By Coexpression of CD80 1-Mediated T Cell Suppression Is Overcome

Tumor Cell Programmed Death Ligand 1-Mediated T Cell Suppression Is Overcome by Coexpression of CD80

This information is current as Samuel T. Haile, Jacobus J. Bosch, Nnenna I. Agu, Annette of September 29, 2021. M. Zeender, Preethi Somasundaram, Minu K. Srivastava, Sabine Britting, Julie B. Wolf, Bruce R. Ksander and Suzanne Ostrand-Rosenberg J Immunol 2011; 186:6822-6829; Prepublished online 9 May

2011; Downloaded from doi: 10.4049/jimmunol.1003682 http://www.jimmunol.org/content/186/12/6822

Supplementary http://www.jimmunol.org/content/suppl/2011/05/09/jimmunol.100368 http://www.jimmunol.org/ Material 2.DC1 References This article cites 51 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/186/12/6822.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Tumor Cell Programmed Death Ligand 1-Mediated T Cell Suppression Is Overcome by Coexpression of CD80

Samuel T. Haile,*,1 Jacobus J. Bosch,*,†,1 Nnenna I. Agu,* Annette M. Zeender,* Preethi Somasundaram,* Minu K. Srivastava,* Sabine Britting,† Julie B. Wolf,* Bruce R. Ksander,‡,x and Suzanne Ostrand-Rosenberg*

Programmed death ligand 1 (PDL1, or B7-H1) is expressed constitutively or is induced by IFN-g on the cell surface of most human cancer cells and acts as a “molecular shield” by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma), we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1-mediated immune suppression by tumor cells and restores T cell activation. Mechanistically, CD80 mediates its effects through its extracellular domain, which blocks the cell surface expression of PDL1 but does not prevent intracellular Downloaded from expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating antitumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression. The Journal of Immunology, 2011, 186: 6822– 6829. umor-induced immune suppression is a major obstacle mor cell-expressed PDL1 (8). PDL1 may also tolerize tumor-

for therapies aimed at activating an individual’s immune reactive T cells by reverse signaling through CD80 on T cells, http://www.jimmunol.org/ T system to eliminate autologous cancer cells. This immune as has been shown in in vitro T cell activation systems (9) and suppression can be mediated by nonmalignant host cells such as T in vivo in an oral tolerance system (10). Regardless of the regulatory cells (1) and myeloid-derived suppressor cells (2) that mechanism by which tumor cell-expressed PDL1 promotes tumor are driven by tumor-secreted factors, as well as by malignant cells growth, blocking PDL1–PD1 interactions with anti-PDL1 or PD1 that express immune inhibitory molecules. Programmed death li- Abs improves activation of tumor-reactive T cells and reduces gand 1 (PDL1, also known as B7 homolog 1 [B7-H1] or CD274) tumor progression (4, 11–13), confirming that tumor cell-expressed is such an inhibitory molecule that is either induced or constitu- PDL1 is a major obstacle for cancer immunotherapies. tively expressed by many malignant cells (3–6). Tumor cell- Many cancer immunotherapies are designed to stimulate the expressed PDL1 facilitates tumor progression by at least two production of cytokines and chemokines that mobilize tumor- by guest on September 29, 2021 mechanisms: (i) tumor-reactive T cells are tolerized by PDL1 reactive, cell-mediated effector cells such as T lymphocytes, NK binding to its receptor programmed death 1 (PD1; CD279) on cells, and M1 macrophages (14–17). A key cytokine in this process T cells (3, 7); and (ii) tumor cells are rendered resistant to T cell- is IFN-g, which polarizes macrophages toward an M1 phenotype mediated and FasL-mediated lysis by PD1 signaling through tu- (18), activates NK cells (19), and facilitates the development of type 1 helper CD4+ T cells and cytotoxic CD8+ T cells (20). However, IFN-g is also a potent inducer of PDL1 (21). Therefore, *Department of Biological Sciences, University of Maryland Baltimore County, cancer immunotherapies that optimally activate desirable effector Baltimore, MD 21250; †Department of Medicine 5, Hematology/Oncology, Univer- sity of Erlangen-Nu¨rnberg, D-91054 Erlangen, Germany; ‡Schepens Eye Research cells may concomitantly upregulate tumor cell expression of Institute, Boston, MA 02114; and xDepartment of Ophthalmology, Harvard Medical PDL1 and thereby limit their own effectiveness. School, Boston, MA 02115 We have generated tumor cell-based cancer vaccines that induce 1S.T.H. and J.J.B. contributed equally to this work. high levels of IFN-g and are potent activators of tumor-reactive Received for publication November 8, 2010. Accepted for publication April 5, 2011. T cells. Our “MHC class II” vaccines were designed to activate + This work was supported by National Institutes of Health Grants R01CA115880, tumor-reactive type 1 CD4 Th cells and consist of tumor cells R01CA84232 (to S.O.-R.), and R01EY016486 (to B.R.K.). S.T.H. was partially transfected/transduced with genes encoding the costimulatory mol- supported by a Graduate Assistance in Areas of National Need predoctoral fellowship and by the National Institutes of Health (T32 GM066706 and R25-GM55036). J.J.B. ecule CD80 and syngeneic HLA-DR molecules. In vitro studies was partially supported by Rotterdamse Vereniging Blindenbelangen, Stichting with three human solid tumor cell lines demonstrated that the Blindenhulp, Stichting Blinden-Penning, Stichting Dondersfonds, Stichting Nelly MHC class II (MHC II) vaccines are potent activators of a diverse Reef Fund, Gratama Stichting, Stichting Admiraal van Kinsbergen Fonds, and Foun- + dation “De Drie Lichten” fellowships and by Deutsche Forschungsgemeinschaft DFG- repertoire of tumor-reactive and tumor-specific CD4 T cells SFB643 (C8). (22–25), and in vivo studies with three mouse tumor systems Address correspondence and reprint requests to Dr. Suzanne Ostrand-Rosenberg, confirmed that the vaccines prolong survival of mice with estab- Department of Biological Sciences, University of Maryland Baltimore County, lished primary and/or metastatic disease (26). In contrast to ac- 1000 Hilltop Circle, Baltimore, MD 21250. E-mail address: [email protected] cepted dogma (27, 28), T cell activation and tumor rejection The online version of this article contains supplemental material. required vaccine cell expression of CD80 in the boost stage, Abbreviations used in this article: CD80DR, CD80tr with the 14 terminal aa of HLA- DRa–chain at its C-terminal end; CD80tr, CD80 truncated for its C-terminal 19 aa; suggesting that CD80 was playing a role other than as a costimu- hPD1–Fc, human PD1–Fc fusion protein; mDC, mature dendritic cell; MHC II, MHC latory molecule to prime naive T cells. We now report that CD80 class II; miRNA-513, microRNA-513; mPD1–Fc, mouse PD1–Fc fusion protein; PD1, also prevents constitutive and IFN-g–induced tumor cell expres- programmed death 1; PDL1, programmed death ligand 1; qPCR, quantitative PCR. sion of PDL1 and thereby facilitates tumor immunity by inhibiting Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 PDL1-mediated immune suppression. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003682 The Journal of Immunology 6823

