Constitutive Expression of Functional 4-1BB (CD137) 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, , 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 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 (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 (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 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 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). Primers for human ␤-actin (5Ј-GTG GGG (Life Technologies, Gaithersburg, MD) was added for4hat37°C. The CGC CCC AGG CAC CA-3Ј, nucleotides 144–163 and 5Ј-CTC CTT AAT cells were then shocked with 10% DMSO in PBS. Cells were then cultured GTC ACG CAC GAT TC-3Ј, nucleotides 660–683) served as control. in 10% FBS/DMEM and used 48 h after transfection. After denaturation at 94°C for 5 min, 40 PCR cycles were performed, each consisting of a denaturation step (94°C, 1 min), an annealing step (52°C, 2 Abs, enzymes, and fusion proteins min), and an elongation step (72°C, 2 min). The PCR products (expected size 465 bp for human 4-1BBL, 539 bp for human actin) were separated by Anti-human CD3 Ab G19.4 was obtained internally from Bristol-Myers electrophoresis on 1% agarose gel and visualized by staining with ethidium Squibb (New York, NY). Goat anti-rabbit FITC conjugate was from Bio- bromide. Source International (Camarillo, CA). Goat anti-human PE conjugate was Downloaded from from Jackson ImmunoResearch (West Grove, PA). Mouse anti-human Northern blot hybridization HLA class I PE, rat anti-mouse CD45 PE conjugate, and the isotype con- trols rat IgG2b PE conjugate and mouse IgG1 PE were from PharMingen Samples of mRNA (2 ␮g) were fractionated according to size in 3% form- (San Diego, CA). The nonstimulatory anti-CD137 Ab clone BBK-2 was aldehyde/1.2% agarose gels and then transferred to nylon membranes. The from Neo Markers (Fremont, CA); the clone 4B4-1 was from Ancell (Bay- membranes were prehybridized for1hinhybridization solution (Express port, MN). All other reagents were obtained from Sigma (St. Louis, MO). hyb; Clonetech, Palo Alto, CA) at 65°C, then hybridized for 2 h with a Rabbit polyclonal antisera to 4-1BBL were prepared by immunizing random primed [␣-32P]dCTP-labeled (Random Primed DNA Labeling Kit; http://www.jimmunol.org/ New Zealand white rabbits with the peptide CHTEARARHAWQLTQ rep- Boehringer Mannheim, Indianapolis, IN) human 4-1BBL cDNA fragment resenting aa 217–230 of the 4-1BBL polypeptide sequence, conjugated to or a GAPDH cDNA fragment as a control (2 ϫ 106 cpm/ml). Membranes keyhole limpet hemocyanin. This sequence of the peptide is predicted to were washed twice in 2ϫ SSC with 0.05% SDS at room temperature and form part of the extracellular domain of 4-1BBL. Polyclonal antisera were twice in 0.1ϫ SSC with 0.1% SDS at 65°C and exposed to x-ray film with purified by affinity chromatography on protein A-Sepharose. After elution, intensifying screens at Ϫ70°C. the antisera were dialyzed into PBS, filter sterilized (0.22 ␮m), and stored at 4°C. The soluble 4-1BB fusion protein (human 4-1BB with a human Western blot analysis IgG1 tail) (33) and soluble B7-2 fusion protein (human B7-2 with a human Tumor cells were isolated by dissociation with RPMI 1640 containing 5 IgG1 tail) (34) were constructed and prepared as previously described. mM EDTA and washed twice with PBS, and the cell pellets were then frozen at Ϫ70°C. Cell pellets (3 ϫ 107 total cells) were lysed at 1 ϫ 108

Disaggregation of solid tumor tissue by guest on October 1, 2021 cells/ml in cell lysis buffer (33) containing 1% Triton X-100 and protease Tumors were excised and pooled, and 2–3 g of tumor tissue was minced in inhibitors. Cell lysates were clarified by centifugation at 15,000 rpm in a 7 ml HBSS. A tumor cell suspension was obtained by adding enzymes to microfuge for 10 min at 4°C and mixed with an equal volume of 2ϫ SDS a final concentration of 0.02% DNase, 0.3% collagenase, and 0.4% hyal- Laemmli sample buffer containing 2-ME. Samples were heated at 95°C for uronidase, and incubating for2hat37°C. Cells were washed three times 5 min before separation by SDS-PAGE on 8–16% acrylamide gels. Gels in HBSS and passed three times through a 25-gauge needle. After centrif- were then transferred to polyvinylidene difluoride membranes by Western ugation, cells were resuspended in RPMI 1640 containing 10% FCS. blotting; the membranes were blocked with buffer containing 5% BSA and 10% nonfat dried milk, probed with anti-4-1BBL polyclonal antiserum Flow cytometry (1 ␮g/ml), and visualized by probing with HRP goat anti-rabbit IgG and Western blot chemiluminescence reagent (NEN Life Science Products, For the FACS analysis of 4-1BBL expression on carcinoma cell lines, cells Boston, MA). were washed once in PBS and 106 cells were incubated at 4°C in 100 ␮l staining buffer (RPMI 1640 medium, 2% FCS, 2.5 mM EDTA, mouse IgG ELISA at 250 ␮g/ml) containing 10 ␮g/ml 4-1BB-Ig or 10 ␮g/ml B7-2-Ig