Systemic Agonistic Anti-CD40 Treatment of Tumor-Bearing ...or Nox2 / mice were injected into the tail vein of tumor-free CD45.2þCd40 / mice. Mice were subsequently inoculated i.p

Research Article

Systemic Agonistic Anti-CD40 Treatment ofTumor-Bearing Mice Modulates HepaticMyeloid-Suppressive Cells and CausesImmune-Mediated Liver DamageJos�e Medina-Echeverz1, Chi Ma1, Austin G. Duffy1, Tobias Eggert1, Nga Hawk2,David E. Kleiner3, Firouzeh Korangy1, and Tim F. Greten1

Abstract

Immune-stimulatory mAbs are currently being evaluated asantitumor agents. Although overall toxicity from these agentsappears to bemoderate, liver toxicities have been reported and arenot completely understood. We studied the effect of systemicCD40 antibody treatment onmyeloid cells in the spleen and liver.Na€�ve and tumor-bearing mice were treated systemically withagonistic anti-CD40 antibody. Immune cell subsets in the liverand spleen, serum transaminases, and liver histologies wereanalyzed after antibody administration. Nox2�/�, Cd40�/�, andbonemarrow chimericmicewere used to study themechanismbywhich agonistic anti-CD40 mediates its effects in vivo. Suppressorfunction of murine and human tumor-induced myeloid-derivedsuppressor cells (MDSC) was studied upon CD40 ligation. Ago-nistic CD40 antibody caused liver damage within 24 hours afterinjection in two unrelated tumor models and mice strains. Usingbone marrow chimeras, we demonstrate that CD40 antibody–

induced hepatitis in tumor-bearing mice was dependent on thepresence of CD40-expressing hematopoietic cells. AgonisticCD40 ligation–dependent liver damage was induced by thegeneration of reactive oxygen species. Furthermore, agonisticCD40 antibody resulted in increased CD80-positive and CD40-positive liver CD11bþGr-1þ immature myeloid cells. CD40ligation on tumor-induced murine and human CD14þHLA-DRlow peripheral blood mononuclear cells from patients withcancer reduced their immune suppressor function. Collectively,agonistic CD40 antibody treatment activated tumor-inducedmyeloid cells, caused myeloid-dependent hepatotoxicity, andameliorated the suppressor function of murine and humanMDSC. Collectively, our data suggest that CD40 may matureimmunosuppressive myeloid cells and thereby cause liverdamage in mice with an accumulation of tumor-inducedhepatic MDSC. Cancer Immunol Res; 3(5); 557–66. �2015 AACR.

IntroductionThe TNF receptor family member CD40 is a stimulatory mol-

ecule constitutively expressed on a large variety of cells, includingdendritic cells, B cells, macrophages, and endothelial cells (1–5).CD40 engagement of antigen-presenting cells provides the"license" to T-cell help and enhances T-cell activation (6, 7).Agonistic CD40 antibodies were shown to overcome T-cell tol-erance in tumor-bearing mice and facilitate development ofpotent cytotoxic T-cell responses by enhancing the effects ofcancer vaccines (8–12). Recently, immune-modulatory regi-mens—cytokine therapy (1–5, 13–17), radiotherapy (6, 7, 18–20), chemotherapy (8–12, 21–23), kinase inhibitors (24), ormonoclonal antibodies (25)—have been shown to synergizewith

agonistic CD40 antibodies, leading to tumor rejection in animalmodels.

However, systemic administration of immunostimulatoryCD40 antibodies has been associated with cytokine release syn-drome, lymphopenia, and liver toxicity in clinical trials (1, 3–5).In preclinical models, Fransen and colleagues (6) observed thatintravenous delivery of high- or low-dose agonistic CD40 anti-body increased liver toxicity in mice bearing virally transformedtumors. Agonistic anti-CD40 biodistribution experiments bySandin and colleagues (9) showed that systemic administrationled to higher antibody concentrations in the liver compared withlocal delivery. However, the reason why systemic agonistic CD40antibody causes liver toxicity remained unknown.

Tumor-induced myeloid-derived suppressor cells (MDSC)constitute one of the main players in tumor-induced immunesuppression. They comprise a heterogeneous population ofmyeloid cells of diverse differentiation status, whose mainfeature is the suppression of innate and adaptive immuneresponses (8). Our laboratory and others have previouslydescribed that tumor-induced CD11bþGr-1þ MDSC accumu-late in the liver of mice (13, 15, 17) and in patients withhepatocellular carcinoma (18, 20). In addition, hepatic MDSChave been reported to promote the generation of liver metas-tasis (21), and CD11bþCCR2þ cells have been detected in livermetastasis from patients with colorectal cancer (26). However,little is known about the biology of tumor-induced hepaticMDSC.

1Gastrointestinal Malignancy Section, Thoracic and GastrointestinalOncology Branch, Center for Cancer Research, National Cancer Insti-tute, NIH, Bethesda, Maryland. 2Experimental Transplantation andImmunology Branch, NIH, Bethesda, Maryland. 3Laboratory of Pathol-ogy, National Cancer Institute, NIH, Bethesda, Maryland.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Tim F. Greten, NIH/NCI, 10 Center Drive, Building 10,Room 12N226, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-451-4723;Fax: 49-511-220027203; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0182

�2015 American Association for Cancer Research.

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Published OnlineFirst January 30, 2015; DOI: 10.1158/2326-6066.CIR-14-0182

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In the study reported herein, we investigated the effect ofagonistic anti-CD40 antibody injection on hepatic and splenicMDSC in tumor-bearing mice. Although agonistic anti-CD40treatment led to severe, MDSC-mediated hepatitis in mice,we also provide evidence suggesting that MDSC mature intoa proinflammatory cell type with less arginase activity. Theseresults are recapitulated in human CD14þHLA-DRlow MDSC,which also lose arginase expression and thereby suppressorfunction in vitro.

Materials and MethodsMice and cell lines

Eight- to 10-week-old female BALB/c, C57BL/6-CD45.2, andBL6-CD45.1 were purchased from NCI/Frederick. H-2Kb

OVA257-264 TCR transgenic OT-I, Cd40�/� (purchased from TheJackson Laboratories), andNox2�/�mice (a kind gift from RobertMumford, NCI) were bred at NCI/Frederick. Bone marrow chi-meric mice were generated as previously described (27). Bonemarrow chimerism was confirmed 4 weeks after bone marrowtransplant and was above 80%. EL4 and B16 GM-CSF cells were akind gift of Dr. Drew Pardoll (The Johns Hopkins University,Baltimore, MD) and previously used (27). 4T1 cells were kindlyprovided by Christopher A. Klebanoff (NCI). RIL-175 hepatocel-lular carcinoma cell line was obtained from Dr. Lars Zender(University Hospital of T€ubingen, Germany) and used recently(13, 28). All tumor cell lines used were tested negative formycoplasma using a MycoAlert Plus kit (Lonza) routinely. Lasttest was performed on December 2014. Mice were injected sub-cutaneously in the flank with 1� 106 tumor cells. Tumor size wasmeasured twice a week. Metastatic tumors were established in theliver by intrasplenic injection of 3 � 105 EL4 cells (29). Micereceived antibody treatment 3 weeks after tumor cell inoculationinto the spleen. All mice were handled, fed, and housed inaccordance with the U.S. Department of Health and HumanServices institutional guidelines.

