytes,neurons and endothelial cells [67]. Mesenchymal stem cells have beenfound in the tumor stroma of: melanoma, colorectal carcinoma, pancre-atic ductal adenocarcinoma, lung adenocarcinoma and glioblastoma, toname a few [67]. MSCs are highly tropic toward tumor microenviron-ments, in part because bone marrow-derived MSCs migrate towardgradients of cytokines and/or chemokines that are expressed andreleased by inflammatory cells during wound repair processes [68].Once in the tumor stroma, MSCs can differentiate into a variety oftumor-supportive cell types including pericytes – which serve asendothelial progenitor cells – and cancer-associated fibroblasts(described above). In addition to the contributions to tumor-associated angiogenesis and stromal remodeling stemming from theseMSC differentiated cell types, MSCs may directly promote metastasesof adjacent tumor cells within the stroma. A landmark study fromthe Weinberg lab demonstrated that bone-marrow-derived MSCs(BM-MSCs), co-implanted with weakly metastatic mammary

adenocarcinoma cells result in a profound increase inmetastatic poten-tial and tumor aggressiveness [69].MSC-secreted CCL5paracrine activa-tion of surrounding mammary adenocarcinoma cells was found to beresponsible for the bulk of themetastatic induction byMSC. These find-ings highlight the potential importance of MSC-derived cytokines andchemokines in malignant disease progression. Intriguingly, MIF isconsistently one of the highest expressed cytokines/chemokines foundin human bone marrow-, cord blood- and placental-derived MSCs [70]and hypoxia induces MIF expression and secretion beyond its alreadyhigh steady state levels [71]. Hypoxia-induced MIF reportedly providessimilar evasion from cell senescence in MSCs [71] as that which isobserved in fibroblasts [56] although it utilizes an Akt-dependent pro-survival pathway to accomplish this as opposed to an inhibitory effecton tumor suppressor p53 [56].

In separate studies, extracellular MIF or mAb-mediated CD74activation serves to inhibit the motility of MSCs consistent with MIF'soriginal activity as a “migration inhibitory factor” [72,73]. Although it'snot clear what functional contribution, if any, MIF provides to MSC-mediated tumor progression, one could speculate that tumor- and/orMSC-derived MIF may actively promote MSC survival while antagoniz-ing MSC motility out of the tumor microenvironment. Beyond that, it istempting to speculate that MIF – and/or D-DT – may provide somefunctional contribution(s) to MSC differentiation processes in normaland/or malignant disease processes. Given that MIF has been found tocontribute to differentiation processes in other cell types [74–76] and,in fact, regulates the expression of MSC lineage specifying transcriptionfactors Oct3/4 and Sox2 in MSCs [71], it is not unlikely that MIF familymembers may participate in MSC differentiation. It should be notedthat MIF participates in both epithelial–mesenchymal transition(EMT) and mesenchymal–epithelial transition (MET) [77,78] — twodifferentiation-like processes that serve to coordinate metastaticdissemination and distal secondary tumor growth, respectively [79].Despite the significant, albeit largely anecdotal, evidence to support arole forMIF and/or D-DT inMSC-dependentmalignant disease progres-sion, a great deal of study is still needed to clarify whether: 1) MSC vs.tumor-derived MIF/D-DT provides functional contributions to diseaseprogression, 2) whether MIF/D-DT participates in MSC differentiationprocesses and, if so, which ones, 3) whether MIF/D-DT is functionallyinvolved in hypoxia/HIF-dependent MSC stromal processes, and 4) iftherapeutic targeting of MSC-associated MIF and/or D-DT providesclinically efficacious responses.

5. Myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneouspopulation of immature myeloid cells with suppressive propertiesthat preferentially expand in cancer. MDSCs suppress T-cell prolif-eration and cytotoxicity, inhibit NK cell activation, and induce the differ-entiation and expansion of regulatory T cells (Treg). There isalso evidence that this cell subset is involved in an array of non-immunological functions, such as promotion of angiogenesis, tumorinvasion and metastases [80,81]. MDSCs accumulate in peripheralblood, lymphoid tissues as well as draining tumor sites of cancer-bearing hosts [82].

Mouse MDSCs are characterized by the expression of Gr-1 andCD11b. CD11b+Gr-1+ cells represent approximately 2 to 4% of allnucleated splenocytes, but can increase to up to 50% in tumor-bearingmice [83,84]. These cells are a mixture of immature myeloid cells,immature granulocytes, monocytes-macrophages, dendritic cells(DCs) and myeloid progenitor cells. Murine MDSCs are furthersubdivided into two major groups: CD11b+Gr-1high granulocytic MDSC(also identified as CD11b+Ly6-G+/Ly6Clow MDSC) and CD11b+Gr-1low

monocytic MDSC (also identified as CD11b+Ly6-G−/Ly6C+ MDSC) [84]. Recently, murine MDSCs have been further sub-divided into 5 differentclasses dependent on the relative expression of CD11b and Gr-1 [85].

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Murine MDSCs suppress T cell responses by multiple mechanisms.L-Arginine represents an important molecule central to the immunesuppressive function of mouse MDSCs. L-Arginine serves as a substratefor both inducible nitric oxide synthase (iNOS) and arginase-1(ARG1), both of which are highly expressed in MDSCs isolated fromtumor-bearing mice. While iNOS utilizes L-arginine as a substratefor nitric oxide (NO) generation, L-arginine catabolic pathways serveto suppress T cell function in several ways [86]. For example, depletionof L-arginine (and L-cysteine, in some cases) causes the downregulationof the ζ-chain in the T cell receptor (TCR) complex resulting in prolifer-ative arrest of antigen-activated T cells [87]. Reactive oxygen species(ROS) represent another T cell suppressive mechanism. Nitric oxide,superoxide and peroxynitrite – formed from the cooperative activitiesof iNOS, NADPH oxidase and ARG1 overexpressed in MDSCs – preventlymphocyte responses in several ways including via T cell receptorand CD8 nitrosylation [88].

Using an implantable syngeneic mouse metastatic breast cancermodel, Simpson et al. showed that tumors derived from MIF shRNA-expressing 4T1 cells contain significantly fewer monocytic MDSCsthan control tumors [89]. Importantly, reconstitution of MIF-depletedcells with wild-typeMIF restoresMDSC tumor infiltration and increasesthe metastatic potential of the tumors. In contrast, a tautomerase-inactive MIF variant fails to reconstitute MDSC tumor infiltration andmetastatic tumor burden. Based on these findings, the authors concludethat the tautomerase activity of tumor-derived MIF is important for itseffects on MDSCs and tumor metastasis [89].

We recently identified that splenic MDSCs isolated frommelanoma-bearing MIF-deficient mice are less immunosuppressive than thoseisolated from MIF wild-type mice and these phenotypes correspond tosignificantly reduced primary and metastatic melanoma growth andprogression [35]. Importantly, 4-iodo-6-phenylpyrimidine (4-IPP —

our previously discovered small molecule MIF tautomerase inhibitor)fully recapitulates MIF-deficiency in vitro and in vivo and serves toattenuate MDSC immunosuppression and melanoma disease progres-sion in mice [35]. Our current efforts are focused on delineating themolecular mechanisms driving the MDSC-dependent tumor-promotingeffects of MIF. Recent studies demonstrate, for example, that in vitrodifferentiated bone marrow-derived MDSCs require MIF for maximalARG1 expression and MIF-deficient bone marrow-derived monocyticMDSCs possess reducedMDSC immunosuppressive activity (unpublishedresults).

The importance of a hypoxic tumor microenvironment in dictatingthe differentiation and functional properties of tumor-infiltratingMDSCs has recently been established [55]. Hypoxia-induced HIF-1α dramatically alters the function of MDSC in the tumor micro-environment and serves to redirect MDSC differentiation towardtumor-associated macrophages (TAMs), providing a mechanistic linkbetween different myeloid suppressive cells in the tumor stroma [55].Functionally, hypoxia-induced HIF1-α inMDSC promotes T lymphocyteimmune suppression via ARG1 and iNOS transcription [55]. One criticalquestion that remains unanswered is what role – if any – does MIF playin influencing the hypoxia/HIF1-α-dependent MDSC differentiation/function [56].