fusion proteins. After incubation, cells were washed twice with PBS and incu- assays were performed by ELISA according to the manufactur- bated at 4°C for 30 min in 100 ␮lofa10␮g/ml solution of PE-conjugated er’s instructions using OptEIA sets from PharMingen. Cytokine concen- goat anti-human IgG Ab in staining buffer and then washed further. Al- trations in supernatants are expressed as mean Ϯ SEM of quadruplicates. ternatively, 10 ␮g/ml rabbit anti-4-1BBL polyclonal antiserum or 10 ␮g/ml Where indicated, results were compared using the Student t test. rabbit IgG followed by 100 ␮lof20␮g/ml goat anti-rabbit FITC-conju- gated Abs were used under the same conditions. When the rabbit antiserum Results was used, cells were counterstained with propidium iodide, and positive Expression of 4-1BBL on carcinoma cells cells were gated out to ensure only live cells were evaluated. For analysis of cells derived from solid tumors growing in athymic mice, 106cells were The expression of 4-1BBL on the surface of the cell lines A 2780, stained with anti-4-1BBL rabbit polyclonal antiserum or control rabbit IgG, Colo 205, HCT 116, HT 29, LX 1, PC 3, and SKBR 3 was inves- as described above. After washing twice in PBS, a second staining step was tigated by flow cytometry. Analysis of the staining of cells with a ␮ incorporated using 10 g/ml mouse anti-human HLA class I PE conjugate, human 4-1BB-human IgG1 Fc fusion protein (4-1BBIg) compared or 10 ␮g/ml mouse IgG1 PE as the isotype control, in 100 ␮l of cell suspension. With this procedure, by gating on the HLA-positive cells, only with a B7-2-Ig fusion protein (B7-2Ig) revealed that the cells ex- the human tumor cells were investigated for 4-1BBL expression. Alterna- pressed 4-1BBL at various densities. The highest levels of expres- tively, cells were counterstained with anti-mouse CD45 and positive cells sion were seen on the HCT 116 and Colo 205 cell lines (Fig. 1A, were gated out. Samples were analyzed on a FACScan (Becton Dickinson, panels 2 and 3), with lower levels in HT 29 cells (Fig. 1A, panel Mountain View, CA) by monitoring for forward vs sideward scatter and 4). Very similar results were found when the cells were analyzed FL2 or FL1 and FL2, respectively. with a polyclonal rabbit anti-4-1BBL antiserum compared with RNA extraction normal rabbit IgG (Fig. 1B). A previous report indicated that Total RNA was extracted from tumor cells using the RNeasy Midi kit from 4-1BBL was expressed on T lymphocytes only following stimu- Qiagen (Valencia, CA), according to the manufacturer’s instructions. For lation (16). In concurrence, no expression could be detected on The Journal of Immunology 2905 Downloaded from http://www.jimmunol.org/ FIGURE 1. FACS analysis of the expression of 4-1BBL. A, A total of 1 ϫ 106 cells of the different carcinoma cell lines was stained with human 4-1BB Ig (shaded histograms) or human B7-2Ig (open histograms), followed by staining with a secondary PE-conjugated goat anti-human Ig polyclonal Ab. Cell lines were as follows: panel 1, A 2780; 2, Colo 205; 3, HCT 116; 4,HT29;5,LX1;6,PC3;7, SKBR 3. B, A total of 1 ϫ 106 cells was stained with rabbit anti-human 4-1BBL polyclonal antiserum (shaded histograms) or purified rabbit IgG (open histograms), followed by staining with a secondary FITC-conjugated goat anti-rabbit polyclonal Ab. Cells were counterstained with propidium iodide to exclude dead cells. The cell lines were as given in A. C, Resting peripheral T cells from two different donors (panels 1 and 2) were stained as described in B; Colo 205 (panel 3) and HCT 116 (panel 4) cells were stained with rabbit anti-4-1BBL after prior addition of 4-1BBIg (dotted line) or B7-2Ig (shaded histograms), as described in B. D, Solid tumors from athymic mice were disaggregated and then stained with either anti-4-1BBL polyclonal antiserum or rabbit IgG together with an anti-human HLA class I PE conjugate for the selection of human cells. Gates were set to include only the HLA-positive cells in the analysis. Tumor cells were as follows: panel

1, HCT 116; 2, L 2987; 3, LX 1. 4, taxol-resistant human ovarian tumor; 5, human pancreatic tumor. by guest on October 1, 2021

freshly isolated peripheral T cells (Fig. 1C, panels 1 and 2). Ad- evaluated using RT-PCR. Each of the tumor cell lines expressed dition of 4-1BBIg before staining with the 4-1BBL antiserum re- mRNA for 4-1BBL, as revealed by the expected products of 465 duced the detectable levels of 4-1BBL expressed on both HCT 116 bp (Fig. 2A). The level of expression of 4-1BBL differed on the and Colo 205 cells, respectively (Fig. 1C, panels 3 and 4). various cell lines. The RT-PCR results were confirmed by North- To determine whether cell surface expression of 4-1BBL was ern blot analysis using the cDNA encoding for the extracellular the consequence of long-term in vitro culture of cell lines, two domain of 4-1BBL as a hybridization probe. One larger and two approaches were taken. First, tumor