A Soluble Form of CD80 Enhances Antitumor Immunity by ...Mice Breeding stock for C57BL/6 and CD28-deﬁcient C57BL/6 (CD28 /) mice was obtained from The Jackson Laboratory. Mice were

Priority Brief

A Soluble Form of CD80 Enhances Antitumor Immunity byNeutralizing Programmed Death Ligand-1 andSimultaneously Providing Costimulation

Samuel T. Haile, Lucas A. Horn, and Suzanne Ostrand-Rosenberg

AbstractTumor cells use various methods of immunosuppression to overcome antitumor immunity. One such

method is that of programmed death ligand-1 (PD-L1 or B7-H1), which upon binding its receptor PD-1 onT cells triggers apoptotic death of the activated T cells. Overexpression of the costimulatory moleculeCD80 on PD-L1þ tumor cells, or inclusion of a soluble form of CD80 (CD80-Fc), maintains the activationof PD-1þ–activated T cells. Using T cells from CD28-deficient mice and antibodies to block CD28 onhuman T cells, we now report that a soluble form of CD80 mediates this effect by simultaneouslyneutralizing PD-1–PD-L1-mediated immunosuppression and by providing CD80–CD28 costimulation, andis more effective than antibodies to PD-L1 or PD-1 in maintaining IFNg production by PD-1þ activatedT cells. Therefore, soluble CD80 may be a more effective therapeutic than these checkpoint antibodies forfacilitating the development and maintenance of antitumor immunity because it has the dual functionsof preventing PD-L1–mediated immunosuppression and simultaneously delivering the second signal forT-cell activation. Cancer Immunol Res; 2(7); 610–5. �2014 AACR.

IntroductionProgrammed death ligand-1 (PD-L1), also known as B7

homolog 1 (B7-H1) or CD274, is expressed by many humanand mouse tumor cells either constitutively or in response toexposure to IFNg (1, 2). Expression of PD-L1 results in thesuppression of antitumor immunity throughmultiplemechan-isms. PD-L1 renders tumor cells resistant to CTL- and FasL-mediated lysis (3). It also induces apoptosis of activated T cellsby signaling through its receptor PD-1. PD-L1 also reversessignals through T cell–expressed CD80 to anergize T cells, andits expression promotes the induction and expansion of reg-ulatory T cells (Treg; refs. 1, 4–7). Some T and B cells, dendriticcells, Tregs, macrophages, and myeloid-derived suppressorcells may also express PD-L1 (8–10), and thus contribute tothe inhibition of antitumor immunity.

Human and mouse tumor cells modified to expressCD80 as an integral membrane protein prevent PD-L1 frombinding to its receptor PD-1 (11, 12). As a result, PD-1þ Tcells remain activated. Treatment with a fusion protein

consisting of the extracellular domains of CD80 fused tothe Fc region of human immunoglobulin G1 (IgG1; CD80-Fc) similarly maintains the viability of activated PD-1þ Tcells (12).

In addition to overcoming suppression by PD-L1, mem-brane-bound CD80 or CD80-Fc has the potential to costi-mulate T-cell activation via T cell–expressed CD28 (13). Wenow report that CD80-Fc maintains the activation of PD-1þ

T cells by simultaneously preventing PD-1–PD-L1 sup-pression and by providing costimulation through CD28, andis more effective than antibodies to PD-L1 or PD-1 formaintaining IFNg production by activated T cells. Thesefindings suggest the potential of CD80-Fc as a therapeuticagent to overcome immunosuppression and sustain antitu-mor immunity.

Materials and MethodsCell lines and transfections

Human cutaneous melanoma cell line C8161 was kindlyprovided byDr. Elisabeth Seftor (Children'sMemorial ResearchCenter, NorthwesternUniversity, Evanston, IL) in 2011 andwascultured as described previously (11). Because C8161 cells werenot obtained from a cell bank, they cannot be authenticated;however, the line has maintained a unique profile by shorttandem repeat (STR) analysis and is routinely tested forMycoplasma infection. C8161/CD80 transfectants were gener-ated andmaintained as described previously (11). Cell lines andprocedures with human materials were approved by theUniversity of Maryland Baltimore County (UMBC; Baltimore,MD) Institutional Review Board.