Materials and Methods amplified using a one-step RT-PCR kit (Qiagen). PDL1 forward primer, Cell lines 59-AAACAATTAGACCTGGCTG-39, and PDL1 reverse primer, 59-TC- TTACCACTCAGGACTTG-39. cDNA was amplified in a PTC-200 Peltier Primary uveal melanoma cell lines MEL202 and MEL270, metastatic Thermal Cycler (MJ Research) under the following conditions: reverse uveal melanoma line OMM2.3 (29), and cutaneous melanoma cell lines transcription for 30 min at 50˚C, denature for 15 min at 95˚C, 40 cycles of MEL1011 (30), C8161 (31), and 624MEL (32) were obtained from the 94˚C for 30 s, 50˚C for 30 s, 72˚C for 1 min, followed by a final extension cited sources except for 624MEL and C8161, which were obtained from at 72˚C for 10 min. PCR products were analyzed on a 1.0% agarose gel F. Marincola (National Cancer Institute, National Institutes of Health) and stained with ethidium bromide (BioRad, Hercules, CA). E. Seftor (Children’s Memorial Research Center, Chicago, IL), respecti- For quantitative PCR (qPCR), 1.0 mg RNA/sample was amplified using vely. These lines were cultured in RPMI 1640 (BioSource, Rockville, MD) the Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent supplemented with 10% heat-inactivated FCS (Hyclone, Logan, UT), 10 Technologies, Santa Clara, CA) in a Step One Real Time PCR System mM HEPES (Invitrogen, Grand Island, NY), 1% penicillin/streptomycin (Applied Biosystems, Foster City, CA). PDL1 forward primer, 59-TA- (BioSource, Rockville, MD), 1% 2-mercaptoethanol (VWR, West Chester, TGGTGGTGCCGACTACAA-39, and PDL1 reverse primer, 59-TGGCT- PA), 2 mM Glutamax (BRL/Life Sciences, Grand Island, NY), 0.1% CCCAGAATTACCAAG-39; 18S forward primer, 59-ACCGATTGGATG- gentamicin (BioSource), and 5 mg/ml Prophylactic Plasmocin (InvivoGen, GTTTAGTGAG-39, and 18S reverse primer, 59-CCTACGGAAACCTTG- San Diego, CA). MCF10CA1 (hereafter called MCF10) (33) mammary TTACGAC-39 (Metabion, Martinsried, Germany). Samples were dena- carcinoma and bronchioloalveolar adenocarcinoma H358 (34) cell lines tured for 15 min at 95˚C, subsequently 40 cycles including denaturation at were obtained from the American Type Culture Collection and cultured as 95˚C for 5 s and annealing at 60˚C for 15 s, followed by continuous described (25, 35) except 5 mg/ml Prophylactic Plasmocin was included in fluorescence measurement during heating from 60˚C to 90˚C (0.1˚C/s). the media. Human mature dendritic cells (mDCs) were generated as de- 18S to PDL1 signal ratios are presented. scribed (36) from PBMCs derived from apheresis of healthy donors, except media was supplemented with 10% human AB serum instead of autolo- Western blots gous serum. Cell lines and procedures with human materials were ap- Western blots were performed as described (22) with the following mod- Downloaded from proved by the institutional review boards of the participating institutions. ifications. Cell lysates were resuspended in sample buffer without 2-mercaptoethanol. After electrophoresis on 10% SDS-PAGE gels, proteins were IFN-g treatment transferred to polyvinylidene difluoride membranes (Amersham, Piscat- Cells (3 3 105/8 ml/T25 flask) were incubated at 37˚C for 48 h in their away, NJ) using a Bio-Rad PowerPac HC (100 V for 70 min) and blocked culture medium supplemented with 50–100 U/ml recombinant human IFN- with 4% nonfat dry milk in TBST. PDL1 and actin were detected using 1 g (Pierce Biotechnology, Rockford, IL) and then washed with excess mg/ml PDL1 mAb (clone MIH1; eBioscience) and 0.05 mg/ml b-actin culture medium. mAb, respectively, followed by 1:5000 dilution of goat anti-mouse HRP http://www.jimmunol.org/ (BD Biosciences). Plasmids, transfections, and transfectants PBMC activation The human CD80 molecule (accession no. P33681; www.uniprot.org/uniprot/ P33681) contains 288 aa of which the C-terminal ∼21 aa constitute the PBMCs were obtained and then primed and boosted with vaccine cells as cytoplasmic domain (37–39) (structure prediction programs at http://ca. described (22, 23) except they were expanded for 7 d with IL-15 (20 ng/ml; 3 5 expasy.org/tools/). The CD80tr construct was generated from the pLHCX/ PeproTech, Rocky Hill, NJ). Alternatively, PBMCs (1 10 /200 ml) were CD80 plasmid (22) construct by PCR using the primers 59-AAAAG- cocultured with 5 mg/ml PHA (Sigma-Aldrich, St. Louis, MO) and irra- GATCCTATGGGCCACACACGGAGG-39 and 59-AGCGAAGCTTTTAT- diated tumor cells (C8161 and C8161/CD80, or MEL202 and MEL202/ GGGGCAAAGCAGTA-39, which amplified all but the 19 aa of the C- CD80, 5000 and 10,000 rads, respectively) at 37˚C, 5% CO2 for 72 h. Re- terminal cytoplasmic domain. The final construct was confirmed by DNA combinant human and mouse PD1–Fc fusion proteins (R&D Systems) by guest on September 29, 2021 sequence analysis. To facilitate construction of additional C-terminal variants were added to some coculture experiments. IFN-g production was mea- of CD80, the CD80tr construct was modified to include a BsmI site over- sured by ELISA (23). lapping the terminal proline codon. The HLA-DRa–chain cytoplasmic re- Microscopy gion was added to this modified construct by ligation of the double-stranded oligonucleotide adapter 59-GGAGTGCGCAAAAGCAATGCAGCAGAA- Tumor cells were cultured in 6-well plates for 48 h after transfection, then CGCAGGGGGCCTCTGTAA-39 and 59-AGCTTTACAGAGGCCCCCT- washed with excess PBS containing 2% FCS (2% PBS), labeled with CD80- GCGTTCTGCTGCATTGCTTTTGCGCACTCCCG-39 to a BsmI-digested PE*Cy7 (clone 2D10) for 30 min, and again washed with excess 2% PBS. CD80tr construct. The resulting fusion molecule (CD80DR) contains the Live fluorescence images were captured with an Olympus IX-81 (Olym- C-terminal sequence NH2-GVRKSNAAERRGPL-COOH and was confirmed pus, Center Valley, PA) microscope using Slidebook software (Intelligent by DNA sequence analysis. The pCMV6-AC-PDL1-GFP (PDL1-GFP) Imaging Innovations, Denver, CO). A minimum of 150 cells/sample was plasmid was obtained from Origene (Rockville, MD). counted. MEL202, MEL202/DR1, MEL202/CD80, MEL202/DR1/CD80, MCF10, MCF10/DR7/CD80, H358, H358/CD80, H358/DR7, and H358/DR7/CD80 Statistical analysis were described previously (22, 23, 25, 35). Stable C8161/CD80 and 624MEL/CD80 transfectants were generated by Amaxa nucleofection with SD and Student t test were calculated using Microsoft Excel version 2008. the pLHCX/CD80 plasmid (22) and were selected and maintained in media Mann–Whitney U test was performed using http://faculty.vassar.edu/lowry/ supplemented with 400 mg/ml hygromycin (Calbiochem, San Diego, CA). VassarStats.html. C8161/CD80tr, C8161/CD80DR, and MEL202/DR1/CD80/PDL1-GFP tran- sient transfectants were generated as per stable transfectants but were not drug Results selected and were used 48 h after transfection. Tumor cell expression of CD80 facilitates the boosting of Abs and flow cytometry primed T cells Flow cytometry Abs CD80–FITC (clone L307.4), PD1–PE (clone MIH4), Priming of naive T cells by professional APCs requires two signals, PDL1–PE (clone MIH1), mouse IgG1–FITC (clone X40), and mouse an Ag-specific signal delivered by a peptide–MHC complex and IgG1–PE (clone MOPC-21) mAbs were from BD Pharmingen (San Diego, a costimulatory signal. Boosting of primed T cells by professional CA). PDL1–allophycocyanin (clone 29E.2A3), PDL1–PE (clone 27A2), and mouse IgG2b–allophycocyanin were from BioLegend (San Diego, APCs requires an Ag-specific signal but does not require a co- CA), Medical & Biological Laboratories (Woburn, MA), and eBioscience stimulatory signal (40). To determine if costimulation is optional (San Diego, CA), respectively. Cells were stained for cell surface and in the boosting phase of CD4+ T cells when tumor cells are the intracellular expression by flow cytometry as described (22, 24) and ana- APCs, we used uveal melanoma (MEL202) and lung adenocar- lyzed using a Beckman Coulter Cyan ADP flow cytometer and Summit V4.3.02 software. cinoma (H358) tumor cells transfected with genes encoding HLA- DR and the costimulatory molecule CD80 (B7.1; MEL202/DR1/ PCR CD80 and H358/DR7/CD80, respectively). Because of their + For RT-PCR, total RNA was isolated using an RNeasy Mini kit according ability to activate tumor-reactive CD4 T cells that do not react to the manufacturer’s directions (Qiagen), and 1.3 mg RNA/sample was with nonmalignant tissue, we have called these genetically 6824 CD80 BLOCKS PDL1 EXPRESSION AND FUNCTION modified tumor cells “MHC II vaccines” (22, 25, 41). Priming and boosting HLA-DR7+ or HLA-DR1+ PBMCs with HLA-DR syngeneic MHC II lung adenocarcinoma (Fig. 1A) or uveal melanoma vaccine cells (Fig. 1B), respectively, yielded high levels of IFN-g. In contrast, priming with syngeneic HLA-DR+CD80+ tumor cells and boosting with HLA-DR+ tumor cells lacking CD80 induced significantly less IFN-g. Therefore, if APCs are tumor cells, then costimulation in the boosting phase is essential for optimal T cell activation. Expression of PDL1 on the cell surface of tumor cells is reduced by transfection of HLA-DR and CD80 Many tumor cells constitutively express or are induced by IFN-g to express PDL1 on their cell surface (21), raising the possibility that MHC II vaccines may induce T cell apoptosis due to PDL1 expression. To assess this possibility, H358/DR7/CD80 and a mammary carcinoma MHC II vaccine (MCF10/DR7/CD80) (24, 35) and their parental cells were stained for CD80 and PDL1 (mAb