cells were inoculated into smaller mRNA transcripts of sizes 4.4, 3, and 1.6 kb were found, athymic mice and the resulting xenografts were excised, the cells similar to that previously described (16). The level of mRNA ex- then isolated by enzyme digestion and analyzed by FACS for ex- pression was highest in HCT 116 and Colo 205 cell lines, in ac- pression of 4-1BBL using the rabbit polyclonal antiserum. To ex- cordance with the results obtained in the FACS analysis (Fig. 2B). clude the analysis of mouse stromal cells and tumor-infiltrating The expression of 4-1BBL protein was determined by Western lymphocytes, the tumor cells were also stained with anti-human blot analysis of Triton X-100 detergent extracts of the tumor cells HLA class I mAb and gates were set to include only these HLA- (1 ϫ 108/ml). A major protein band of about 60 kDa was detected positive cells. Cells of solid tumors obtained from three different using the IgG fraction of the anti-4-1BBL antiserum. The 4-1BBL carcinoma cell lines (HCT 116, L 2987, LX 1) were evaluated by was detected in all of the cell lines, with the highest expression in this procedure and showed expression of 4-1BBL on the cell sur- the extract from HCT 116 cells (Fig. 2C, lane 3). No band was face (Fig. 1D, panels 1–3). In addition, analysis of the cell surface detected in extracts from unactivated peripheral T cells. The anti- expression of 4-1BBL on tumor cells derived from patient tumors serum also recognized soluble 4-1BBL expressed as a fusion pro- cultured in athymic mice with the anti-4-1BBL antiserum revealed tein with a mouse CD8 tag (data not shown). that cells from an ovarian tumor and a pancreatic tumor both ex- pressed 4-1BBL (Fig. 1D, panels 4 and 5). Analysis using an anti- Cytokine release of carcinoma cells induced by reverse mouse CD45 counterstain and gating out all the CD45-positive signaling through 4-1BBL cells showed similar results (data not shown). It has been reported that ligation of 4-1BBL can activate macro- phages and induce cytokine production (32). To determine whether Detection of 4-1BBL mRNA and protein in carcinoma cells 4-1BBL expressed on the tumor cell lines was functionally active, To confirm the expression of 4-1BBL on tumor cell lines, total the cells were incubated for 24 h in the presence of 4-1BBIg, RNA was extracted from the cells and 4-1BBL expression was B7-2Ig, or in medium alone. B7-2Ig did not enhance IL-8 secretion 2906 4-1BB LIGAND ON CARCINOMA CELLS

contrast to the effect on IL-8, treatment of HCT 116, LX 1, or Colo 205 cells with 4-1BBIg did not significantly stimulate the release of IL-6, IL-10, IL-12, TNF-␣, or TGF-␤ into the cell supernatants (data not shown). Stimulation of HCT 116 and LX 1 cells for 24 h with increasing concentrations of 4-1BBIg showed that IL-8 pro- duction was dose dependent (Fig. 3B). Addition of the anti-4-1BB mAb BBK-2 or 4B4-1 (20 ␮g/ml) to the cultures before stimula- tion of the cells with 4-1BBIg significantly (all p Ͻ 0.03) reduced the stimulation of IL-8 release (Fig. 3C), whereas control mouse IgG1 was without effect. This confirms that the 4-1BBIg-induced IL-8 production from the carcinoma cells is mediated through the 4-1BBL/4-1BB receptor interaction.

4-1BBL-expressing carcinoma cells induce T cells to produce IFN-␥ In addition to showing that the tumor cells expressed 4-1BBL that was capable of signaling back into the tumor cells, it was important to determine whether 4-1BBL expressed on the tumor cells was able to activate the costimulatory 4-1BB pathway of human T Downloaded from cells. One response of T cells to ligation of 4-1BB, in conjunction with signals through the TCR, is the production of IFN-␥ (26). Thus, the effect of carcinoma cells on T cell production of IFN-␥ was determined. HCT 116 cells were cultured in 96-well plates, and human T cells were added to some of the cultures, with or without addition of soluble anti-CD3. After 48 h, the culture su- http://www.jimmunol.org/ pernatants were harvested and analyzed for production of IFN-␥ by ELISA. In the absence of anti-CD3, T cells alone or T cells cocultured with HCT 116 cells produced very little IFN-␥ (2.1 pg/ml Ϯ 0.8 and 13.5 pg/ml Ϯ 3.9, respectively). In the presence of soluble anti-CD3, the HCT 116 cells alone only produced very low levels of the cytokine (1.9 pg/ml Ϯ 0.4). Similarly, low levels FIGURE 2. Expression of 4-1BBL mRNA and protein. A, RT-PCR ␥ Ϯ analysis of 4-1BBL mRNA expression in tumor cell lines. Expression was of IFN- (5.9 pg/ml 1.7) were produced when T cells alone were cultured with soluble anti-CD3. In contrast, cocultures of determined by analysis of an equal level of mRNA isolate from each cell by guest on October 1, 2021 line. HL 60 cell cDNA was used as positive and human small intestine tumor cells with T cells in the presence of soluble anti-CD3 in- cDNA as negative control. The mRNA-specific amplification products for duced marked increases in the