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Human Cancer Biology

Programmed Death Ligand 1 Is Expressed by Non–Hodgkin Lymphomas and Inhibits the Activity of Tumor-Associated T Cells

David J. Andorsky, Reiko E. Yamada, Jonathan Said, Geraldine S. Pinkus, David J. Betting and John M. Timmerman
DOI: 10.1158/1078-0432.CCR-10-2660 Published July 2011

Abstract

Purpose: Programmed death ligand 1 (PD-L1) is expressed on antigen-presenting cells and inhibits activation of T cells through its receptor PD-1. PD-L1 is aberrantly expressed on some epithelial malignancies and Hodgkin lymphomas and may prevent effective host antitumor immunity. The role of PD-L1 in non–Hodgkin lymphomas (NHL) is not well characterized.

Experimental Design: PD-L1 expression was analyzed in cell lines and lymphoma specimens by using flow cytometry and immunohistochemistry. Functional activity of PD-L1 was studied by incubating irradiated lymphoma cells with allogeneic T cells with or without anti-PD-L1 blocking antibody; T-cell proliferation and IFN-γ secretion served as measures of T-cell activation. Similar experiments were conducted using cultures of primary lymphoma specimens containing host T cells.

Results: PD-L1 was expressed uniformly by anaplastic large cell lymphoma (ALCL) cell lines, but rarely in B-cell NHL, confined to a subset of diffuse large B-cell lymphomas (DLBCL) with activated B-cell features (3 of 28 cell lines and 24% of primary DLBCL). Anti-PD-L1 blocking antibody boosted proliferation and IFN-γ secretion by allogeneic T cells responding to ALCL and DLBCL cells. In autologous cultures of primary ALCL and DLBCL, PD-L1 blockade enhanced secretion of inflammatory cytokines IFN-γ, granulocyte macrophage colony-stimulating factor, interleukin (IL)-1, IL-6, IL-8, IL-13, TNF-α, and macrophage inflammatory protein-1α. In establishing cell lines from an aggressive PD-L1+ mature B-cell lymphoma, we also noted that PD-L1 expression could be lost under certain in vitro culture conditions.

Conclusions: PD-L1 may thwart effective antitumor immune responses and represents an attractive target for lymphoma immunotherapy. Clin Cancer Res; 17(13); 4232–44. ©2011 AACR.

Translational Relevance

Impaired host immunity is thought to play a role in the pathogenesis and progression of lymphoma. Expression of the negative T-cell regulator programmed death ligand 1 (PD-L1) seems to facilitate immune tolerance of various carcinomas. Here, we describe the spectrum of expression of PD-L1 among non–Hodgkin lymphomas (NHL) and evaluate its functional activity in suppressing T-cell responses. In vitro experiments using established cell lines and primary lymphoma specimens show that both T-cell and B-cell lymphomas express biologically active PD-L1 and that suppression of tumor-associated T cells can be reversed by PD-L1 blockade. Among diffuse large B-cell lymphomas, the most common NHL in adults, we found that PD-L1 is expressed only in the nongerminal center subtype, which carries a poorer prognosis and frequently recurs after conventional chemoimmunotherapy. Our results suggest that targeting PD-L1 may be an effective antilymphoma immunotherapy for certain histologic subtypes.

Introduction

Programmed death 1 (PD-1), a member of the CD28 family, is an inhibitory receptor expressed on the surface of T cells that functions to physiologically limit T-cell activation and proliferation (1). Its ligand, PD-L1 (B7-H1/CD274), is expressed on antigen-presenting cells. Binding of PD-L1 to its receptor inhibits T-cell activation and counterbalances T-cell stimulatory signals, such as the binding of B7 to CD28.

Dysregulation of the PD-1/PD-L1 pathway has been implicated in a wide variety of diseases. Impairment of PD-1/PD-L1 signaling can lead to autoimmune disease in murine systems (2, 3). In humans, several single nucleotide polymorphisms in PD-1 have been associated with an increased risk of developing rheumatologic disease (4). Conversely, upregulation of PD-1 signaling is associated with the persistence of chronic infections, including HIV (5, 6), Helicobacter pylori infection (7), and schistosomiasis (8).

PD-L1 is not expressed by normal epithelial tissues, but it is aberrantly expressed on a wide array of human cancers (9). In this context, PD-L1 may promote cancer progression by disabling the host antitumor response. Its expression on tumor cells has been associated with poorer prognosis in renal cell carcinoma (10–12), breast cancer (13), Wilms' tumor (14), pancreatic cancer (15), ovarian cancer (16), urothelial cancer (17), gastric cancer (18), esophageal cancer (19), and hepatocellular carcinoma (20). In murine systems, melanoma cells engineered to express PD-L1 are resistant to cytotoxic T lymphocyte (CTL)-mediated lysis and exhibit more aggressive tumor growth than wild-type melanoma (21). Moreover, melanoma cells expressing PD-L1 can induce apoptosis in tumor-specific CTLs (9).

Compared with solid tumors, the spectrum of expression and biological activity of PD-L1 in lymphomas is incompletely characterized. Using immunohistochemistry, Brown and colleagues reported PD-L1 expression on 7 of 11 peripheral T-cell lymphomas and 0 of 16 B-cell non-Hodgkin lymphomas (NHL; ref. 22). PD-L1 was detected by reverse transcriptase–PCR in 5 anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) cell lines and by immunohistochemistry in 18 primary ALK+ ALCL specimens (23). Another series reported that PD-L1 was expressed in 4 of 14 diffuse large B-cell lymphomas (DLBCL), 0 of 9 T-cell lymphomas, and 8 of 13 classic Hodgkin lymphoma (HL) cases (24).