In vivo antibody treatmentTumor-free littermates or mice bearing subcutaneous tumors

between 10 and 15 mmmaximum diameter were inoculated i.p.with 100 mg of rat anti-mouse agonist CD40 antibody (cloneFGK-45; BioXCell) or irrelevant rat IgG2a (2A3; BioXCell). Micewere sacrificed 24 hours after injection. Alanine/aspartate ami-notransferase (ALT/AST) levels were determined inmouse sera bybiochemistry analysis in the Department of Laboratory Medicine(NCI). Serum TNFa levels were quantified by ELISA following themanufacturer's instructions (eBioscience). Hematoxylin–eosin-stained liver tissues were analyzed by a pathologist (D.E. Kleiner)in a blinded fashion.

Flow cytometry analysisLiver mononuclear cells were obtained as previously described

(13). Mouse cell samples were stained using antibodies from BDBiosciences and eBioscience (available upon request). Whenindicated, tumor-induced hepatic myeloid cells were isolatedusing CD11b beads followed by magnetically activated cell sep-aration (MACS) separation (Miltenyi Biotec). Purity after enrich-mentwas above 90%. Flow cytometrywas performedonBDFACSCalibur or LSRII using CellQuest Pro or FACS Diva acquisitionsoftware, respectively (Becton Dickinson). Data were analyzedusing FlowJo software (Tree Star).

Functional assays in vitroReactiveoxygen species (ROS)productionwas determinedusing

Carboxy-H2DCFDA (Invitrogen) as described by Corzo and col-leagues (30). DCFDA expression was quantified on gated mouseCD11bþGr-1þ cells from liver mononuclear cells 3 hours afterinjection of 100 mg of either isotype or anti-mouse CD40 antibody.In another setting, DCFDA expression was determined on gatedhuman CD14þHLA-DRhigh and CD14þHLA-DRlow cells after incu-bation of healthy donor peripheral bloodmononuclear cells in thepresence or absence of 0.1mg/mLmegaCD40L (Enzo Life Sciences)for 2 hours. For arginase activity and TNFa determination, hepaticCD11bþ cells were isolated from tumor-bearing mice and culturedovernight alone or in the presence of 0.1 mg anti-mouse CD40antibody. Supernatantswere collected and TNFawas quantified byELISA following the manufacturer's instructions (eBioscience).Arginase activity in cell lysates was determined as described (31).For ovalbumin (OVA) cross-presentation, 1 � 105 CD11bþ cellswere cultured for 24 hours alone or in the presence of 0.1 mg of ratanti-mouseCD40antibody. Cells werewashed twicewith PBS, andOT-I CD8þ T cells were MACS-sorted using a mouse CD8þ T cellisolation kit (Miltenyi Biotec), added to the culture in a 1:1 ratio,and stimulated with 0.1 mg/mL OVA-derived SIINFEKL peptideovernight. IFNg production by OT-I CD8þ T cells was determinedby intracellular staining.

Determination of hepatocyte cytotoxicity by hepaticCD11bþ cells

Luciferase-expressing RIL-175 hepatoma target cells were cul-tured at a 1:50 (target: effector) ratio with EL4-induced hepaticCD11bþ cells isolated frommice 3 hours after treatment with 100mg of either IgG or anti-mouse CD40. After 16 hours, the numberof surviving adherent cells was evaluated using Dual LuciferaseReporter Assay (Promega). H2O2 (2 mmol/L; Invitrogen) and100U/mL catalase (Sigma)were used for apoptosis induction andblocking of ROS release, respectively.

Adoptive cell transferHepatic CD45.1þCD11bþ cells were MACS-isolated from B16

GM-CSF tumor-bearing mice, because GM-CSF–expressingtumors have been shown to support the accumulation of largenumbers of CD11bþGr-1þ cells in spleen and liver (13). CD11bþ

cells (5 � 107) were injected into the tail vein of tumor-freeCD45.2þCd40�/� mice. In another set of experiments, 5 � 107

CD11bþ cells from B16 GM-CSF tumor-bearing wild-type (WT)or Nox2�/� mice were injected into the tail vein of tumor-freeCD45.2þCd40�/� mice. Mice were subsequently inoculated i.p.either with 100 mg of anti-mouse CD40 or isotype control. Micewere sacrificed 16 hours after antibody injection.

Human MDSC studiesPeripheral blood mononuclear cells (PBMC) were obtained

from NIH Blood Bank (healthy donors) and patients with gas-trointestinal-related cancer (see Supplementary Information).Written consent was obtained from all patients before bloodsampling on a research protocol approved by the NCI Institu-tional ReviewBoard. FACS-sortedCD14þHLA-DRhigh andCD14þ

HLA-DRlow cells were purified as previously described (32).Whenindicated, 1 � 105 or 2 � 105 sorted cells were cultured incomplete RPMI medium with or without 0.1 mg/mL megaCD40L(Enzo Life Sciences) or 5 mg/mL anti-human CD40 antibody(clone 82111; R&D systems) for 24 hours. Then, total RNA was

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isolatedusing anRNeasy kit (Qiagen). cDNAsynthesiswas carriedout using an iScript cDNA synthesis kit according to the manu-facturer's instructions (Biorad). Arginase-1 qPCR (primersequences available upon request) was performed using IQ SYBRGreen Supermix and thermal cycler (Applied Biosystems). Trip-licate reactionswere performed for each sample, and expressionoftested gene was normalized relative to levels of GAPDH. To assesshuman MDSC function, CD14þHLA-DRlow cells were isolatedfrom healthy donor PBMC and incubated at different ratios withCD3/CD28 (Miltenyi Biotec)-stimulated CD8þ T cells previouslyisolated from the remaining CD14� fraction (human CD8þ T cellisolation kit; Miltenyi Biotec) in the presence or absence of0.1 mg/mL megaCD40L. Proliferation was measured 72 hourslater by incorporation of 3H Thymidine (Amersham).