levels of IFN-␥ (94.6 pg/ml Ϯ 27.5; 4-1BBL and actin were separated by agarose gel electrophoresis and vi- Fig. 4A). sualized by staining with ethidium bromide. The figure shown is a reverse FACS analysis had revealed that the carcinoma cell lines HCT image. Lanes 1–7, Correspond to the following tumor cells: 1, A 2780; 2, 116 and HT 29 express 4-1BBL at different densities, with HCT Colo 205; 3, HCT 116; 4, HT 29; 5,LX1;6,PC3;7, SKBR 3. B, Northern 116 expressing higher levels than HT 29 cells. Thus, equivalent blot analysis of 4-1BBL mRNA expression in tumor cells. RNA blotting numbers of HCT 116 and HT 29 were cultured in 96-well plates was performed after separation of 2 ␮g mRNA on 1.2% formaldehyde gel and human T cells were added together with soluble anti-CD3. The and transfer to a nitrocellulose membrane. Hybridization was performed using 32P-labeled probes consisting of the cDNA encoding for the extra- cultures were incubated for 48 h, and supernatants were harvested ␥ cellular domain of human 4-1BBL or GAPDH. The tumor cells were: 1, and analyzed for production of IFN- by ELISA. The level of A 2780; 2, Colo 205; 3, HCT 116; 4,HT29;5,LX1;6,PC3;7, SKBR IFN-␥ found in the coculture supernatants of both HCT 116 and 3. C, Western blot analysis of 4-1BBL expression in tumor cell lines. Cells HT 29 cells was dependent on the number of tumor cells present were lysed in Triton lysis buffer at 1 ϫ 108/ml, separated by SDS-PAGE in the culture. By increasing the number of HCT 116 cells from on 8–16% polyacrylamide gels, and transferred to polyvinylidene difluo- 6.25 ϫ 103 cells to 2 ϫ 105 cells in the coculture experiments, the ride membranes; the membranes were blocked and then probed with the level of IFN-␥ (over that in T cells alone) was raised from 2- to polyclonal anti-4-1BBL Ab, followed by goat anti-rabbit IgG-HRP conju- about 20-fold, respectively. The same experiment with HT 29 cells gate. Lanes: 1, A 2780; 2, Colo 205; 3, HCT 116; 4,HT29;5,LX1;6,PC resulted in a 1- to 5-fold increase, respectively (Fig. 4B). In wells 3; 7, SKBR 3. with 2 ϫ 105 tumor cells, the levels of the cytokine were signif- icantly ( p Ͻ 0.01) lower with the HT 29 cells (21.4 pg/ml Ϯ .16.1) than with the HCT 116 cells (100.7 pg/ml Ϯ 29.2, respectively). over that found in untreated cells. Treatment of the cells with Stimulation of T cells with 2 ϫ 105 tumor cells gave a 17-fold 4-1BBIg induced a marked increase in the levels of IL-8 in the cell increase of IFN-␥ levels using HCT 116 cells, and a 5-fold in- supernatants from Colo 205 (4.2-fold), HCT 116 (2.2-fold), and crease using HT 29 cells, compared with that from T cells in the LX 1 (2.4-fold); LX 1 cells were of note because they produced absence of tumor cells. LX 1, which we showed to have interme- high spontaneous levels of the cytokine, and this was further stim- diate levels of 4-1BBL surface expression, stimulated a 7-fold in- ulated. However, the largest increase induced by 4-1BBIg was crease in the levels of IFN-␥ (data not shown). Together, these seen with Colo 205 cells (Fig. 3A). Very little effect of 4-1BBIg on results indicate that under the conditions used, there appears to be IL-8 secretion was observed in A 2780 and SKBR 3 cell lines. PC a dose-dependent relationship among the density of 4-1BBL ex- 3 cells exhibited very high levels of constitutive production of IL-8 pressed on tumor cells, the number of cells, and the levels of IFN-␥ that was not increased further by 4-1BBIg (data not shown). In production. The Journal of Immunology 2907 Downloaded from http://www.jimmunol.org/

FIGURE 3. Induction of IL-8 by 4-1BBIg in carcinoma cell lines. Tumor cells were grown for 48 h on tissue culture plates, then the culture medium was exchanged. A, A total of 10 ␮g/ml soluble 4-1BBIg or B7-2Ig was added to the cultures. After 24 h, 100 ␮l of each supernatant were harvested and levels of IL-8 in 100 ␮l were determined by ELISA. The data shown are means of quadruplicates with SDs from one representative experiment from a total of five. B, Dose-response curves for IL-8 induction by 4-1BBIg. Tumor cell lines LX 1 and HCT 116 were cultured in 48-well plates at 5 ϫ 104/ml. by guest on October 1, 2021 After 48 h, the media were exchanged and the indicated concentrations of 4-1BBIg were added to the cells. The concentration of IL-8 in 25 ␮l of culture supernatant was determined after 24 h. The data shown are means of quadruplicates with SDs from one representative experiment from a total of three. C, Modulation of IL-8 production by addition of anti-4-1BB Abs. Tumor cells were cultured as described in B. After 48 h, the medium was replaced with the addition of 4-1BBIg (10 ␮g/ml) and anti-4-1BB mAb BBK-2, 4-B4-1, or IgG1 (20 ␮g/ml), as indicated. The concentration of IL-8 in 25 ␮l of the culture supernatant was determined by ELISA after 24 h. The data shown are means of quadruplicates with SDs of a representative experiment from a total of three.