In contrast tomurineMDSCs, which arewell defined, humanMDSCsare inadequately characterized. The best marker for human MDSCsremains their suppressor function,which can be either direct or indirectthrough the induction of Treg. Human MDSCs are defined as cells thatexpress the common myeloid markers such as CD14+, CD11b+ andCD33+, but are usually negative/low for HLA-DR and lack expressionof lineage specific antigens (Lin) such as CD3, CD57, and CD19.Monocytic MDSCs are usually characterized by HLA-DRlow/−, CD11b+,CD33+ and CD14+ phenotypes in humans (represented byCD11b+Ly6-G−/Ly6C+ in mice) whereas mature monocytes expresshigh HLA-DR. Human granulocytic MDSCs are generally characterizedby HLADRlow/−, CD11b+, CD33+, and CD15+ phenotypes in humans(CD11b+Ly6-G+/Ly6Clow in mice). Monocytic or granulocytic MDSCs

are present in patients with melanoma [90], multiple myeloma [91],hepatocarcinoma [92], NSCLC [93], renal cell carcinoma [94], andprostate cancer [95] among others. Despite ample evidence supportingthe superior immune suppressive activity of tumor-infiltrating MDSCsin murine tumor models [96], most human studies of MDSCs havefocused on peripheral blood. The accumulation of MDSCs in the periph-eral blood correlates with tumor burden, stage and grade in a variety ofcancers. For example, among stage IV solid-tumor patients, those withextensive metastatic tumor burden have the highest percent andabsolute number of MDSCs [97].

The molecular mechanisms governing the immune-regulatory roleof human tumor-infiltrating and circulating myeloid cells are largelyunexplored. HumanMDSCs in some cancers are shown to have elevatedarginase activity, which is associated with a decreased CD3 ζ-chainexpression on T cells [98,99]. In addition to impairing T cell proliferationin response to TCR triggering, MDSCs can impair the migratory proper-ties of activated T lymphocytes, as reported in patients with headand neck, lung and urinary cancers [100]. Depletion of L-arginine andL-cysteine, increased nitric oxide, superoxide, peroxynitrates and avariety of cytokines have been shown to mediate human MDSC T cell–suppressive function. In head and neck squamous cell carcinomapatients, both tumor infiltrating and circulating MDSC-suppressiveactivity is associated with activated STAT3-mediated events [101].Mao et al. have shown that monocytic MDSCs from melanoma patientssuppress autologous T cell proliferation via COX-2/PGE2 production. Infact, PGE2 is sufficient to induce monocytes to independently suppressproliferation and IFN-γ production in autologous T cells ex vivo [102].

In melanoma circulating MDSCs expressing myeloid markers arequantitatively predominant, while granulocytic MDSCs are rarelydetected [103]. In an attempt to assess if MIF participates in humanmelanoma-induced MDSC differentiation and/or immune suppression,we studied the CD14+CD11b+HLA-DRlow/− monocytic MDSCs — apopulation that is significantly expanded in the periphery in alladvanced melanoma patients [90]. Our findings indicate that smallmolecule MIF inhibitors dramatically attenuate the suppressive proper-ties of circulating CD14+HLADRlow/− MDSCs isolated from late stagemetastatic melanoma patients confirming MIF as a critical mediator ofMDSC-dependent immune suppression in patients with advancedstage melanoma (manuscript in preparation).

An accumulation of phenotypic MDSCs is associated with thedecreased number of DCs in the peripheral blood of patients withhead and neck, lung, or breast cancer [104]. In functional testing,MDSCs isolated from peripheral blood of HLA-A2-positive cancer pa-tients inhibit production of IFN-γ by CD8+ T cells re-stimulated withspecific peptide-pulsed DCs [105]. Thus, accumulation of MDSCs couldbe one of the mechanisms by which a growing tumor may induceantigen-specific CD8+ T-cell unresponsiveness. It seems logical thatelimination of these immune suppressive cells may help to enhancethe anti-tumor immunemechanisms in patients. A promising, clinicallyrelevant approach to reduce the proportion of MDSCs in tumor-bearinghostsmay be the use of agents that promote the differentiation ofMDSCinto DC. Retinoic acids, ligands of the retinoic acid receptors [RAR;retinoid X receptor (RXR)], are among the compounds that have beenshown to stimulate differentiation of myeloid progenitors into myeloidDCs [106,107]. In vivo, parenteral or oral administration of all-transretinoic acid (ATRA) significantly reduces the presence of MDSCs, andeffectively serves to differentiate MDSCs in vivo into CD11c+MHCclass II+ myeloid DCs, macrophages, and granulocytes [108]. Areduction in the number of Lin−HLA-DR−CD33+ cells accompanied byan improvement of tetanus toxoid-specific T-cell response is observedin metastatic renal cell carcinoma patients treated with ATRA [109].A randomized phase II trial is currently testing whether ATRA canenhance the efficacy of DC-based vaccine in patients with late-stagesmall cell lung cancer (NCT00618891).

Initial studies performed by our group using human melanomaMDSC models lend strong support to the rationale that therapeutic

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inhibition ofMIF in humanmelanomaMDSCsmay represent a clinicallyviable approach to enhancing anti-tumor T cell immunity. Twoquestions that are currently being explored are: 1) how to use MIFantagonists therapeutically to eliminate MDSCs in cancer patients,and 2) whether MIF inhibition can be used to effectively inducedifferentiation of MDSCs into DCs with concomitant improvementin myeloid/lymphoid DC ratio, DC function, and antigen-specific T-cell-mediated immune responses in cancer patients.

6. Tumor-associated macrophages

Solid tumors have long been known to be infiltrated by inflammato-ry leukocytes, and evidence clearly demonstrates a strong correlationbetween increased numbers of tumor-infiltrating macrophagesand poor prognosis in a variety of human malignancies [110–112].As such, both the recruitment and activation of tumor-associatedmacrophage (TAMs) are regarded as pivotal to solid tumor progression,and are therefore considered critically important targets for therapeuticintervention. TAMs are derived from circulating monocytes that arerecruited to tumors by chemotactic factors such as CCL2, CCL5, CCL7,CCL8, and CXCL12. Cytokines, including VEGF, platelet-derived growthfactor (PDGF), and IL-10, are also reported to promote macrophagerecruitment [110,113]. Additionally, several lines of evidence indicatethat some proportion of circulating MDSCs is recruited into the tumorstroma where they can differentiate into mature TAMs [114,115].Once incorporated into the tumor stroma, TAMs secrete a variety ofparacrine acting factors that functionally promote tumor-associatedangiogenesis, tumor cell division, metastatic dissemination, immuno-suppression, matrix deposition and matrix remodeling.

Macrophages can be activated by a variety of stimuli and polarized tofunctionally different phenotypes. Two distinct subsets of macrophageshave been proposed, including classically activated (M1) and alter-natively activated (M2) macrophages. M1 macrophages express pro-inflammatory cytokines, chemokines, and effector molecules, such asIL-12, IL-23, TNF-α, iNOS, IFN-γ, IL-1β, IRF5, and MHC class I/II [110,116,117]. In contrast, M2, alternatively activated macrophages expressa wide array of anti-inflammatory molecules including: IL-10, TGF-β,Fizz1, Mrc2 and ARG-1 [111,118]. In most solid cancers, infiltratedmac-rophages are polarized into anM2 phenotype that functionally providesan immunosuppressive, pro-angiogenic, pro-metastatic tumor micro-environment [112,116,119,120]. M2 TAMs promote intratumoralneoangiogenesis through the coordinated expression of VEGF, CCL2,FGF2, CXCL8, CXCL1, and CXCL2 [110,121–123]. TAM-derived proteases,such as matrix metalloproteases (MMP-2 and MMP-9), plasmin, andurokinase plasminogen activator promote matrix remodeling, tumormetastatic dissemination and colonization [124,125]. TAM-derivedcytokines and proteases, such as TGF-β, IL-10, and ARG 1, induceantigen-specific lymphocyte non-responsiveness [110,117,126,127]and skew T cell responses from a pro-tumoral, Th1 phenotype, to ananti-tumoral, Th2 phenotype, through the production of CCL17 [110],CCL18 [128], and CCL22 [129].