Authors' Affiliation: Department of Biological Sciences, University ofMaryland Baltimore County, Baltimore, Maryland

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

Corresponding Author: Suzanne Ostrand-Rosenberg, Department ofBiological Sciences, University of Maryland Baltimore County (UMBC),1000 Hilltop Circle, Baltimore, MD 21250. Phone: 410-455-2237; Fax: 410-455-3875; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0204

�2014 American Association for Cancer Research.

CancerImmunology

Research

Cancer Immunol Res; 2(7) July 2014610

on February 17, 2021. © 2014 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst April 2, 2014; DOI: 10.1158/2326-6066.CIR-13-0204

http://cancerimmunolres.aacrjournals.org/

MiceBreeding stock for C57BL/6 and CD28-deficient C57BL/6

(CD28�/�) mice was obtained from The Jackson Laboratory.Mice were bred and maintained in the UMBC animal facility.All animal procedures were approved by the UMBC Institu-tional Animal Care and Use Committee.

Antibodies, reagents, and flow cytometryMouse CD3-Pacific Blue (clone 17A2), mouse CD28-PE-Cy7

(clone E18), mouse PD-1-APC (clone RMP1–30), mouse PD-L1-PE (clone 10F.9G2), mouse CD152-APC (clone UC10-4B9), andlow endotoxin, azide-free human CD80 (clone 2D10) mono-clonal antibodies (mAb) were from BioLegend. Mouse CD69-FITC (clone H1.2F3) and functional grade mouse IgG1 (cloneP3.6.2.8.1) were from BD Biosciences and eBioscience, respec-tively. Anti-PD-L1 (clone 5H1) was provided by Dr. EugeneKwon (Mayo Clinic, Rochester, MN). Anti-human IgG1-AlexaFluor 488 and anti-mouse IgG-Alexa Fluor 647 polyclonalantibodies were from Invitrogen. Cells were stained for cellsurface expression and subjected to flow cytometry asdescribed previously (14, 15), and analyzed using a BeckmanCoulter CyAn ADP flow cytometer and Summit V4.3. Forinternal staining, cells were fixed with 2% paraformaldehydeand permeabilized before staining.

Human PBMC activationCryopreserved peripheral blood mononuclear cells (PBMC)

were obtained from healthy human donors as described pre-viously (16). PBMCs (6� 104) and tumor cells (50Gy-irradiated;3 � 104) were cocultured in 96-well plates as describedpreviously (11). HumanCD80-Fc, humanTROY-Fc (TNF recep-tor superfamily member) recombinant fusion proteins (R&DSystems), and antibodies to human PD-L1 (clone 29E.2A3;BioLegend) and human PD-1 (clone PD1.3.1.3; Miltenyi Biotec)were added to some wells at 10 mg/mL. Mouse IgG1 (cloneP3.6.2.8.1), and antibodies to human CD80 (clone 2D10) werefrom eBioscience and BioLegend, respectively. Human IFNgproduction was measured by ELISA on day 3 as describedpreviously (16).

CD28-Fc and PD-1-Fc bindingC8161/CD80 cells were incubated in the presence of 10

or 100 mg/mL anti-human CD80 (BioLegend) or mouse

Figure 1. CD80-mediated costimulation is not exclusively responsible formaintaining activated T cells in the presence of PD-L1þ human tumorcells. A, CD80-transfected tumor cells coexpress CD80 and PD-L1,and do not bind to PD-1. Human cutaneous melanoma C8161 andC8161/CD80 cells were stained for cell surface CD80 and PD-L1(mAb 5H1; top), or incubated with or without recombinant human PD-1-Fc followedbystainingwith anti-human IgG-AlexaFluor 488 (bottom) andanalyzed by flow cytometry. B, anti-CD80 mAbs block CD28-Fc