In this report, we describe the pattern of expression of PD-L1 in a large series of primary human lymphoma specimens (n = 110) and NHL cell lines (n = 34). Using both cell lines and primary tumor specimens, we show that PD-L1 expressed on tumor cells is immunologically active in suppressing the activation of tumor-associated T cells. These results suggest PD-L1 blockade as a potentially useful strategy for lymphoma immunotherapy.

Materials and Methods

Cell lines and clinical sample preparation

Raji, Ramos, and Daudi human Burkitt lymphoma, and Jurkat T-cell lymphoblastic leukemia cell lines were obtained from the American Type Culture Collection (ATCC). SU-DHL-1, SU-DHL-4, SU-DHL-6, SU-DHL-8, SU-DHL-9, SU-DHL-16, BCBL-1, Karpas 299, DEL, Hut78, and SUP-M2 were gifts from Dr. Linda Baum (UCLA, Los Angeles, CA). Granta-519, JeKo-1, and REC-1 were gifts from Dr. William Matsui (Johns Hopkins University, Baltimore, MD). OCI-Ly-2, -3, -7, -10, -19, HBL-1, SU-DHL-2, and U2932 were gifts from Dr. Louis Staudt (National Cancer Institute, Bethesda, MD). SU-DHL-5, SU-DHL-7, SU-DHL-10, NU-DHL-1, and USC-DHL-1 were gifts from Dr. Alan Epstein (University of Southern California, Los Angeles, CA). RC-K8 and MC116 cells were gifts from Dr. Izidore Lossos (University of Miami). BJA-B was a gift from Dr. Elliott Kieff (Harvard, Boston, MA). Unless otherwise specified, tumor cells were cultured in RPMI 1640 medium (Invitrogen) plus 10% heat-inactivated fetal calf serum (FCS; Omega Scientific), 100 units/mL penicillin/streptomycin, 2 mmol/L l-glutamine, and 50 μmol/L β-mercaptoethanol (“RPMI complete medium”; all supplements from Invitrogen), at 37°C in 5% CO2. SU-DHL-6 and SU-DHL-8 cells were cultured in RPMI complete medium plus 20% FCS. Hut78 cells were cultured in Isocove's modified Dulbecco's medium (IMDM; Invitrogen) plus 20% FCS. The OCI-Ly series, SU-DHL-2, U2932, and HBL-1 were cultured in IMDM complete medium plus 20% fresh human plasma (heparinized) instead of FCS.

Primary lymphoma specimens were obtained from lymph node biopsies or involved peripheral blood after written informed consent approved by the UCLA Institutional Review Board, enriched by Ficoll–Hypaque sedimentation (GE Healthcare), and cryopreserved in liquid nitrogen. For analysis, specimens were thawed quickly in a 37°C water bath and washed twice with warm RPMI complete medium before use.

Cytokine stimulation of B-cell lines

Ramos, Daudi, SU-DHL-4, and SU-DHL-6 cells were cultured in RPMI complete medium containing 2,000 RU/mL IFN-γ (R&D Systems), or 10 μg/mL CpG oligodeoxynucleotide (ODN) 10103 (sequence 5′-TCGTCGTTTTTCGGTCGTTTT-3′; Coley Pharmaceuticals Group) plus interleukin (IL)-4 at 2 ng/mL (R&D Systems) for 24 to 48 hours. Daudi, Ramos, SU-DHL-4, SU-DHL-6, OCI-Ly-3, and HBL-1 cells were cultured in complete medium containing IL-6, IL-10, or both at 50 ng/mL (R&D Systems) for 24 to 72 hours. PD-L1 expression was analyzed by flow cytometry.

Flow cytometry

Monoclonal antibodies (mAb) used to measure expression of cell surface markers by flow cytometry included phycoerythrin (PE)-conjugated anti-human PD-L1/B7-H1 (clone MIH1), PD-L2/B7-DC PE (clone MIH18), and PD-1 PE (clone MIH4) from eBioscience; and CD3 fluorescein isothiocyanate (FITC; clone HIT3a), CD3 PE (clone UCHT1), CD4 PE (clone RPA-T4), CD8 PE (clone RPA-T8), CD20 FITC (clone L27), CD30 PE or CD30 FITC (clone BerH8), EMA/CD227/MUC1 FITC (clone HMPV), and appropriate isotype controls, all from BD Biosciences. Stained tumor cells were analyzed using a BD FACSCaliber flow cytometer (BD Biosciences) with FCS Express software (De Novo Software).

Immunohistochemistry

Frozen sections were cut at 2 to 4 μm and immediately fixed in cold acetone for 20 minutes at 4°C. After air drying for 10 minutes, slides were incubated overnight with mouse anti-PD-L1 Ab (clone MIH1; eBioscience). Slides were then incubated with DakoCytomation Envision+ System horseradish peroxidase (HRP)-labeled polymer anti-mouse for 30 minutes (DAKO), followed by the diaminobenzidine (DAB) reaction. The sections were counterstained with hematoxylin.

