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NKG2D is an important immunoreceptor expressed on the surface of NK cellsand some T cells. NKG2D recognizes a set of ligands typically expressed oninfected or transformed cells, but recent studies have also documented NKG2Dligands on subsets of host non-tumor cells in tumor-bearing animals andhumans. Here we show that in transplanted tumors and genetically engineeredmouse cancer models, tumor-associated macrophages are induced to express theNKG2D ligand RAE-1[delta]. We find that a soluble factor produced by tumorcells is responsible for macrophage RAE-1[delta] induction, and we identifytumor-derived colony-stimulating factor-1 (CSF-1) as necessary and sufficientfor macrophage RAE-1[delta] induction in vitro and in vivo. Furthermore, weshow that induction of RAE-1[delta] on macrophages by CSF-1 requires PI3Kp110[alpha] kinase signaling. Thus, production of CSF-1 by tumor cellsleading to activation of PI3K p110[alpha] represents a novel cellular andmolecular pathway mediating NKG2D ligand expression on tumor-associatedmacrophages.




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NKG2D is a lectin-like cell surface immunoreceptor expressed on all NK cellsand some T cell subsets (Raulet, 2003 (36)). NKG2D recognizes a diverse setof MHC-like proteins. In mice, these include the RAE-1 family (includingisoforms [alpha], [beta], [gamma], [delta], and [epsilon]), the H60 family(a, b, c), and MULT1. Human NKG2D ligands include the ULBP family (withisoforms 1--6) and the MICA and MICB proteins (Raulet et al., 2013 (35)).


Here we show that the NKG2D ligand RAE-1[delta] is induced ontumor-associated macrophages but not other cells that infiltrate severalmodels of transplanted and autochthonous cancer. Unexpectedly, we find thatthe cytokine colony-stimulating factor-1 (CSF-1) is released by tumor cellsand is necessary and sufficient to induce RAE-1[delta] at the mRNA and cellsurface levels on macrophages in vitro and on tumor-associated macrophages invivo. Furthermore, we show that the p110[alpha] catalytic subunit of PI3K isrequired for CSF-1-mediated macrophage RAE-1[delta] induction. Thus, tumorcell secretion of CSF-1 is sensed by macrophages through CSF-1R and PI3Kp110[alpha], leading to induction of the NKG2D ligand RAE-1[delta].


A limited number of studies have described NKG2D ligand expression on subsetsof tumor-associated hematopoietic cells (Crane et al., 2014 (3); Deng et al.,2015 (4); Nausch et al., 2008 (30)). To further investigate this phenomenon,we used flow cytometry to analyze NKG2D ligands on hematopoietic cellsinfiltrating several transplant tumor models. First, WT C57BL/6 mice wereinjected subcutaneously with a high dose (1 x 106) of B16-BL6 melanoma cells, hereafter referred to as B16. Once establishedat approximately 1 cm in diameter (10--17 days post-injection), tumors weredissociated and stained with lineage markers and monoclonal antibodies forNKG2D ligands, including RAE-1[delta], RAE-1[epsilon], MULT1, or a polyclonalantibody that recognizes multiple H60 isoforms. As RAE-1 molecules are quitesimilar, we validated the specificity of the antibodies by staining B16 cellstransduced with RAE-1[delta] or RAE-1[epsilon] with the antibodies targetingthese ligands (Figure 1--figure supplement 2A), and we previously confirmedisoform-specific blocking by these antibodies (Thompson et al., 2017 (39)).


Tumor-associated macrophages (hereafter called TAMs) are an important subsetof myeloid cells identified as CD45-pos; CD11b-hi; Ly6G-neg; F4/80-hi (Figure1--figure supplement 1A). Interestingly, TAMs in B16 tumors expressedRAE-1[delta] but not other NKG2D ligands (Figure 1A). In addition to strongexpression on TAMs, RAE-1[delta] was weakly expressed on monocytes in B16tumors (identified as CD45-pos; CD11b-hi; Ly6G-neg; F4/80-low; Ly6C-hi --gating strategy in Figure 1--figure supplement 3A) -- but negligible on otherhematopoietic cells (Figure 1B). Importantly, RAE-1[delta] staining on TAMswas completely absent in RAE-1-KO mice, which contain frameshift mutations inthe genes encoding RAE-1[delta] and RAE-1[epsilon], confirming thespecificity of the RAE-1[delta] staining (Figure 1--figure supplement 1B). Incontrast to robust TAM expression of RAE-1[delta], splenic macrophages,peritoneal macrophages, and blood monocytes in mice bearing B16 tumorsexpressed little to no RAE-1[delta] (Figure 1--figure supplements 1C and3B,C). These data indicate that macrophages within the B16 tumormicroenvironment are induced to express the NKG2D ligand RAE-1[delta].Expression of RAE-1[delta] in TAMs within B16 tumors was similar at variousstages of tumor growth (Figure 1--figure supplement 2B). Gating strategiesfor blood monocytes and peritoneal macrophages are shown in Figure 1--figuresupplement 3.


Figure 1. RAE-1[delta] is induced on tumor-associated macrophagesin subcutaneously transferred and spontaneous tumors. (A) Established B16 S.C. tumors were dissociated and analyzed for NKG2D ligandexpression on tumor-associated macrophages. (B) RAE-1[delta] expression (left) and MFI quantification (right) on theindicated cell types in B16 tumors. (C) RAE-1[delta] expression on TAMs in spontaneous KP sarcoma, but not inspontaneous TRAMP prostate adenocarcinoma or transferred RMA-S lymphoma. Dataare representative of >3 independent experiments.