Statistical analysisAnalyses were performed with GraphPad Prism software. Data

are provided as mean � SEM, unless indicated otherwise. Forcomparisons between two groups, statistical analyses were per-formed using a Student t test, and P < 0.05 was consideredsignificant. For comparisons involving three groups or more, aone-way ANOVA using the Kruskal–Wallis test was performed(P < 0.05was considered statistically significant).When analyzingthe response of two populations to two or more treatments, two-way ANOVA was performed (P < 0.05 was considered statisticallysignificant). Symbols indicating statistical significance are asfollows (unless indicated otherwise): n.s., not statistically signif-icant; �, P < 0.05; ��, P < 0.01; ��, P < 0.005.

ResultsSystemic agonistic anti-CD40 induces immune-mediated acutehepatitis in tumor-bearing mice

We treated na€�ve tumor-free and EL4 tumor–bearing mice withagonistic anti-CD40 or isotype IgG control. Systemic CD40 anti-body treatment resulted in a significant increase of serum ALT andAST levels in tumor-bearing mice (ALT, 5,529 � 647 U/L; AST,6,687 � 1,166 U/L) compared with tumor-free mice (ALT, 528 �170 U/L; AST, 973 � 302 U/L) and with tumor-bearing micetreatedwith IgG (ALT, 122� 21U/L; AST, 465� 108U/L; Fig. 1A).Similarly, in BALB/cmice (Fig. 1B), transaminases were elevated in4T1 tumor-bearing mice treated with agonistic anti-CD40 (ALT,170� 20 U/L; AST, 415� 62 U/L) compared with tumor-bearingmice treatedwith isotype control (ALT, 57� 11U/L; AST, 220� 21U/L). Time course studies demonstrated a peak for ALT and ASTserum levels after 24hours in EL4 tumor-bearingmice and a returnto baseline within 72 hours (Supplementary Fig. S1A and S1B).Histologic analysis of liver sections from EL4 tumor-bearing micetreated with agonistic anti-CD40 antibody showed signs of severehepatitis with inflammatory cell infiltration and confluent paren-chymal necrosis (Fig. 1C and D). Similar results were obtained in4T1 tumor-bearing BALB/c littermates (Supplementary Fig. S1BandS1C). Furthermore, in amodel of EL4-induced livermetastases(Supplementary Fig. S2A), systemic anti-CD40 agonist increasedserum ALT and AST levels (ALT, 2,096 � 759 U/L; AST, 5,645 �1,320 U/L) compared with IgG-treated mice (ALT, 414 � 92 U/L;AST, 2,542 � 619 U/L; Supplementary Fig. S2B).

CD40 is not only expressed by liver-infiltrating immune cellsbut also by parenchymal cells such as hepatocytes (33). Therefore,we generated Cd40 knockdown bone marrow chimeras to disso-ciate the relative contribution of the liver immune cell compart-

ment to CD40 antibody–mediated liver damage from directligation of CD40 antibody on CD40-expressing hepatocytes.Transaminases were clearly elevated in EL4 tumor-bearingCd40�/� mice reconstituted with WT bone marrow upon CD40ligation (ALT, 437 � 62 U/L; AST, 1,207 � 230 U/L; Fig. 1E). Incontrast, tumor-bearing WT mice reconstituted with Cd40�/�

bone marrow cells showed lower transaminases (ALT, 175 �21U/L; AST, 827�199U/L). TNFa is a proinflammatory cytokineproduced upon liver damage (34). Consistently, elevated serumTNFa levels were only observed in Cd40�/� tumor-bearing miceafter transfer of WT bone marrow cells but not in WT tumor-bearingmice after transfer of bonemarrow fromCd40�/� counter-parts (Supplementary Fig. S1D).

Agonistic CD40 antibody causes ROS-mediated hepatitis intumor-bearing mice

ROS produced by mononuclear phagocytes are importantmediators of drug-induced hepatotoxicity (35). We studied therole of ROS upon anti-CD40 treatment in tumor-bearing miceusing phagocytic NADPH oxidase 2 (Nox2)-deficient mice. Lowertransaminases were observed in EL4 tumor-bearingNox2�/�mice(ALT, 973 � 474 U/L; AST, 1,700 � 678 U/L) than WT littermatecontrols upon agonistic anti-CD40 administration (ALT, 4,452�1,266 U/L; AST, 5,830 � 1,841 U/L; Fig. 2A). As expected, onlymoderate ALT elevations were found in tumor-bearing Nox2�/�

mice treated with agonistic anti-CD40, possibly due to the directeffect of the antibody on parenchymal cells. In addition, nodifferences in serum TNFa levels were observed between WT andNox2�/� tumor-bearing mice upon anti-CD40 agonist treatment(data not shown). Histologic studies revealedmilder immune cellinfiltrate, but still evidence of endothelial inflammation andinjury in agonistic CD40 antibody–treated Nox2�/� mice(Fig. 2B and C). Although the pattern of liver injury was similarin WT and Nox2�/� tumor-bearing mice, Nox2�/� mice showedless confluent parenchymal necrosis involving only 2% to 3% ofthe cross-sectional area. Furthermore, fewer fibrin thrombi wereobserved in the outflow veins. In line with this finding, systemicCD40 agonist treatment resulted in a significant increase of ROSproduction by hepatic CD11bþGr-1þ cells in tumor-bearingmicecompared with tumor-free mice and with tumor-bearing micetreated with control IgG (Fig. 2D). To find out the contribution ofhepaticmyeloid cells in hepatocyte cell death, we isolated hepaticCD11bþ cells from tumor-bearing mice 3 hours after treatmenteither with agonistic anti-CD40 or isotype control and coculturedthem with luciferase-expressing hepatoma cells. Using luciferaseexpression as readout of cell viability, luciferase signal wasdecreased when hepatoma cells were incubated with hepaticCD11bþ cells from agonistic anti–CD40-treated mice (Fig. 2E).CD11bþ-mediated cell death was blocked in the presence of theROS inhibitor catalase, suggesting that CD40 ligation exacerbatesROS-mediated liver cell killing by tumor-induced hepatic mye-loid cells. In summary, ROS release plays a role in systemic CD40agonist–mediated hepatotoxicity.

Agonistic CD40 antibody modulates tumor-induced hepaticCD11bþGr-1þ cells

Next, we studied CD11bþGr-1þ cells after agonistic anti-CD40treatment. We found that the absolute cell number of hepaticCD11bþGr-1þ cells increased inEL4 tumor-bearingmice24hoursafter agonistic anti-CD40 injection (tumor-free 4.4� 106� 1.4�106 vs. tumor-bearing 1.1� 107 � 1.9� 106, P¼ 0.052; Fig. 3A).