For formalin-fixed specimens, histologic sections from paraffin-embedded tissue blocks were subjected to heat-induced epitope retrieval by using a steamer at 95°C for 25 minutes in 0.01 mol/L citrate buffer, pH 6.0 (for PD-L1), or at 115°C for 3 minutes in 0.1 mmol/L EDTA, pH 8.0, for CD10, BCL6, and MUM1. Sections were incubated with mouse mAbs to CD10 (Vector laboratories), BCL6 (DAKO), and MUM1 (DAKO), followed by antibody localization using the DakoCytomation Envision+ System HRP-labeled polymer (DAKO). After 10 minutes of incubation with DAB, sections were counterstained with hematoxylin. Staining for PD-L1 in paraffin sections was conducted using a mAb (clone 5H1, provided by Dr. Lieping Chen, Johns Hopkins University) at BioPillar Laboratories, using previously described methods (11) or using a polyclonal rabbit antiserum (Lifespan Biosciences), followed by DAKO DakoCytomation Envision+ HRP-labeled polymer anti-rabbit detection.

Allogeneic T-cell proliferation assays

T cells were enriched from whole blood obtained from healthy donors who gave informed consent, using the RosetteSep T-cell enrichment cocktail (StemCell Technologies), following the manufacturer's protocol. Enriched T cells (2 × 105) were cultured in RPMI complete medium at a 10:1 ratio with irradiated (3000R) Karpas 299 cells in 96-well U-bottom plates (Nunc). Cells were fed every 2 days with fresh medium containing 10 IU/mL IL-2 (Chiron). After 1 week, T cells were harvested, counted, and replated in quadruplicate with fresh Karpas 299 cells (irradiated 3000R) at effector:target (E:T) ratios of 2:1 and 1:1 with 2 × 104 tumor cells per well in 96-well U-bottom plates with or without 10 μg/mL anti-PD-L1 (clone MIH1) or mouse IgG1 isotype control mAbs (eBioscience). After 4 days, anti-PD-L1 and control mAbs were replenished before cells were pulsed with 1 μCi/well 3[H]-thymidine (MP Biomedicals); cells were harvested 16 hours later. Incorporated radioactivity (counts per minute, cpm) was measured using a β-liquid scintillation analyzer (PerkinElmer), and results from quadruplicate cultures reported as arithmetic means ± SD.

Derivation of PD-L1–expressing lymphoma cell lines LC-96 and RS-27

An 18-year-old women (LC-96) presented with rapidly progressive cervical and abdominal lymphadenopathy, ascites, and pleural effusions. Cervical lymph node biopsy confirmed ALK+ ALCL. Malignant ascites fluid was collected at therapeutic paracentesis, and then cells were isolated by centrifugation and cryopreserved. Cell-free ascites fluid was obtained by centrifugation and 0.45 μm filtration. A sample of unmanipulated ascites fluid was placed into immediate culture supplemented 1:1 with Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS, 100 units/mL penicillin/streptomycin, 2 mmol/L l-glutamine, and 50 μmol/L β-mercaptoethanol (Invitrogen) at 37°C in 5% CO2. As tumor cells slowly grew over a period of 1 month, supplementation of the culture with cell-free ascites fluid was gradually decreased (from 40% to 5%), until the resulting LC-96 cell line could grow in DMEM containing 15% FCS. The surface immunophenotypes of the primary ascites cells and the resulting LC-96 cell line were determined by flow cytometry as described above.

Peripheral blood mononuclear cells (PBMC) were obtained from a patient (RS-27) with peripheral blood involvement with an aggressive DLBCL. Flow cytometry showed a monomorphic B-cell population expressing CD19, CD20, CD22, and FMC7, with surface κ light chain restriction. The cells did not express BCL1, CD5, CD10, or CD38, and FISH was negative for t(11;14) and c-Myc translocations (data not shown). After Ficoll–Hypaque isolation, PBMCs were cryopreserved in liquid nitrogen. Thawed cells were initially cultured in DMEM complete medium containing 20% FCS plus 10% fresh human serum and 10% 0.45-μm-filtered LC-96 ascites fluid, as described in Results.

Cytokine analyses

For allogeneic experiments, supernatants from cocultures of Karpas 299 cells and healthy donor T cells, as described earlier under allogeneic T-cell proliferation assays, were collected after 4 days of incubation and analyzed for IFN-γ by ELISA (R&D Systems). Ninety-six-well Maxisorp plates (Nunc) were coated with mouse anti-human IFN-γ antibody and then washed and blocked with 1% BSA in PBS for 1 hour. Supernatants were added and incubated for 2 hours, followed by biotinylated goat anti-human IFN-γ antibody (50 ng/mL). Detection was conducted using streptavidin-conjugated HRP and hydrogen peroxide–tetramethylbenzidine substrate, and absorbance was determined at 450 nm/570 nm with a SPECTRAmax Plus 384 microplate reader (Molecular Devices). Recombinant human IFN-γ was used to generate a standard curve.

For autologous tumor cell–T-cell cocultures, cryopreserved LC-96 and RS-27 primary tumor specimens were thawed at 37°C, washed twice with warm RPMI complete medium, and 2–2.5 × 105 cells per well were plated in 6 replicates in a 96-well U-bottom plate in RPMI complete medium. Phytohemagluttinin (PHA; Sigma) was added at 0, 0.5, or 1 μg/mL with or without anti-PD-L1 or mouse IgG1 isotype control mAbs at 10 μg/mL. Cells were incubated for 5 days at 37°C in a 5% CO2 humidified incubator. Supernatants were collected and analyzed for IFN-γ by ELISA.

Cytokine multiplex analysis was conducted on cell-free malignant LC-96 ascites fluid, spent media from the LC-96 cell line, and primary LC-96 cells treated as above with PHA and anti-PD-L1 or isotype control antibody. Supernatants were analyzed for levels of 16 cytokines [granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-γ, IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, IL-13, thymus and activation regulated chemokine (TARC), IFN-α, stromal cell-derived factor-1β (SDF-1β), macrophage inflammatory protein-1α (MIP-1α), and monokine induced by interferon-γ (MIG)], using SearchLight protein array multiplex sandwich-ELISA (Pierce Biotechnology).