binding to CD80þ tumor cells. C8161/CD80 human tumor cells wereincubated with 10 or 100 mg/mL of CD80 blocking mAbs or IgG1 controlfollowed by incubation with recombinant human CD28-Fc, staining withanti-human IgG-Alexa Fluor 488, and analysis by flow cytometry. C,CD80 blockade diminishes, but does not eliminate the ability of CD80 tomaintain T-cell activation. PHA-activated PBMCs from healthy humandonors were cocultured with human C8161 or C8161/CD80 cells. Ten,50, or 100 mg/mL of CD80 blocking mAb or IgG1 control were included insome wells. IFNg production was measured by ELISA. Statisticalevaluation was performed by the Mann–Whitney test. D, human PBMCsfrom healthy donors were undepleted, CD8-depleted, CD4-depleted, orCD8- and CD4-depleted, and subsequently incubated with PHA for 72hours. IFNg productionwasmeasuredbyELISA.Data are from three, two,two, and two independent experiments for A, B, C, and D, respectively.Slightly modified versions of this experiment using five different tumorcells and different quantities of CD80-Fc have been done with PBMCsfrom six different healthy donors.

CD80 Costimulates and Prevents PD-L1 Immunosuppression

www.aacrjournals.org Cancer Immunol Res; 2(7) July 2014 611




IgG1 (eBioscience) mAbs, followed by incubation with afusion protein consisting of the extracellular region ofCD28 fused to the Fc region of human IgG1 (CD28-Fc;R&D Systems), and detected by anti-human IgG-AlexaFluor 488 (Invitrogen). C8161 and C8161/CD80 cells wereincubated in the presence of human PD-1-Fc, followed by

anti-human IgG-Alexa Fluor 488 (Invitrogen) as describedpreviously (11).

Mouse splenocyte activationSplenocytes fromC57BL/6 andCD28�/�micewere depleted

of red blood cells and cultured at 37�C, 5%CO2 in 96-well plates

Figure 2. T cell–expressed PD-L1suppresses T-cell activation andis overcome by CD80-Fc. A,activated CD4þ and CD8þ T cellsupregulate PD-1 and PD-L1.Human PBMCs from healthydonors were cultured for 72 hourswith or without PHA, subsequentlystained for CD3, CD4, CD8, andeither PD-1 or PD-L1 mAbs, andanalyzed by flow cytometry. B, Tcell–expressed PD-1 or PD-L1suppresses T-cell activation.Human PBMCs were activated asin A in the presence or absence of10 mg/mL IgG isotype control,TROY-Fc, PD-L1 blocking mAb,PD-1 blocking mAb, or CD80-Fc.IFNg production was measured byELISA. Data are from three and twoto three independent experimentsfor A andB, respectively. Statisticalevaluation was performed by one-way ANOVA followed by the TukeyHSD test. Similar results wereobtained in additional experimentsusing four other mAbs to PD-L1(MIH1, 5H1, MIH3, and 130021)and four other mAbs to PD-1(EH12.1, EH12.2H7, J116,and MIH4).

Haile et al.

Cancer Immunol Res; 2(7) July 2014 Cancer Immunology Research612




at 1� 105 cells per 200 mL per well with 20 ng/mL phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) plus 1 mg/wellionomycin (EMD Millipore) in the presence or absence ofmouse TROY-Fc or CD80-Fc (R&D Systems) at 1, 5, and 10mg/mL. IFNg production was measured on day 2 by ELISAaccording to the manufacturer's directions (BioLegend).

Statistical analysisSD, one-way ANOVA, and Tukey honestly significant differ-

ence (HSD) test were performed using Excel version 2010. TheMann–Whitney testwas performed using http://faculty.vassar.edu/lowry/VassarStats.html.