29E.2A3) and analyzed by ﬂow cytometry (Fig. 2A). Parental Downloaded from H358 and MCF10 cells constitutively express PDL1; however, H358/DR7/CD80 and MCF10/DR7/CD80 vaccine cells did not have detectable PDL1. To conﬁrm that the lack of PDL1 on the vaccine cells was not a peculiarity of the 29E.2A3 PDL1 mAb, H358 and H358/DR7/CD80 cells were stained with two other

PDL1 mAbs (mAbs MIH1 and 27A2) (Supplemental Fig. 1A). As http://www.jimmunol.org/ for the MIH1and 27A2 mAbs, most vaccine cells did not express PDL1, whereas most parental cells constitutively expressed PDL1. To determine if MHC II vaccine cells were refractory to IFN-g FIGURE 2. Expression of PDL1 at the cell surface of tumor cells is reduced by transfection of HLA-DR and CD80. A, Expression of HLA-DR induction of PDL1, uveal melanoma MEL202 and its corre- and CD80 blocks constitutive expression of PDL1. Parental H358 (ade- g sponding vaccine (MEL202/DR1/CD80) were cultured in IFN- nocarcinoma) and MCF10 (mammary adenocarcinoma) cells and their and subsequently stained for CD80 and PDL1 (mAb 29E.2A3) HLA-DR7 and CD80 transductants were stained for cell surface CD80 and PDL1 (mAb 29E.2A3) and analyzed by ﬂow cytometry. B, Tumor cells expressing HLA-DR and CD80 are not induced by IFN-g to express PDL1.