Agonistic Anti-CD40 Activates Tumor-Induced CD11bþGr-1þ Cells

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This increase was significantly higher in the CD11bþGr-1low

monocytic cell subset (M-MDSC) than in the CD11bþGr-1high

granulocytic subset (G-MDSC; Supplementary Fig. S3A and S3B).

Apart from CD11bþGr-1þ cell changes, a moderate decrease ofCD3�CD19þ B cells and an increase of CD11cþ dendritic cellswere observed upon agonistic CD40 antibody injection

Figure 1.Systemic agonistic anti-CD40 inducesimmune-mediated liver damage intumor-bearing (TB) mice. Tumor-free(TF) and tumor-bearingmice receivedi.p. either 100 mg agonistic CD40antibody (CD40 Ab) or control IgG(IgG). A, serum ALT and AST levels inC57BL/6 tumor-free and EL4 tumor-bearing mice (n ¼ 6–8 mice/group)were determined 24 hours afterantibody injection. Cumulative dataexpressed as mean � SEM,representative of three independentexperiments. B, serum ALT and ASTlevels in BALB/c tumor-free mice(n ¼ 4 mice/ group) and 4T1 tumor-bearingmice (n¼ 8mice/group)weredetermined 24 hours after antibodyinjection. Cumulative data from twoindependent experiments areexpressed as mean � SEM.Representative hematoxylin andeosinstaining of liver sections from EL4tumor-bearingmice 24 hours after IgG(C) or CD40 Ab injection (D). Imagesshow �20 magnification and yellowbar ¼ 0.2 mm. Tumor-bearing bonemarrow chimeric mice (donor !recipient) received i.p. either CD40Abor IgG (n ¼ 4 mice/group). SerumALT and AST (E) levels weremeasured 24 hours after antibodyinjection. Cumulative data expressedas mean� SEM, representative of twoindependent experiments. n.s.,not statistically significant;� , P < 0.05; ��, P < 0.01; ��, P < 0.005:Student t test, (E) one-way ANOVA.






(Supplementary Fig. S3C–S3G). Interestingly, agonistic anti-CD40 treatment significantly increased the absolute number ofCD40- andCD80-expressingCD11bþGr-1þ cells (SupplementaryFig. S4 and Fig. 3B and C). Similar results were observed in 4T1BALB/c tumor-bearing mice (Supplementary Fig. S3I and S3J). Toaddress whether the effects of agonistic anti-CD40 were restrictedto hepatic myeloid cells or whether this was a systemic effect, wealso studied splenic CD11bþGr-1þ cells in our system.An increasein the absolute numbers of CD11bþGr-1þ cells and more CD40-andCD80-expressingCD11bþGr-1þ cells was also seen in spleensof tumor-bearingmice treatedwith agonistic anti-CD40 (Fig. 3D–

F). We further analyzed MDSC subsets and found that bothhepatic G-MDSC and M-MDSC accumulated over time afteranti-CD40 agonist injection (Supplementary Fig. S5A and S5B).However, hepaticG-MDSCproducedmore ROS thanM-MDSC inresponse to CD40 ligation in vivo (Supplementary Fig. S5C).

Finally, we studied the effects of agonistic anti-CD40onhepaticCD11bþ cells in vitro. Here, a significant decrease in their arginaseactivity was seen upon anti-CD40 treatment (Fig. 3G). Moreover,anti-CD40 treatment improved their ability to induce antigen-specific IFNg release by CD8þ T cells (Fig. 3H). Finally, significantlevels of TNFawere detected in cell supernatants after incubationof tumor-induced hepatic myeloid cells with CD40 antibody(Fig. 3I). In summary, CD40 ligation on tumor-induced hepaticmyeloid cells results in enhanced maturation and activation invivo and in vitro.

Tumor-induced hepatic CD11b cells mediate liverinflammation upon systemic agonistic CD40 antibody

To provide a direct link between the presence of hepaticmyeloid cells and anti–CD40-mediated liver inflammation, wetransferred WT tumor-induced hepatic CD45.1þCD11bþ cells,

Figure 2.Agonistic CD40 antibody exacerbatesliver inflammation via oxidative stress.Tumor-bearing (TB) WT and Nox2�/�

mice (n¼4mice/group)were injectedeither with CD40 Ab or IgG. SerumALT and AST levels (A) weremeasured 24 hours after injection.Cumulative data shown as mean �SEM are representative of twoindependent experiments.Representative hematoxylin and eosinstaining of liver sections from WTtumor-bearing (B) or Nox2�/� tumor-bearing (C) 24 hours after systemicCD40 Ab injection. Images show�60 magnification and yellow bar ¼0.1 mm. D, tumor-free (TF) and EL4tumor-bearing mice received i.p.either 100 mg agonistic CD40 Ab orcontrol IgG (n ¼ 2 mice/group). Meanfluorescence intensity (MFI) of DCFDAgated on hepatic CD11bþGr-1þ cellswas used to quantify ROS production3 hours after treatment. Data shown asmean � SEM are representative ofthree independent experiments.E, luminescence intensity byluciferase-expressing RIL-175 cellscultured with or without hepaticCD11bþ cells derived from EL4 tumor-bearing mice 3 hours after injectionof either IgG or anti-CD40 agonist(n ¼ 2 mice/group). Catalase(100 U/mL) was used to block ROSproduction, and 2 mmol/L H2O2 wasset as positive control. Data areexpressed as mean � SEM,representative of two independentexperiments. n.s., not statisticallysignificant; � , P < 0.05; ��, P < 0.01;�� , P < 0.005; (A and D) Studentt test, (E) one-way ANOVA.






where 80% were CD11bþGr-1þ MDSC (Supplementary Fig. S6Aand S6B), into CD45.2þCd40�/�-na€�ve recipients followed bysystemic injection of the agonistic anti-CD40. Although agonisticCD40 antibody did not cause inflammation to regular Cd40�/�

na€�ve mice, transfer of tumor-induced hepatic CD45.1þ myeloidcells and subsequent agonistic anti-CD40 injection to the CD40knockouts resulted in ALT and AST serum elevation (Fig. 4A andB). In addition, an increase in TNFa serum levels was observed(Fig. 4C). Consequently, CD40 ligation on tumor-induced hepat-ic myeloid cells resulted in enhanced inflammation. To addressthe role of myeloid-derived ROS in this setting, we transferredeither WT or Nox2�/� tumor-induced hepatic CD11bþ cells into

Cd40�/� recipients and then challenged themwith agonistic anti-CD40. Transfer of WT tumor-induced hepatic myeloid cells andsubsequent agonistic anti-CD40 injection to the Cd40 knockoutsresulted in higher ALT serum levels compared with Nox2�/�

tumor-induced hepatic CD11bþ cell transfer (Fig. 4C).

