Impaired gp100-specific CD8 + T-Cell Responses in the Presence of Myeloid-Derived Suppressor Cells in a Spontaneous Mouse Melanoma Model

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1 ORIGINAL ARTICLE Impired gp1-speifi CD8 + T-Cell Responses in the Presene of Myeloid-Derived Suppressor Cells in Spontneous Mouse Melnom Model Dvid G. Mirhofer 1, Dniel Ortner 1, Christoph H. Tripp 1,2, Sndr Shffenrth 1,2, Viktor Fleming 1,3, Luks Heger 1,3, Kerstin Komend 1, Dniel Reider 1,2, Din Dudzik 3, Suzie Chen 4, Jürgen C. Beker 5, Vinent Flher 1,6 nd Ptrizi Stoitzner 1 Murine models tht losely reflet humn diseses re importnt tools to investigte rinogenesis nd immunity. The trnsgeni (tg) mouse strin tg(grm1)epv develops spontneous melnom due to etopi overexpression of the metbotropi glutmte reeptor 1 (Grm1) in melnoytes. In the present study, we hrterized the immune sttus nd funtionl properties of immune ells in -bering mie. Melnom development ws ompnied by redution in the perentges of CD4 + T ells inluding regultory T ells (Tregs) in CD45 + leukoytes present in tissue nd drining lymph nodes (LNs). In ontrst, the perentges of were unhnged, nd these ells showed n tivted phenotype in mie. Endogenous melnom-ssoited ntigen glyoprotein 1 (gp1)-speifi were not deleted during development, s reveled by pentmer stining in the skin nd drining LNs. They, however, were unresponsive to ex vivo gp1-peptide stimultion in lte-stge mie. Interestingly, immunosuppressive myeloidderived suppressor ells (MDSCs) were reruited to tissue with preferentil umultion of grnuloyti MDSC (s) over monoyti MDSC (s). Both subsets produed Arginse-1, induible nitri oxide synthse (inos), nd trnsforming growth ftor-β nd suppressed T-ell prolifertion in vitro. In this work, we desribe the immune sttus of spontneous melnom mouse model tht provides n interesting tool to develop future immunotherpeutil strtegies. Journl of Investigtive Dermtology (215) 135, ; doi:1.138/jid ; published online 3 July 215 INTRODUCTION Clinilly relevnt niml models of melnom tht mimi the humn sitution re ruil for investigtions of etiology nd immunology. Chen et l. (1996) me ross spontneous melnom model when they tried to generte 1 Deprtment of Dermtology nd Venereology, Medil University of Innsbruk, Innsbruk, Austri; 2 Onotyrol, Center for Personlized Cner Mediine, Innsbruk, Austri; 3 Deprtment of Dermtology, Lbortory of DC- Biology, Friedrih-Alexnder University of Erlngen-Nürnberg, University Hospitl of Erlngen, Erlngen, Germny; 4 Deprtment of Chemil Biology, Lb for Cner Reserh, Rutgers University, Pistwy, New Jersey, USA nd 5 Deprtment for Trnsltionl Dermto-Onology, Center for Medil Biotehnology, University Hospitl Essen, Essen, Germny Correspondene: Ptrizi Stoitzner, Deprtment of Dermtology nd Venereology, Medil University of Innsbruk, Anihstrsse 35, Innsbruk A-62, Austri. E-mil: ptrizi.stoitzner@i-med..t 6 Current ddress: CNRS UPR 3572, Lbortory of Immunopthology nd Therpeuti Chemistry/Lbortory of Exellene MEDALIS, Institut de Biologie Moléulire et Cellulire, Strsbourg, Frne. Abbrevitions: BMDCs, bone mrrow-derived dendriti ells; gp, glyoprotein; s, grnuloyti myeloid derived suppressor ells; Grm1, metbotropi glutmte reeptor1; inos, induible nitri oxide synthse; LN, lymph node; MDSC, myeloid-derived suppressor ell; s, monoyti myeloid derived suppressor ells; qpcr, quntittive PCR; tg, trnsgeni; TGF-β, trnsforming growth ftor-β; Tregs, regultory T ells Reeived 18 Deember 214; revised 1 June 215; epted 11 June 215; epted rtile preview online 29 June 215; published online 3 July 215 mouse with dipoyte dysfuntion. This strin, nmed LLA- TG3, possessed multiple tndem insertions of trnsgene in the gene Grm1 enoding the metbotropi glutmte reeptor 1 (Grm1), resulting in systemi overexpression. This led to the persistene of melnoytes in skin res without fur, e.g. er nd til skin, followed by hyperprolifertion nd subsequent melnom development (Chen et l., 1996; Cohen-Soll et l., 21). The signifine of Grm1 in igenesis ws onfirmed by the genertion of the tg(grm1)epv mouse model, in whih overexpression of Grm1 is driven by the melnoyte-speifi dophrome tutomerse promoter (Pollok et l., 23). These mie spontneously develop melnom with 1% penetrne t the ge of 4 6 months in skin regions suh s er nd til skin. Tumors in tg(grm1) EPv mie do metstsize, s the presene of melnom ells in lymph nodes (LNs) hs lredy been reported (Pollok et l., 23). Grm1-positive ells ould lso be deteted in lymphti orgns nd in lung s well s liver; however, most of these ells were non-pigmented (Shiffner et l., 212). The relevne of this pthwy for humn igenesis ws substntited by the ft tht 46% of humn melnom smples overexpress Grm1 (Nmkoong et l., 27). Cner immunosurveillne s proposed in 1957 by Burnet ontrols both initil steps of rinogenesis nd ner 215 The Soiety for Investigtive Dermtology