ResultsCD80-mediated costimulation is not exclusivelyresponsible for maintaining activated T cells in thepresence of PD-L1þ human tumor cells

Coexpression of membrane-bound CD80 by PD-L1þ tumorcells prevents the binding of PD-1 to tumor cell–expressed PD-L1 (Fig. 1A), suggesting that CD80may sustain T-cell activationby preventing PD-1–PD-L1–mediated immunosuppression. Inaddition to binding PD-L1, CD80 also functions as a costimu-latory molecule by binding to T cell–expressed CD28 (13). Wehave shown previously that PD-L1þ human and mouse tumorcells modified to express CD80 or treated with a solublerecombinant CD80 (CD80-Fc) facilitate the activation of Tcells (11), an effect that could be due to either blocking ofPD-1–PD-L1 interactions and/or CD80–CD28 costimulation.To test the latter possibility, PD-L1þ human melanoma C8161cells transfectedwithCD80 (C8161/CD80)were incubatedwith10 or 100 mg/mL anti-CD80 blocking mAb or IgG1 isotypecontrol followed by human CD28-Fc. Binding of CD28-Fc wasdetected by staining with fluorescently labeled goat anti-human IgG and analysis by flow cytometry (Fig. 1B). Pretreat-mentwith 10 or 100mg/mL anti-CD80mAbdecreased CD28-Fcbinding by 73% and 94%, respectively, whereas pretreatmentwith 10 or 100 mg/mL of control IgG mAb decreased thebinding by no more than 4%. Because treatment with 100mg/mL of anti-CD80 antibodies inhibited the binding of CD28,it is reasonable to expect that CD80 antibody–treated PD-L1þ

cells cannot bind to CD28 and costimulate.To determine whether CD80–CD28 costimulation was

exclusively responsible for maintaining T-cell activation in thepresence of PD-L1þ tumor cells, phytohemagglutinin (PHA)-activated human PBMCs were cocultured with PD-L1þ C8161or with C8161/CD80 cells in the presence or absence ofincreasing concentrations of either anti-CD80 blocking mAbor IgG1 isotype control antibody (Fig. 1C). T-cell activationwasmeasured by the production of IFNg . If CD80 only costimu-lates, then cultures of C8161/CD80 cells with anti-CD80 mAbswill display suppression similar to cultures containing CD80�

C8161 cells. C8161 cells suppressed the activation, whereasC8161/CD80 cells increased IFNg production. Inclusion of anti-CD80 mAb, but not IgG1 isotype control, at 10, 50, or 100 mg/mL, decreased the activation to levels similar to those ofactivated PBMCs alone, butmore than that of activated PBMCsplus C8161 cells. IFNg was produced by CD4þ and CD8þT cellsas depletion of these cells eliminated IFNg production (Fig.1D). Therefore, anti-CD80 mAbs partially decreased T-cellactivation by C8161/CD80 cells, suggesting that in the absenceof costimulation, CD80 maintains T-cell activation by prevent-ing PD-1–PD-L1–mediated suppression. This latter conclusionis supported by our finding that CD80þ PD-L1þ tumor cells donot bind to PD-1 (Fig. 1A).

CD80-Fc overcomes suppression by PD-L1þ PBMCs,including PD-L1–expressing activated CD4þ and CD8þ

T cellsIn addition to tumor cells, activated CD4þ and CD8þ T cells

as well as non-CD3þ PBMCs express PD-1 and PD-L1 (Fig. 2A),

Figure3. MurineTcells activatedwithPMAplus ionomycin expressCD69,PD-1, PD-L1, and CTLA-4. Splenocytes from wild-type and CD28�/�

C57BL/6 mice were activated for 48 to 72 hours with PMA plusionomycin, subsequently surface-stained with CD3 and either CD28,CD69, PD-1, PD-L1, or CTLA-4 mAbs, and analyzed by flow cytometry.Splenocytes were also fixed and permeabilized and stained forintracellular CTLA-4. Data are from one of three (CD69, PD-1, and PD-L1)or two (CTLA-4) independent experiments.






