Constitutive Expression of Functional 4-1BB (CD137) Ligand on Carcinoma Cells Helmut R. Salih, Steven G. Kosowski, Vanessa F. Haluska, Gary C. Starling, Deryk T. Loo, Francis Lee, Alejandro A. This information is current as Aruffo, Pamela A. Trail and Peter A. Kiener of October 1, 2021. J Immunol 2000; 165:2903-2910; ; doi: 10.4049/jimmunol.165.5.2903 http://www.jimmunol.org/content/165/5/2903 Downloaded from References This article cites 42 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/165/5/2903.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Constitutive Expression of Functional 4-1BB (CD137) Ligand on Carcinoma Cells1 Helmut R. Salih,* Steven G. Kosowski,* Vanessa F. Haluska,* Gary C. Starling,* Deryk T. Loo,* Francis Lee,† Alejandro A. Aruffo,* Pamela A. Trail,† and Peter A. Kiener2* Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a Downloaded from costimulatory molecule for the production of cytokines (most notably IFN-␥) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction. The Journal of Immunology, 2000, 165: 2903–2910. he 4-1BB is a member of the TNF-R gene family, which It has been reported that human 4-1BBL is constitutively ex- includes an increasing number of proteins involved in pressed on several types of APC, such as activated B cells, mono- T regulation of cell proliferation, differentiation, and pro- cytes, and splenic dendritic cells, and can be induced on T lym- http://www.jimmunol.org/ grammed cell death (1, 2, 3). Members include the low-affinity phocytes (21, 16, 28, 29, 23). In addition, not surprisingly given nerve growth factor receptor (4), TNF-RI (5), TNF-RII (6), CD40 their derivation, expression of 4-1BBL has also been found on (7), the Hodgkin’s Ag CD30 (8), CD27 (9), Fas/APO-1 (10), tumor cells of lymphoid or myeloid origin. Studies with 4-1BBLϩ 4-1BB (11), and several others. These receptors recognize soluble APC have shown that the interaction of 4-1BB with its ligand or cell-bound ligands such as nerve growth factor (3), TNF (12), stimulates cell proliferation and production of IL-2 and IL-4 by CD40L (13), CD27L (14), Fas ligand (FasL)3 (15), and 4-1BBL CD4 T cells. Moreover, it has been reported that anti-4-1BB mAb (16), which share C-terminal amino acid homology. Expression of stimulate the production of IFN-␥ by CD8 T cells (26). The role some members of the TNF-R/ligand family on tumor cells has for 4-1BBL in the development of TH1 and TH2 cells is reported by guest on October 1, 2021 previously been reported (17, 18). For example, FasL on tumor to be most apparent in the absence of a strong B7-CD28 interaction cells has been implicated in the escape of tumors from immune (23, 24). Likewise, Saoulli and coworkers (30) have demonstrated surveillance by inducing apoptosis in tumor-infiltrating lympho- that isolated 4-1BBL can costimulate resting T cells via a CD28- cytes (19, 20). independent pathway. Several ligands of the TNF superfamily Recently, a number of studies that used either transfected ligand have been shown to be able to signal in both directions, through the (21) or a soluble form of the 4-1BB receptor to block receptor/ respective receptor and into the cell that expresses the ligand. Re- ligand interactions (22, 23, 24) have demonstrated a role for the verse signaling following cross-linking of 4-1BBL has been shown TNF/TNF-R family member 4-1BB/4-1BBL in T cell activation. to inhibit proliferation, to induce apoptosis, and to up-regulate ex- mAbs against 4-1BB have been shown to eradicate established pression of Fas (CD95) on lymphocytes (31), and to stimulate tumors in a mouse model (25), and to preferentially induce pro- macrophages to release IL-8 (32). liferation of CD8 T cells compared with CD4 T cells. This