2 CD45 + ells in skin d FoxP3 Tumor free CD25 Erly Skin Lte Lymph node 42.6% 22.1% b Perentge T ells in skin * CD4 + T ells e Tregs of CD45 + ells Skin Perentge T ells in LN LN CD4 + T ells Erly Figure 1. The perentges of CD4 + T ells nd Tregs re redued in lte-stge s of tg(grm1)epv mie. Cell suspensions from the skin nd drining lymph nodes (LNs) of -free, erly-stge, nd lte-stge tg(grm1)epv mie were nlyzed by flow ytometry. () Perentges of CD45 + leukoytes re shown fter pregting on vible ells (n = 14 mie). For ll further nlyses, the perentges of ells were lulted bsed on CD45 + vible ells. (b nd ) CD4 + nd were nlyzed in (b) the skin (n = 8 mie) nd () the drining LNs (n = 15 mie). (d) Representtive FACS plots for FoxP3 + CD25 + Tregs in the skin nd drining LNs of -free mie. (e) Summry grph for the perentges of FoxP3 + CD25 + Tregs in the skin nd drining LNs (n 8 mie). progression (Burnet, 1957). The immune system is ble to reognize nd eliminte trnsformed ells, s demonstrted by enhned suseptibility to ner when the immune system is impired (Dunn et l., 22). The blne between effetive immunity nd espe mehnisms deides on the elimintion or progression of ner (Shreiber et l., 211). Cytotoxi effetor ells suh s T nd nturl killer ells infiltrte tissue to fight ells (Vesely et l., 211; Gjewski et l., 213). One prerequisite for effetive nti immunity is the presenttion of ntigens by ells nd their reognition by infiltrting ytotoxi. A problem enountered with mny -ssoited ntigens is tht they re poorly immunogeni non-mutted self-ntigens. A reson for their poor immunogeniity is tolerne indution ssoited with the deletion or nergy of self-retive CD8 + T ells during igenesis (Willimsky nd Blnkenstein, 27). In ddition, s employ omplex immunosuppressive network to espe immune reognition. Suppressive ells, suh s regultory T ells (Tregs) nd myeloid-derived suppressor ells (MDSCs), infiltrte s nd prevent nti- immune responses (Nishikw nd Skguhi, 21; Gbrilovih et l., 212). These vrious immune evsion mehnisms eventully result in n impired funtion of ytotoxi nd nturl killer ells trnslting in poor prognosis for the ptients (Shreiber et l., 211; Motz nd Coukos, 213). Despite this, speifi forssoited self-ntigens, suh s glyoprotein 1 (gp1), tyrosinse, nd MART, do exist in ptients with mlignnt melnom (Bkker et l., 1994; Kwkmi et l., 2; Boon et l., 26). For this reson, linil studies re using melnosoml ntigens for vintion pprohes (Rosenberg et l., 24). In the present study, we hrterized the immunologil fetures of spontneous mouse melnom model bsed on Grm1 overexpression tht n lso be observed in bout 6% of humn melnom smples. Our results suggest tht s grow progressively despite the presene of tivted T ells. We found possible explntions for impired immunity, inluding gp1-speifi CD8 + T-ell nergy nd the infiltrtion of immunosuppressive MDSCs into the miroenvironment. RESULTS The perentges of CD4 + T ells nd Tregs re redued in lte-stge s of tg(grm1)epv mie The immunologil sttus of the tg(grm1)epv mouse ws hitherto unknown; hene, we hrterized the immune ells present in tissue nd drining LNs. For this purpose, we ompred -free mie (2 3 months of ge) with mie bering erly-stge (5 6 months) nd lte-stge s (8 9 months; Supplementry Figure S1 online). Immunofluoresene stinings of lte-stge setions reveled tht CD4 + nd were present in the nd tht proportion of CD4 + T ells o-expressed FoxP3- mrker for Tregs (Supplementry Figure S2 online). For quntifition of immune ells present in -free nd -bering mie, er skin nd drining LNs were enzymtilly digested, nd CD45 + ells were nlyzed by flow ytometry. Beuse growth in tg(grm1)epv mie ours progressively over severl months, we lso monitored young (2 3 months) nd old (8 9 months) C57BL/6 mie in order to rule out tht these observed hnges were not solely used by ging (Supplementry Figure S3 online). There ws no hnge in the overll perentges of CD45 + ells in the skin of -free, erly-stge, nd lte-stge mie (Figure 1), similr to wht we observed in C57BL/6 mie (Supplementry Figure S3 online). Less CD4 + T ells were present in lte-stge s (Figure 1b), feture tht ws lerly bsent from old C57BL/6 mie (Supplementry Figure 2786 Journl of Investigtive Dermtology (215), Volume 135