and may contribute to suppression. To test this possibility,human PBMCs were activated for 72 hours with PHA in thepresence of anti-PD-L1 mAb (clone 29E.2A3), anti-PD-1 mAb(PD1.3.1.3), or an isotype control (Fig. 2B). IFNg productionwasmeasured by ELISA. Antibodies to both PD-L1 and PD-1increased the activation relative to the untreated or IgGcontrols, confirming that T cell–expressed PD-L1 suppressesactivation. We have shown previously that a soluble form ofCD80 (CD80-Fc) is equally effective as membrane-bound CD80inmaintaining the activation of bothmouse and human T cells(12), and therefore included CD80-Fc and a negative controlrecombinant protein (TROY-Fc) in this experiment. CD80-Fcenhanced activation more efficiently than treatment witheither antibody (Fig. 2B). Experiments with four other anti-PD-L1 mAbs (MIH1, 5H1, MIH3, and 130021) and four otheranti-PD-1 mAbs (EH12.1, EH12.2H7, J116, and MIH4) gavesimilar results. Therefore, expression of PD-L1 by activatedT cells and other PBMCs also reduces T-cell activation, and T-cell production of IFNg is more effectively enhanced by CD80-Fc than by antibodies to PD-L1 or PD-1.

CD80 increases T-cell activation independent ofcostimulation

The experiments depicted in Figs. 1 and 2 demonstrated thatCD80, either as a membrane-bound protein or as a solublerecombinant protein, contributes to maintaining T-cell acti-vation, and that its effect is partially due to the costimulationvia CD28. To test the function of CD80 in the absence ofcostimulation, wild-type and CD28�/� C57BL/6 mouse sple-nocytes were treated with PMA plus ionomycin for 48 hours,

and the resulting T cells were analyzed by flow cytometry forthe activationmarker CD69 and for the expression of PD-1 andPD-L1. Activated splenocytes were also tested for CTLA-4expression because CD80 can cause anergy/apoptosis by bind-ing to T cell–expressed CTLA-4 (17). Because cell surfaceCTLA-4 has a short half-life, both intracellular and cell surfaceCTLA-4 were measured (Fig. 3 and Supplementary Fig. S1).Both wild-type and CD28�/� T cells displayed increased CD69and PD-1 expression following treatment with PMA plusionomycin, demonstrating that both cell populations wereactivated. Mouse T cells also upregulated PD-L1 after activa-tion, indicating that activated mouse T cells, like activatedhuman T cells, may also contribute to PD-L1–mediated immu-nosuppression. CTLA-4 was also expressed by activatedmouseT cells.

To test whether murine T cell–expressed PD-L1 decreasedT-cell activation, splenocytes from C57BL/6 wild-type andCD28�/� mice were activated with PMA plus ionomycin andcultured for 48 hours with either CD80-Fc or control TROY-Fc.T-cell activation was assessed by measuring IFNg production(Fig. 4A). If CD80-Fc prevents suppression by blocking PD-1–PD-L1 interactions, then CD80-Fc will increase IFNg produc-tion by CD28�/� T cells. However, if CD80-Fc only increases T-cell activation by costimulation, then there will be no effectwhen CD80-Fc is added to the CD28�/� T cells. Inclusion ofCD80-Fc, but not TROY-Fc, increased IFNg production by bothwild-type and CD28�/� T cells, and the dose-dependentincrease was most dramatic for the CD28�/� T cells (Fig. 4Aand B). IFNg production was increased by CD80-Fc despite theexpression of CTLA-4 on activatedT cells. Thisfinding suggests

Figure 4. CD80-Fc increases T-cellactivation independent ofcostimulation. A, PMA plusionomycin-activated splenocytesfrom wild-type and CD28�/�

C57BL/6 mice were cultured for 48hours in thepresenceor absenceof10 mg/mL of mouse CD80-Fc orTROY-Fc. IFNg production wasmeasured by ELISA. Statisticalevaluation was performed by one-way ANOVA followed by the TukeyHSD test. B, murine wild-type andCD28�/� splenocytes wereactivated as in A in the presence orabsence of increasing quantities ofCD80-Fc or TROY-Fc. IFNgproduction was measured byELISA. Data are from one of twoindependent experiments.Statistical evaluation wasperformed by the Mann–Whitneytest.

Haile et al.

Cancer Immunol Res; 2(7) July 2014 Cancer Immunology Research614




that CD80-Fc is not inducing T-cell anergy/apoptosis viaCTLA-4 or that such interactions are minimal. Collectively,these results demonstrate that, similar to the effects of CD80on human T cells, CD80 also sustains the activation of murineT cells through a costimulation-independent mechanism.