Results

PD-L1 is widely expressed by ALCL but uncommonly among B-cell NHL cell lines

We screened a large panel of human lymphoma cell lines for PD-L1 expression by flow cytometry (Table 1). Representative histograms are shown in Figure 1A. Three of the 28 B-cell lymphoma cell lines tested expressed PD-L1. Two of the positive lines (OCI-Ly-10 and HBL-1) have been classified as DLBCL of the activated B-cell (ABC) subtype on the basis of gene expression profiling (25), whereas the third (RC-K8) is known to have constitutive upregulation of the nuclear factor κB (NF-κB) pathway, a hallmark of the ABC phenotype (26). In contrast, 5 of 6 T-cell lymphoma cell lines exhibited expression of PD-L1, including all 4 ALCL lines tested. PD-L1 expression was strongest amongst the ALCL lines, with 3 of 4 lines showing 2-log increases over isotype controls, whereas Jurkat cells (T-cell acute lymphoblastic leukemia) had low-level expression of PD-L1, and Hut78 (Sezary syndrome) was negative for PD-L1. PD-L1 mRNA expression correlated with protein expression, with the highest levels found in ALCL and a subset of ABC DLBCL lines (Supplementary Table S1). In addition, all cell lines were screened for PD-1 and PD-L2 expression. Only Jurkat cells expressed PD-1 (data not shown), and no cell line expressed PD-L2.

Figure 1.
Figure 1.

High-level PD-L1 expression among ALCL but not among most B-cell NHL cell lines. A, flow cytometric analysis of PD-L1 expression is shown for 8 representative NHL cell lines (among 34 described in Table 1). Consistent high-level PD-L1 expression is a feature of ALCL cell lines but not of B-cell lines. B, low-level expression of PD-L1 and PD-L2 is induced in Ramos B-cell lymphoma cells after incubation with CpG plus IL-4 for 48 hours. Bold black line represents PD-L1 or PD-L2 staining, blue line isotype control, and shaded histogram unstained.

View this table:
Table 1.

Expression of PD-L1 among 34 human lymphoma cell lines

We then attempted to induce PD-L1 expression in B-cell lymphoma lines that did not constitutively express it, as stimulation of some tumor cells with IFN-γ can induce PD-L1 expression (9,27). Ramos, Daudi, SU-DHL-4, and SU-DHL-6 were incubated with IFN-γ or CpG plus IL-4 for 24 to 48 hours, and PD-L1 expression was monitored by flow cytometry. No cell line responded to IFN-γ, and only the Ramos cell line showed modestly increased PD-L1 expression 48 hours after stimulation with CpG plus IL-4 (Fig. 1B). In ALCL, PD-L1 expression is induced by STAT3 signaling (23), and IL-6 and IL-10 are both potent inducers of STAT3 (25). Therefore, we asked whether IL-6 and IL-10 stimulation of NHL lines could upregulate PD-L1. Daudi, Ramos, SU-DHL-4, SU-DHL-6, OCI-Ly-3, and HBL-1 cells were cultured with IL-6, IL-10, or both at 50 ng/mL for 24 to 72 hours, but none showed significant increase in PD-L1 expression (data not shown). Thus, PD-L1 expression is not readily altered in cultured lymphoma cells by exogenous cytokines.

PD-L1 is expressed by a subset of primary human DLBCLs

We next tested 68 lymphoma tissue specimens for expression of PD-L1 [Table 2 (A and B)]. Thirty-three DLBCLs, 3 primary mediastinal B-cell lymphomas (PMBCL) and 9 HLs were analyzed by immunohistochemistry in frozen specimens. Single-cell suspensions of 23 additional B-cell NHL specimens, including 16 follicular lymphomas (FL), were analyzed by flow cytometry. Expression of PD-L1 among B-cell NHL specimens was heterogeneous. Twenty-seven percent of DLBCL specimens showed expression of PD-L1. In contrast, all 3 PMBCL specimens were PD-L1+. PD-L1 was not expressed in any cases of FL (n = 16), small lymphocytic lymphoma (n = 2), marginal zone lymphoma (n = 3), or single cases of Burkitt or mantle cell lymphoma. Eight of 9 HLs expressed PD-L1 in Reed–Sternberg cells, in concordance with previous observations (28).

View this table:
Table 2.

Expression of PD-L1 in primary lymphoma tissue specimens

DLBCLs were classified into germinal-center B (GCB) or non-GCB subtype on the basis of the immunohistochemical markers CD10, BCL6, and MUM1, which correlate with cell of origin subtype as determined by gene expression profiling (29). Of 33 evaluable frozen cases, 19 were GCB and 14 were non-GCB. Only 1 of the GCB tumors expressed PD-L1. In contrast, 8 of 14 (57%) of non-GCB tumors expressed PD-L1 (P = 0.0004 by Fisher's exact test). This pattern of expression parallels that seen in our cell lines, where PD-L1 expression was found in 3 of 6 DLBCL lines with ABC features but in 0 of 7 GCB DLBCL cell lines (Table 1).