3 S3b online). Cutneous were unltered by development (Figure 1b), despite showing slight inrese over ging in C57BL/6 mie (Supplementry Figure S3b online). However, in LN drining either lte-stge of tg (Grm1)EPv mie (Figure 1) or the skin of ged C57BL/6 mie (Supplementry Figure S3 online), we observed omprble redution in CD4 + T ells, suggesting tht this hnge might be ge relted. In ddition, we were ble to detet CD4 + Tells expressing the Treg mrker FoxP3 in the skin nd lymphoid tissue of tg(grm1)epv mie (Figure 1d). The perentges of Tregs in the totl CD45 + ell pool were redued in lte-stge tissue nd the drining LNs (Figure 1e), wheres these proportions remined the sme in young nd old C57BL/6 mie (Supplementry Figure S3d online). Altogether, our dt suggest -relted redution of CD4 + T ells inluding Tregs in tissue. Ativted T ells re present in lte-stge mie nd n produe IFN-γ Beuse of the ft tht perentges of T ells differed between -free nd lte-stge tg(grm1)epv mie, we ssessed the phenotype nd funtionl properties of T ells in drining LNs of these mie. Tumor development ws ompnied by signifint upregultion of the tivtion mrker CD44 nd redution in the homing reeptor L-seletin (CD62L) on CD4 + nd, refleting enhned differentition into CD44 hi CD62L low effetor memory T ells (Figure 2). In ddition, more T ells upregulted the tivtion mrker CD69 in drining LNs when ompred with -free mie (Figure 2b). Next, we determined whether formtion would lter the bility of T ells to produe the ytokine IFN-γ. For this purpose, LN ell suspensions were restimulted in vitro with ntibodies ginst CD3 nd CD28. CD4 + T ells showed little IFN-γ, nd prodution ws unhnged upon development. In ontrst, more produed IFN-γ in drining LNs of lte-stge mie (Figure 2). Although similir tivtion ptterns were deteted in LNs of ged C57BL/6 mie inditing possible ge-relted effets (Supplementry Figure S4 online), it is still interesting to note tht tivted nd funtionl T ells re present in the drining LNs of -bering tg(grm1)epv mie. Endogenous gp1-speifi re present in mie but re funtionlly impired upon ntigen-speifi restimultion Despite the presene of tivted T ells in -bering tg (Grm1)EPv mie, growth nnot be ontrolled. To better understnd the mehnisms behind impired immunity, we investigted T ells by pentmer stining with fous on the presene nd funtionl properties of gp1- speifi (Figure 3). We found tht, in the skin nd drining LNs of tg(grm1)epv mie, the perentges of gp1-speifi were not ffeted by development (Figure 3b). In order to test the funtionl properties of gp1-speifi, we performed ntigen-speifi in vitro restimultion of totl LN ells with gp1 peptide. The perentges of gp1-responsive CD8 + CD44 hi CD62L low T ells in LN b CD69 + T ells in LN IFN-γ + T ells in LN CD4 + T ells CD4 + T ells CD4 + T ells T ells were determined by ompring the proportion of produing IFN-γ upon gp1-peptide restimultion relted to the proportion of lbeled by mjor histoomptibility omplex I/gp1 pentmer. This lultion ws neessry beuse IFN-γ produing CD8 + T ells downregulte their TCR, whih n therefore no longer be deteted in relible wy by pentmer stining. In free tg(grm1)epv mie, 6% of pentmer + produed IFN-γ, wheres T ells in lte-stge mie were inpble of produing IFN-γ (Figure 3 nd d). These dt suggest tht, in lte-stge tg(grm1)epv mie, gp1-speifi re nergi to the melnoytederived ntigen gp1. MDSCs umulte in the skin nd drining lymph nodes of lte-stge mie The redution of Tregs in lte-stge s of tg(grm1)epv mie suggested negligible role for inhibition of nti- * * Figure 2. Ativted T ells re present in lte-stge mie nd produe IFN-γ. Cell suspensions from drining lymph nodes (LNs) of -free nd lte-stge tg(grm1)epv mie were nlyzed by flow ytometry. All nlyses used pregte on CD45 + vible ells. CD4 + nd CD8 + T-ell subsets in drining LNs were nlyzed for the following: () CD44 hi CD62L low T ells (n = 11 mie) nd (b) CD69 + tivted T ells (n = 8 mie). () IFN-γ-produing T ells were nlyzed fter restimultion of LN ells with ntibodies ginst CD3 nd CD28 for 48 h (n 6 mie)