DiscussionPreviously, we have demonstrated that treatment of PD-L1þ

tumor cells with a soluble form of the extracellular domains ofCD80 or coexpression of membrane-bound CD80 maintainsthe activation of both human and mouse T cells (11, 12). Inthese previous studies, it was unclear if and how much of theeffect of CD80 was due to its ability to costimulate throughCD28 versus its ability to prevent PD-1–PD-L1–mediatedimmune evasion. The present report demonstrates that bothmechanisms contribute to the ability of CD80-Fc to maintainIFNg production by mouse and human PD-1þ T cells in thepresence of PD-L1þ cells. As recent clinical trials have dem-onstrated, PD-L1–mediated suppression is a potent mecha-nism used by many tumor cells to evade antitumor immunity,and antibody blocking of PD-L1 or PD-1 significantly increasespatient responses and survival times (18, 19). In the presentstudies, CD80-Fc was more effective than antibodies to PD-L1or PD-1 at maintaining T-cell production of IFNg . Thisenhanced activity may be because of the dual action ofCD80-Fc in simultaneously blocking PD-1–PD-L1 suppression

and providing costimulation and, therefore, CD80-Fc may be amore effective therapeutic than mAbs to PD-L1 and/or PD-1for restoring and maintaining antitumor immunity in patientswith cancer.

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

Authors' ContributionsConception and design: S.T. Haile, S. Ostrand-RosenbergDevelopment of methodology: S.T. HaileAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.T. Haile, L.A. Horn, S. Ostrand-RosenbergAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.T. Haile, L.A. Horn, S. Ostrand-RosenbergWriting, review, and/or revision of the manuscript: S.T. Haile, L.A. Horn,S. Ostrand-RosenbergAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.T. HaileStudy supervision: S. Ostrand-Rosenberg

Grant SupportThis study was financially supported by NIH RO1 CA84232 and CA115880 (to

S. Ostrand-Rosenberg). L.A. Horn was partially supported by GAANN P200A12–17 and S.T. Haile by NIH R25GM55036

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 14, 2013; revised February 28, 2014; accepted March 19,2014; published OnlineFirst April 2, 2014.

References1. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al.

Tumor-associated B7-H1 promotes T-cell apoptosis: a potentialmechanism of immune evasion. Nat Med 2002;8:793–800.

2. Lee SJ, Jang BC, Lee SW, Yang YI, Suh SI, Park YM, et al. Interferonregulatory factor-1 is prerequisite to the constitutive expression andIFN-gamma-induced upregulation of B7-H1 (CD274). FEBS Lett2006;580:755–62.

3. Azuma T, Yao S, Zhu G, Flies AS, Flies SJ, Chen L. B7-H1 is aubiquitous antiapoptotic receptor on cancer cells. Blood 2008;111:3635–43.

4. Latchman YE, Liang SC,Wu Y, Chernova T, Sobel RA, KlemmM, et al.PD-L1–deficient mice show that PD-L1 on T cells, antigen-presentingcells, and host tissues negatively regulates T cells. Proc Natl Acad SciU S A 2004;101:10691–6.

5. Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ. Pro-grammed death-1 ligand 1 interacts specifically with the B7–1 costi-mulatory molecule to inhibit T cell responses. Immunity 2007;27:111–22.

6. Park JJ, Omiya R, Matsumura Y, Sakoda Y, Kuramasu A, AugustineMM, et al. B7-H1/CD80 interaction is required for the induction andmaintenance of peripheral T-cell tolerance. Blood 2010;116:1291–8.

7. Francisco LM, Salinas VH, Brown KE, Vanguri VK, Freeman GJ,Kuchroo VK, et al. PD-L1 regulates the development, maintenance,and function of induced regulatory T cells. The J Exp Med 2009;206:3015–29.

8. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, Tanno Y, et al.Expression of programmeddeath 1 ligands bymurine T cells and APC.J Immunol 2002;169:5538–45.