The requirement for 4-1BBL in these cultures was verified by 10.3 vs 164.3 pg/ml ϩ 47.3, respectively). This increase was mark- culturing HCT 116 cells with T cells and anti-CD3 in the absence edly inhibited ( p Ͻ 0.02) by addition of the nonstimulatory anti- or presence of two different nonstimulatory anti-4-1BB mAb, 4-1BB mAb BBK-2 (44.1 pg/ml ϩ 18.4) to the cultures (Fig. 4D). BBK-2, and 4B4-1. In the absence of tumor cells, T cells cultured with anti-CD3 together with either of these two anti-4-1BB mAb did not produce IFN-␥, whereas IFN-␥ production was stimulated Discussion with several other anti-4-1BB mAb (data not shown). Cocultures Several reports have suggested an important role for 4-1BB in the were set up as described, but in the presence of 10 ␮g/ml of the stimulation or costimulation of T cells (22–30, 36). These results BBK-2 or 4B4-1 anti-4-1BB mAb or purified mouse IgG. After indicate that 4-1BB is primarily a costimulatory molecule for CD8 48 h, supernatants were harvested and assayed by ELISA. Produc- T cells (26) that functions independently of CD28. In addition, tion of IFN-␥ was significantly inhibited by both BBK-2 ( p Ͻ 4-1BB may also promote long-term T cell survival (27). Expres- 0.01) and 4B4-1 ( p Ͻ 0.05) mAb, whereas addition of mouse IgG sion of 4-1BBL has been reported on lymphocytes, macrophages, was without effect (Fig. 4C). and cell lines of lymphoid and myeloid origin (22–30, 32). To additionally verify that the induction of IFN-␥ production In the study reported in this work, we show that the 4-1BBL is was due to expression of 4-1BBL on the tumor cells, COS cells expressed and functional on carcinoma cells. Analysis by FACS growing in 48-well plates were transfected with cDNA to obtain revealed expression of 4-1BBL at different densities on several 4-1BBL-expressing cells. After 48 h, the plates were washed and different carcinoma cell lines, on cells of human tumor cell lines medium alone or medium containing 2 ϫ 106 T cells was added to growing as solid tumors in athymic mice, and on human tumor transfected or mock-transfected cells; additionally, anti-CD3 Ab cells derived from patient samples after biopsy and propagation in (2 ␮g/ml) was added to each well. After an additional 48 h, su- athymic mice. In human tumors, infiltrating APC or lymphocytes pernatants were harvested and analyzed by ELISA. Compared with might be expected to express 4-1BBL, which would complicate mock-transfected cells, COS cells transfected with 4-1BBL showed sig- detection of 4-1BBL expression on the carcinoma cells. Thus, the nificantly ( p Ͻ 0.01) enhanced IFN-␥ production (11.3 pg/ml ϩ human tumors were propagated in athymic mice to remove human 2908 4-1BB LIGAND ON CARCINOMA CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. 4-1BBL expression on carcinoma cells induces IFN-␥ production from human T cells in coculture. A, HCT 116 cells were grown for 48 h in 96-well plates. After the exchange of media, 1 ϫ 105 human T cells were added together with 2 ␮g/ml anti-CD3 mAb G 19.4, where indicated. Plates were incubated for 48 h, and the supernatants were then harvested and assayed by ELISA. Additionally, HCT 116 cells and T cells alone or by guest on October 1, 2021 together Ϯ anti-CD3 mAb were cultured as indicated. Samples were assayed in quadruplicate. The data shown, mean with SD, are from one experiment. Similar results were obtained with T cells from eight different donors each in two independent experiments. B, HCT 116 or HT 29 cells were plated at the indicated density and grown for 48 h in 96-well plates. After exchange of medium, primary human T cells (1 ϫ 105/well) and 2 ␮g/ml CD3 Ab were then added. Plates were incubated for 48 h, then supernatants were harvested and quantitated by ELISA. Samples were assayed in quadruplicate. Similar results were obtained with T cells from six different donors in two independent experiments each. C, Equal numbers of HCT 116 cells were grown on 96-well plates, and the culture medium was then discarded and 1 ϫ 105 T cells/well in fresh medium alone or medium containing anti-4-1BB mAb BBK-2, 4B4-1, or purified mouse IgG, at a final concentration of 10 ␮g/ml together with anti-CD3 Ab to a final concentration of 2 ␮g/ml were added. Supernatants were harvested after 48 h and quantitated in quadruplicate by ELISA. D, COS cells growing in 48-well plates were transfected with cDNA encoding for the extracellular domain of 4-1BBL fused to the cDNA for the intracellular and cytoplasmic domains of CD40L (COS*). Control cells were treated identically, except that no cDNA was added (COS). After 48 h, the plates were washed twice with PBS and medium alone vs medium containing 2 ϫ 106 T cells was added to transfected and untransfected cells. Anti-CD3 mAb to a final concentration of 2 ␮g/ml was added to each well. After 48 h, supernatants were harvested and assayed in quadruplicate by ELISA after 48 h.