4 Pentmer k 1 k 15 k 2 k 25 k SSC-W Skin 24.6% Lymph node.27% 5 k 1 k 15 k 2 k 25 k b pentmer Skin LN pentmer Untreted gp1 restimultion 1.1% 5.15% d IFN-γ 1 3 CD gp1-responsive IFN-γ + Figure 3. Endogenous glyoprotein 1 (gp1)-speifi re present in lte-stge s but re funtionlly impired upon ntigen-speifi restimultion. Cell suspensions from the skin nd drining lymph nodes (LNs) of -free nd lte-stge tg(grm1)epv mie were nlyzed by flow ytometry for gp1-speifi. All nlyses used pregte on CD45 + vible ells. () Representtive FACS plots from -free tg(grm1)epv mouse for pentmer + in the skin nd drining LNs, pregte on vible. (b) Summry grph for pentmer + in the skin nd drining LNs (n 9 mie). () Representtive FACS plots from -free tg(grm1)epv mouse for IFN-γ prodution of CD8 + LN ells ultured without (untreted) or with gp1 peptide for 48 h. (d) Summry grph for gp1-responsive IFN-γ + CD8 + ells lulted s following: ((men of IFN-γ + CD8 + ells restimulted with gp1 peptide) (men of IFN-γ + CD8 + ells ultured without gp1 peptide))/(men of unstimulted pentmer + CD8 + ells), n 4 mie. immune responses by this ell type. Another popultion of immunosuppressive ells, nmely MDSCs, re pble of suppressing immune responses ginst s, thereby llowing espe (Gbrilovih et l., 212). We investigted CD11b + Gr-1 + MDSCs in the skin nd drining LNs of -free, erly-stge, nd lte-stge mie. In the skin, MDSCs umulted in lte-stge s, nd, similr trend ws observed in the -drining LNs, lbeit not s pronouned s in the skin (Figure 4 nd b). In n effort to hrterize CD11b + Gr-1 + MDSC subsets, we nlyzed the perentges of CD11b + Gr-1 inter Ly-6C high monoyti MDSCs (s) nd CD11b + Gr-1 high Ly-6C low grnuloyti MDSCs (s) in the skin nd drining LNs of -free nd lte-stge mie. Tumor development led to n umultion of both MDSC subsets in the skin nd drining LNs of tg(grm1)epv mie (Figure 4 nd d). We exmined dditionl mrkers on MDSCs nd deteted higher levels of M-CSF reeptor (CD115) nd mjor histoomptibility omplex lss II on s thn on s (Supplementry Figure S5 online). In order to rule out tht MDSC infiltrtion ws ge relted, we nlyzed both MDSC subsets in young nd old C57BL/6 mie. No MDSC umultion ws deteted in both the skin nd drining LNs of ged C57BL/6 mie (Supplementry Figure S6 online). Thus, umultion of CD11b + Gr-1 + ells in lte-stge s of tg(grm1)epv mie suggested n immunosuppressive miroenviroment tht fvors growth. MDSCs in the re potent suppressors of T-ell tivity In order to test the pity of MDSCs to suppress T-ell tivity, we flow ytometrilly sorted MDSC subsets from the skin of lte-stge tg(grm1)epv mie (Supplementry Figure S7 online). We purified gp1-speifi from pmel-1 mie nd ultured them with gp1-peptide loded mture bone mrrow derived dendriti ells (BMDCs). The gp1-speifi proliferted in response to peptide-loded BMDCs, nd the ddition of sorted s or s signifintly redued T-ell prolifertion (Figure 5). The suppression of T-ell funtion ws dependent on the number of MDSCs (Supplementry Figure S7b online). Next, we ttempted to eluidte ftors responsible for MDSC-medited immunosuppression nd investigted the expression of Arginse-1 nd induible nitri oxide synthse (inos) in s nd s sorted from lte-stge s. In ddition, we exmined trnsforming growth ftorβ (TGF-β), ytokine tht is onsidered to be one of the key immunosuppressive ftors relesed by s nd MDSCs (Gbrilovih nd Ngrj, 29). Quntittive PCR (qpcr) nlysis of sorted MDSCs from s reveled high mrna levels for ll three moleules nd low to undetetble levels in ells nd non-mdsc immune ells (Figure 5b d). In ddition, we observed different expression ptterns in the two MDSC subsets, suh tht s expressed higher mrna levels of inos (Figure 5) nd TGF-β (Figure 5d) but lower levels of Arginse-1 ompred with s (Figure 5b). This ws onfirmed by intrellulr flow ytometri nlysis showing tht more s were positive for inos thn for Arginse-1 (Figure 5e h). From this we onlude tht s s the predominnt immunosupressive popultion in s of tg(grm1)epv mie most likely suppress T-ell funtion by inos, TGF-β, nd Arginse-1 prodution Journl of Investigtive Dermtology (215), Volume 135

