Programmed death ligand 1 (PD-L1) is expressed by non...

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Programmed death ligand 1 (PD-L1) is expressed by non-Hodgkin lymphomas

and inhibits the activity of tumor-associated T cells

*David J. Andorsky,1 *Reiko E. Yamada,1 Jonathan Said,2 Geraldine S. Pinkus,3

David J. Betting,1 and John M. Timmerman1

1Division of Hematology & Oncology, Department of Medicine and 2Department of Pathology

& Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, 3Department

of Pathology, Harvard Medical School, Boston, MA.

*D.J.A. and R.E.Y. are co-first authors.

Running Title: Functional PD-L1 expression by non-Hodgkin lymphomas

Key Words: lymphoma, programmed death ligand 1, immunotherapy, T cells, immunomodulation Grant Support Supported by a grant from Gabrielle's Angel Foundation for Cancer Research (to JMT) and an American Society of Clinical Oncology Young Investigator Award (to DJA). JMT is a Damon Runyon Clinical Investigator supported in part by the Damon Runyon Cancer Research Foundation (CI-26-05). Address correspondence to: John M. Timmerman Division of Hematology & Oncology Center for Health Sciences, Room 42-121 10833 LeConte Avenue University of California, Los Angeles Medical Center Los Angeles, CA 90095-1678 Phone: 310-794-4820 Fax: 310-206-5511 Email: [email protected] Authors’ Contributions D.J.A. and R.E.Y. contributed equally in designing and performing research, analyzing data, and writing the paper. J.S. designed and performed research, and analyzed data. G.S.P. designed and performed research. D.J.B. designed and performed research, and analyzed data. J.M.T. designed research and wrote the paper. Disclosure of Potential Conflicts of Interest None to declare

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Translational statement Impaired host immunity is thought to play a role in the pathogenesis and progression of

lymphoma. Expression of the negative T cell regulator PD-L1 appears to facilitate immune

tolerance of various carcinomas. Here, we describe the spectrum of expression of PD-L1 among

non-Hodgkin lymphomas and evaluate its functional activity in suppressing T cell responses. In

vitro experiments using established cell lines and primary lymphoma specimens demonstrate 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 non-Hodgkin lymphoma in adults, we found that PD-L1 is

expressed only in the non-germinal center subtype, which carries a poorer prognosis and

frequently recurs after conventional chemoimmunotherapy. Our results suggest that targeting

PD-L1 may be an effective anti-lymphoma immunotherapy for certain histologic subtypes.




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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 anti-tumor

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

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 performed 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, GM-CSF, IL-1, IL-6, IL-8, IL-13, TNF-α,

and MIP-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 anti-tumor immune responses and represents an

attractive target for lymphoma immunotherapy.




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Introduction

PD-1, a member of the CD28 family, is an inhibitory receptor expressed on the surface of

T cells which 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 rheumatological disease.4 Conversely, upregulation of PD-1 signaling

is associated with the persistence of chronic infections, including HIV,5,6 Helicobacter pylori,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 anti-tumor 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 to solid tumors, the spectrum of expression and biological activity of PD-L1 in

lymphomas is incompletely characterized. Using immunohistochemistry, Brown et al reported

PD-L1 expression on 7 of 11 peripheral T cell lymphomas and 0 of 16 B cell non-Hodgkin




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lymphomas (NHL).22 PD-L1 was detected by RT-PCR in 5 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 demonstrate 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.




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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 (Manassas, VA).

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, SUDHL-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, Miami, FL). BJA-B was a gift from Dr. Elliott Kieff (Harvard, Boston,

MA). Unless otherwise specified, tumor cells were cultured in RPMI 1640 medium (Invitrogen,

Carlsbad, CA) plus 10% heat-inactivated fetal calf serum (FCS)(Omega Scientific, Tarzana,

CA), 100 Units/ml penicillin/streptomycin, 2 mM L-glutamine, and 50 μM 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, Piscataway, NJ), and




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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 IFN-γ 2000 RU/ml (R&D Systems, Minneapolis, MN), or CpG

oligodeoxynucleotide (ODN) 10103 10 μg/ml (sequence 5’-TCGTCGTTTTTCGGTCGTTTT-

3’)(Coley Pharmaceuticals Group, Wellesley, MA) plus IL-4 at 2 ng/ml (R&D Systems) for 24-

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 (mAbs) used to measure expression of cell surface markers by

flow cytometry included: Phycoerythrin (PE)-conjugated anti-human PD-L1/B7-H1 (clone

M1H1), PD-L2/B7-DC PE (clone M1H18), and PD-1 PE (clone M1H4) from eBioscience (San

Diego, CA); 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 (Carlsbad, CA). Stained tumor cells were analyzed using a

BD FACSCaliber flow cytometer (BD Biosciences) with FCS Express software (De Novo

Software, Los Angeles, CA).