Parental MEL202 (uveal melanoma) cells and their HLA-DR1 and CD80 by guest on September 29, 2021 transductants were either untreated or cocultured in IFN-g for 48 h, stained for cell surface CD80 and PDL1 (mAb 29E.2A3), and analyzed by ﬂow cytometry. Data are representative of four independent experiments.

(Fig. 2B). IFN-g treatment induced cell surface expression of PDL1 on MEL202 cells, whereas MEL202/DR1/CD80 cells did not have detectable cell surface PDL1. To ascertain that the absence of cell surface PDL1 on IFN-g–treated MEL202/DR1/CD80 cells was not a peculiarity of the 29E.2A3 mAb, parental and vaccine cells were also stained with the MIH1 and 27A2 mAbs to PDL1 (Supplemental Fig. 1B). In agreement with Fig. 2B, cell surface PDL1 was not detected. PDL1 was similarly induced on the cell surface of uveal melanoma MEL270 and its metastatic derivative OMM2.3 but not detected by the MIH1 mAb if the tumor cells were transfected with CD80 and HLA-DR (data not shown). Therefore, human tumor cells transfected with CD80 and HLA-DR do not constitutively express, nor are they induced by IFN-g to express, cell surface PDL1. CD80 inhibits the constitutive and IFN-g–induced expression FIGURE 1. Tumor cell expression of CD80 facilitates the boosting of of cell surface PDL1 on tumor cells primed T cells. A, PBMCs from an HLA-DR7+ healthy donor were primed The absence of detectable PDL1 on the surface of DR+CD80+ in vitro with irradiated H358 lung adenocarcinoma cells transduced with vaccine cells could be mediated by either HLA-DR or CD80 or HLA-DR7 and CD80 (H358/DR7/CD80) and boosted with either H358/ could be the result of the transfection, transduction, or drug se- DR7/CD80, single gene transductants (H358/DR7 or H358/CD80), or lection. To distinguish these possibilities, MEL202 cells express- parental cells. B, PBMCs from an HLA-DR1+ healthy donor were primed g with irradiated uveal melanoma cells transduced with HLA-DR1 and ing HLA-DR1 or CD80 were treated with IFN- and subsequently CD80 (MEL202/DR1/CD80) and boosted with either MEL202/DR1/ labeled with mAbs to CD80 and PDL1 (29E.2A3 mAb) (Fig. 3A). CD80, single gene transductants (MEL202/DR1 or MEL202/CD80), or MEL202/DR1 cells had detectable PDL1, whereas MEL202/ parental cells. IFN-g production was measured by ELISA. Data are rep- CD80 cells did not; indicating that IFN-g–induced cell surface resentative of three independent experiments. expression of PDL1 was inhibited by coexpression of CD80 and The Journal of Immunology 6825

pression of cell surface PDL1 (Fig. 3B; mAb 29E.2A3). Similar to MEL202/CD80, 624MEL/CD80 cells did not have detectable cell surface PDL1 after IFN-g stimulation, whereas parental 624MEL cells did. Another cutaneous melanoma, C8161, which constitutively expresses PDL1, was also negative for cell surface PDL1 when transfected with CD80 (Fig. 3C). Likewise, two other PDL1 mAbs (MIH1 and 27A2) failed to detect cell surface PDL1 on IFN-g–treated 624MEL/CD80 (Supplemental Fig. 2B)and C8161/CD80 cells (Supplemental Fig. 2C) cells. Therefore, coexpression or pre-expression of CD80 inhibits the constitutive and IFN-g–induced cell surface expression of PDL1 on human tumor cells. CD80 binds to PDL1 (9, 42) raising the possibility that PDL1 may be sterically blocked from binding PDL1 Abs but retain the ability to function and bind PD1. To assess this possibility, C8161 and C8161/CD80 cells were incubated with recombinant human PD1–Fc fusion protein. Binding of the fusion protein was detected by staining with ﬂuorescently labeled anti-PD1 mAb or goat anti-