Intratumoral TAMs can be re-educated to potentiate anti-tumorimmunity by various immune-regulatory cues [130–132]. This hasspurred significant interest in developing therapies aimed at skewingTAMs from a pro-tumoral M2 phenotype toward an anti-tumoral M1-like phenotype [133]. However, to date, very few target moleculeshave been identified that can orchestrate this process and be therapeu-tically targeted. Studies performed by our group indicate that stromalmacrophage-derived MIF (as opposed to tumor cell-derived MIF)polarizes TAMs toward an M2 phenotype that – in turn – promotesan immunosuppressive, pro-angiogenic microenvironment withinmalignant melanoma lesions [35]. Implantation of high MIF-expressing melanoma cell lines into syngeneic MIF-deficient miceresults in significant reductions in both subcutaneous melanomaoutgrowth and metastatic melanoma lung colonization compared toMIFwild-typemice. Correspondingwith the reducedmelanomadisease

phenotypes in MIF-deficient mice is an attenuation of TAM alternativeactivation markers and immunosuppressive activities. Moreover, MIF-deficient TAMs exhibit significant reductions in pro-angiogenic growthfactor expression and angiogenic potential consistent with a reducedalternative activation phenotype. Importantly, 4-IPP [134], recapitulatesMIF-deficiency in vitro and in vivo and can rapidly re-polarizeTAMs from an M2, alternative activation phenotype toward a pro-inflammatory, reduced angiogenic, M1 classically activated phenotype[35]. Consistent with a role for MIF in contributing to the angiogenicphenotype of alternatively activated TAMs, lung adenocarcinoma-derived MIF promotes CXCL8 (IL-8) and VEGF expression in humanmonocytes [135,136]. More recently, stromal macrophage MIF wasfound to be an important contributor to intratumoral angiogenesisrequired for murine teratoma formation [137]. Moreover, a study bythe Dranoff group revealed that melanoma patients showing durableanti-melanoma immune responses to an experimental therapeutichad high levels of anti-MIF auto-antibodies that specifically neutralizedMIF-dependent Tie-2 and MMP-9 expression in TAMs leading todisrupted tumoral vasculature, lymphocyte/granulocyte infiltratesand, by extension, a significantly improved prognosis [138]. Despitethese advances, the precise melanoma-promoting angiogenic andadaptive immune effector cell requirements – and the respective rolesthat tumor-derived MIF vs. TAM-derived MIF plays in them – are stilllargely unresolved.

TAMs preferentially localize to hypoxic areas of tumors [139,140].Hypoxia has profound effects on TAM functions including their migra-tion into tumors and patterns of gene expression, especially thoseencoding pro-angiogenic cytokines and enzymes [141]. Hypoxia in-duces gene expression in TAMs through up-regulation of HIF-1α andHIF-2α and subsequently a wide array of HIF target genes in hypoxic/necrotic areas of human tumors [141]. Importantly, hypoxia is a potentinducer of both VEGF and MMP7 in TAMs, both of which are knownto support tumor angiogenesis, invasion, and metastasis. In addition,hypoxia up-regulates the expression of M2 macrophage markers likeIL-10, arginase, and PGE2 [142]. It is possible that the expression/function of MIF in M2 polarized TAMs is regulated through thishypoxia-HIF1-α/HIF2-α circuitry.

In addition to HIF-1α, NF-κB has been identified as master regulatorof TAM transcriptional programs, and evidence suggests that modula-tion of NF-κB activity in these cells is an important mechanism bywhich their pro-tumoral functions can be controlled [110]. TAMs fromadvanced tumors show defective NF-κB activation in response todifferent pro-inflammatory signals [111,117,126]. This defective NF-κBactivation in TAMs correlates with impaired expression of NF-κB-dependent inflammatory mediators including TNF-α, IL-1β, and IL-12[110]. Importantly, restoration ofNF-κB activity in TAMs fromadvanced tu-mors results in increased expression of inflammatory cytokines (e.g., TNF-α) and is associated with a delay in tumor growth [117].

Emerging results indicate that signaling through the energy homeo-static AMPK pathway may inhibit the inflammatory responses inducedby NF-κB in alternatively activated M2 TAMs [118]. AMPK-α is thecatalytic AMPK subunit of a kinase complex consisting of 3 AMPKsubunits and its activity is tightly regulated by phosphorylation on aconserved threonine residue at position 172 within the activationloop. Several recent studies demonstrate an important and centralregulatory role for MIF in promoting stress-response AMPK activation[143–145]. Interestingly, activated AMPK antagonizes NF-κB pro-inflammatory signaling [146] while activating Foxo3 [147], likelyresulting in immunosuppressive signaling [148]. Preliminary resultsfromour group indicate thatMIF-deficient and 4-IPP-treatedmelanomaTAMs have significantly reduced AMPK pathway activation comparedto control macrophages, which suggests that MIF may control TAMM2 polarization by promoting AMPK activity. Studies are ongoing toidentify the precisemechanisms of action for TAMalternative activationin the context of MIF and AMPK focusing on both upstream anddownstream MIF and AMPK effectors.

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7. NK Cells

Natural killer (NK) cells are lymphocytes that are part of the innateimmune system. They are an important component of the first line ofdefense that protects the body from pathogen invasion and malignanttransformation. NK cells comprise ~5–10% of peripheral blood lympho-cytes and are also found in the liver, spleen, bone marrow, and lymphnodes in humans. Strikingly, high activity of peripheral blood NK cellsis associated with a 10% lower incidence of tumors for men and 4%for women [149], and their infiltration into certain tumor tissues is anindicator for better disease prognosis [150]. NK cells are characterizedby strong cytolytic activity against susceptible target cells and by theability to release several cytokines. Unlike cytotoxic T lymphocytes(CTLs), NK cells kill without prior sensitization via the polarized releaseof cytotoxic granules, which are loaded with perforin and granzymes[151]. Cytolysis requires the formation of a complex immunologicalsynapse between the target cell and the NK cell, in a highly organizedmanner [152].

Over the past two decades, major advances have been made in thedefinition of NK cell function including the molecular mechanismsenabling NK cells to selectively kill tumor or virus-infected cells whilesparing normal cells. A conceptually important advance was proposedin 1990 by Ljunggren and Karre [153] in their ‘missing-self’ hypothesis.According to this hypothesis, one of the functions of NK cells is to recog-nize and eliminate cells that fail to express self major histocompatibilitycomplex (MHC) class I molecules or human leukocyte antigen (HLA)class I — that is to say, when the cells are missing expression of self-molecules, which are usually expressed on healthy tissue. The findingimplied that NK cells worked like T cells by recognizing foreign antigenson the target cell, and are strongly influenced by the expression ofMHC/HLA class I molecules on the target cell. Two models were proposed toexplain the role of class I molecules controlling target cell resistance/susceptibility to NK cell lysis. The first model, the receptor inhibitionmodel, states that a putative receptor specific for MHC/HLA class Imolecules on the NK cell will transmit an inhibitory signal that willturn off NK cell activation. The second model – the target interferencemodel – postulates that ligands on target cells for activating NK cellreceptors will be masked by the expression of MHC class I molecules,making themunable to trigger NK cell activation. The parallel identifica-tion, in the early 1990s, of MHC class I-specific inhibitory and activatingreceptors in mice [154] and humans [155] provided themolecular basisunderlying the missing-self hypothesis. The balance of activating andinhibitory receptor stimulation determines NK cell activation.

The inhibitory receptors on NK cells (iNKRs) comprise receptorsthat mostly recognize MHC/HLA class I molecules on the surfaceof target cells. Inhibitory receptors on human NK cells include thepromiscuous immunoglobulin-like transcript (ILT)2 receptors, the killerimmunoglobulin-like receptors (KIRs), which recognize differentallelic groups of HLA-A, HLA-B, and mainly HLA-C molecules and theCD94–NKG2A receptor, which recognizes HLA-E [156]. The activatingreceptors of human NK cells trigger cytolytic activity mainly againsttumor cells and virus-infected cells [150,157]. NK cells express severalactivating receptors, including natural-killer group 2, member D(NKG2D), DNAX accessory molecule (DNAM)-1 and 2B4 [158]. As it isnecessary for NK cells to be turned ‘off’ to prevent the NK-mediatedkilling of normal MHC/HLA class I+ autologous cells, an ‘on’ signalmust occur when NK cells interact with target cells. This ‘on’ signalcan be readily detected whenever NK cells interact with MHC/HLAclass I− target cells. The receptors involved in NK cell activation duringthis process of natural cytotoxicity are collectively termed ‘naturalcytotoxicity receptors’ (NCRs), they are represented by NKp46, NKp44andNKp30. The importance of these activating receptors is underscoredby the fact that the surface density of NCRs is correlatedwith the degreeof NK cell-mediated cytotoxicity toward tumor cells [159,160].Moreover, blocking of NCRs results in significantly decreased killing oftumor cells in vitro [161].