We next conducted immunohistochemistry for PD-L1 expression in a separate set of 42 formalin-fixed, paraffin-embedded lymphoma specimens [Table 2 (C)], which required a different mAb (5H1), previously used to stain PD-L1 in paraffin sections (10, 30). Cases were considered positive when the majority of tumor cells stained for PD-L1. All 7 FLs were negative for PD-L1, whereas 4 of 5 ALCLs and 6 of 30 DLBCLs were positive for PD-L1. Representative images are shown in Supplementary Figure S1. Of note, tumor-associated histiocytes stained positive for PD-L1 in 9 of 24 (38%) DLBCLs in which tumor cells were negative. Similarly, PD-L1+ histiocytes were also found in FLs surrounding tumor cell follicles. Of DLBCL specimens, 11 were GCB and 19 were non-GCB. None of the GCB DLBCL stained for PD-L1, whereas 6 (32%) of the non-GCB DLBCLs were positive for PD-L1 (P = 0.061), consistent with results we obtained in frozen sections. Comparative results of the frozen and paraffin DLBCL series are shown in Table 2 (D).

Of note, we also stained 91 formalin-fixed, paraffin-embedded lymphoma specimens, using a polyclonal rabbit anti-PD-L1 antiserum (Lifespan Biosciences). Using this methodology (31), 18 of 22 (82%) DLBCLs expressed PD-L1 (data not shown). Because of discordance between paraffin and frozen section results, we tested the polyclonal anti-PD-L1 antibody on cell pellets of several PD-L1–negative B-cell lines and a Daudi lymphoma xenograft. As several of these negative controls stained positive, we concluded that this antibody was unreliable for detecting PD-L1 expression in lymphomas.

PD-L1 expressed by ALCL inhibits the proliferation and cytokine secretion of allogeneic T cells

We next asked whether PD-L1 expressed by lymphoma cells was biologically active in attenuating host immune responses. Because PD-L1 was strongly expressed in both ALCL cell lines and tumor specimens, we chose this as our initial in vitro model. We hypothesized that antibody blockade of PD-L1 would result in greater T-cell activity, showing that the presence of PD-L1 on target tumor cells serves to inhibit T-cell responses. In cultures of donor allogeneic T cells primed for 7 days with irradiated ALCL cells, both T-cell proliferation and IFN-γ secretion were markedly increased in the presence of a blocking anti-PD-L1 antibody (Fig. 2A). In contrast, anti-PD-L1 did not alter proliferation or IFN-γ secretion of T cells incubated in the absence of tumor targets. As a control, irradiated SU-DHL-4 cells, which do not express PD-L1, were used as targets. In this case, PD-L1 blockade did not alter the degree of T-cell proliferation or IFN-γ secretion (Fig. 2B). Even in 5-day cultures of unprimed normal donor T cells plus irradiated ALCL target cells, IFN-γ secretion was uniformly increased in the presence of anti-PD-L1 (Fig. 2C). The differences seen in T-cell proliferation were smaller and not consistently statistically significant. Nonetheless, these results show functional expression of immunosuppressive PD-L1 by ALCL cells.

Figure 2.
Figure 2.

PD-L1 blockade enhances the activation of T cells cocultured with allogeneic ALCL cells. A, irradiated Karpas 299 ALCL cells (PD-L1+), were stimulated for 1 week with T cells from 2 healthy donors and then incubated with freshly irradiated Karpas 299 target cells at 2:1 and 1:1 E:T ratios in the presence of media alone, anti-PD-L1 antibody, or control antibody. After 4 days, supernatants were collected for IFN-γ measurement (bottom), or antibody was replenished and cells pulsed with [3H]-thymidine overnight to measure T-cell proliferation (top). Data are represented as mean ± SD of quadruplicate cultures. P values shown are for anti-PD-L1 antibody versus isotype control antibody by one-way ANOVA. B, PD-L1 blockade does not affect T cells incubated with PD-L1–negative tumor cells. Incubation of healthy donor T cells with a B-cell lymphoma line that does not express PD-L1 (SU-DHL-4) show that anti-PD-L1 antibody does not significantly alter allospecific proliferation (top) or IFN-γ secretion (bottom). C, PD-L1 blockade augments activation of allogeneic T cells directly stimulated with ALCL cells. Irradiated Karpas 299 cells were incubated for 5 days with T cells from 3 healthy donors at 4:1 and 2:1 E:T ratios in the presence of anti-PD-L1 antibody, control antibody, or media alone. Supernatants were collected for IFN-γ measurement by ELISA (bottom) or cells were pulsed with [3H]-thymidine overnight to measure T-cell proliferation (top). Proliferation data are represented as mean ± SD of quadruplicate cultures. P values shown are for anti-PD-L1 antibody versus isotype control antibody.

PD-L1 expression by primary ALCL attenuates the activity of tumor-associated T cells

To further study tumor–T-cell interactions, cryopreserved malignant ascites from a patient with newly diagnosed ALK+ ALCL (LC-96; see Materials and Methods), containing approximately equivalent proportions of PD-L1–expressing tumor cells and tumor-associated T cells, was used as an autologous system (Fig. 3A). The primary tumor cells within the ascites (PD-L1+, EMA+, and CD30+) were associated with a mixture of CD4+ and CD8+ T cells.

Figure 3.
Figure 3.