APC. To ensure that only human tumor cells were investigated for potent survival signal and to induce activation of the cells to re- expression of 4-1BBL and not, for example, tumor-infiltrating lease IL-8 (32). mouse lymphocytes, cells were also stained with mAb for human There are several reports indicating that both colonic epithelial HLA class I. Only HLA class I-positive cells were evaluated. Sim- cells and other epithelial cells such as bronchial epithelia and gas- ilar results were obtained if the cells were stained with anti-4- tric carcinoma cells can produce IL-8 (40). In our experiments, 1BBL and anti-mouse CD45, and the population expressing CD45 activation of the tumor cells with 4-1BBIg enhanced IL-8 produc- was gated out. Expression of 4-1BBL in tumor cells was confirmed tion from the tumor cells. This cytokine is a potent neutrophil, T by RT-PCR analysis of total RNA and Northern blot analysis of cell, and basophil chemoattractant, and release of IL-8 is thought mRNA extracted from tumor cells, and additionally by Western to amplify an ongoing acute immune response (39). Thus, it is blot analysis of tumor cell lysates. likely that reverse signaling through 4-1BBL on the carcinoma There have been reports of bidirectional signaling for several cells may influence the immune response at the site of the tumor. ligands of this family (37, 38), and recent studies have addressed There did not appear to be a direct correlation between the levels the effects of reverse signaling through 4-1BBL. Cross-linking of of 4-1BBL expression on tumor cells and the IL-8 production in- 4-1BBL expressed on T lymphocytes inhibited the proliferation of duced by 4-1BBIg. This might be due to a differing ability of the the cells and induced apoptosis in activated T cells in a Fas-inde- 4-1BBL on the tumor cells to transduce the appropriate signal into pendent manner (31). Reverse signaling through 4-1BBL has also the cell after engagement with 4-1BBIg. At present, the mecha- been observed in monocytes, and this has been shown to deliver a nism for 4-1BBL signaling back into cells is unknown. The Journal of Immunology 2909

The ability of 4-1BBL expressed on the carcinoma cells to de- years (e.g., Refs. 17 and 18). Many reports have addressed the role liver a signal to T cells through 4-1BB was also evaluated. Because of FasL on tumor cells in host-tumor interaction (e.g., Refs. 19 and it has been reported that T cells treated with anti-CD3 and anti-4- 20), and these reports indicate that in some situations, FasL ex- 1BB mAb produce IFN-␥ (26), the ability of the tumor cells to pressed on tumors may be involved in the escape of tumors from stimulate T cells was assessed. Purified peripheral T cells were immune surveillance. The finding that carcinoma cells can express cocultured with HCT 116 cells, which showed high expression of 4-1BBL indicates that tumor cell/T cell interactions are indeed 4-1BBL, and this resulted in the release of IFN-␥ into the culture very complex. The role of 4-1BBL on carcinoma cells remains to medium. Similar but more variable results were obtained for the be fully elucidated. While treatment of tumor cells with 4-1BBIg induction of IL-2 (data not shown). Monocultures of T cells or increased IL-8 production, preliminary results showed that this tumor cells, with or without anti-CD3, produced only low levels of treatment had no major effect on the expression of 4-1BBL on the IFN-␥. Similarly, only low levels of IFN-␥ were found in super- cells. The effect of ligation of 4-1BBL on the tumor cells on sub- natants of cocultures of T cells and tumor cells in the absence of sequent MHC and costimulatory molecule expression and Ag pre- anti-CD3. This suggests that 4-1BBL on the carcinoma cells was sentation to T cells is currently under study. Among the various able to deliver a costimulatory signal, which also required activa- carcinoma cells, we have observed significant differences in the tion of the TCR. A comparison between IFN-␥ induction in the levels of MHC class I, CD80, and CD86. Preliminary analysis has coculture with the HT 29 tumor cell line (low expression of revealed no major changes in the levels of these surface molecules 4-1BBL), LX 1 cells (intermediate expression), and HCT 116 cells following stimulation of the cells with 4-1BBIg; however, this is (high expression) showed that the HCT 116 cells induced the most the subject of further studies. of cytokine, followed by LX 1 and HT 29, indicating that IFN-␥ Stimulation of IFN-␥ production in T cells by the tumor cells production was related to the level of 4-1BBL expressed on tumor through 4-1BB might be expected to both increase MHC expres- Downloaded from cells. Furthermore, IFN-␥ induction was dose dependent on the sion and to enhance T cell cytolytic activity, thus stimulating an number of the tumor cells in the cocultures. immune response. Indeed, Melero and coworkers (41) and Guinn The level of IFN-␥ produced by the T cells was markedly re- and coworkers (42) have shown that transfection of 4-1BBL into duced by the addition of neutralizing anti-4-1BB mAb to the cul- mastocytoma or lymphoma cells resulted in protective immunity tures, which suggests that binding of 4-1BBL was indeed respon- against these tumors. Thus, it is unclear why carcinoma cells sible for the induction of the cytokine in the cultures. Other 4-1BB would express 4-1BBL. It is possible that down-regulation of the http://www.jimmunol.org/ mAb enhanced rather than inhibited IFN-␥ production from iso- expression of the 4-1BBL occurs within the tumor; however, this lated T cells (data not shown); most likely, these mAb are able, seems unlikely because surface expression was detected on cells even when soluble, to activate directly the 4-1BB receptor on T from human tumors. Possibly, 4-1BBL signaling back into the tu- cells. No expression of the costimulatory molecules B7-1 and B7-2 mor cell may stimulate an as yet unrecognized pathway, which is could be detected on the HCT 116 cells by FACS analysis. In advantageous for the growth or survival of the carcinoma cell. addition, incorporation of B7-Ig into the cultures had no effect on Although reverse signaling through 4-1BBL