human IgG and analysis by flow cytometry (Fig. 3D). Parental Downloaded from C8161 cells, but not C8161/CD80 cells, bound PD1–Fc. There- fore, coexpression of CD80 not only blocks Ab-mediated detection of PDL1 but also prevents binding of PDL1 to its receptor PD1. CD80 blocks PDL1 suppressive activity and restores T cell http://www.jimmunol.org/ activation The preceding experiments demonstrate that CD80 prevents detection of PDL1 but do not establish if tumor cell expression of CD80 overcomes PDL1-mediated immune suppression. To test functionality, we compared IFN-g production by PHA-activated PBMCs with and without various numbers of MEL202, MEL202/ CD80, C8161, or C8161/CD80 cells (Fig. 4A). PBMCs cocultured with CD80 transfected tumor cells produced more IFN-g, consistent with the concept that CD80 prevented PDL1-mediated by guest on September 29, 2021 suppression. To confirm that CD80 increased PBMC activation by inhibiting PDL1-mediated suppression, we compared IFN-g production by PHA-activated PBMCs cocultured with C8161 or C8161/CD80 in the presence of increasing quantities of recombinant human PD1–Fc (hPD1–Fc) fusion protein (Fig. 4B). FIGURE 3. CD80 inhibits the constitutive and IFN-g–induced expres- Recombinant mouse PD1–Fc (mPD1–Fc) was used as a negative sion of cell surface PDL1 on tumor cells. A, CD80 is sufficient to prevent control. Inclusion of hPD1–Fc, but not mPD1–Fc, significantly cell surface expression of PDL1 on uveal melanoma cells. MEL202 increased IFN-g production in cultures with C8161 cells. In transductants expressing HLA-DR1, CD80, or DR1 plus CD80 were cul- contrast, there was no significant difference in IFN-g production tured with IFN-g for 48 h, stained for cell surface CD80 and PDL1 (mAb in cultures containing C8161/CD80 cells with hPD1–Fc versus 29E.2A3), and analyzed by flow cytometry. B, CD80 inhibits IFN-g–in- mPD1–Fc. Therefore, blocking PDL1 has the same effect as CD80 duced expression of PDL1. IFN-g–treated 624MEL (cutaneous melanoma) parental cells and CD80 transfectants (624MEL/CD80) were stained for expression, consistent with the concept that CD80 facilitates T cell cell surface CD80 and PDL1 (mAb 29E.2A3) and analyzed by flow activation by inhibiting PDL1–PD1 interactions. cytometry. C, Expression of CD80 blocks constitutive expression of PDL1. CD80 does not inhibit intracellular expression of PDL1 C8161 (cutaneous melanoma) parental cells and CD80 transfectants were stained for cell surface CD80 and PDL1 (mAb 29E.2A3). D, Expression of To determine if CD80 inhibits detection and function of PDL1 by CD80 prevents PDL1 binding by PD1. C8161 and C8161/CD80 cells were blocking PDL1 transcription, we performed RT-PCR and qPCR on incubated with or without recombinant human PD1–Fc fusion protein tumor cells that constitutively express PDL1 (C8161) or are in- followed by staining with either goat anti-human IgG–PE (GaH IgG–PE) duced by IFN-g to express PDL1 (MEL202). MEL1011 cells, or PE-labeled mAb to PD1 (anti-PD1–PE) and analyzed by flow cytom- which neither constitutively express nor are induced to express etry. Data are representative of two, three, four, and three independent PDL1, and mDCs, which constitutively express PDL1, served experiments for A, B, C, and D, respectively. as negative and positive controls, respectively (Fig. 5A). PDL1 mRNA was present in constitutive expressers and after IFN-g was not due to drug selection or the transfection or transduction induction regardless of CD80 expression, indicating that CD80 process. The absence of detectable PDL1 on IFN-g–treated does not prevent transcription of PDL1. MEL202/CD80 cells was confirmed by staining with the two other To determine if CD80 inhibits translation of PDL1 mRNA, PDL1 mAbs (Supplemental Fig. 2A, MIH1 and 27A2 mAbs). To C8161, MEL202, and their CD80 transfectants were fixed, per- confirm that the effect of CD80 was not limited to uveal mela- meabilized, and stained for CD80 and PDL1 (mAb 29E.2A3) to noma cells, parental and CD80-transfected 624MEL cutaneous visualize intracellular PDL1 protein (Fig. 5B). In contrast to the melanoma cells were treated with IFN-g and analyzed for ex- absence of PDL1 at the cell surface, C8161/CD80 and IFN-g– 6826 CD80 BLOCKS PDL1 EXPRESSION AND FUNCTION Downloaded from http://www.jimmunol.org/ FIGURE 4. CD80 blocks PDL1 suppressive activity and restores T cell activation. A, T cell suppression is reversed by CD80 inhibition of PDL1 expression. PBMCs were activated with PHA in the presence or absence of varying numbers of irradiated MEL202, MEL202/CD80, C8161, or C8161/CD80 cells, and IFN-g production was measured by ELISA. B, PBMCs were activated with PHA and analyzed for IFN-g production as in A in the presence or absence of 1.5–6 mg/ml human or mouse recombinant PD1–Fc fusion protein and irradiated C8161 or C8161/CD80 cells. PBMC/ tumor cell ratio is 1:0.5. Data are representative of three and two in- by guest on September 29, 2021 dependent experiments for A and B, respectively. *p , 0.009. induced MEL202/CD80 contained intracellular PDL1, and there was no difference in the level of intracellular PDL1 in CD80+ versus CD802 cells. Staining with the MIH1 and 27A2 PDL1 mAbs confirmed intracellular PDL1 in C8161/CD80 and IFN-g– treated MEL202/CD80 cells (Supplemental Fig. 3A,3B, respectively). To examine further intracellular expression of PDL1 in CD80+ tumor cells, MEL202/DR1/CD80 cells were transiently transfected with a retroviral PDL1–GFP construct and 48 h later stained for cell surface CD80 and analyzed by fluorescence mi- FIGURE 5. CD80 does not inhibit intracellular expression of PDL1. A, croscopy (Fig. 5C). PDL1 was present intracellularly and on the 2 + RNA from MEL1011 (cutaneous melanoma), C8161, and C8161/CD80 cell surface of CD80 cells. In contrast, CD80 cells contained cells was subjected to RT-PCR using PDL1 primers (left). RNA from lower levels of intracellular PDL1 and no PDL1 at the cell surface. mDCs, MEL202, MEL202/CD80, and IFN-g–treated MEL202 and In agreement with the intracellular staining and fluorescence mi- MEL202/CD80 cells was subjected to qPCR using 18S and PDL1 primers croscopy, Western blot analysis revealed PDL1 protein in IFN-g– (right). B, Fixed and permeabilized C8161, C8161/CD80, MEL202, and treated MEL202 and MEL202/CD80 cells (Fig. 5D). These results MEL202/CD80 cells were internally stained for CD80 and PDL1 (mAb demonstrate that although CD80+ tumor cells do not have de- 29E.2A3) and analyzed by flow cytometry. MEL202 and MEL202/CD80 tectable cell surface PDL1, they contain intracellular PDL1 pro- cells were treated with IFN-g for 48 h prior to fixation, permeabilization, tein. and mAb labeling. C, MEL202/DR1/CD80 cells were transiently transfected with a PDL1–GFP plasmid, plated in 6-well plates, stained with PDL1 expression is predominately regulated by the CD80–PE–Cy7 (mAb 2D10), and analyzed by fluorescence microscopy. extracellular domains of CD80 CD80 is false-colored red. Of 167 cells counted, 18% were CD80+PDL1+, 54% were CD80+PDL12, and 28% were CD802PDL1+. D, Western blots CD80 signals intracellularly to modulate dendritic cell (43) and of untreated (2) or IFN-g–treated (+) MEL202 and MEL202/CD80 cells T cell (44, 45) function raising the possibility that CD80 may probed for PDL1 (mAb MIH1) and b-actin. mDCs and T2 cells served as downregulate PDL1 cell surface expression in tumor cells via its PDL1+ and PDL12 controls, respectively. Data are representative of three cytoplasmic domain. To test this possibility, two retroviral con- independent experiments. structs were made (Fig. 6A). One construct encoded CD80 truncated for 19 of the 21 aa of the C-terminal end cytoplasmic domain (CD80truncated, or CD80tr). The second construct The Journal of Immunology 6827