After cell-to-cell contact, NK cells integrate signals from itssurface receptors in seconds, resulting in either target-cell attack or noresponse. It is well known that tumors often express low levels ofMHC/HLA class I molecules. Over 85% of human metastatic carcinomasdisplay deficient HLA class I expression. In the case of downregulationof all MHC/HLA class I molecules, lysis of tumor cells can be mediatedby all mature NK cells because of the insufficient engagement of thevarious iNKRs. In the case of downregulation of individual class I alleles(e.g. loss of single alleles or of one full haplotype), only KIR+ NK cellswould be involved. NK cells also kill malignantly transformed cellsafter interaction of induced or over-expressed ligands with theiractivating receptors, the most common one being NKG2D. NKG2Drecognizes several well-defined ligands on the target cells includingMHC class I chain-related protein (MIC)A, MICB, and UL16-bindingproteins (ULBPs) [158]. In healthy adult cells, the expression ofNKG2D ligands is restricted to the thymic epithelium and to thegastrointestinal mucosa. However, MICA/B and other ligands are upreg-ulated on the surface of many tumor cell types. Ligand overexpressionhas been detected in solid tumors ofmultiple origins and in lymphopro-liferative malignancies. Some oncogenes have been reported to directlyupregulate the expression of NKG2D ligands. The upregulation of NKcell ligands might be a cell-intrinsic protective mechanism in order torender altered/tumor cells susceptible to killing, and on the contraryreduced ligand expression or shedding of the ligands is beneficial totumor cells in order to prevent NK cell activation.

Tumor cells employ many tricks to actively bypass detection andelimination by NK cells of the immune system. Persistent expressionof activating ligands and sustained triggering of NKG2D leads to hypo-responsiveness and decreased cytotoxicity due to decrease in NKG2Dexpression and reduced IFN- γ production and also, tumor-releasedcytokines such as TGF-β and IFN-γ repress MICA/B expression anddown-modulate NKG2D expression in NK cells. In this context,ovarian-carcinoma-derived MIF contributes to tumoral immuneevasion by directly inhibiting NK cell killing of ovarian cancer cells.MIF is overexpressed in ovarian carcinoma cells and this expression cor-relateswith disease severity and the presence of ascites [162]. Secretionof MIF by the carcinoma cells decreases NKG2D levels in both CD8+ Tcells and NK cells [162]. Mechanistically, MIF appears to exert its effectson the NK cells in an immediate and profoundmanner by inhibiting thetranscription of NKG2D mRNA, whereas other tumor-derived suppres-sive mediators such as TGF-β and MMPs appear to inhibit NKG2Dexpression in a more delayed fashion mainly via post-transcriptionalmechanisms [163]. Neutralization of MIF in the ovarian carcinomacells restores NKG2D expression and anti-tumor cytolysis of NK cellsexposed MIF-deficient tumor cells in vitro [162].

Various studies have revealed that NK cells infused into cancerpatients are particularly efficient in the eradication of metastasizingtumor cells and small tumors. Rosenberg's group pioneered NK cell-based immunotherapy by administration of autologous IL-2-activatedNK cells to patients with advanced cancer [164]. In fact, allogeneic andhaplo-identical NK cells transduced with NKG2D can target humanmalignancies in a superior way and are attractive for cell-based immu-notherapy because of minimal toxicities and negligible interaction withstandard cancer treatments. Since MIF is present ubiquitously in theperiphery and is highly expressed in most malignancies targetingMIF may represent an attractive combinatorial immunostimulatoryapproach to render tumors more susceptible to NK cell-based immuno-therapy of human cancers.

8. Cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs), also known as killer T cells, providea cell-mediated response to specific foreign antigens associated withcells. CTLs (or effector CD8+ T cells) respond to foreign antigenspresented in the context of MHC-1 expressed on the cell surface. CTLsdo not respond to soluble antigens, but induce apoptosis in viral-

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infected cells and in cancer cells. Most CTLs express T-cell receptors(TCRs) that can recognize a specific antigen. Once released into the pe-riphery, naive T cells constantly survey and sample antigen-presentingcells (APCs) in secondary lymphoid tissues in search of cognatepeptide-MHC-1 molecules. T cell–APC interactions, in the context ofinfection or inflammation, drive the activation and clonal expansion ofnaïve T cells to become effector cells that exhibit potent cytolyticfunction [165]. CTLs induce apoptosis in the target cell primarily bytwo pathways; one involving perforin-mediated apoptosis and theother involving Fas/Fas-ligand interaction. Activated CTLs releaseperforin proteins that integrate into the membrane of the targetcell and organize to form a membrane pore. This allows the proteasegranzyme to enter the cell and activate the apoptotic/proteolyticcascade, and also allows other effector molecules to cross the cellmembrane and trigger osmotic lysis of the target cell membrane [165].

Although a variety of host immune effector cells participate in tumorcell killing, tumor antigen-specific CTLs are highly effective inmediatingtumor destruction. An important goal of current immunotherapyresearch is to induce durable and long-lasting functional CTLs that canmediate cytotoxic effects on tumor cells. To attain this goal, there arefour distinct steps that must be achieved. To initiate an effective CTL-mediated anti-tumor immune response, mature DCs must captureantigens derived from tumors. Next, tumor-antigen-loaded DCs mustactivate CTLs in lymphoid organs (also called cross-priming). Subse-quently, activated CTLs must enter the tumor microenvironment [thencalled tumor-infiltrating lymphocytes (TILs)] to perform their effectorfunctions, at which point a variety of negative regulatory signalssuppress the immune response. Finally, CTL-mediated cytotoxic effectsmust overcome the tolerance induced by tumor cells. Each step is acomplex process thatmay be disrupted inmanyways and the constant-ly changing tumor/stromal characteristics alongside tumor growthdemand a continuous adaptation of the immune system.

In a tumor-bearing host, DCs play an important role in the immune-surveillance by initiating the primary anti-tumor effector T-cell re-sponses. Both tumor cell- and stromal cell-derived factors such as

Fig. 1.MIF andD-DT contributions to tumor-stromal interactions.MIF andD-DTare highly exprecrine and stromal cell-derived autocrine MIF and D-DT promote the phenotypes as indicated f

VEGF, TGF-β, and IL-10 induce functional defects in DCs. DefectiveDCs express substantially lower levels of MHC molecules, adhesionmolecules, and co-stimulatorymolecules andhave impaired capabilitiesfor antigen uptake, diminished cell motility, and an impaired ability toprime naïve T cells. Cumulatively, these defects may ultimately resultin CD8+ T cell tolerance to tumor antigens [166]. As discussed earlier,hypoxic microenvironments within the tumor stroma stimulate theaccumulation of immunosuppressive TAMs. TAM-derived soluble TGF-β, IL-10, and PGE2 can directly inhibit the effector functions of anti-tumor CTLs [167] while many tumor cells/stromal cells express cellsurface-associated programmed death ligand 1 (PD-L1), that servesto directly inhibit CTL activation [167]. Additionally, tumor-derivedsoluble factors can induce and attract immunosuppressive cell typessuch as Treg and MDSCs into the tumor microenvironment. In cancerpatients, CD4+CD25+ Treg induce CD8+ T cell tolerance via directsuppressive functions on T cells or via the secretion of immunosuppres-sive cytokines such as IL-10 and TGF-β [167]. MDSCs inhibit CTL activa-tion directly in an antigen-specific or non-specific manner, or indirectlyby (i) altering the peptide presenting ability ofMHC class Imolecules ontumor cells, (ii) inhibiting DC differentiation, and (iii) expanding thenumbers of Treg [167].

MIF is necessary for both in vitro and in vivo Th2 subset of CD4+ Thelper responses [168]. Both mitogen- and antigen-induced Th2lymphocyte activation depend upon autocrine production of MIF.Mitogen- or antigen-activated T cells express significant quantities ofMIF mRNA and protein, and neutralization of MIF inhibits IL-2 produc-tion and T cell proliferation in vitro and decreases the Th2 cell responseto soluble antigen in vivo [168]. Abe et al. reported that splenocytecultures treated with neutralizing anti-MIF antibody results in a signifi-cant increase in CTL activity and a concomitant increase in IFN-γproduction. This increase in CTL activity is associated with increasedexpression of the commonγc-chain of the IL-2 receptor that is necessaryfor CD8+ T cell survival [169]. These reports suggest that MIF plays animportant role in the regulation of anti-tumor T lymphocytes in vivo,and may exert its pro-tumorigenic effects by regulating T lymphocyte

ssed inmost solid cancers and overexpressed in response to hypoxia. Tumor-derivedpara-or each stromal cell within solid tumor microenvironments.

2976 R.A. Mitchell, K. Yaddanapudi / Cellular Signalling 26 (2014) 2969–2978

responses to tumors. Lending further support to this concept are resultsfrom Johnson's group showing that MIF-deficient neuroblastoma cellsgenerate a much more robust CD8+ T cell-mediated, anti-tumorresponses than MIF-abundant cells when injected into syngeneic mice[170]. Vaccination with MIF-deficient cells resulted in a significantincrease in the number of IFN-γ-secreting CD8+ T cells in the lymphoidtissues of vaccinated mice. Consequently, MIF-deficient neuroblastomacells could be more effectively rejected in immune-competent micewhen compared to MIF-expressing tumor cells [170].