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Immunohistochemistry

Frozen sections were cut at 2-4 microns 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 labeled polymer horseradish peroxidase (HRP)-anti-mouse for 30 minutes

(DAKO, Carpinteria, CA), 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 using a steamer at 95˚C for 25 minutes in 0.01

M citrate buffer, pH 6.0 (for PD-L1), or at 115˚C for 3 minutes in 0.1 mM EDTA pH 8.0 for

CD10, BCL6 and MUM1. Sections were incubated with mouse mAbs to CD10 (Vector

laboratories, Burlingame, CA), BCL6 (DAKO), and MUM1 (DAKO), followed by antibody

localization using the DakoCytomation Envision+ System-HRP-labeled polymer (DAKO).

After a 10 minute incubation with DAB, sections were counterstained with hematoxylin.

Staining for PD-L1 in paraffin sections was performed using a mAb (clone 5H1, provided by Dr.

Lieping Chen, Johns Hopkins University) at BioPillar Laboratories (Monmouth Junction, New

Jersey) using previously described methods,11 or using a polyclonal rabbit antiserum (Lifespan

Biosciences, Seattle, WA), followed by DAKO DakoCytomation Envision+ labeled polymer

HRP-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,

Vancouver, BC), following the manufacturer’s protocol. 2 x 105 enriched T cells were cultured




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in RPMI complete medium at a 10:1 ratio with irradiated (3000R) Karpas 299 cells in 96-well U-

bottom plates (Nunc, Rochester, NY). Cells were fed every 2 days with fresh medium

containing 10 IU/ml IL-2 (Chiron, Emeryville, CA). After 1 week, T cells were harvested,

counted and re-plated in quadruplicate with fresh Karpas 299 cells (irradiated 3000R) at

effector:target (E:T) ratios of 2:1 and 1:1 with 2 x 104 tumor cells per well in 96-well U-bottom

plates with or without 10 μg/ml anti-PD-L1 (clone M1H1) 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, Solon, OH); cells were harvested 16

hours later. Incorporated radioactivity (counts per minute, cpm) was measured using a β-liquid

scintillation analyzer (PerkinElmer, Waltham, MA), and results from quadruplicate cultures

reported as arithmetic means ± standard deviation.

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

An 18-year old female (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, 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 Medium (DMEM)

containing 10% FCS, 100 Units/ml penicillin/streptomycin, 2 mM L-glutamine, and 50 μM β-

mercaptoethanol (Invitrogen) at 37˚C in 5% CO2. As tumor cells slowly grew over a period of

one month, supplementation of the culture with cell-free ascites fluid was gradually decreased

(from 40% to 5%), until the resulting LC-96 cell line was able to grow in DMEM containing




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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 demonstrated 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, PBMC

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 co-cultures of Karpas 299 cells and

healthy donor T cells, as described above under allogeneic T cell proliferation assays, were

collected after 4 days incubation and analyzed for IFN-γ by enzyme-linked immunosorbent assay

(ELISA; R&D Systems). 96-well Maxisorp plates (Nunc) were coated with mouse anti-human

IFN-γ antibody, 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 performed using streptavidin-conjugated HRP and hydrogen peroxide-

tetramethylbenzidine substrate, and absorbance determined at 450 nm/570 nm with a

SPECTRAmax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA). Recombinant

human IFN-γ was used to generate a standard curve.

For autologous tumor cell-T cell co-cultures, cryopreserved LC-96 and RS-27 primary

tumor specimens were thawed at 37˚C, washed twice with warm RPMI complete medium, and 2-




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2.5 x 105 cells per well were plated in six replicates in a 96-well U-bottom plate in RPMI

complete medium. Phytohemagluttinin (PHA; Sigma, St. Louis, MO) 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 performed 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

(GM-CSF, IFN-γ, IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, IL-13, TARC, IFN-α, SDF-

1β, MIP-1α, and MIG) using SearchLight® protein array multiplex sandwich-ELISA (Pierce

Biotechnology, Woburn, MA).




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Results

PD-L1 is widely expressed by ALCL but uncommonly among B cell non-Hodgkin

lymphoma cell lines

We screened a large panel of human lymphoma cell lines for PD-L1 expression using

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 based on gene

expression profiling,25 while the third (RC-K8) is known to have constitutive upregulation of the

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 (Supplemental Table 1). 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.