tumor cells against T cell-mediated elimination through its de- livery of apoptotic signals to tumor-reactive T cells (3). The results reported in this article using seven cell lines representing four histologically distinct solid tumors (lung, breast, cutaneous melanoma, and uveal melanoma) demonstrate that human tumor cell coexpression of CD80 reverses PDL1-mediated immune suppression, enables tumor cell boosting of primed tumor-speciﬁc T cells, and restores T cell activation during priming of tumor- reactive T cells. CD80 mediates this effect by preventing the expression of PDL1 protein at the cell surface of the tumor cells and without altering PDL1 transcription or intracellular expression. Many cells contain PDL1 mRNA but do not contain PDL1 protein (46). Gong and colleagues (47) have shown that PDL1 protein can be regulated by microRNA-513 (miRNA-513). miRNA-513 inhibits translation of PDL1 mRNA and is downregulated by IFN-g, consistent with the ability of IFN-g to upregulate PDL1 expression. miRNA-513, however, is unlikely to play a role in CD80-mediated regulation of PDL1 because CD80-

transfected tumor cells that constitutively express PDL1 or are Downloaded from IFN-g–treated to express PDL1 contain intracellular PDL1 protein. Our studies demonstrate that mechanistically, the extracellular domains of CD80 regulate PDL1 expression. CD80 and PD1 bind to the same region of PDL1 (9, 42), raising the possibility that