induces apoptosis in IFN-␥ release (data not shown). These results indicate that the T lymphocytes in a Fas-independent manner (31), we did not see observed IFN-␥ release of T cells most likely was not due to a any induction of cell death in the cell lines mediated by 4-1BBIg by guest on October 1, 2021 pathway involving CD28/B7. under the specified culture conditions (data not shown). The ability of 4-1BBL on carcinoma cells to stimulate the T The overall outcome of the interaction between T cells and solid cells was confirmed by the transfection of COS cells with a con- tumors is dependent on the interplay between cell surface receptors struct containing the extracellular domain of 4-1BBL, with the and their ligands on the tumor cells. Our results indicate that the membrane spanning and cytoplasmic domains of CD40L. In the 4-1BB receptor/ligand system is likely to play a significant role in presence of anti-CD3, these transfectants, unlike the mock trans- this host-tumor interaction. fectants, were able to stimulate production of IFN-␥ in a T cell coculture assay. This induction of IFN-␥ by the 4-1BBL-trans- References fected COS cells was greatly reduced by addition of neutralizing 1. Smith, C. A., T. Farrah, and R. G. Goodwin. 1994. The TNF receptor superfamily anti-4-1BB mAb, confirming that 4-1BBL was responsible for the of cellular and viral proteins: activation, costimulation, and death. Cell 76:959. activation of the T cells. 2. Beutler, B., and C. van Huffel. 1994. Unraveling function in the TNF ligand and receptor families. Science 264:667. The delivery of a costimulatory signal has been shown to be 3. Mallett, S., and A. N. Barclay. 1991. A new superfamily of cell surface proteins critical for the activation of T cells and for the prevention of in- related to the nerve . Immunol. Today 12:220. duction of either cell death or an anergic state. Previous studies 4. Johnson, D., A. Lanahan, C. R. Buck, A. Sehgal, C. Morgan, E. Mercer, M. Bothwell, and M.Chao. 1986. Expression and structure of the human NGF have shown that, while unprimed T cells require 4–5 days to re- receptor. Cell 47:545. spond to anti-4-1BB Abs, only 24 h are required for T cells to 5. Loetscher, H., Y.-C. Pan, H.-W. Lahm, R. Gentz, M. Brockhaus, H. Tabuchi, and produce IL-2 in response to 4-1BBL in the absence of CD28 co- W. Lesslauer. 1990. Molecular cloning and expression of a receptor for human . Cell 61:361. stimulation (30). This suggests that the natural ligand for 4-1BB 6. Smith, C., T. Davis, D. Anderson, L. Solam, M. Beckman, R. Jerzy, S. Dower, provides a more potent signal than that delivered by Ab ligation. In D. Cosman, and R. Goodwin. 1990. A receptor for tumor necrosis factor defines our experiments, we were able to detect elevated levels of IFN-␥ an unusual family of cellular and viral proteins. Science 248:1019. 7. Stamenkovic, I., E. A. Clarc, and B. Seed. 1989. A B-lymphocyte activation in supernatants of resting T cells cocultured with the 4-1BBL- molecule related to the nerve growth factor receptor and induced by cytokines in expressing carcinoma cells in the presence of anti-CD3 as early as carcinomas. EMBO J. 8:1403. 8. Duerkop, H., U. Latza, M. Hummel, F. Eitelbach, B. Seed, and H. Stein. 1992. after 16 h, while strongest stimulation was seen after 48 h (data not Molecular cloning and expression of a new member of the nerve growth factor shown). A relatively high concentration of anti-CD3 (2 ␮g/ml) was receptor family that is characteristic for Hodgkin’s disease. Cell 68:421. needed in these experiments to provide optimal stimulation. 9. Camerini, D., G. Walz, W. A. Loenen, J. Borst, and B. Seed. 1991. The T cell activation antigen CD27 is a member of the nerve growth factor/tumor necrosis Saoulli and coworkers (30) have shown that with optimal signaling factor receptor gene family. J. Immunol. 147:3165. through the TCR, 4-1BBL was as effective as anti-CD28 Ab, while 10. Itoh, N., S. Yonehara, A. Ishii, M. Yonehara, S.-I. Mizushima, M. Sameshima, at low doses of anti-CD3, anti-CD28 was more effective. A. Hase, Y. Seto, and S. Nagata. 1991. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66:233. The expression and role of other members of the TNF family in 11. Kwon, B. S., and S. M. Weissman. 1989. cDNA sequences of two inducible tumor cell function have been intensively discussed in the recent T-cell genes. Proc. Natl. Acad. Sci. USA 86:1963. 2910 4-1BB LIGAND ON CARCINOMA CELLS

12. Beutler, B., and A. Cerami. 1989. The biology of cachectin/TNF-␣ primary me- 27. Takahashi, C., R. S. Mittler, and A. T. Vella. 1999. Cutting edge: 4-1BB is a bona diator of the host response. Annu. Rev. Immunol. 7:625. fide CD8 T cell survival signal. J. Immunol. 162:5037. 13. Armitage, R. J., W. C. Fanslow, L. Strockbine, T. A. Sato, K. N. Clifford, 28. DeBenedette, M. A., N. R. Chu, K. E. Pollok, J. C. Hurtado, W. S. Wade, B. M. Macduff, D. Anderson, S. G. Gimpel, T. Davis-Smith, and B. S. Kwon, and T. H. Watts. 1995. Role of 4-1BB ligand in costimulation of T C. R. Maliszewski. 1992. Molecular and biological characterization of a murine lymphocyte growth and its up-regulation on M12 B lymphoma by cAMP. J. Exp. ligand for CD40. Nature 357:80. Med. 181:985. 14. Goodwin, R. G., M. R. Alderson, C. A. Smith, R. J. Armitage, T. VandenBos, 29. Pollok, K. E., Y.-J. Kim, J. Hurtado, Z. Zhou, K. K. Kim, and B. S. Kwon.1994. R. Jerzy, T. W. Tough, M. A. Schoenborn, T. Davis-Smith, K. Hennen, et al. 4-1BB T cell antigen binds to mature B cells and macrophages and costimulates 1993. Molecular and biological characterization of a ligand for CD27 defines a anti ␮-primed splenic B cells. Eur. J. Immunol. 