CD40 ligation impairs immunosuppressive function of humanCD14þHLA-DRlow MDSC

We isolated human CD14þHLA-DRlow MDSC from PBMC ofhealthy controls (Fig. 5A) or patients (Fig. 5B) with gastrointes-tinal cancer (Supplementary Table S1). CD40 engagement usingmultivalent CD40L reduced arginase-1 mRNA expression in

Figure 3.Systemic anti-CD40 modulates tumor-induced suppressive myeloid cells. C57BL/6 tumor-free (TF) and EL4 tumor-bearing (TB) mice (n ¼ 4–8 mice/group)injected i.p. either with CD40 Ab or IgG. A–C, absolute number of hepatic CD11bþGr-1þ cells (A) expressing CD40 (B) and CD80 (C) 24 hours after injection.D–F, absolute number of splenic CD11bþGr-1þ cells (D) expressing CD40 (E) and CD80 (F). A–F, data expressed as mean � SEM are representative of threeindependent experiments. EL4-induced liver CD11bþ cells (1 � 106; n ¼ 3/mice) were cultured in the presence or absence of 0.1 mg CD40 Ab. Arginase activity (G)determinedby urea release in the cell lysateswasmeasuredwith a colorimetric assay 16 hours after incubation. H, EL4-induced liver CD11bþ cells (1� 105; n¼ 3/mice)were cultured in the presence or absence of 0.1 mg CD40 Ab. After 24 hours, cells were washed with PBS and incubated overnight with 1 � 105 sortedCD8þ OT-I OVA–specific T cells in the presence of 0.1 mg/mL OVA. Intracellular IFNg production by CD8þ T cells is shown. Data shown as mean � SEM arerepresentative of two independent experiments. EL4-induced liver CD11bþ cells (1 � 106; n¼ 3/mice) were cultured in the presence or absence of 0.1 mg CD40 Ab.I, TNFa from the cell culture supernatants was determined by ELISA. Data shown as mean � SEM are representative of two independent experiments. n.s., notstatistically significant; � , P < 0.05; �� , P < 0.01; �� , P < 0.005: (A–G, I) Student t test, (H) one-way ANOVA.






human CD14þHLA-DRlow MDSC in both healthy controls andpatients with cancer. This was not observed in CD14þHLA-DRhigh

controls (Fig. 5A and B). Similar results were obtained usingagonistic anti-human CD40 antibody (Supplementary Fig. S7).Next, we isolated CD14þHLA-DRlow cells from PBMC and testedtheir suppressor function after CD40L treatment. CD40 ligationimpaired the suppressor function of human CD14þHLA-DRlow

MDSC (Fig. 5C). Interestingly, incubation of PBMC in the pres-ence of CD40L resulted in enhanced ROS production by CD14þ

HLA-DRlow cells (Fig. 5D).

DiscussionThe approval of ipilimumab by the FDA in 2011, and very

recently pembrolizumab and nivolumab, has sparked great inter-est in immune checkpoint inhibitors in oncology in recent years(36). Similarly, agonistic antibodies to TNF receptor molecules,such as activating CD137, OX40, and CD40 antibodies, haveshown promising results in both preclinical and early clinicalsettings (5, 37, 38). Different mechanisms of action have beendescribed for agonistic anti-CD40 antibody therapy in cancer(37). Here, we studied the effect of systemic anti-CD40 treatmentin tumor-bearing mice on hepatic and splenic MDSC. We foundthat agonistic CD40 antibody triggered immune-mediated andROS-dependent acute liver damage in tumor-bearing mice by

activating hepatic CD11bþGr-1þ cells. Further studies providepreliminary evidence suggesting that agonistic anti-CD40 treat-ment causesmaturation and loss of suppressor functionof hepaticand systemic murine as well as human MDSC.

Various cells in the liver, including hepatocytes, Kupffer cells,and different myeloid cells, express CD40 (39). Previously, trans-aminitis had been reported in patients treated with agonistic anti-CD40 in a phase I trial (1). Similarly, liver toxicity upon agonisticanti-CD40 treatment has been observed in preclinical modelsupon intravenous administration, but this was reduced when theantibody was injected peritumorally (6). This adverse event wasinitially attributed to a direct effect of agonistic anti-CD40 onCD40-expressing hepatocytes (33). Our findings suggest a poten-tial alternative explanation, namely the engagement of hepaticCD11bþGr-1þ MDSC and ultimately ROS release leading tohepatocyte death upon anti-CD40 treatment. There is a prefer-ential accumulation of MDSC in the liver of tumor-bearing mice,which are in larger numbers than othermyeloid populations suchas Kupffer cells (13, 15, 17, 28). These cells express low levels ofCD40 (40–42), which can be further enhanced upon incubationwith IFNg (43) as well as in a setting of acute inflammation (datanot shown). Our data using bone marrow chimeric mice clearlysuggest a pivotal role for CD40-expressingmyeloid cells in CD40-mediated liver damage in tumor-bearing mice. First, serum trans-aminases and TNFa serum levels were higher in Cd40�/� mice

Figure 4.Tumor-induced myeloid cells enhanceliver inflammation in vivo uponsystemic agonistic anti-CD40treatment. B16 GM-CSF–induced WTliver CD45.1þCD11bþ cells (5 � 107;n ¼ 3 mice) were injected i.v. intotumor-free (TF) CD45.2þ Cd40�/�

mice (n ¼ 6–8 mice/group). Theneither CD40Ab or IgGwas injected i.p.Tumor-free WT and Cd40�/� micereceived CD40 Ab as control (n ¼ 3mice/group). Serum ALT and AST (A)levels as well as serum TNFa (B) weremeasured 16 hours after injection.A, cumulative data shown as mean �SEM are representative of twoindependent experiments. B, datashown as mean � SEM arerepresentative of two independentexperiments. C, 5 � 107 B16 GM-CSF–induced WT or Nox2�/� liver CD11bþ

cells (n¼ 2mice/group) were injectedi.v. into tumor-free CD45.2þ Cd40�/�

mice (n ¼ 6–7 mice/group). Theneither CD40Ab or IgGwas injected i.p.Tumor-free Cd40�/� mice receivedCD40 Ab as control (n ¼ 2 mice).Serum ALT levels were measured 16hours after injection. Cumulativedata shown as mean � SEM arerepresentative of two independentexperiments. n.s., not statisticallysignificant; � , P < 0.05: one-wayANOVA.






reconstituted with WT bone marrow than in WT mice afterreconstitution with Cd40�/� bone marrow. Second, adoptivetransfer of WT MDSC into Cd40�/� mice followed by agonisticanti-CD40 treatment resulted in increased transaminases andTNFa serum levels.