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 24-48 hours, and PD-L1 expression 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 (Figure 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




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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 diffuse large B cell lymphomas

We next tested 68 lymphoma tissue specimens for expression of PD-L1 (Table 2A-B).

Thirty-three DLBCL, 3 primary mediastinal B cell lymphomas (PMBCL) and 9 HL 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 demonstrated 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 HL expressed PD-L1 in Reed-Sternberg cells, in concordance

with previous observations.28

DLBCL were classified into germinal-center B (GCB) or non-GCB subtype based on 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).




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We next performed immunohistochemistry for PD-L1 expression in a separate set of 42

formalin-fixed, paraffin-embedded lymphoma specimens (Table 2C), 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 FL were negative, while 4 of

5 ALCL and 6 of 30 DLBCL were positive. Representative images are shown in Supplemental

Figure 1. Of note, tumor-associated histiocytes stained positive for PD-L1 in 9 of 24 (38%)

DLBCL in which tumor cells were negative. Similarly, PD-L1+ histiocytes were also found in

FL 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 DLBCL

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 2D.

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




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antibody blockade of PD-L1 would result in greater T cell activity, demonstrating 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

(Figure 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 (Figure 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 (Figure 2C). The differences seen in T cell proliferation were smaller

and not consistently statistically significant. Nonetheless, these results demonstrate functional

expression of immunosuppressive PD-L1 by ALCL cells.

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 Methods), containing approximately equivalent

proportions of PD-L1-expressing tumor cells and tumor-associated T cells, was used as an

autologous system (Figure 3A). The primary tumor cells within the ascites (PD-L1+, EMA+, and

CD30+) were associated with a mixture of CD4+ and CD8+ T cells.

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 assayed for IFN-γ secretion as an indicator of T cell stimulation

(Figure 3B). Without the addition of PHA (media alone), even with addition of IL-2 (10 μg/ml),




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no IFN-γ secretion was seen. Yet in the presence of PHA (0.5 μg/ml or 1.0 μg/ml), anti-PD-L1

provoked a marked increase in IFN-γ secretion (P < 0.0001 by one-way ANOVA compared to

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 Methods). The immunophenotype of the LC-96 cell line mirrored that of

the primary ascites tumor cells (Figure 4A, lower panel), 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) 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), demonstrating that T cells,

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 using multiplex ELISA. First, we measured the cytokines present

in cell-free ascites fluid, which would reflect the tumor environment in situ (Supplemental

Figure 2A). 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

(Supplemental Figure 2B), 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 (Supplemental Figure 2C). 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 to control antibody or media alone. The




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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 demonstrate that PD-L1 can also be immunologically active when expressed by B cell

lymphomas, analogous experiments were performed using a tumor sample from a patient with an

aggressive DLBCL (RS-27), which lacked expression of CD10, consistent with non-GCB

phenotype. Circulating tumor cells strongly co-expressed PD-L1 and CD20, as measured by

flow cytometry (Figure 4A). Unmanipulated RS-27 PBMCs containing approximately 75%

tumor cells and 20% CD3+ T cells (Figure 4A), were cultured with PHA in the presence or

absence of anti-PD-L1. After 5 days, supernatants were assayed for IFN-γ secretion (Figure

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.

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 can be lost during in vitro culture (Figure 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




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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 (Supplemental Figure 3). 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 (Figure 4D). When grown in media

supplemented with 20% human plasma, the cells displayed bright expression of PD-L1 (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.




19

Discussion

Cancers use multiple mechanisms to thwart endogenous host anti-tumor 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 has 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

demonstrated 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 demonstrated 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 is

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 NHL.22 Marzec

and colleagues reported PD-L1 staining in 100% of 18 ALCLs,23 although the polyclonal anti-




20

PD-L1 antibody used in this study possibly yielded false-positive cases (see below). Wilcox and

colleagues reported PD-L1 expression in 15% of 131 T cell lymphomas, including 3 of 9

ALCL.30 Xerri and colleagues reported PD-L1 expression in 4 of 14 DLBCL (including 2

PMBCL), but not in follicular (n=8), mantle cell (n=4), marginal zone (n=4), or Burkitt

lymphomas (n=3),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, since

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 appears 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 1-2). The ABC DLBCL subtype

identified by gene expression profiling is associated with inferior survival compared to 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 DLBCL, and we suggest this hypothesis be tested in a large

series of molecularly-classified DLBCL with associated clinical outcome data. The aggressive

B cell lymphoma from which we derived the PD-L1+ RS-27 cell line (Figure 4) was indeed

virulent; the patient died of CNS 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




21

establishment of other human lymphoma cell lines, as appears to be the case in melanoma.