has led Although 4-1BBL is expressed on cells of hemopoietic origin, to the suggestion that 4-1BB is primarily a costimulatory molecule its expression on carcinoma cells has not been examined. In this for CD8 T cells (26). Most recently, studies have shown that in study, we show that 4-1BBL is expressed to varying extents on addition to providing costimulation, 4-1BB may also promote several human carcinoma cell lines as well as on cells obtained long-term T cell survival especially of CD8 T cells (27). from patient solid tumors. We demonstrate that the 4-1BBL is functional in that reverse signaling through the 4-1BBL by a 4-1BB-Ig fusion protein induces tumor cells to produce IL-8. Fur- Departments of *Immunology, Inflammation, and Pulmonary, and †Oncology Drug Discovery, Bristol-Myers Squibb, Pharmaceutical Research Institute, Princeton, thermore, we show that coculture of anti-CD3-activated T lym- NJ 08543 phocytes with tumor cells expressing 4-1BBL results in production Received for publication April 4, 2000. Accepted for publication June 14, 2000. of IFN-␥ by the activated T cells. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 H.R.S. was supported by a grant of Dr. Mildred Scheel Stiftung fuer Krebsfors- Cell preparation and culture chung, Germany. Human T cells were isolated from healthy donors by standard protocols. In 2 Address correspondence and reprint requests to Dr. Peter A. Kiener, Department brief, PBMC were prepared from heparinized whole blood by Ficoll den- of Immunology and Inflammation, Bristol-Myers Squibb, Pharmaceutical Research sity-gradient sedimentation; T cells were then isolated by rosetting with Institute, Princeton, NJ 08543. E-mail address: [email protected] SRBC. In some experiments, the T cells were further purified by passage 3 Abbreviations used in this paper: FasL, Fas ligand; 4-1BBL, 4-1BB ligand. through nylon wool columns or by a second round of rosetting. The human Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 2904 4-1BB LIGAND ON CARCINOMA CELLS tumor cell lines A 2780 (ovarian), Colo 205 (colon), HT 29 (colon), L 2987 extraction of mRNA, the Fast Track 2.0 kit from Invitrogen (Carlsbad, CA) (lung), LX 1 (lung), PC 3 (prostate), and HL 60 (promyelocytic leukemia) was used as instructed by the manufacturer. The cDNA library for human were routinely cultured in RPMI 1640 medium supplemented with 10% small intestine was internally obtained from Bristol-Myers Squibb. heat-inactivated FCS and 1% penicillin/streptomycin. HCT 116 (colon) and SKBR 3 (breast) cell lines were grown in McCoy’s medium with the RT-PCR same supplements. cDNA was prepared using the Superscript Preamplification System for COS cell transfection First Strand cDNA Synthesis (Life Technologies, Rockville, MD) using 3 ␮g total RNA per sample. Aliquots of 5 ␮l of the cDNA were then am- To obtain COS cells expressing surface 4-1BBL, the cDNA encoding for plified with 44 ␮l of PCR supermix high fidelity (Life Technologies) and the extracellular domain of 4-1BBL (accession no. U03398) was fused to 0.5 ␮l of each primer (final concentration, 0.5 ␮M). For detection of human the cDNA for the intracellular and cytoplasmic domains of CD40L (ac- 4-1BBL cDNA, the primers used for amplification were human 4-1BBL cession number Z 15017) in pCDM7Ϫ. COS cells were transfected by sense primer corresponding to nucleotides 484–513 (5Ј-GTT TCA CTT using DEAE-dextran. Briefly, cells were grown in 48-well plates, then 1 GCG CTG CAC CTG CAG CCA CTG-3Ј) and antisense primer comple- ␮g/ml DNA in the presence of DEAE-dextran 400 ␮g/ml plus Chloroquine mentary to nucleotides 926–949 (5Ј-GGC TCT AGA TAT CAA GGT (100 ␮M) in 5% NuSerum (Becton Dickinson, Bedford, MA) in DMEM CCA ACT TGG GGA AGG-3Ј).