PD-L1 blockade enhances the activation of T cells in the presence of autologous ALCL cells that express PD-L1. A, immunophenotyping of malignant ascites from a patient with newly diagnosed ALK+ ALCL and cultured cell line (LC-96) from the same patient. The top 2 rows show results for the primary tumor (ascites), with far left panel displaying forward and side scatter plots for analysis of tumor (large cell) and lymphocyte (small cell) gates. The red curve in each histogram represents the surface marker, and unstained cells are shown in gray. Tumor cells (top row) were mostly negative for CD3, CD4, CD8, and PD-1 but expressed PD-L1, EMA, and CD30. The lymphocyte gate (middle row) contains predominantly CD3+ T cells, with a mixture of CD4+ and CD8+ T cells having low-level expression of PD-1, PD-L1, and CD30. The immunophenotype of the derived LC-96 cell line is shown in the bottom row. Tumor cells are strongly positive for PD-L1, EMA, and CD30, without expression of CD3, CD4, CD8, or PD-1. B, LC-96 primary ascites cells, containing approximately equivalent proportions of PD-L1–expressing tumor cells and tumor-associated T cells, were incubated for 5 days with PHA to activate T cells in the presence of media alone, anti-PD-L1 antibody, or isotype control antibody. In the presence of 0.5 or 1.0 μg/mL PHA, cells incubated with anti-PD-L1 antibody secreted more IFN-γ than controls (P < 0.0001 by one-way ANOVA). Results shown are representative of 3 independent experiments.

Cells from the ascites were incubated for 5 days with anti-PD-L1, isotype control antibody, or media alone, plus different concentrations of PHA, to serve as a polyclonal T-cell activator, and supernatants were assayed for IFN-γ secretion as an indicator of T-cell stimulation (Fig. 3B). Without the addition of PHA (media alone), even with the addition of IL-2 (10 μg/mL), no IFN-γ secretion was seen. Yet in the presence of PHA (0.5 or 1.0 μg/mL), anti-PD-L1 provoked a marked increase in IFN-γ secretion (P < 0.0001 by one-way ANOVA compared with isotype control antibody or media alone). Thus, PD-L1 expressed by fresh, primary ALCL cells can suppress the function of tumor-associated autologous T cells.

Ascites cells were serially passaged in cell culture to derive a new ALCL cell line, designated LC-96 (see Materials and Methods). The immunophenotype of the LC-96 cell line mirrored that of the primary ascites tumor cells (Fig. 3A, bottom), with strong expression of PD-L1, EMA, and CD30 but without expression of PD-1, CD3, CD4, or CD8. FISH analysis revealed a t(2;5)(p23;q35) nucleophosmin (NPM)–ALK translocation, which was also observed in the primary clinical specimen (data not shown). When LC-96 cells were incubated with PHA, with or without anti-PD-L1 antibody, there was no secretion of IFN-γ (data not shown), showing that T cells and not tumor cells are the source of IFN-γ in the primary ascites cultures after PD-L1 blockade.

To further characterize the T-cell response induced by PD-L1 blockade, we quantitated the secretion of 16 cytokines by using multiplex ELISA. First, we measured the cytokines present in cell-free ascites fluid, which would reflect the tumor environment in situ (Supplementary Fig. S2A). Interestingly, the fluid contained high levels of IL-6, as well as IL-10, and SDF-1β. We next surveyed cytokines in spent culture media from the established LC-96 cell line (Supplementary Fig. S2B) to discern which might be products of tumor cells themselves. High levels of IL-8, IL-10, and SDF-1β were observed, indicating these as likely products of primary tumor cells in vivo.

Next, primary ascites cells were incubated with or without PHA and anti-PD-L1 antibody, and the cytokine profile determined (Supplementary Fig. S2C). Without PHA, most cytokines were secreted at low levels. However, the addition of anti-PD-L1 resulted in increased secretion of IL-6, IL-8, TNF-α, and MIP-1α compared with control antibody or media alone. The addition of PHA resulted in further enhancement of cytokine secretion, including GM-CSF, IFN-γ, IL-1, IL-6, IL-8, TNF-α, IL-13, and MIP-1α. Levels of IL-2, IL-4, IL-5, IL-10, IFN-α, TARC, SDF-1β, and MIG were not altered by PD-L1 blockade (data not shown).

Functional PD-L1 expression by an aggressive primary B-cell lymphoma

To show that PD-L1 can also be immunologically active when expressed by B-cell lymphomas, analogous experiments were conducted using a tumor sample from a patient with an aggressive DLBCL (RS-27), which lacked expression of CD10 and BCL6, consistent with non-GCB phenotype. Circulating tumor cells strongly coexpressed PD-L1 and CD20, as measured by flow cytometry (Fig. 4A). Unmanipulated RS-27 PBMCs containing approximately 75% tumor cells and 20% CD3+ T cells (Fig. 4A) were cultured with PHA in the presence or absence of anti-PD-L1. After 5 days, supernatants were assayed for IFN-γ secretion (Fig. 4B). As described above with the LC-96 ALCL tumor cell–T-cell mixture, PD-L1 blockade resulted in increased IFN-γ secretion by tumor-associated T cells (P = 0.009 by one-way ANOVA), indicating functional inhibition of T cells by PD-L1 expressed by the B-cell lymphoma.

Figure 4.
Figure 4.

PD-L1 expressed in primary B-cell lymphoma is biologically active but readily lost during in vitro culture. A, PBMCs from a patient with aggressive mature B-cell lymphoma were analyzed by flow cytometry. Malignant cells coexpress CD20 and PD-L1 and comprise 75% of the total. The specimen also contains 11% CD4+ T cells and 9% CD8+ T cells. B, PD-L1 blockade enhances the activation of T cells in the presence of autologous B-cell lymphoma expressing PD-L1. Patient PBMCs were incubated in triplicate for 5 days with PHA 0.5 μg/mL in the presence of media alone, anti-PD-L1 antibody, or isotype control antibody, and IFN-γ was measured in supernatants by ELISA. Data are represented as mean ± SD of triplicate cultures. Results are representative of 2 independent experiments. C, PD-L1 expression by primary B-cell lymphoma can be lost during serial in vitro passage. Primary tumor cells obtained directly from peripheral blood coexpress PD-L1 and CD20. Cells were initially cultured in vitro with human serum, and weaned either rapidly or gradually to medium containing FCS, while monitoring PD-L1 and CD20 expression by flow cytometry. D, PD-L1 expression may be attenuated on the basis of cell culture conditions. OCI-Ly-10 cells, which express PD-L1, were cultured in 20% human plasma or 20% FCS. PD-L1 expression was substantially lower when the cells were grown in 20% FCS (MFI = 72) than in 20% human plasma (MFI = 465).