5 CD11b + Gr-1 + ells in skin MDSC subsets in skin Erly b.8 *.6 Tumor free Erly Lte CD11b + Gr-1 + ells in LN MDSC subsets in LN Tumor free Erly Lte Figure 4. Myeloid-derived suppressor ells (MDSCs) umulte in the skin nd drining lymph nodes (LNs) of lte-stge mie. Cell suspensions of -free, erly-stge, nd lte-stge tg(grm1)epv mie were nlyzed by flow ytometry for the presene of CD11b + Gr-1 + MDSCs in () the skin nd (b) drining LNs (n = 6 mie). MDSCs were further subdivided into CD11b + Gr-1 inter Ly-6C high monoyti MDSCs (s) nd CD11b + Gr-1 high Ly-6C low grnuloyti MDSCs (s) in -free nd lte-stge tg(grm1)epv mie for () the skin nd (d) the drining LNs (n = 1 mie). All nlyses used pregte on CD45 + vible ells. DISCUSSION Trnsgeni (tg) mouse models bsed on geneti berrtions re essentil for n improved understnding of melnom biology nd immunology (Beker et l., 21). Here, we hve investigted the immune sttus of murine melnom model bsed on etopi overexpression of Grm1 in melnoytes. This overexpression results in spontneous development nd estblished link between metbotropi glutmte signling in melnoytes nd melnom development, refleting the relevne of this mouse model nd the funtionl importne of the glutmte pthwy in ner (Pollok et l., 23; Prikett nd Smuels, 212). In line with this, studies on humn mteril reveled tht Grm1 is berrntly expressed in 6% of humn melnom smples (Nmkoong et l., 27). Over the lst dede, severl studies mde use of this murine melnom model in order to better understnd the metstti behvior of s nd to test future immunotherpeutil pprohes (Mrin et l., 26; Xio et l., 211; Shiffner et l., 212). However, nothing ws known bout the immune sttus of these mie. In this work, we desribe the omposition of immune ells present in the skin nd drining LNs of -free nd -bering mie nd provide some lues on their funtionl role in immunity. We observed immunosuppression on two levels: (i) CD8 + T ells seemed to be nergi to the melnom-ssoited ntigen gp1; (ii) MDSCs with the bility to suppress gp1- speifi CD8 + T-ell response represented the dominting immune ell popultion in lte-stge tissue mediting their suppressive effet by inos, TGF-β, nd Arginse-1 prodution. d.4.2. * High prevlene of T ells in s is n inditor for longer overll survivl of dvned-stge melnom ptients (Hnen et l., 26; vn Houdt et l., 28). Therefore, we hrterized the omposition of immune ells present in the miroenvironment nd the drining LNs to whih immune ells might relote. When we investigted the CD45 + ell pool, we observed tht the CD8 + T-ell frequenies were not ffeted by growth, wheres the perentges of CD4 + T ells inluding Tregs were signifintly redued in lte-stge mie. The CD4 + T-ell redution in the drining LNs ws lso detetble in ged C57BL/6 mie, inditing n ge-relted effet on immune ell omposition; however, ll other ltertions in the preentges of immune ells in tg(grm1)epv mie were ssoited. Thus, our findings indite tht erly- nd lte-stge s re not infiltrted by T ells orrelting with less immunogeni miroenvironment (Gjewski et l., 213). The observed redution in Tregs fits to n erlier report on the ret-tg mouse model for melnom tht demonstrted reruitment of Tregs in erly-stge s followed by derese in Tregs in lte-stge s (Kimpfler et l., 29). The uthors lso showed tht depletion of Tregs did not dely development, inditing n lterntive soure of immunosuppression. In ontrst, in trnsplntble B16 melnom mouse model, the depletion of Tregs used prtil regression of estblished s (Klges et l, 21). Hene, the mehnisms of immunosuppression might differ between the vrious mouse models for melnom. The undisturbed growth in the tg(grm1)epv mouse model led us to hypothesize tht the remining lymphoytes re ompromised in mounting proper immune response ginst the. In line with this, studies performed in humn melnom ptients nd vrious murine melnom models suggested tht lymphoytes re dysfuntionl (Ldnyi et l., 24; Boon et l., 26; Ahmdzdeh et l., 29; Fourde et l., 21; Lndsberg et l., 21). Therefore, we hrterized the tivtion sttus of T ells present in the drining LNs of lte-stge mie. These mie showed elevted perentges of CD44 hi CD62L low CD4 + nd. Enhned tivtion ws further onfirmed by higher levels of the erly tivtion mrker CD69 on both T-ell subsets. This implied tht, similr to the ret-tg melnom mouse model (Umnsky et l., 28), growth is ompnied by T-ell differentition into effetor memory T ells. In ddition, from LN drining lte-stge s were ble to produe IFN-γ upon in vitro restimultion, inditing tht they re not funtionlly impired. However, we nnot exlude tht ging hs some effet due to similr findings in old C57BL/6 mie nd reports tht showed n tivted phenotype in T ells of ged mie (Akbr nd Henson, 211). Nevertheless, the ft tht growth nnot be ontined by the immune system suggested potent immune espe mehnisms. Adptive immunity ginst the is onduted by ntigen-speifi T ells. In the se of melnom, the ntigen gp1 is expressed by melnoytes nd upregulted during growth. This protein represents -ssoited