Oxidative stress via ROS release by mononuclear phagocytesplays a pivotal role in inflammatory liver injury (35). Treatmentof mice with free radical scavengers decreased ALT in a model ofimmune-mediated hepatitis (44). Using phagocytic Nox2-defi-cient mice to study the potential contribution of ROS (30)in agonistic CD40 antibody–mediated liver damage, weobserved that knockout tumor-bearing mice had lower ALT/AST levels than tumor-bearing littermate controls, suggesting aROS-mediated liver cell damage in our model. We providefurther biologic evidence by showing that tumor-inducedhepatic myeloid cells produce ROS upon CD40 ligation, whichin turn induces hepatocyte cell death.

Our data show that systemic administration of agonisticanti-CD40 increased the accumulation of CD11bþGr-1þ cells inthe liver of tumor-bearing mice. Our experiments did not addressa possible role for liver-infiltrating neutrophils, which accumulatein murinemodels of immune-mediated hepatitis bymarginationof neutrophils through sinusoids (45). Inflammatory neutrophilsexpress CD11b and high levels of Gr-1 similar to the granulocyticsubset of tumor-induced hepatic MDSC. However, we providemultiple lines of evidence suggesting that tumor-induced hepaticCD11bþGr-1þ cells and not inflammatory neutrophils wereresponsible for agonistic anti–CD40-mediated liver toxicity:(i) Transaminase levels were higher in tumor-bearing mice (in

which hepatic MDSC accumulate) than in na€�ve mice after ago-nistic anti-CD40 treatment; (ii) significant accumulationof CD11bþGr-1low monocytic-like MDSC rather than CD11bþ

Gr-1high granulocytic-like MDSC was observed in tumor-bearingmice upon agonistic CD40 antibody–driven liver toxicity; and(iii) transfer of tumor-induced hepaticmyeloid cells intoCd40�/�

mice caused ALT/AST elevation upon agonistic anti-CD40. In thisexperiment, only tumor-induced MDSC expressed CD40.

Our data suggest that tumor-induced hepatic and—to a lesserextent—splenic CD11bþGr-1þ cells increase CD40 and CD80surface marker expression upon agonist CD40 antibody treat-ment. This study, along with others, shows that tumor-inducedCD11bþGr-1þ MDSC express low levels of CD40 (2, 46) andCD80 (7, 47). Because CD40 ligation induces upregulation ofCD80 (10–12, 48), our results suggest that hepatic tumor-inducedCD11bþGr-1þ cells may mature and get activated by CD40ligation in vivo. However, in the absence of specific markers toclearly separate tumor-induced myeloid cells with suppressorfunction from other innate immune cells, which migrate to theliver in acute inflammatory settings (14, 16, 45), we could notformally prove the plasticity of hepatic MDSC.

CD14þHLA-DRlow MDSC accumulate in the peripheral bloodand tumors frompatients with different types of cancer, includingliver cancer (18–20, 49). Although an accumulation of CD11bþ

CCR2þ cells has beendescribed in sectionsobtained frompatientswith colorectal liver metastasis (22, 23, 26), it is not known so farwhether MDSC also accumulate in tumor-free livers of patientswith cancer. Therefore, at this point, one can only speculatewhether hepatic MDSC caused ALT/AST elevations in patients

Figure 5.CD40 ligation impairs suppressor function in human CD14þHLA-DRlow MDSC. A total of 1 � 105 sorted CD14þHLA-DRhigh or CD14þHLA-DRlow cells either fromhealthy controls (A) or patients with cancer (B) were incubated overnight in the presence or absence of human CD40L trimer. Results show the fold changeinduction in arginase-1 (ARG1) mRNA expression. Data expressed as mean � SEM are representative of two independent experiments. C, CD14þHLA-DRlow MDSCwere incubated at different ratios with CD8þ T cells isolated from the remaining CD14� fraction, stimulated with anti-CD3/CD28, and in the presence or absence ofhuman CD40L trimer for 48 hours. Proliferation was assessed by thymidine incorporation. Data expressed as mean � SEM are representative of twoindependent experiments. D, isolated PBMCs (1 � 107) from healthy donors (n ¼ 5) were cultured in complete RPMI medium in the presence or absence ofmegaCD40L for 2 hours. ROS production was quantified by measuring DCFDA mean fluorescence intensity (MFI) either on CD14þHLA-DRhigh or CD14þHLA-DRlow

cells. Cumulative data are representative of two independent experiments. n.s., not statistically significant; � , P < 0.05; �� , P < 0.01. (A–C) Student t test,(D) two-way ANOVA.






treated with agonistic anti-CD40, and further studies are clearlyneeded.

Tumor-induced mouse myeloid cells and human CD14þHLA-DRlow MDSC both express high arginase levels and can suppressT-cell function through an arginase-dependent mechanism(24, 50). Both our murine and human data clearly suggest thatagonistic anti-CD40 treatment may impair the suppressor func-tion of MDSC and would therefore represent a potential novelapproach to target MDSC as also recently suggested by others(25, 51).

Immune-related adverse effects in clinical trials have beenassociated with the systemic use of immune-modulatory drugs(5). Much of anti-CD40 treatment–dependent toxicity can beavoided by local delivery in a slow-release vehicle,maintaining itsantitumor effects in animal models (6, 9). However, it is not clearwhether such treatment would also induce MDSC maturation.Therefore, further studies are needed to investigate new admin-istration routes and compounds to stabilize andprolong the effectof immune-modulatory compounds with the aim to mitigateimmunotherapy-related toxicity and potentially target MDSC.

Overall, our data indicate that liver toxicity caused by systemicCD40 antibody in mice is due to activation of tumor-inducedhepatic CD11bþGr-1þ cells and provide preliminary evidencesuggesting a reprogramming of tumor-inducedmyeloid cells intoproinflammatory myeloid subsets without suppressor function.Finally, our studies not onlyprovide anovel potential explanation

for anti–CD40-induced hepatotoxicity observed in early clinicaltrials, but may also open new opportunities for the targeting ofimmunosuppressive MDSC in patients with cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Medina-Echeverz, F. Korangy, T.F. GretenDevelopment of methodology: J. Medina-Echeverz, N. HawkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Medina-Echeverz, C. MaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Medina-Echeverz, C. Ma, T. Eggert, D.E. Kleiner,T.F. GretenWriting, review, and/or revision of the manuscript: J. Medina-Echeverz,A. Duffy, T. Eggert, D.E. Kleiner, F. Korangy, T.F. GretenStudy supervision: J. Medina-Echeverz, T.F. Greten

Grant SupportThis work was supported by the Intramural Research Program of the NCI,

NIH.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 29, 2014; revised January 9, 2015; accepted January 22,2015; published OnlineFirst January 30, 2015.