PDL1 is present on the cell surface, but the extracellular domains http://www.jimmunol.org/ of CD80 sterically block binding of anti-PDL1 mAb and PD1. The dissociation constant (KD) for CD80–PDL1 binding (∼1.4 mM) is FIGURE 6. PDL1 expression is regulated by the extracellular domains higher than the KD for PD1–PDL1 binding (∼0.77 mM) (42), in- of CD80. A, Amino acid sequence and predicted transmembrane and cy- dicating that PD1 could compete with CD80 for binding to PDL1. toplasmic domains of wild-type human CD80, CD80 with the truncated However, PD1–Fc fusion protein did not bind to CD80-expressing cytoplasmic domain (CD80tr), and CD80 with the cytoplasmic domain of cells, and CD80+ cells facilitated T cell boosting, suggesting that HLA-DRa–chain (CD80DR). Numbers indicate the amino acid position, PDL1 is physically absent from the cell membrane. Furthermore, with the first amino acid of the structural protein being position 1. B, if CD80 is mediating its effect by steric interference, then it is C8161 cells were transiently transfected with plasmids encoding wild-type unclear why CD80 does not also obscure PDL1 detection in the by guest on September 29, 2021 CD80, CD80tr, or CD80DR. Forty-eight hours after transfection, the cells cytoplasm of CD80+ cells. If CD80 is not sterically blocking were stained for cell surface CD80 and PDL1 (mAb 29E.2A3) and analyzed by flow cytometry. Data are representative of two independent PDL1 at the cell surface, it may mediate its effect by restricting experiments. trafficking of PDL1 to the cell surface or by facilitating the rapid turnover of cell surface PDL1. T cell activation is also inhibited by the binding of CD80 to the encoded the truncated CD80 gene ligated to a DNA fragment negative regulator CTLA-4 (48). CTLA-4 and PDL1 share a par- encoding the C-terminal 14 aa of the cytoplasmic domain of HLA- tially overlapping binding site on CD80 (42). As a result of this DRa-chain (CD80DR). Constitutive PDL1-expressing C8161 common binding site, CD80 can bind either CTLA-4 or PDL1, but cells were transiently transfected with these constructs and 48 h not both molecules concurrently. Therefore, if CD80 is bound to later stained for CD80 and PDL1 (mAb 29E.2A3) (Fig. 6B). PDL1, then it may not be able to suppress via CTLA-4. Our data CD80tr transfectants expressed less cell surface CD80 and a lower showing MHC II activation of T cells from healthy donors and percentage of cells were transfected compared with C8161 cells cancer patients is consistent with the concept that the vaccines are transfected with the wild-type CD80 construct. This decreased potent T cell activators because they deliver activation signals and expression is most likely due to the known role of the cytoplasmic do not suppress via either PDL1–PD1 or CD80–CTLA-4 path- domain of CD80 in regulating plasma membrane localization of ways. CD80 (38). However, C8161 cells expressing the CD80tr and The cytoplasmic domain of CD80 is required for CD80 co- CD80DR proteins were significantly downregulated for cell stimulation through CD28 (38) and is needed for correct sub- surface PDL1, with maximum downregulation observed in the cellular localization of CD80 and resultant signaling to CD28 transfectants expressing the highest quantity of CD80. Similar (37). It is also needed for PDL1 reverse signaling and resulting results were obtained using the MIH1 mAb (Supplemental Fig. 4). T cell suppression through CD80 (10). However, the CD80 cy- In contrast, C8161 cells transiently transfected with the same toplasmic domain is not required for the downregulation of PDL1 vector containing a CD80 gene mutated in its N-terminal extra- on the cell surface of tumor cells. This discrepancy raises the cellular domain were not downregulated for PDL1 expression possibility that CD80-mediated downregulation of PDL1 could be (data not shown). These results demonstrate that the extracellular functionally separated from the costimulatory effects of CD80 and domains and not the cytoplasmic region of CD80 predominantly therefore might be exploited therapeutically to inhibit PDL1- regulate cell surface expression of PDL1. mediated tumor cell-induced immune suppression. CD80-mediated blocking of PDL1 at the cell surface appears Discussion to be a tumor-specific effect because nonmalignant cells such as PDL1 is expressed constitutively or is induced by IFN-g on the cell dendritic cells and macrophages simultaneously express both surface of most human cancer cells and is credited with protecting molecules (S. Haile, J. Bosch, and S. Ostrand-Rosenberg, unpub- 6828 CD80 BLOCKS PDL1 EXPRESSION AND FUNCTION lished observations, and Refs. 49, 50). PDL1 downregulation is 17. Ma, J., L. Liu, G. Che, N. Yu, F. Dai, and Z. You. 2010. The M1 form of tumor- associated macrophages in non-small cell lung cancer is positively associated also proportional to the level of CD80. Quantity of CD80 may with survival time. BMC Cancer 10: 112. also explain why some human leukemias with low-level expres- 18. Anderson, C. F., and D. M. Mosser. 2002. A novel phenotype for an activated sion of CD80 have serologically detectable PDL1 (51). macrophage: the type 2 activated macrophage. J. Leukoc. Biol. 72: 101–106. 19. Itoh, K., K. Shiiba, Y. Shimizu, R. Suzuki, and K. Kumagai. 1985. Generation of Regardless of how CD80 deters PDL1 expression, PDL1 is not activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration serologically detectable and, more importantly, is functionally with interferon-gamma (IFN-gamma). J. Immunol. 134: 3124–3129. absent from the cell surface of tumor cells transfected with CD80. 20. Scott, P. 1991. 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