PD-L1 expression may be lost or attenuated during serial in vitro passage of lymphoma cells

In deriving the new RS-27 B-cell lymphoma line, we discovered that expression of PD-L1 could be lost during in vitro culture (Fig. 4C). After thawing, primary tumor cells from patient RS-27 were initially cultured in medium containing 20% FCS plus 10% fresh human serum and 10% 0.45-μm-filtered LC-96 ascites fluid (as a source of lymphoma-derived growth factors). Tumor cells slowly expanded under these conditions, and when weaned slowly from LC-96 ascites and fresh human serum, continued to express high levels of PD-L1 and CD20. However, if cells were weaned rapidly (over 2 weeks) into media containing 20% FCS and 5% pooled human AB serum, PD-L1 expression was almost entirely lost. Accordingly, T-cell proliferation and IFN-γ production were only increased by PD-L1 blockade when allogeneic T cells were incubated with RS-27 PD-L1–positive cells (Supplementary Fig. S3). Culture of PD-L1–negative RS-27 cells for 48 hours in media containing 10% fresh human serum, or CpG plus IL-4, could not restore PD-L1 expression (data not shown). Thus, PD-L1 expression by B-cell lymphomas can easily be lost upon tumor cell establishment and serial passage in vitro.

We also observed attenuation of PD-L1 expression under different culture conditions in the established OCI-Ly-10 ABC DLBCL cell line (Fig. 4D). When grown in media supplemented with 20% human plasma, the cells displayed bright expression of PD-L1 [mean fluorescence intensity (MFI) = 465]. However, when the same cells were transferred to media containing 20% FCS, the cells appeared less healthy, as evidenced by increased numbers of dead cells with lower forward scatter, and PD-L1 expression diminished in the viable cells (MFI = 72). When the cells were transferred back to 20% human plasma, PD-L1 expression returned to its previous level (MFI = 664; data not shown). Therefore, culture conditions can alter the expression of PD-L1 even among well-established lymphoma cell lines.

Discussion

Cancers use multiple mechanisms to thwart endogenous host antitumor immunity (32). While accumulating data indicate that expression of the negative T-cell regulatory molecule PD-L1 by tumor cells or tumor-associated antigen-presenting cells represents an important pathway whereby cancers evade host immunity (1), only limited data have been available regarding the expression of PD-L1 among common NHL subtypes and its ability to suppress autologous T-cell functions.

We studied the spectrum of PD-L1 expression among human lymphomas and showed its capacity to impair the function of tumor-associated T cells in both T- and B-cell lymphomas. This is the largest reported series of PD-L1 expression in human lymphoma cell lines and primary tumors and encompasses the most common B-cell NHL subtypes. We observed near uniform expression of PD-L1 in ALCL cell lines and primary tumors. In contrast, we found that among B-cell lymphomas, PD-L1 expression is essentially confined to a subset of the clinically important ABC/non-GCB subtype of DLBCL. We further showed that PD-L1 expressed by lymphoma cells is biologically active, with antagonist antibody blockade resulting in increased activation of adjacent T cells. This was true in allogeneic models using ALCL or DLBCL tumor cells as targets, as well as in primary tumor specimens of ALCL and DLBCL containing mixtures of lymphoma cells and autologous lymphocytes. Further studies will be required to confirm whether PD-L1 blockade results in similar effects in primary NHL specimens from lymph nodes or other extranodal sites of tumor.

Our observations regarding the pattern of expression of PD-L1 in human lymphomas are consistent with other reports in smaller series. Brown and colleagues reported PD-L1 expression in 7 of 11 peripheral T-cell lymphomas, including ALCL, and in 0 of 16 B-cell NHLs (22). Marzec and colleagues reported PD-L1 staining in 100% of 18 ALCLs (23), although the polyclonal anti-PD-L1 antibody used in this study possibly yielded false-positive cases (see later). Wilcox and colleagues reported PD-L1 expression in 15% of 131 T-cell lymphomas, including 3 of 9 ALCLs (30). Xerri and colleagues reported PD-L1 expression in 4 of 14 DLBCLs (including 2 PMBCLs) but not in follicular (n = 8), mantle cell (n = 4), marginal zone (n = 4), or Burkitt (n = 3) lymphomas (24), similar to our own results. PD-L1 is frequently expressed in HL within Hodgkin and Reed–Sternberg cells, as reported in a series of 4 cases by Yamamoto and colleagues (28), consistent with our findings. PD-L1 expression by PMBCL is not surprising, as gene expression profiling has revealed increased PD-L1 and PD-L2 mRNAs in this lymphoma and given the close biologic relationship of this disease to HL (33).