9. 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.

10. Lin PY, Sun L, Thibodeaux SR, Ludwig SM, Vadlamudi RK, Hurez VJ,et al. B7-H1–dependent sex-related differences in tumor immunity andimmunotherapy responses. J Immunol 2010;185:2747–53.

11. HaileST,Bosch JJ, AguNI, ZeenderAM,SomasundaramP,SrivastavaMK, et al. Tumor cell programmed death ligand 1-mediated T cellsuppression is overcome by coexpression of CD80. J Immunol 2011;186:6822–9.

12. Haile ST, Dalal SP, Clements V, Tamada K, Ostrand-Rosenberg S.Soluble CD80 restores T cell activation and overcomes tumor cellprogrammed death ligand 1–mediated immune suppression. J Immu-nol 2013;191:2829–36.

13. JenkinsM, Taylor P, Norton S, Urdahl K. CD28 delivers a costimulatorysignal involved in antigen-specific IL-2 production by human T cells.J Immunol 1991;147:2461–7.

14. Dissanayake SK, Thompson JA, Bosch JJ, Clements VK, Chen PW,Ksander BR, et al. Activation of tumor-specific CD4(þ) T lympho-cytes by major histocompatibility complex class II tumor cell vac-cines: a novel cell-based immunotherapy. Cancer Res 2004;64:1867–74.

15. Thompson JA, Srivastava MK, Bosch JJ, Clements VK, Ksander BR,Ostrand-Rosenberg S. The absence of invariant chain inMHC II cancervaccines enhances the activation of tumor-reactive type 1 CD4þ Tlymphocytes. Cancer Immunol Immunother 2008;57:389–98.

16. Bosch JJ, Thompson JA, Srivastava MK, Iheagwara UK, Murray TG,Lotem M, et al. MHC class II–ransduced tumor cells originating in theimmune-privileged eye prime and boost CD4(þ) T lymphocytes thatcross-react with primary andmetastatic uvealmelanoma cells. CancerRes 2007;67:4499–506.

17. Walunas TL, LenschowDJ,BakkerCY, Linsley PS, FreemanGJ,GreenJM, et al. CTLA-4 can function as a negative regulator of T cellactivation. Immunity 1994;1:405–13.

18. Brahmer JR, Tykodi SS, Chow LQ, HwuWJ, Topalian SL, Hwu P, et al.Safety and activity of anti-PD-L1 antibody in patients with advancedcancer. N Engl J Med 2012;366:2455–65.

19. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDer-mott DF, et al. Safety, activity, and immune correlates of anti-PD-1antibody in cancer. N Engl J Med 2012;366:2443–54.






2014;2:610-615. Published OnlineFirst April 2, 2014.Cancer Immunol Res Samuel T. Haile, Lucas A. Horn and Suzanne Ostrand-Rosenberg Providing CostimulationNeutralizing Programmed Death Ligand-1 and Simultaneously A Soluble Form of CD80 Enhances Antitumor Immunity by

Updated version

10.1158/2326-6066.CIR-13-0204doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerimmunolres.aacrjournals.org/content/suppl/2014/04/03/2326-6066.CIR-13-0204.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerimmunolres.aacrjournals.org/content/2/7/610.full#ref-list-1

This article cites 19 articles, 10 of which you can access for free at:

Citing articles

http://cancerimmunolres.aacrjournals.org/content/2/7/610.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerimmunolres.aacrjournals.org/content/2/7/610To request permission to re-use all or part of this article, use this link



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-13-0204

http://cancerimmunolres.aacrjournals.org/content/suppl/2014/04/03/2326-6066.CIR-13-0204.DC1

http://cancerimmunolres.aacrjournals.org/content/2/7/610.full#ref-list-1

http://cancerimmunolres.aacrjournals.org/content/2/7/610.full#related-urls

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/content/2/7/610


Documents

A Soluble Form of CD80 Enhances Antitumor Immunity by ...Mice Breeding stock for C57BL/6 and CD28-deﬁcient C57BL/6 (CD28 /) mice was obtained from The Jackson Laboratory. Mice were