Immunotherapeutic strategies targeting immune tolerance incancer patients have generally focused on re-activating adaptive, Tcell-mediated immune responses byneutralizing lymphocyte inhibitorypathways induced by malignant cells. For example, CTLA-4 [171],programmed death-1 (PD-1) [172] and Treg [173,174] are all beingtargeted with varying degrees of success. The penultimate success ofthese approaches depends upon a robust CD8+ T effector cell responsefollowing alleviation of the tumor suppressor pathway being targeted.Since tumor antigen-specific IFN-γ-producing CD8+ T cells have beenshown to be highly effective in mediating anti-tumor immunity, evenwhen antigen density is low on the target cells, the enhancement ofCD8+ T effector responses through the inhibition of MIF may be anattractive strategy for increasing the efficacy of immunotherapy forMIF-producing tumors. The mechanism by which tumor-derived MIFregulates CD8+ T cell immunity is still unclear. Furthermore, how orwhether stromal MIF contributes to this phenotype is currently notknown. Because MIF-expressing TAMs and MDSCs, within the tumorstroma, can functionally and differentially dictate tumor infiltratinglymphocyte proliferation, and CTL tolerance, it will be important toelucidate the contributions of MIF-dependent MDSC/TAM polarizationto the CTL effector functions in the cancer microenvironment.

9. Conclusions

During the last 20 years, studies on MIF have gone from beingprimarily focused on its role as an innate immune-acting cytokine/chemokine to its evaluation on direct phenotypic effects in malignantcells back to, more recently, its phenotypic contributions to bothimmune and non-immune tumor stromal cell phenotypes. Fig. 1 depictsthe complex array of MIF and D-DT expression characteristics and theirautocrine- and paracrine-mediated effects on both malignant andstromal cells within solid tumor microenvironments. There remaina number of outstanding questions regarding mechanisms of action,intracellular vs. extracellular phenotypes, the synergy between MIFand D-DT in immune suppressive stromal mechanisms and whetherthere are distinctions between tumor-derived vs. stromal cell-derivedMIF/D-DT phenotypes. Despite these questions, the cumulative currentdata strongly indicate that therapeutic targeting of MIF and/or D-DTmay provide substantial clinical efficacy by neutralizing bothmalignantand stromal processes involved in dictating disease progression in latestage cancer patients (Fig. 1).

References

[1] Flieger O, Engling A, Bucala R, Lue H, Nickel W, Bernhagen J. 2003;551:78–86.[2] Sugimoto H, Taniguchi M, Nakagawa A, Tanaka I, Suzuki M, Nishihira J. 1999;38:

3268–3279.[3] Sun HW, Bernhagen J, Bucala R, Lolis E. 1996;93:5191–5196.[4] Rosengren E, Bucala R, Aman P, Jacobsson L, Odh G, Metz CN, Rorsman H. 1996;2:

143–149.[5] Rosengren E, Aman P, Thelin S, Hansson C, Ahlfors S, Bjork P, Jacobsson L, Rorsman

H. 1997;417:85–88.[6] Matsunaga J, Sinha D, Pannell L, Santis C, Solano F, Wistow GJ, Hearing VJ. 1999;

274:3268–3271.[7] Bendrat K, Al Abed Y, Callaway DJ, Peng T, Calandra T, Metz CN, Bucala R. 1997;36:

15356–15362.[8] Lubetsky JB, Swope M, Dealwis C, Blake P, Lolis E. 1999;38:7346–7354.[9] Swope M, Sun HW, Blake PR, Lolis E. 1998;17:3534–3541.

[10] Fingerle-Rowson G, Kaleswarapu DR, Schlander C, Kabgani N, Brocks T, ReinartN, Busch R, Schutz A, Lue H, Du X, Liu A, Xiong H, Chen Y, Nemajerova A, HallekM, Bernhagen J, Leng L, Bucala R. 2009;29:1922–1932.

[11] Bloom BR, Bennett B. 1966;153:80–82.[12] David JR. 1966;56:72–77.[13] Leng L, Metz CN, Fang Y, Xu J, Donnelly S, Baugh J, Delohery T, Chen Y, Mitchell

RA, Bucala R. 2003;197:1467–1476.[14] Borghese F, Clanchy FI. 2011;15:237–251.[15] Shi X, Leng L,Wang T,WangW, Du X, Li J, McDonald C, Chen Z, Murphy JW, Lolis

E, Noble P, Knudson W, Bucala R. 2006;25:595–606.[16] Binsky I, Haran M, Starlets D, Gore Y, Lantner F, Harpaz N, Leng L, Goldenberg

DM, Shvidel L, Berrebi A, Bucala R, Shachar I. 2007;104:13408–13413.[17] Bernhagen J, Krohn R, Lue H, Gregory JL, Zernecke A, Koenen RR, Dewor M,

Georgiev I, Schober A, Leng L, Kooistra T, Fingerle-Rowson G, Ghezzi P, KleemannR, McColl SR, Bucala R, Hickey MJ, Weber C. 2007;13:587–596.

[18] Schwartz V, Lue H, Kraemer S, Korbiel J, Krohn R, Ohl K, Bucala R, WeberC, Bernhagen J. 2009;583:2749–2757.

[19] Klasen C, Ohl K, Sternkopf M, Shachar I, Schmitz C, Heussen N, Hobeika E,Levit-Zerdoun E, Tenbrock K, Reth M, Bernhagen J, El BO. 2014;192:5273–5284.

[20] Tarnowski M, Grymula K, Liu R, Tarnowska J, Drukala J, Ratajczak J, MitchellRA, Ratajczak MZ, Kucia M. 2010;8:1328–1343.

[21] Merk M, Mitchell RA, Endres S, Bucala R. 2012.[22] Gore Y, Starlets D, Maharshak N, Becker-Herman S, Kaneyuki U, Leng L, Bucala

R, Shachar I. 2008;283:2784–2792.[23] Coleman AM, Rendon BE, Zhao M, Qian MW, Bucala R, Xin D, Mitchell RA. 2008;

181:2330–2337.[24] Xin D, Rendon BE, Zhao M, Winner M, Coleman A, Mitchell RA. 2010.[25] Pasupuleti V, Du W, Gupta Y, Yeh IJ, Montano M, Magi-Galuzzi C, Welford

SM. 2014;289:3713–3723.[26] Odh G, Hindemith A, Rosengren AM, Rosengren E, Rorsman H. 1993;197:619–624.[27] White ES, Flaherty KR, Carskadon S, Brant A, Iannettoni MD, Yee J, Orringer

MB, Arenberg DA. 2003;9:853–860.[28] McClelland M, Zhao L, Carskadon S, Arenberg D. 2009.[29] Brock SE, Rendon BE, Yaddanapudi K, Mitchell RA. 2012;287:37917–37925.[30] Brock SE, Rendon BE, Xin D, Yaddanapudi K, Mitchell RA. 2014;9:e99795.[31] Miller EJ, Li J, Leng L,McDonaldC, Atsumi T, Bucala R, Young LH. 2008;451:578–582.[32] Qi D, Atsina K, Qu L, Hu X, Wu X, Xu B, Piecychna M, Leng L, Fingerle-Rowson G,

Zhang J, Bucala R, Young LH. 2014.[33] Iwata T, Taniguchi H, Kuwajima M, Taniguchi T, Okuda Y, Sukeno A, Ishimoto

K, Mizusawa N, Yoshimoto K. 2012;7:e33402.[34] O'Neill LA, Hardie DG. 2013;493:346–355.[35] K. Yaddanapudi, K. Putty, B.E. Rendon, G.J. Lamont, J.D. Faughn, A. Satoskar,

A. Lasnik, J.W. Eaton, R.A. Mitchell, J. Immunol. 190 (2013) 2984–2993.[36] Kalluri R, Zeisberg M. 2006;6:392–401.[37] Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem

R, Carey VJ, Richardson AL, Weinberg RA. 2005;121:335–348.[38] Chang HY, Chi JT, Dudoit S, Bondre C, van de Rijn M, Botstein D, Brown PO. 2002;

99:12877–12882.[39] Simian M, Hirai Y, Navre M, Werb Z, Lochter A, Bissell MJ. 2001;128:3117–3131.[40] Onodera S, Kaneda K, Mizue Y, Koyama Y, Fujinaga M, Nishihira J. 2000;275:

444–450.[41] Frenette G, Legare C, Saez F, Sullivan R. 2005;11:575–582.[42] Mitchell RA, Metz CN, Peng T, Bucala R. 1999;274:18100–18106.[43] Liao H, Bucala R, Mitchell RA. 2003;278:76–81.[44] Swant JD, Rendon BE, Symons M, Mitchell RA. 2005;280:23066–23072.[45] O. Petrenko, G. Fingerle-Rowson, T. Peng, R.A. Mitchell, C.N. Metz, J. Biol. Chem. 278

(2003) 11078–11085.[46] Fokas E, McKenna WG, Muschel RJ. 2012;31:823–842.[47] Ino Y, Yamazaki-Itoh R, Oguro S, Shimada K, Kosuge T, Zavada J, Kanai Y,

Hiraoka N. 2013;8:e55146.[48] E.M. De Francesco, R. Lappano, M.F. Santolla, S. Marsico, A. Caruso, M. Maggiolini,

Breast Cancer Res. 15 (2013) R64.[49] Meijer TW, Kaanders JH, Span PN, Bussink J. 2012;18:5585–5594.[50] Covello KL, Kehler J, Yu H, Gordan JD, Arsham AM, Hu CJ, Labosky PA, Simon MC,

Keith B. 2006;20:557–570.[51] Soeda A, Park M, Lee D, Mintz A, Androutsellis-Theotokis A, McKay RD,

Engh J, Iwama T, Kunisada T, Kassam AB, Pollack IF, Park DM. 2009;28:3949–3959.

[52] Semenza GL. 2013;123:3664–3671.[53] Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, Royer RE, Vander Jagt

DL, Semenza GL, Dang CV. 2010;107:2037–2042.[54] Doedens AL, Stockmann C, Rubinstein MP, Liao D, Zhang N, DeNardo DG, Coussens

LM, Karin M, Goldrath AW, Johnson RS. 2010;70:7465–7475.[55] C.A. Corzo, T. Condamine, L. Lu, M.J. Cotter, J.I. Youn, P. Cheng, H.I. Cho, E. Celis,

D.G. Quiceno, T. Padhya, T.V. McCaffrey, J.C. McCaffrey, D.I. Gabrilovich, J. Exp. Med.207 (2010) 2439–2453.

[56] S.M. Welford, B. Bedogni, K. Gradin, L. Poellinger, M. Broome Powell, A.J. Giaccia,Genes Dev. 20 (2006) 3366–3371.

[57] Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, Giaccia AJ. 1996;379:88–91.

[58] Graeber TG, Peterson JF, Tsai M, Monica K, Fornace AJ, Jr., Giaccia AJ. 1994;14:6264–6277.

[59] Hudson JD, Shoaibi MA, Maestro R, Carnero A, Hannon GJ, Beach DH. 1999;190:1375–1382.

[60] Mitchell RA, Liao H, Chesney J, Fingerle-Rowson G, Baugh J, David J, Bucala R. 2002;99:345–350.

[61] Winner M, Leng L, Zundel W, Mitchell RA. 2007;435:355–369.[62] Rendon BE, Willer SS, Zundel W, Mitchell RA. 2009.

2977R.A. Mitchell, K. Yaddanapudi / Cellular Signalling 26 (2014) 2969–2978

[63] Gaber T, Schellmann S, Erekul KB, Fangradt M, Tykwinska K, Hahne M,Maschmeyer P, Wagegg M, Stahn C, Kolar P, Dziurla R, Lohning M, BurmesterGR, Buttgereit F. 2011;186:764–774.

[64] Gilkes DM, Bajpai S, Chaturvedi P, Wirtz D, Semenza GL. 2013;288:10819–10829.[65] Fiaschi T, Marini A, Giannoni E, Taddei ML, Gandellini P, De DA, Lanciotti M, Serni S,

Cirri P, Chiarugi P. 2012;72:5130–5140.[66] Girard E, Strathdee C, Trueblood E, Queva C. 2012;107:1498–1505.[67] Barcellos-de-Souza P, Gori V, Bambi F, Chiarugi P. 2013;1836:321–335.[68] Direkze NC, Forbes SJ, BrittanM, Hunt T, Jeffery R, Preston SL, Poulsom R, Hodivala-

Dilke K, Alison MR, Wright NA. 2003;21:514–520.[69] Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, Richardson AL,

Polyak K, Tubo R, Weinberg RA. 2007;449:557–563.[70] J.H. Hwang, S.S. Shim, O.S. Seok, H.Y. Lee, S.K. Woo, B.H. Kim, H.R. Song, J.K. Lee,

Y.K. Park, J. Korean Med. Sci. 24 (2009) 547–554.[71] S. Palumbo, T.L. Tsai, W.J. Li, Stem Cells Dev. 23 (2014) 852–865.[72] B.L. Barrilleaux, D.G. Phinney, B.W. Fischer-Valuck, K.C. Russell, G. Wang,

D.J. Prockop, K.C. O'Connor, Tissue Eng. A 15 (2009) 2335–2346.[73] B.L. Barrilleaux, B.W. Fischer-Valuck, J.K. Gilliam, D.G. Phinney, K.C. O'Connor, In

Vitro Cell. Dev. Biol. Anim. 46 (2010) 566–572.[74] X. Zhang, L. Chen, Y. Wang, Y. Ding, Z. Peng, L. Duan, G. Ju, Y. Ren, X. Wang, Int. J.

Biol. Sci. 9 (2013) 1108–1120.[75] Kanzler I, Tuchscheerer N, Steffens G, Simsekyilmaz S, Konschalla S, Kroh A, Simons

D, Asare Y, Schober A, Bucala R, Weber C, Bernhagen J, Liehn EA. 2013;108:310.[76] Lee J, Jung E, Kim YS, Roh K, Jung KH, Park D. 2010;285:32647–32656.[77] Funamizu N, Hu C, Lacy C, Schetter A, Zhang G, He P, Gaedcke J, Ghadimi MB, Ried

T, Yfantis HG, Lee DH, Subleski J, Chan T, Weiss JM, Back TC, Yanaga K, Hanna N, Al-exander HR, Maitra A, Hussain SP. 2012.

[78] Morris KT, Nofchissey RA, Pinchuk IV, Beswick EJ. 2014;9:e98656.[79] Tsai JH, Yang J. 2013;27:2192–2206.[80] V. Bronte, M. Wang, W.W. Overwijk, D.R. Surman, F. Pericle, S.A. Rosenberg, N.P.

Restifo, J. Immunol. 161 (1998) 5313–5320.[81] S. Ostrand-Rosenberg, Cancer Immunol. Immunother. 59 (2010) 1593–1600.[82] J.I. Youn, S. Nagaraj,M. Collazo, D.I. Gabrilovich, J. Immunol. 181 (2008) 5791–5802.[83] F. Zhao, S. Obermann, R. von Wasielewski, L. Haile, M.P. Manns, F. Korangy, T.F.

Greten, Immunology 128 (2009) 141–149.[84] J.I. Youn, D.I. Gabrilovich, Eur. J. Immunol. 40 (2010) 2969–2975.[85] V. Greifenberg, E. Ribechini, S. Rossner, M.B. Lutz, Eur. J. Immunol. 39 (2009)

2865–2876.[86] D.I. Gabrilovich, S. Nagaraj, Nat. Rev. Immunol. 9 (2009) 162–174.[87] P.C. Rodriguez, D.G. Quiceno, A.C. Ochoa, Blood 109 (2007) 1568–1573.[88] S. Nagaraj, K. Gupta, V. Pisarev, L. Kinarsky, S. Sherman, L. Kang, D.L. Herber,

J. Schneck, D.I. Gabrilovich, Nat. Med. 13 (2007) 828–835.[89] K.D. Simpson, D.J. Templeton, J.V. Cross, J. Immunol. 189 (2012) 5533–5540.[90] I. Poschke, D. Mougiakakos, J. Hansson, G.V. Masucci, R. Kiessling, Cancer Res. 70

(2010) 4335–4345.[91] M.K. Brimnes, A.J. Vangsted, L.M. Knudsen, P. Gimsing, A.O. Gang, H.E. Johnsen,

I.M. Svane, Scand. J. Immunol. 72 (2010) 540–547.[92] B. Hoechst, L.A. Ormandy, M. Ballmaier, F. Lehner, C. Kruger, M.P. Manns, T.F. Greten,

F. Korangy, Gastroenterology 135 (2008) 234–243.[93] C.Y. Liu, Y.M.Wang, C.L.Wang, P.H. Feng, H.W. Ko, Y.H. Liu, Y.C.Wu, Y. Chu, F.T. Chung,

C.H. Kuo, K.Y. Lee, S.M. Lin, H.C. Lin, C.H. Wang, C.T. Yu, H.P. Kuo, J. Cancer Res. Clin.Oncol. 136 (2010) 35–45.