24:367. new family of cytokines with homology to tumor necrosis factor. Cell 75:1169. 30. Saoulli, K., S. Y. Lee, J. L. Cannons, W. C. Yeh, A. Santana, M. D. Goldstein, 15. Suda, T., T. Takahashi, P. Golstein, and S. Nagata. 1993. Molecular cloning and N. Bangia, M. A. DeBenedette, T. W. Mak, Y. Choi, and T. H. Watts. 1998. expression of the Fas ligand, a novel member of the tumor necrosis factor family. CD28-independent,TRAF2-dependent costimulation of resting T cells by 4-1BB Cell 75:1169. ligand. J. Exp. Med. 187:1849. 16. Alderson, M. R., C. A. Smith, T. W. Tough, T. Davis-Smith, R. J. Armitage, 31. Michel, J., S. Pauly, P. Langstein, P. H. Krammer, and H. Schwarz. 1999. B. Falk, E. Roux, E. Baker, G. R. Sutherland, W. S. Din, and R. G. Goodwin. CD137-induced apoptosis is independent of CD95. Immunology 98:42. 1994. Molecular and biological characterization of human 4-1BB and its ligand. Eur. J. Immunol. 24:2219. 32. Langstein, J., J. Michel, J. Fritsche, M. Kreutz, R. Andreesen, and H. Schwarz. 17. Paulie, S., B. Ehlin-Henriksson, H. Mellstedt, H. Koho, H. Ben-Aissa, and 1998. CD137 (ILA/4-1BB), a member of the TNF receptor family, induces P. Perlmann. 1985. A p50 surface antigen restricted to human urinary bladder monocyte activation via bi-directional signaling. J. Immunol. 160:2488. carcinomas and B lymphocytes. Cancer Immunol. Immunother. 20:23. 33. Rankin, B. M., S. A. Yocum, R. S. Mittler, and P. A. Kiener. 1993. Stimulation 18. Hahne, M., D. Rimoldi, M. Schroeter, P. Romero, M. Schreier, L. E. French, of tyrosine phosphorylation and calcium mobilization by Fc␥ receptor cross- P. Schneider, T. Bornand, A. Fonatan, D. Lienard, et al. 1996. Melanoma cell linking. J. Immunol. 150:605. expression of Fas (APO-1/CD95) ligand: implications for tumor immune escape. 34. Loo, D. T., N. J. Chalupny, J. Bajorath, W. W. Shuford, R. S. Mittler, and Science 274:1363. A. Aruffo. 1997. Analysis of 4-1BBL and laminin binding to murine 4-1BB, a 19. Walker, P. R., P. Saas, and P.-Y. Dietrich. 1997. Role of Fas ligand (CD95L) in member of the tumor necrosis factor receptor superfamily, and comparison with immune escape: the tumor cell strikes back. J. Immunol. 158:4521. human 4-1BB. J. Biol. Chem. 272:6448. 20. Shiraki, K., N. Tsuji, T. Shioda, K. J. Isselbacher, and H. Takahashi. 1997. Ex- 35. Linsley, P. S., J. L. Greene, W. Brady, J. Bajorath, J. A. Ledbetter, and R. Peach. Downloaded from pression of Fas ligand in liver metastases of human colonic adenocarcinomas. 1994. Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but Proc. Natl. Acad. Sci. USA 94:6420. distinct kinetics to CD28 and CTLA-4 receptors. Immunity 1:793. 21. Goodwin, R. G., W. S. Din, T. Davis-Smith, D. M. Anderson, S. D. Gimpel, 36. Hurtado, J. C., Y.-J. Kim, and B. S. Kwon. 1997. Signals through 4-1BB are T. A. Sato, C. R. Maliszewski, C. I. Brannan, N. G. Copeland, N. A. Jenkins, et costimulatory to previously activated splenic T cells and inhibit activation-in- al. 1993. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a duced cell death. J. Immunol. 158:2600. member of an emerging family of cytokines with homology to tumor necrosis 37. Van Essen, D., H. Kikutani, and D. Gray. 1995. CD40 ligand-transduced co- factor. Eur. J. Immunol. 23:2631. stimulation of T cells in the development of helper function. Nature 378:620.

22. Hurtado, J. C., S. H. Kim, K. E. Pollok, Z. H. Lee, and B. S. Kwon. 1995. http://www.jimmunol.org/ 38. Wiley, R. W., R. G. Goodwin, and C. A. Smith. 1996. Reverse signaling via Potential role of 4-1BB in T cell activation: comparison with the costimulatory CD30 ligand. J. Immunol. 157:3635. molecule CD28. J. Immunol. 155:3360. 23. DeBenedette, M. A., A. Shahinian, T. W. Mak, and T. H. Watts. 1997. Costimu- 39. Eckmann, L., H. C. Jung, C. Schuerer-Maly, A. Panja, E. Morzycka-Wroblewska, lation of CD28Ϫ T lymphocytes by 4-1BB ligand. J. Immunol. 158:551. and M. F. Kagnoff. 1993. Differential cytokine expression by human intestinal 24. Chu, N. R., M. A. DeBenedette, B. J. N. Stiernholm, B. H. Barber, and epithelial cell lines: regulated expression of interleukin-8. Gastroenterology 105: T. H. Watts. 1997. Role of IL-12 and 4-1BB ligand in cytokine production by 1689. CD28ϩ and CD28Ϫ T cells. J. Immunol. 158:3081. 40. Schuerer-Maly, C.-C., M. Eckmann, M. F. Kagnoff, M. T. Falco, and F.-E. Maly. 25. Melero, I., W. W. Shuford, S. A. Newby, A. Aruffo, J. A. Ledbetter, 1994. Colonic epithelial cell lines as a source of interleukin-8: stimulation by K. E. Hellstrom, R. S. Mittler, and L. Chen. 1997. Monoclonal antibodies against inflammatory cytokines and bacterial lipopolysaccharide. Immunology 81:85. the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med. 41. Melero, I., N. Bach, K. E. Hellstrom, A. Aruffo, R. S. Mittler, and L. Chen. 1998. 3:682. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB

26. Shuford, W. W., K. Klussman, D. D. Tritchler, D. T. Loo, J. Chalupny, ligand: synergy with the CD28 co-stimulatory pathway. Eur. J. Immunol. 28: by guest on October 1, 2021 A. W. Siadak, T. J. Brown, J. Emswiler, H. Raecho, C. P. Larsen, et al. 1997. 1116. 4-1BB costimulatory signals preferentially induce CD8ϩ T cell proliferation and 42. Guinn, B. A., M. A. DeBenedette, T. H. Watts, and N. L. Berinstein. 1999. lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 4-1BBL cooperates with B7-1 and B7-2 in converting a B cell lymphoma cell line 186:47. into a long-lasting antitumor vaccine. J. Immunol. 162:5003.