It seems that PD-L1 expression in B-cell lymphomas is uncommon. Interestingly, we found that among DLBCL cell lines and primary tumor specimens, PD-L1 expression was almost entirely confined to the ABC/non-GCB subtype (Tables 1 and 2). The ABC DLBCL subtype identified by gene expression profiling is associated with inferior survival compared with the GCB subtype (34), even in cohorts of patients treated with rituximab (35, 36). ABC DLBCL is characterized biologically by upregulation of NF-κB (37), but numerous other differences from GCB DLBCL exist. It is possible that PD-L1 expression is one of several “virulence factors” that lead to the inferior prognosis among ABC DLBCLs, and we suggest this hypothesis be tested in a large series of molecularly classified DLBCLs with associated clinical outcome data. The aggressive B-cell lymphoma from which we derived the PD-L1+ RS-27 cell line (Fig. 4) was indeed virulent; the patient died of central nervous system disease despite high-dose chemotherapy and allogeneic stem cell transplantation. Finally, the low frequency of PD-L1 expression we observed in DLBCL cell lines (50% among ABC-type; Table 1) may underestimate the true prevalence in primary DLBCL. In establishing the RS-27 cell line, we observed that PD-L1 expression was easily lost during serial passage of the cells. Thus, loss of PD-L1 expression may have occurred during the establishment of other human lymphoma cell lines, as seems to be the case in melanoma. Although virtually all primary melanomas express PD-L1, most melanoma cell lines do not (9).

PD-L1 expressed by leukocytes within the tumor microenvironment may play a role in host immune suppression even when not expressed by the tumor cells themselves. We observed that 38% of PD-L1 negative DLBCLs were infiltrated by PD-L1+ histiocytes. Increased numbers of lymphoma-associated macrophages have been associated with worse prognosis in FL (38) and HL (39), although the same association is not seen in DLBCL (40, 41). Thus, further studies examining the relationships between lymphoma-associated macrophages, PD-L1 expression, and clinical outcome are warranted. For such studies, it will be important to utilize the mAbs MIH1 or 5H1. In our experience, the polyclonal anti-PD-L1 antisera used by Marzec and colleagues (23) seems to be less specific for PD-L1 than reported (see Results).

Several investigators have also shown that the PD-1/PD-L1 axis can also influence the function of lymphoma-infiltrating T cells in cases of PD-L1–negative human lymphomas. Yang and colleagues found that B-cell lymphoma–associated Treg cells could express PD-L1 and suppress the function of PD-1+ tumor–associated T cells, an effect partially reversible by PD-L1 blockade (42). Similarly, Nattamai and Neelapu found that PD-1 was markedly upregulated on tumor-derived and peripheral blood T cells in FL in association with impaired Th1 cytokine secretion (43). Antibody blockade of PD-1 improved proliferation of tumor-derived T cells and promoted the activation of natural killer cells (44).

As in other series, we found near uniform expression of PD-L1 in ALCL, a rare but clinically distinct T-cell neoplasm. Nonetheless, PD-L1 is not a feature of all peripheral T-cell lymphomas. Wilcox and colleagues (30) found that PD-L1 was expressed by T-cell lymphoma tumor cells in only a minority of cases, yet often expressed by tumor-associated stromal histiocytes, just as we observed in DLBCL. Our report adds to these findings in showing the ability of PD-L1 on ALCL and DLBCL cells to suppress the responses of both allogeneic and autologous tumor-associated T cells (Figs. 2–4 and Supplementary Figs. S2 and S3).

In our cultures of primary ALCL ascites cells containing autologous T cells, PD-L1 blockade enhanced the production of IFN-γ, as well as GM-CSF, IL-1, IL-6, IL-8, TNF-α, IL-13, and MIP-1α. We also noted that the ascites fluid representing the tumor microenvironment of this case contained high levels of IL-6, IL-10, and SDF-1β whereas the established LC-96 cell line secreted these same cytokines, plus IL-8. These cytokines likely play roles in the pathogenesis and clinical manifestations of ALCL, having been detected in ALCL and other lymphomas (45–48). Intriguingly, SDF-1 (CXCL12) is associated with cancer metastasis (49) and angiogenesis (41), and as most ALCL express the SDF-1 receptor CXCR4 (50), this suggests a possible autocrine feedback loop in this lymphoma subtype.

In conclusion, the current work adds to a growing body of literature documenting the important role of PD-1/PD-L1 signaling in the pathogenesis of NHL. PD-L1 is highly expressed in ALCL, HL, and some poor prognosis DLBCLs of the ABC/non-GCB subtype, where it acts to negatively regulate adjacent T cells. Targeting the PD-1/PD-L1 pathway using antagonistic mAbs may thus be an attractive approach to lymphoma immunotherapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

The study is supported by a grant from Gabrielle's Angel Foundation for Cancer Research (to J.M. Timmerman) and an American Society of Clinical Oncology Young Investigator Award (to D.J. Andorsky). J.M. Timmerman is a Damon Runyon Clinical Investigator supported in part by the Damon Runyon Cancer Research Foundation (CI-26-05).

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.

Authors' Contributions

D.J. Andorsky and R.E. Yamada contributed equally in designing and conducting research, analyzing data, and writing the manuscript. J. Said designed and conducted research and analyzed data. G.S. Pinkus designed and conducted research. D.J. Betting designed and conducted research and analyzed data. J.M. Timmerman designed research and wrote the manuscript.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • D.J. Andorsky and R.E. Yamada are co-first authors.

  • Received October 1, 2010.
  • Revision received February 28, 2011.
  • Accepted April 13, 2011.

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Clinical Cancer Research: 17 (13)
July 2011
Volume 17, Issue 13

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Programmed Death Ligand 1 Is Expressed by Non–Hodgkin Lymphomas and Inhibits the Activity of Tumor-Associated T Cells
David J. Andorsky, Reiko E. Yamada, Jonathan Said, Geraldine S. Pinkus, David J. Betting and John M. Timmerman
Clin Cancer Res July 1 2011 (17) (13) 4232-4244; DOI: 10.1158/1078-0432.CCR-10-2660
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