References1. Vonderheide RH, Flaherty KT, Khalil M, Stumacher MS, Bajor DL, Hutnick

NA, et al. Clinical activity and immune modulation in cancer patientstreated with CP-870,893, a novel CD40 agonist monoclonal antibody.J Clin Oncol 2007;25:876–83.

2. Grewal IS, Flavell RA. CD40 and CD154 in cell-mediated immunity. AnnuRev Immunol 1998;16:111–35.

3. BeattyGL, TorigianDA,Chiorean EG, Saboury B, Brothers A, Alavi A, et al. Aphase I study of an agonist CD40 monoclonal antibody (CP-870,893) incombinationwith gemcitabine in patients with advanced pancreatic ductaladenocarcinoma. Clin Cancer Res 2013;19:6286–95.

4. Vonderheide RH, Burg JM, Mick R, Trosko JA, Li D, Shaik MN, et al. Phase Istudy of the CD40 agonist antibody CP-870,893 combined with carbo-platin and paclitaxel in patients with advanced solid tumors. Oncoimmu-nology 2013;2:e23033.

5. Gangadhar TC, Vonderheide RH. Mitigating the toxic effects of anticancerimmunotherapy. Nat Rev Clin Oncol 2014;11:91–9.

6. FransenMF, SluijterM,MorreauH, Arens R,Melief CJM. Local activation ofCD8 T cells and systemic tumor eradication without toxicity via slowrelease and local delivery of agonistic CD40 antibody. Clin Cancer Res2011;17:2270–80.

7. Lanzavecchia A. Immunology. Licence to kill. Nature 1998;393:413–4.8. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of

myeloid cells by tumours. Nat Rev Immunol 2012;12:253–68.9. Sandin LC,OrlovaA,GustafssonE, Ellmark P, TolmachevV, T€ottermanTH,

et al. Locally delivered CD40 agonist antibody accumulates in secondarylymphoid organs and eradicates experimental disseminated bladder can-cer. Cancer Immunol Res 2014;2:80–90.

10. French RR, Chan HT, Tutt AL, Glennie MJ. CD40 antibody evokes acytotoxic T-cell response that eradicates lymphoma and bypasses T-cellhelp. Nat Med 1999;5:548–53.

11. Diehl L, Boer den AT, Schoenberger SP, van der Voort EI, Schumacher TN,Melief CJ, et al. CD40 activation in vivo overcomes peptide-inducedperipheral cytotoxic T-lymphocyte tolerance and augments anti-tumorvaccine efficacy. Nat Med 1999;5:774–9.

12. Sotomayor EM, Borrello I, TubbE, Rattis FM, BienH, LuZ, et al. Conversionof tumor-specific CD4þ T-cell tolerance to T-cell priming through in vivoligation of CD40. Nat Med 1999;5:780–7.

13. Kapanadze T, Gamrekelashvili J, Ma C, Chan C, Zhao F, Hewitt S, et al.Regulation of accumulation and function of myeloid derived suppressorcells in different murine models of hepatocellular carcinoma. J Hepatol2013;59:1007–13.

14. Zhang M, Yao Z, Dubois S, Ju W, M€uller JR, Waldmann TA. Interleukin-15combined with an anti-CD40 antibody provides enhanced therapeuticefficacy for murine models of colon cancer. Proc Natl Acad Sci U S A 2009;106:7513–8.

15. ConnollyMK,Mallen-St Clair J, Bedrosian AS,Malhotra A, Vera V, IbrahimJ, et al. Distinct populations of metastases-enabling myeloid cells expandin the liver of mice harboring invasive and preinvasive intra-abdominaltumor. J Leukoc Biol 2010;87:713–25.

16. Weiss JM, Back TC, Scarzello AJ, Subleski JJ, Hall VL, Stauffer JK, et al.Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediatedmitigation of an immunosuppressive tumormicroenvironment.Proc Natl Acad Sci U S A 2009;106:19455–60.

17. Ilkovitch D, Lopez DM. The liver is a site for tumor-induced myeloid-derived suppressor cell accumulation and immunosuppression. CancerRes 2009;69:5514–21.

18. Hoechst B,Ormandy LA, BallmaierM, Lehner F, Kr€uger C,MannsMP, et al.A new population of myeloid-derived suppressor cells in hepatocellularcarcinoma patients induces CD4(þ)CD25(þ)Foxp3(þ) T cells. Gastroen-terology 2008;135:234–43.

19. Honeychurch J, Glennie MJ, Johnson PWM, Illidge TM. Anti-CD40mono-clonal antibody therapy in combinationwith irradiation results in aCD8T-cell-dependent immunity to B-cell lymphoma. Blood 2003;102:1449–57.

20. Duffy A, Zhao F, Haile L, Gamrekelashvili J, Fioravanti S, Ma C, et al.Comparative analysis of monocytic and granulocytic myeloid-derivedsuppressor cell subsets in patients with gastrointestinal malignancies.Cancer Immunol Immunother 2013;62:299–307.

21. Van den Eynden GG, Majeed AW, Illemann M, Vermeulen PB, Bird NC,Høyer-Hansen G, et al. The multifaceted role of the microenvironment inliver metastasis: biology and clinical implications. Cancer Res 2013;73:2031–43.

22. Nowak AK, Robinson BWS, Lake RA. Synergy between chemotherapy andimmunotherapy in the treatment of established murine solid tumors.Cancer Res 2003;63:4490–6.






23. Qu X, FelderMAR, Perez Horta Z, Sondel PM, Rakhmilevich AL. Antitumoreffects of anti-CD40/CpG immunotherapy combined with gemcitabine or5-fluorouracil chemotherapy in the B16 melanoma model. Int Immuno-pharmacol 2013;17:1141–7.

24. Jiang Q, Weiss JM, Back T, Chan T, Ortaldo JR, Guichard S, et al. mTORkinase inhibitor AZD8055 enhances the immunotherapeutic activity of anagonist CD40 antibody in cancer treatment. Cancer Res 2011;71:4074–84.

25. Scheidt von B, Leung PSK, Yong MCR, Zhang Y, Towne JE, Smyth MJ, et al.Combined anti-CD40 and anti-IL-23 monoclonal antibody therapyeffectively suppresses tumor growth and metastases. Cancer Res 2014;74:2412–21.