While 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 has been associated with worse prognosis in FL38 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 monoclonal antibodies

M1H1 or 5H1. In our experience, the polyclonal anti-PD-L1 antisera used by Marzec et al 23

appears 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, Neelapu et al 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 NK 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 colleagues30 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 demonstrating




22

the ability of PD-L1 on ALCL and DLBCL cells to suppress the responses of both allogeneic and

autologous tumor-associated T cells (Figures 2-4 and Supplemental Figures 2-3).

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, while 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 metastasis49 and angiogenesis,41 and

since 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 DLBCL of the ABC/non-GCB subtype, where it acts to

negatively regulate adjacent T cells. Targeting the PD-1/PD-L1 pathway using antagonistic

monoclonal antibodies may thus be an attractive approach to lymphoma immunotherapy.




23

References

1. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and Its Ligands in Tolerance and Immunity. Annu Rev Immunol. 2008;26:677-704. 2. Ansari MJ, Salama AD, Chitnis T, et al. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med. 2003;198:63-69. 3. Nishimura H, Nose M, Hiai H, Minato N, Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-151. 4. Okazaki T, Honjo T. PD-1 and PD-1 ligands: from discovery to clinical application. Int Immunol. 2007;19:813-824. 5. D'Souza M, Fontenot AP, Mack DG, et al. Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction. J Immunol. 2007;179:1979-1987. 6. Zhang JY, Zhang Z, Wang X, et al. PD-1 up-regulation is correlated with HIV-specific memory CD8+ T-cell exhaustion in typical progressors but not in long-term nonprogressors. Blood. 2007;109:4671-4678. 7. Beswick EJ, Pinchuk IV, Das S, Powell DW, Reyes VE. Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells. Infect Immun. 2007;75:4334-4341. 8. Smith P, Walsh CM, Mangan NE, et al. Schistosoma mansoni worms induce anergy of T cells via selective up-regulation of programmed death ligand 1 on macrophages. J Immunol. 2004;173:1240-1248. 9. Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793-800. 10. Thompson RH, Dong H, Kwon ED. Implications of B7-H1 expression in clear cell carcinoma of the kidney for prognostication and therapy. Clin Cancer Res. 2007;13:709s-715s. 11. Thompson RH, Kuntz SM, Leibovich BC, et al. Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up. Cancer Res. 2006;66:3381-3385. 12. Thompson RH, Webster WS, Cheville JC, et al. B7-H1 glycoprotein blockade: a novel strategy to enhance immunotherapy in patients with renal cell carcinoma. Urology. 2005;66:10-14. 13. Ghebeh H, Mohammed S, Al-Omair A, et al. The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors. Neoplasia. 2006;8:190-198. 14. Routh JC, Ashley RA, Sebo TJ, et al. B7-H1 expression in Wilms tumor: correlation with tumor biology and disease recurrence. J Urol. 2008;179:1954-1959; discussion 1959-1960. 15. Nomi T, Sho M, Akahori T, et al. Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer. Clin Cancer Res. 2007;13:2151-2157. 16. Hamanishi J, Mandai M, Iwasaki M, et al. Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. Proc Natl Acad Sci U S A. 2007;104:3360-3365. 17. Nakanishi J, Wada Y, Matsumoto K, Azuma M, Kikuchi K, Ueda S. Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers. Cancer Immunol Immunother. 2007;56:1173-1182.