6 Prolifertion (%) e Count Negtive trl Positive trl * * * * Isotype Arginse-1 f rginse-1 + MDSC in skin b Arginse-1 mrna levels reltive to TBP CD45 + CD11b CD ginse-1 + MDSC in LN inos mrna levels reltive to TBP g Count CD45 + CD11b Isotype inos CD45 h inos + MDSC in skin d TGF-β mrna levels reltive to TBP ,5 1, 5 CD45 + CD11b 8 CD inos + MDSC in LN Figure 5. Myeloid-derived suppressor ells (MDSCs) in the re potent suppressors of T-ell tivity. Sorted MDSC subsets from er nd til skin of ltestge tg(grm1)epv mie were used to suppress CD8 + T ell prolifertion. () glyoprotein 1 (gp1)-speifi pmel-1 (2 1 4 ) were purified, CFSE-lbeled (.4 μm), nd ultured for 3 dys together with gp1 peptide pulsed bone mrrow derived dendriti ells (BMDCs; ). Sorted s (1 1 4 ) or s (1 1 4 ) were dded to ultures, nd prolifertion of T ells ws mesured (n = 3 experiments). (b-d) Quntittive PCR nlysis of sorted grnuloyti MDSC (s) nd monoyti MDSC (s) from six lte-stge tg(grm1)epv mie in omprison with CD45 ells nd CD45 + CD11b leukoytes ws performed for (b) Arginse-1, () induible nitri oxide synthse (inos), nd (d) trnsforming growth ftor-β (TGF-β). (e h) Representtive histogrms from skin MDSCs nd summry grphs of the perentges of positive MDSCs in the skin nd drining LNs from lte-stge tg(grm1)epv mie re shown for (e nd f) Arginse-1 nd (g nd h) inos, (n = 6 mie). ntigen tht n be reognized by gp1-speifi CD8 + T ells (Bkker et l., 1994; Boon et l., 26; Rizzuto et l., 29). In -free tg(grm1)epv mie, o.5% of CD8 + T ells were speifi for gp1 in the drining LNs s opposed to 1 15% gp1-speifi in the skin. Tumorigenesis hd no effet on the perentges of gp1- speifi ; however, from LNs of lte-stge mie were unresponsive to restimultion to gp1 peptide in vitro. These findings suggest n nergi stte due to tolerne indution to the differentition ntigen gp1. Similr to gp1, mny -ssoited ntigens re non-mutted self-proteins tht represent ttrtive trgets for immunotherpy s they re shred between ptients. However, stte of funtionl tolerne pprently exists (Boon et l., 26). Nevertheless, it hs been reported tht immuniztion with gp1 n indue ytotoxi T-ell responses in murine melnom models (Overwijk et l., 23; Xio et l., 211) nd hs been used in linil trils to tret ner ptients (Rosenberg et l., 24). In order to generte nti- immune response ginst gp1, potent immuniztion pprohes need to be employed, suh s virl vetors. In the se of the tg(grm1)epv mouse model, it hs been reported tht immuniztion with virl vetors before onset ould fully protet from melnom development, nd dely in growth ould be hieved in bering mie (Xio et l., 211). The self-tolerne to gp1 ntigen might explin the inbility of the immune system to prevent development in the tg(grm1)epv mouse; however, in ddition, we observed tht the employs nother immune espe mehnism to suppress immunity (Gjewski et l., 213). MDSCs re heterogeneous popultion of ells derived from the myeloid linege endowed with strong immunosuppressive tivities. Formtion of these ells is triggered under hroni inflmmtory onditions or in ner, where they infiltrte the ffeted tissue nd suppress innte nd dptive immune responses (Gbrilovih nd Ngrj, 29). In tg(grm1)epv mie, few MDSCs were deteted in the skin nd drining LNs of -free nd erly-stge mie. However, strong infiltrtion of MDSCs ould be observed in lte-stge s. This ws not ge relted s old C57BL/6 mie showed no MDSC umultion. In ddition, shift towrd more s s ompred with s ws pprent, mimiking the sitution previously desribed in other murine models nd in humn ner ptients (Ze et l., 25; Youn et l., 28). It hs been reported tht both subsets effiiently suppress ntigen-driven CD8 + T-ell prolifertion in vitro (Youn et l., 28). We hd similr findings with s nd s sorted from lte-stge s tht proved to be ble to suppress gp1-speifi CD8 + T-ell prolifertion in vitro. Arginse-1 nd inos hve been desribed s key meditors of the suppressive pity of MDSCs in s (Gbrilovih nd Ngrj, 29). Quntittive PCR nlysis of sorted s nd s from tissue of lte-stge tg(grm1)epv mie reveled high mrna levels of Arginse-1 nd inos, nd expression ws onfirmed by flow ytometri nlyses. Moreover, TGF-β, moleule tht hs been linked to initition, progression, nd metstsis (Gbrilovih nd 279 Journl of Investigtive Dermtology (215), Volume 135