[94] H. van Cruijsen, A.A. van der Veldt, L. Vroling, D. Oosterhoff, H.J. Broxterman,R.J. Scheper, G. Giaccone, J.B. Haanen, A.J. van den Eertwegh, E. Boven, K.Hoekman, T.D. de Gruijl, Clin. Cancer Res. 14 (2008) 5884–5892.

[95] S. Vuk-Pavlovic, P.A. Bulur, Y. Lin, R. Qin, C.L. Szumlanski, X. Zhao, A.B. Dietz,Prostate 70 (2010) 443–455.

[96] G. Gallina, L. Dolcetti, P. Serafini, C. De Santo, I. Marigo, M.P. Colombo, G. Basso,F. Brombacher, I. Borrello, P. Zanovello, S. Bicciato, V. Bronte, J. Clin. Invest. 116(2006) 2777–2790.

[97] C.M. Diaz-Montero, M.L. Salem, M.I. Nishimura, E. Garrett-Mayer, D.J. Cole,A.J. Montero, Cancer Immunol. Immunother. 58 (2009) 49–59.

[98] J. Schmielau, O.J. Finn, Cancer Res. 61 (2001) 4756–4760.[99] A.H. Zea, P.C. Rodriguez, M.B. Atkins, C. Hernandez, S. Signoretti, J. Zabaleta,

D.McDermott, D. Quiceno, A. Youmans, A. O'Neill, J.Mier, A.C. Ochoa, Cancer Res. 65(2005) 3044–3048.

[100] S. Brandau, S. Trellakis, K. Bruderek, D. Schmaltz, G. Steller, M. Elian, H. Suttmann,M. Schenck, J. Welling, P. Zabel, S. Lang, J. Leukoc. Biol. 89 (2011) 311–317.

[101] D. Vasquez-Dunddel, F. Pan, Q. Zeng, M. Gorbounov, E. Albesiano, J. Fu, R.L. Blosser,A.J. Tam, T. Bruno, H. Zhang, D. Pardoll, Y. Kim, J. Clin. Invest. 123 (2013)1580–1589.

[102] Y. Mao, I. Poschke, E. Wennerberg, Y. Pico de Coana, S. Egyhazi Brage, I. Schultz,J. Hansson, G. Masucci, A. Lundqvist, R. Kiessling, Cancer Res. 73 (2013)3877–3887.

[103] F. Hillen, C.I. Baeten, A. van de Winkel, D. Creytens, D.W. van der Schaft,V. Winnepenninckx, A.W. Griffioen, Cancer Immunol. Immunother. 57 (2008)97–106.

[104] B. Almand, J.R. Resser, B. Lindman, S. Nadaf, J.I. Clark, E.D. Kwon, D.P. Carbone,D.I. Gabrilovich, Clin. Cancer Res. 6 (2000) 1755–1766.

[105] B. Almand, J.I. Clark, E. Nikitina, J. van Beynen, N.R. English, S.C. Knight, D.P. Carbone,D.I. Gabrilovich, J. Immunol. 166 (2001) 678–689.

[106] D.I. Gabrilovich, M.P. Velders, E.M. Sotomayor, W.M. Kast, J. Immunol. 166 (2001)5398–5406.

[107] L.M. Hengesbach, K.A. Hoag, J. Nutr. 134 (2004) 2653–2659.[108] S. Kusmartsev, F. Cheng, B. Yu, Y. Nefedova, E. Sotomayor, R. Lush, D. Gabrilovich,

Cancer Res. 63 (2003) 4441–4449.

[109] N. Mirza, M. Fishman, I. Fricke, M. Dunn, A.M. Neuger, T.J. Frost, R.M.Lush, S. Antonia, D.I. Gabrilovich, Cancer Res. 66 (2006) 9299–9307.

[110] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, A. Sica, Trends Immunol. 23 (2002)549–555.

[111] A. Sica, T. Schioppa, A. Mantovani, P. Allavena, Eur. J. Cancer 42 (2006) 717–727.[112] L. Bingle, N.J. Brown, C.E. Lewis, J. Pathol. 196 (2002) 254–265.[113] Condeelis J, Pollard JW. 2006;124:263–266.[114] S. Kusmartsev, D.I. Gabrilovich, J. Immunol. 174 (2005) 4880–4891.[115] S. Kusmartsev, S. Nagaraj, D.I. Gabrilovich, J. Immunol. 175 (2005) 4583–4592.[116] S.K. Biswas, L. Gangi, S. Paul, T. Schioppa, A. Saccani, M. Sironi, B. Bottazzi, A. Doni,

B. Vincenzo, F. Pasqualini, L. Vago, M. Nebuloni, A. Mantovani, A. Sica, Blood 107(2006) 2112–2122.

[117] A. Saccani, T. Schioppa, C. Porta, S.K. Biswas, M. Nebuloni, L. Vago, B. Bottazzi, M.P.Colombo, A. Mantovani, A. Sica, Cancer Res. 66 (2006) 11432–11440.

[118] A. Sica, V. Bronte, J. Clin. Invest. 117 (2007) 1155–1166.[119] Y. Sakai, M. Honda, H. Fujinaga, I. Tatsumi, E. Mizukoshi, Y. Nakamoto, S. Kaneko,

Cancer Res. 68 (2008) 10267–10279.[120] A.H. Beck, I. Espinosa, B. Edris, R. Li, K. Montgomery, S. Zhu, S. Varma, R.J. Marinelli,

M. van de Rijn, R.B. West, Clin. Cancer Res. 15 (2009) 778–787.[121] Y. Luo, H. Zhou, J. Krueger, C. Kaplan, S.H. Lee, C. Dolman, D. Markowitz, W. Wu, C.

Liu, R.A. Reisfeld, R. Xiang, J. Clin. Invest. 116 (2006) 2132–2141.[122] F. Bolat, F. Kayaselcuk, T.Z. Nursal, M.C. Yagmurdur, N. Bal, B. Demirhan, J. Exp. Clin.

Cancer Res. 25 (2006) 365–372.[123] T. Ueno, M. Toi, H. Saji, M. Muta, H. Bando, K. Kuroi, M. Koike, H. Inadera, K.

Matsushima, Clin. Cancer Res. 6 (2000) 3282–3289.[124] G. Solinas, S. Schiarea, M. Liguori, M. Fabbri, S. Pesce, L. Zammataro, F. Pasqualini,

M. Nebuloni, C. Chiabrando, A. Mantovani, P. Allavena, J. Immunol. 185 (2010)642–652.

[125] E.F. Redente, L.D. Dwyer-Nield, D.T. Merrick, K. Raina, R. Agarwal, W. Pao, P.L. Rice,K.R. Shroyer, A.M. Malkinson, Am. J. Pathol. 176 (2010) 2972–2985.

[126] A. Sica, A. Saccani, B. Bottazzi, N. Polentarutti, A. Vecchi, J. van Damme, A.Mantovani,J. Immunol. 164 (2000) 762–767.

[127] M. Munder, K. Eichmann, M. Modolell, J. Immunol. 160 (1998) 5347–5354.[128] E. Schutyser, S. Struyf, P. Proost, G. Opdenakker, G. Laureys, B. Verhasselt, L.

Peperstraete, I. Van de Putte, A. Saccani, P. Allavena, A. Mantovani, J. VanDamme, J. Biol. Chem. 277 (2002) 24584–24593.

[129] T.J. Curiel, G. Coukos, L. Zou, X. Alvarez, P. Cheng, P. Mottram, M. Evdemon-Hogan,J.R. Conejo-Garcia, L. Zhang, M. Burow, Y. Zhu, S. Wei, I. Kryczek, B. Daniel, A.Gordon, L. Myers, A. Lackner, M.L. Disis, K.L. Knutson, L. Chen, W. Zou, Nat. Med.10 (2004) 942–949.

[130] K. Movahedi, D. Laoui, C. Gysemans, M. Baeten, G. Stange, J. Van den Bossche,M. Mack, D. Pipeleers, P. In't Veld, P. De Baetselier, J.A. Van Ginderachter, CancerRes. 70 (2010) 5728–5739.

[131] F. Pucci, M.A. Venneri, D. Biziato, A. Nonis, D. Moi, A. Sica, C. Di Serio, L. Naldini,M. De Palma, Blood 114 (2009) 901–914.