Figure 1--figure supplement 1. Gating strategy and RAE1[delta]expression on tumor-associated macrophages and monocytes in mice with B16tumors. (A) Gating strategy to identify tumor-associated macrophages in B16 tumors. (B) Expression of RAE-1[delta] on TAMs in B16 S.C. tumors in WT and RAE-1-KOmice. (C) RAE-1[delta] expression on TAMs, splenic macrophages, blood monocytes, orperitoneal macrophages in WT mice with established B16 S.C. tumors. Data arerepresentative of >3 independent experiments.


Figure 1--figure supplement 2. RAE-1 antibody validation andRAE-1[delta] staining on TAMs in different tumors. (A) B16 cells transduced to stably express RAE-1[delta] or RAE-1[epsilon] werestained with biotinylated antibodies against RAE-1[delta] or RAE-1[epsilon],followed by fluorophore-conjugated streptavidin. (B) Expression of RAE-1[delta] on TAMs in B16 S.C. tumors in WT mice at theindicated time after injection of 1 x 106 tumor cells. (C) Expression of RAE-1[delta] on TAMs in S.C. B16 and RMA-S tumors andautochthonous KP and TRAMP tumors. Data are compiled from several independentexperiments.


In contrast to the findings with B16 tumors, RAE-1[delta] staining wasnegligible or very low on TAMs in similarly sized S.C. tumors generated byinjection of the RMA-S T cell lymphoma cell line (5 x 106 cells injected) (Figure 1C). We next sought to analyze NKG2D ligands ontumor-associated cells in spontaneous tumor models. In the KP sarcoma modeldriven by lentiviral-Cre activation of oncogenic Kras and deletion of Trp53 (DuPage et al., 2009 (7)), TAMs in primary tumors expressed robustRAE-1[delta] (Figure 1C). In contrast, TAMs within primary TRAMP prostatetumors -- a spontaneous adenocarcinoma model driven by expression of SV40 Tantigens (Greenberg et al., 1995 (14)) -- mostly lacked RAE-1[delta] (Figure1C). Together, these data indicate that TAMs, but not other hematopoieticcells, are induced to express RAE-1[delta] in some transplant and spontaneoustumors (B16 tumors and primary KP sarcomas), but not in others (RMA-S tumorsand primary TRAMP adenocarcinomas) (see Figure 1--figure supplement 2C forcomparisons). Thus, tumor microenvironments are differentially capable ofinducing NKG2D ligand expression by macrophages.


To interrogate the mechanism of RAE-1[delta] induction on TAMs, we began bytesting the hypothesis that a soluble factor released from tumor cellsinduces macrophage RAE-1[delta]. Resident macrophages were obtained fromnaïve WT mice by peritoneal lavage and cultured ex vivo with concentratedcell culture medium from B16 cells (diluted 1:1 with fresh medium) orsimilarly diluted concentrated fresh medium as a control. Macrophagescultured in the control medium showed little to no RAE-1[delta] expression,but culture with B16-conditioned medium led to a robust induction of cellsurface RAE-1[delta] (Figure 2A). RAE-1[delta] was similarly induced byculture medium from a KP sarcoma cell line (Figure 2B). These resultsindicated that a soluble factor(s) produced by B16 tumor cells and KP sarcomacells is sufficient to induce RAE-1[delta] on macrophages ex vivo.


Figure 2. B16 and KP cell line conditioned medium and CSF-1induces RAE-1[delta] on macrophages. (A) Peritoneal wash cells were cultured with a 1:1 mixture of fresh medium plus20X concentrated fresh medium or 20X concentrated B16 cell culturesupernatants, and macrophage RAE-1[delta] was analyzed by flow cytometry 48hr later. (B) Peritoneal wash cells were stimulated 48 hr ex vivo with a 1:1 mixture offresh medium supplemented with fresh medium or conditioned medium fromcultures of a KP sarcoma cell line generated from a primary KP sarcoma, andmacrophage RAE-1[delta] was analyzed 48 hr later by flow cytometry. (C) Peritoneal wash cells were cultured with or without 10 ng/ml CSF-1, withthe addition of control Ig or CSF-1R antibody (1 [mu]g/ml), and macrophageRAE-1[delta] was analyzed 48 hr later by flow cytometry. (D) Peritoneal macrophage Raet1d mRNA 48 hr after stimulation with or without the addition of CSF-1 (10ng/ml). (E) Peritoneal macrophage expression of the indicated NKG2D ligands 48 hr afterstimulation with CSF-1 or control medium. Data are representative of >3independent experiments.


Figure 2--figure supplement 1. Peritoneal macrophage CSFR1expression and dose-dependent RAE-1[delta] induction by CSF-1, and bonemarrow macrophage stimulation with CSF-1 or GM-CSF. (A) CSF-R1 expression on peritoneal macrophages ex vivo. (B) RAE-1[delta] expression on peritoneal macrophages cultured 48 hr with theindicated concentration of CSF-1. (C) RAE-1[delta] expression on bone-marrow-derived-macrophages generated using10 ng/ml CSF-1 or GMCSF. Data are representative of 2--3 independentexperiments. 041b061a72


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