7 Ngrj, 29), ws lso elevted t the mrna level in sorted MDSCs from tissue. Our dt suggest tht s most likely suppress immunity by inos, TGF-β, nd to lesser extent with Arginse-1. The high levels of TGF-β produed by s but lso s hve n importnt role in progression s reported erlier (Yng et l., 21). Altogether, we desribe here the immune sttus of mie developing melnom spontneously due to the etopi overexpression of Grm1. Despite the presene of tivted T ells with memory phenotype, the immune system fils to mount n effetive immune response to ontrol melnom development. The resons for impired dptive immunity re the nergy of melnom-ntigen-ssoited nd the infiltrtion of immunosuppressive MDSCs to the miroenvironment. This spontneous mouse melnom model is highly relevnt for reserh on humn melnom due to the sme berrnt overexpression of Grm1, whih n be observed in 6% of humn melnom smples (Pollok et l., 23). The relevne is further substntited by the ft tht the immune omposition of s is similr to tht in humns, refleted by the infiltrtion of immunosuppressive ells (Senovill et l., 212). Tken together, these fetures mke this mouse model n interesting nd n importnt tool to test future immunotherpeutil pprohes. MATERIALS AND METHODS Mie Breeding pirs of the tg mouse strin tg(grm1)epv (Pollok et l., 23) were provided by Jürgen C. Beker (Deprtment of Dermtology nd Venereology, Medil University Grz, Austri). Tumor-free mie were used before onset (2 3 months) nd ompred with mie bering erly-stge s (5 6 months) nd lte-stge s (8 9 months). C57BL/6 mie were originlly derived from Chrles River (Sulzfeld, Germny). The T-ell reeptor tg pmel-1 mie (Overwijk et l., 23) were kindly provided by Thoms Tüting (Universitätsklinikum Bonn, Germny). All mouse strins were bred t the niml fility of the Deprtment of Dermtology nd Venereology t the Medil University of Innsbruk. All experimentl protools were pproved by the Austrin Federl Ministry of Siene nd Reserh nd performed ording to institutionl guidelines. Flow ytometri nlyses of the skin nd drining LN ell suspensions Er skin from -free nd -bering tg(grm1)epv mie, s well s C57BL/6 mie, ws ut into smll piees nd digested with.15 mg ml 1 Liberse (Rohe Dignostis, Mnnheim, Germny) nd.12 mg ml 1 DNAse (Rohe) for 45 min t 37 C nd pressed through 1-μm ell striners (BD Biosienes, Frnklin Lkes, NJ). Auriulr LNs from -free nd -bering tg(grm1)epv mie, s well s C57BL/6 mie were digested with.125 mg ml 1 ollgense D (Rohe) nd.12 mg ml 1 DNAse (Rohe) for 25 min t 37 C nd pressed through 1-μm ell striners (BD Biosienes). All ntibody stining steps were performed for 15 min t 4 C. Nonspeifi FR-medited ntibody stining ws bloked by inubtion for 15 min with nti-cd16/32 Ab (2.4G2, in-house from hybridom superntnt nd BD Biosienes). For intrellulr IFN-γ prodution, LN ells were restimulted with 3 μgml 1 nti-cd3 (lone 17A2, BD Biosienes) plus 3 μgml 1 nti-cd28 (lone 37.51, BD Biosienes) for 48 h t 37 C. Cytokine relese ws bloked with 1 μgml 1 Brefeldin A (BD Biosienes) during the lst 4 h of restimultion. The ells were fixed using the BD Biosienes Cytofix/Cytoperm Kit (BD Biosienes) ording to the mnufturer s protool. FoxP3 stining ws performed with the FoxP3/ trnsription ftor stining buffer set from ebiosiene (Sn Diego, CA). The following ntibodies were used: nti-ly-6c-fitc (lone AL-21), nti-ifn-γ-apc (lone XMG1.2), nd nti-cd3-fitc (lone 17A2) ll from BD Biosienes; nti-cd45-fitc (lone 3.F11), nti- FoxP3-PE (lone FJK-16s), nd nti-inos-pe (lone CXNFT) ll from ebiosiene; nti-cd115-pe (lone AFS98), nti-cd62l-percp- Cy5.5 (lone MEL-14), nti-cd3-percp-cy5.5 (lone 17A2), ntimjor histoomptibility omplex II-PerCP-Cy5.5 (lone M5- / ), nti-cd69-pe-cy7 (lone H1.2F3), nti-cd25-pe-cy7 (lone PC61), nti-cd44-apc (lone IM7), nti-gr-1-apc (lone RB6-8C5), nti-cd11b-bv421 (lone M1/7), nti-cd4-bv421 (lone RM4-5), nti-cd45-bv51 (lone 3-F11), nd nti-cd8- BV51 (lone ) ll from Biolegend (Sn Diego, CA); nti- Arginse-1-PE from R&D Systems (Minnepolis, MN). Ded ells were exluded with 7AAD (BD Biosienes) or fixble vibility dye efluor 78 (ebiosiene) stining. FACS nlyses were performed on FACS Cnto II (BD Biosienes). Dt nlysis ws performed using FlowJo softwre (Tree Str, Ashlnd, OR). Pentmer stining of endogeneous nd IFN-γ prodution For the detetion of endogenous gp1-speifi, we used n H-2K b -KVPRNQDWL pentmer (ProImmune, Oxford, UK). Skin nd drining LNs were nlyzed for the presene of H-2K b - KVPRNQDWL pentmer-positive. The ells were stined ording to the mnufturer s protool, nd unspeifi pentmer binding ws exluded by pregting on vible CD19 (lone 1D3, BD Biosienes) NKp46 (lone 29A1.4, Biolegend) ells. For the detetion of IFN-γ, we inubted ell suspensions from drining LNs with 1 μm gp1 peptide (KVPRNQDWL, Peptide&Elephnts, Potsdm, Germny) for 48 h t 37 C. IFN-γ relese nd ntibody stining were performed s desribed bove. For the lultion of gp1-responsive IFN-γ + CD8 + ells, we used this formul: ((men of IFN-γ + CD8 + ells restimulted with gp1 peptide) (men of IFN-γ + CD8 + ells ultured without gp1 peptide))/(men of unstimulted pentmer + CD8 + ells). MDSC suppression ssy For purifition of MDSC subsets from tissue, er nd til skin of 4 5 lte-stge mie were digested with.15 mg ml 1 Liberse (Rohe) nd.12 mg ml 1 DNAse (Rohe) for 45 min t 37 C nd pressed through 1-μm ell striners (BD Biosienes). CD45 + ells were enrihed with g ml 1 Histodenz density grdient ording to the mnufturer s protool (Sigm-Aldrih, St. Louis, MO). The s nd s were purified by flow ytometri ell sorting with FACS Ari s follows: CD45 + CD11b + ells were distinguished with Ly-6C nd Gr-1 into Gr-1 inter Ly-6C high s nd Gr-1 high Ly-6C low s. were isolted from LNs nd spleen of pmel-1 mie before the onset of spontneous vitiligo (2 3 months). Purifition ws performed with nti-mouse-cd8-α (Ly-2) MiroBeds (Miltenyi Biote, Bergish Gldbh, Germny). In order to trk T-ell prolifertion, ells

8 were stined with.4 μm CFSE for 5 min t room temperture. For suppression ssys, BMDCs loded with 1 μm gp1 peptide (KVPRNQDWL, Peptide&Elephnts) for 1 h t 37 C. These ells were ultured with CFSE-lbeled from pmel-1 mie nd or sorted s/s for 3 dys. T-ell prolifertion ws mesured by nlysis of CFSE dilution by flow ytometry. qpcr nlysis of the skin nd smples The totl RNA of er skin, smples, sorted MDSC subsets, CD45 ells, nd CD45 + CD11b leukoytes ws isolted using TRIzol Regent (Life Tehnologies, Crlsbd, CA) ording to the mnufturer s instrutions nd nlyzed by eletrophoresis on 1.5% grose gels for RNA integrity (Sigm-Aldrih) fter smple preprtion in 2 RNA Loding Dye (Thermo Fisher Sientifi, Wlthm, MA). Totl RNA ws reverse trnsribed into DNA with rndom hexmers nd SuperSript II Reverse Trnsriptse (Life Tehnologies). qpcr nlysis ws performed on BioRd CFX96 using Brillint III Ultr-Fst qpcr nd qrt-pcr Mster Mix (Agilent tehnologies, Boeblingen, Germny). The following sequenes for probes nd primers speifi for different lignds were purhsed from Life Tehnologies: Mm475988_m1 (Arginse-1), Mm4452_m1 (inos), nd Mm117882_m1 (TGF-β1). Sequenes for probes nd primers speifi for mouse TATA-binding protein were seleted by Primer Express softwre (Applied Biosystems) nd synthesized by Mirosynth (Blgh, Switzerlnd). Sttistil nlyses All experiments involved groups of two to six mie nd were performed t lest twie with similr results. Sttistil nlysis ws performed using PRISM 5. (GrphPd Softwre, L Joll, CA). Unpired Student s t-test ws used to determine the sttistil signifine of the men perentges of ell types between free nd -bering mie (Figures 2, 3b, 4, 4d, 5f nd h; Supplementry Figure S3, S4 nd S6 online). One-wy nlysis of vrine followed by post ho Tukey s test ws used to ompre mens mong three or more independent groups (Figures 1, 1b, 1, 1e, 4, 4b nd 5). A P-vlue of o.5 ws onsidered sttistilly signifint (*), o.1 very signifint (), nd o.1 highly signifint (). The ext number of mie used per experiment (n = number of mie) is indited in the orresponding legend of eh figure. Error brs represent SEM (br grph) or rnge from minimum to mximum vlues (box nd whiskers). CONFLICT OF INTEREST The uthors stte no onflit of interest. ACKNOWLEDGMENTS This work ws supported by the Austrin Siene Fund (FWF) with grnts to PS: FWF-P21487-B13 nd FWF-W111-B15 (DGM is dotorl student finned by this FWF-funded PhD progrm) nd the Tyrolen Cner Soiety (Projet number 41/211). DD ws funded by the Bvrin Genome Network (ByGene) nd the Germn Reserh foundtion (DU548/2-1, RTG166), LH ws supported within RTG166, nd VF by the PROMOS progrm of the DAAD (Germn Ademi Exhnge Servie). Additionl grnts to SC from the New Jersey Commission for Cner Reserh CCR-E, NIH R1CA7477, nd NIH R1CA helped perform this study. 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