[132] B.Z. Qian, J.W. Pollard, Cell 141 (2010) 39–51.[133] M. De Palma, R. Mazzieri, L.S. Politi, F. Pucci, E. Zonari, G. Sitia, S. Mazzoleni, D. Moi,

M.A. Venneri, S. Indraccolo, A. Falini, L.G. Guidotti, R. Galli, L. Naldini, Cancer Cell 14(2008) 299–311.

[134] M. Winner, J. Meier, S. Zierow, B.E. Rendon, G.V. Crichlow, R. Riggs, R. Bucala,L. Leng, N. Smith, E. Lolis, J.O. Trent, R.A.Mitchell, Cancer Res. 68 (2008) 7253–7257.

[135] E.S. White, S.R. Strom, N.L. Wys, D.A. Arenberg, J. Immunol. 166 (2001)7549–7555.

[136] E.S. White, K.R. Flaherty, S. Carskadon, A. Brant, M.D. Iannettoni, J. Yee,M.B. Orringer, D.A. Arenberg, Clin. Cancer Res. 9 (2003) 853–860.

[137] X.Wang, T. Chen, L. Leng, J. Fan, K. Cao, Z. Duan, X. Zhang, C. Shao,M.Wu, I. Tadmori,T. Li, L. Liang, D. Sun, S. Zheng, A.Meinhardt,W. Young, R. Bucala, Y. Ren, Cancer Res.72 (2012) 2867–2878.

[138] J. Schoenfeld, M. Jinushi, Y. Nakazaki, D. Wiener, J. Park, R. Soiffer, D. Neuberg,M. Mihm, F.S. Hodi, G. Dranoff, Cancer Res. 70 (2010) 10150–10160.

[139] C.E. Lewis, J.W. Pollard, Cancer Res. 66 (2006) 605–612.[140] R.D. Leek, K.L. Talks, F. Pezzella, H. Turley, L. Campo, N.S. Brown, R. Bicknell,

M. Taylor, K.C. Gatter, A.L. Harris, Cancer Res. 62 (2002) 1326–1329.[141] C. Murdoch, C.E. Lewis, Int. J. Cancer 117 (2005) 701–708.[142] C. Murdoch, A. Giannoudis, C.E. Lewis, Blood 104 (2004) 2224–2234.[143] E.J. Miller, J. Li, L. Leng, C. McDonald, T. Atsumi, R. Bucala, L.H. Young, Nature 451

(2008) 578–582.[144] D. Heinrichs, M. Knauel, C. Offermanns, M.L. Berres, A. Nellen, L. Leng, P. Schmitz,

R. Bucala, C. Trautwein, C. Weber, J. Bernhagen, H.E. Wasmuth, Proc. Natl. Acad.Sci. U. S. A. 108 (2011) 17444–17449.

[145] H. Ma, J. Wang, D.P. Thomas, C. Tong, L. Leng, W. Wang, M. Merk, S. Zierow,J. Bernhagen, J. Ren, R. Bucala, J. Li, Circulation 122 (2010) 282–292.

[146] E. Bess, B. Fisslthaler, T. Fromel, I. Fleming, PLoS ONE 6 (2011) e20848.[147] E.L. Greer, P.R. Oskoui, M.R. Banko, J.M.Maniar, M.P. Gygi, S.P. Gygi, A. Brunet, J. Biol.

Chem. 282 (2007) 30107–30119.[148] S.K. Watkins, Z. Zhu, E. Riboldi, K.A. Shafer-Weaver, K.E. Stagliano, M.M. Sklavos,

S. Ambs, H. Yagita, A.A. Hurwitz, J. Clin. Invest. 121 (2011) 1361–1372.[149] K. Imai, S. Matsuyama, S. Miyake, K. Suga, K. Nakachi, Lancet 356 (2000)

1795–1799.[150] E. Vivier, S. Ugolini, D. Blaise, C. Chabannon, L. Brossay, Nat. Rev. Immunol. 12

(2012) 239–252.[151] S.S. Farag, M.A. Caligiuri, Blood Rev. 20 (2006) 123–137.[152] E. Vivier, E. Tomasello, M. Baratin, T. Walzer, S. Ugolini, Nat. Immunol. 9 (2008)

503–510.[153] H.G. Ljunggren, K. Karre, Immunol. Today 11 (1990) 237–244.[154] W.M. Yokoyama, W.E. Seaman, Annu. Rev. Immunol. 11 (1993) 613–635.

2978 R.A. Mitchell, K. Yaddanapudi / Cellular Signalling 26 (2014) 2969–2978

[155] A. Moretta, C. Bottino, D. Pende, G. Tripodi, G. Tambussi, O. Viale, A. Orengo,M. Barbaresi, A. Merli, E. Ciccone, et al., J. Exp. Med. 172 (1990) 1589–1598.

[156] L. Moretta, A. Moretta, EMBO J. 23 (2004) 255–259.[157] L.L. Lanier, Annu. Rev. Immunol. 23 (2005) 225–274.[158] J. Koch, A. Steinle, C. Watzl, O. Mandelboim, Trends Immunol. 34 (2013) 182–191.[159] R.T. Costello, S. Sivori, E. Marcenaro, M. Lafage-Pochitaloff, M.J. Mozziconacci,

D. Reviron, J.A. Gastaut, D. Pende, D. Olive, A. Moretta, Blood 99 (2002) 3661–3667.[160] S. Sivori, D. Pende, C. Bottino, E. Marcenaro, A. Pessino, R. Biassoni, L. Moretta,

A. Moretta, Eur. J. Immunol. 29 (1999) 1656–1666.[161] A. Moretta, C. Bottino, M. Vitale, D. Pende, C. Cantoni, M.C. Mingari, R. Biassoni,

L. Moretta, Annu. Rev. Immunol. 19 (2001) 197–223.[162] M. Krockenberger, Y. Dombrowski, C. Weidler, M. Ossadnik, A. Honig, S. Hausler,

H. Voigt, J.C. Becker, L. Leng, A. Steinle, M.Weller, R. Bucala, J. Dietl, J. Wischhusen,J. Immunol. 180 (2008) 7338–7348.

[163] R. Castriconi, C. Cantoni,M. Della Chiesa,M. Vitale, E.Marcenaro, R. Conte, R. Biassoni,C. Bottino, L.Moretta, A.Moretta, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4120–4125.

[164] S.A. Rosenberg, M.T. Lotze, L.M. Muul, S. Leitman, A.E. Chang, S.E. Ettinghausen,Y.L. Matory, J.M. Skibber, E. Shiloni, J.T. Vetto, et al., N. Engl. J. Med. 313 (1985)1485–1492.

[165] R. Arens, S.P. Schoenberger, Immunol. Rev. 235 (2010) 190–205.

[166] F. Cavallo, C. De Giovanni, P. Nanni, G. Forni, P.L. Lollini, Cancer Immunol.Immunother. 60 (2011) 319–326.

[167] G.A. Rabinovich, D. Gabrilovich, E.M. Sotomayor, Annu. Rev. Immunol. 25 (2007)267–296.

[168] M. Bacher, C.N. Metz, T. Calandra, K. Mayer, J. Chesney, M. Lohoff, D. Gemsa,T. Donnelly, R. Bucala, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 7849–7854.

[169] R. Abe, T. Peng, J. Sailors, R. Bucala, C.N. Metz, J. Immunol. 166 (2001) 747–753.[170] Q. Zhou, X. Yan, J. Gershan, R.J. Orentas, B.D. Johnson, J. Immunol. 181 (2008)

1877–1886.[171] A. Ribas, Semin. Oncol. 37 (2010) 450–454.[172] J.R. Brahmer, C.G. Drake, I. Wollner, J.D. Powderly, J. Picus, W.H. Sharfman,

E. Stankevich, A. Pons, T.M. Salay, T.L. McMiller, M.M. Gilson, C. Wang, M. Selby,J.M. Taube, R. Anders, L. Chen, A.J. Korman, D.M. Pardoll, I. Lowy, S.L. Topalian,J. Clin. Oncol. 28 (2010) 3167–3175.

[173] M.A. Rasku, A.L. Clem, S. Telang, B. Taft, K. Gettings, H. Gragg, D. Cramer, S.C. Lear,K.M. McMasters, D.M. Miller, J. Chesney, J. Transl. Med. 6 (2008) 12.

[174] K. Mahnke, K. Schonfeld, S. Fondel, S. Ring, S. Karakhanova, K. Wiedemeyer,T. Bedke, T.S. Johnson, V. Storn, S. Schallenberg, A.H. Enk, Int. J. Cancer 120 (2007)2723–2733.