26. Zhao L, Lim SY, Gordon-Weeks AN, Tapmeier TT, Im JH, Cao Y, et al.Recruitment of a myeloid cell subset (CD11b/Gr1 mid) via CCL2/CCR2promotes the development of colorectal cancer liver metastasis. Hepatol-ogy 2013;57:829–39.

27. Medina-Echeverz J, Haile LA, Zhao F, Gamrekelashvili J, Ma C, M�etais J-Y,et al. IFN-g regulates survival and function of tumor-induced CD11bþGr-1(high)myeloid derived suppressor cells bymodulating the anti-apoptoticmolecule Bcl2a1. Eur J Immunol 2014;44:2457–67.

28. Eggert T, Medina-Echeverz J, Kapanadze T, Kruhlak MJ, Korangy F, GretenTF. Tumor induced hepatic myeloid derived suppressor cells can causemoderate liver damage. PLoS ONE 2014;9:e112717.

29. Xu R, Sun X, Tse L-Y, Li H, Chan P-C, Xu S, et al. Long-term expression ofangiostatin suppresses metastatic liver cancer in mice. Hepatology 2003;37:1451–60.

30. Corzo CA, Cotter MJ, Cheng P, Cheng F, Kusmartsev S, Sotomayor E, et al.Mechanism regulating reactive oxygen species in tumor-induced myeloid-derived suppressor cells. J Immunol 2009;182:5693–701.

31. Youn J-I, Collazo M, Shalova IN, Biswas SK, Gabrilovich DI. Characteri-zation of the nature of granulocytic myeloid-derived suppressor cells intumor-bearing mice. J Leukoc Biol 2012;91:167–81.

32. Hoechst B, Gamrekelashvili J, Manns MP, Greten TF, Korangy F. Plasticityof human Th17 cells and iTregs is orchestrated by different subsets ofmyeloid cells. Blood 2011;117:6532–41.

33. Afford SC, Randhawa S, Eliopoulos AG, Hubscher SG, Young LS, AdamsDH.CD40 activation induces apoptosis in cultured humanhepatocytes viainduction of cell surface fas ligand expression and amplifies fas-mediatedhepatocyte death during allograft rejection. J Exp Med 1999;189:441–6.

34. Schwabe RF, Brenner DA. Mechanisms of Liver Injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways. Am J PhysiolGastrointest Liver Physiol 2006;290:G583–9.

35. Jaeschke H. Reactive oxygen andmechanisms of inflammatory liver injury:Present concepts. J Gastroenterol Hepatol 2011;26 Suppl 1:173–9.

36. Pardoll DM. The blockade of immune checkpoints in cancer immuno-therapy. Nat Rev Cancer 2012;12:252–64.

37. Vonderheide RH, Glennie MJ. Agonistic CD40 antibodies and cancertherapy. Clin Cancer Res 2013;19:1035–43.

38. Melero I,Hirschhorn-CymermanD,Morales-KastresanaA, SanmamedMF,Wolchok JD. Agonist antibodies to TNFRmolecules that costimulate T andNK cells. Clin Cancer Res 2013;19:1044–53.

39. Zhou F, Ajuebor MN, Beck PL, Le T, Hogaboam CM, Swain MG. CD154-CD40 interactions drive hepatocyte apoptosis in murine fulminant hep-atitis. Hepatology 2005;42:372–80.

40. Yang R, Cai Z, Zhang Y, YutzyWH, Roby KF, Roden RBS. CD80 in immunesuppression by mouse ovarian carcinoma-associated Gr-1þCD11bþ mye-loid cells. Cancer Res 2006;66:6807–15.

41. Liu Y, Zeng B, Zhang Z, Zhang Y, Yang R. B7-H1 on myeloid-derivedsuppressor cells in immune suppression by a mouse model of ovariancancer. Clin Immunol 2008;129:471–81.

42. Ko HJ, Lee JM, Kim YJ, Kim YS, Lee KA, Kang CY. Immunosuppressivemyeloid-derived suppressor cells can be converted into immunogenicAPCs with the help of activated NKT cells: an alternative cell-basedantitumor vaccine. J Immunol 2009;182:1818–28.

43. Frasca L, Nasso M, Spensieri F, Fedele G, Palazzo R, Malavasi F, et al. IFN-gamma arms human dendritic cells to performmultiple effector functions.J Immunol 2008;180:1471–81.

44. Nakashima H, Kinoshita M, Nakashima M, Habu Y, Shono S, Uchida T,et al. Superoxide produced by Kupffer cells is an essential effector inconcanavalin A-induced hepatitis in mice. Hepatology 2008;48:1979–88.

45. Bonder CS, Ajuebor MN, Zbytnuik LD, Kubes P, Swain MG. Essential rolefor neutrophil recruitment to the liver in concanavalin A-induced hepatitis.J Immunol 2004;172:45–53.

46. Pan PY, Ma G, Weber KJ, Ozao-Choy J, Wang G, Yin B, et al. Immunestimulatory receptor CD40 is required for T-cell suppression and T regu-latory cell activation mediated by myeloid-derived suppressor cells incancer. Cancer Res 2010;70:99–108.

47. Youn J-I, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol 2008;181:5791–802.

48. Eshima K, Choi Y, Flavell RA. CD154-CD40-independent up-regulation ofB7-2 on splenic antigen-presenting cells and efficient T cell priming bystaphylococcal enterotoxin A. Int Immunol 2003;15:817–26.

49. Greten TF, Manns MP, Korangy F. Myeloid derived suppressor cells inhuman diseases. Int Immunopharmacol 2011;11:802–7.

50. Vasquez-Dunddel D, Pan F, ZengQ, GorbounovM, Albesiano E, Fu J, et al.STAT3 regulates arginase-I inmyeloid-derived suppressor cells from cancerpatients. J Clin Invest 2013;123:1580–9.

51. Liljenfeldt L, Dieterich LC, Dimberg A, Mangsbo SM, Loskog ASI. CD40Lgene therapy tilts the myeloid cell profile and promotes infiltration ofactivated T lymphocytes. Cancer Gene Ther 2014;21:95–102.






2015;3:557-566. Published OnlineFirst January 30, 2015.Cancer Immunol Res José Medina-Echeverz, Chi Ma, Austin G. Duffy, et al. Immune-Mediated Liver DamageModulates Hepatic Myeloid-Suppressive Cells and Causes Systemic Agonistic Anti-CD40 Treatment of Tumor-Bearing Mice

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Systemic Agonistic Anti-CD40 Treatment of Tumor-Bearing ...or Nox2 / mice were injected into the tail vein of tumor-free CD45.2þCd40 / mice. Mice were subsequently inoculated i.p