24

18. Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. Immunohistochemical localization of programmed death-1 ligand-1 (PD-L1) in gastric carcinoma and its clinical significance. Acta Histochem. 2006;108:19-24. 19. Ohigashi Y, Sho M, Yamada Y, et al. Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2 expression in human esophageal cancer. Clin Cancer Res. 2005;11:2947-2953. 20. Gao Q, Wang XY, Qiu SJ, et al. Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma. Clin Cancer Res. 2009;15:971-979. 21. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A. 2002;99:12293-12297. 22. Brown JA, Dorfman DM, Ma FR, et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol. 2003;170:1257-1266. 23. Marzec M, Zhang Q, Goradia A, et al. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1). Proc Natl Acad Sci U S A. 2008. 24. Xerri L, Chetaille B, Seriari N, et al. Programmed death 1 is a marker of angioimmunoblastic T-cell lymphoma and B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia. Hum Pathol. 2008;39:1050-1058. 25. Lam LT, Wright G, Davis RE, et al. Cooperative signaling through the signal transducer and activator of transcription 3 and nuclear factor-{kappa}B pathways in subtypes of diffuse large B-cell lymphoma. Blood. 2008;111:3701-3713. 26. Kalaitzidis D, Davis RE, Rosenwald A, Staudt LM, Gilmore TD. The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-kappaB signal transduction pathway. Oncogene. 2002;21:8759-8768. 27. Liu J, Hamrouni A, Wolowiec D, et al. Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and increase expression after stimulation with IFN-{gamma} and TLR ligands via a MyD88-, TRAF6-, and MEK-dependent pathway. Blood. 2007;110:296-304. 28. Yamamoto R, Nishikori M, Kitawaki T, et al. PD-1-PD-1 ligand interaction contributes to immunosuppressive microenvironment of Hodgkin lymphoma. Blood. 2008;111:3220-3224. 29. Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103:275-282. 30. Wilcox RA, Feldman AL, Wada DA, et al. B7-H1 (PD-L1, CD274) suppresses host immunity in T-cell lymphoproliferative disorders. Blood. 2009;114:2149-2158. 31. Andorsky DJ, Yamada R, Steward KK, De Vos S, Said J, Timmerman JM. Spectrum of Expression and Biological Activity of Programmed Death Ligand 1 (PD-L1) in Non-Hodgkin's Lymphomas. ASH Annual Meeting Abstracts. 2008;112:4140. 32. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol. 2007;25:267-296. 33. Rosenwald A, Wright G, Leroy K, et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med. 2003;198:851-862. 34. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503-511.




25

35. Fu K, Weisenburger DD, Choi WWL, et al. Addition of Rituximab to Standard Chemotherapy Improves the Survival of Both the Germinal Center B-Cell-Like and Non-Germinal Center B-Cell-Like Subtypes of Diffuse Large B-Cell Lymphoma. J Clin Oncol. 2008;26:4587-4594. 36. Wilson WH, Dunleavy K, Pittaluga S, et al. Phase II Study of Dose-Adjusted EPOCH and Rituximab in Untreated Diffuse Large B-Cell Lymphoma With Analysis of Germinal Center and Post-Germinal Center Biomarkers. J Clin Oncol. 2008;26:2717-2724. 37. Davis RE, Brown KD, Siebenlist U, Staudt LM. Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J Exp Med. 2001;194:1861-1874. 38. Farinha P, Masoudi H, Skinnider BF, et al. Analysis of multiple biomarkers shows that lymphoma-associated macrophage (LAM) content is an independent predictor of survival in follicular lymphoma (FL). Blood. 2005;106:2169-2174. 39. Steidl C, Lee T, Shah SP, et al. Tumor-Associated Macrophages and Survival in Classic Hodgkin's Lymphoma. N Engl J Med;362:875-885. 40. Hasselblom S, Hansson U, Sigurdardottir M, Nilsson-Ehle H, Ridell B, Andersson PO. Expression of CD68+ tumor-associated macrophages in patients with diffuse large B-cell lymphoma and its relation to prognosis. Pathol Int. 2008;58:529-532. 41. Lenz G, Wright G, Dave SS, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med. 2008;359:2313-2323. 42. Yang ZZ, Novak AJ, Stenson MJ, Witzig TE, Ansell SM. Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma. Blood. 2006;107:3639-3646. 43. Nattamai D, Neelapu S. PD-1 Expression Is Markedly Upregulated on Intratumoral CD4+ and CD8+ T Cells in Follicular Lymphoma and Is Associated with T-Cell Exhaustion. Blood. 2007;110. 44. Chu F, Foglietta M, Qin H, et al. In Vitro and In Vivo Effects of CT-011, a Humanized Anti-PD-1 Monoclonal Antibody, in Combination with Rituximab against Human B-Cell Lymphomas. ASH Annual Meeting Abstracts. 2009;114:724-. 45. Merz H, Fliedner A, Orscheschek K, et al. Cytokine expression in T-cell lymphomas and Hodgkin's disease. Its possible implication in autocrine or paracrine production as a potential basis for neoplastic growth. Am J Pathol. 1991;139:1173-1180. 46. Merz H, Lange K, Gaiser T, et al. Characterization of a novel human anaplastic large cell lymphoma cell line tumorigenic in SCID mice. Leuk Lymphoma. 2002;43:165-172. 47. Willers J, Dummer R, Kempf W, Kundig T, Burg G, Kadin ME. Proliferation of CD30+ T-helper 2 lymphoma cells can be inhibited by CD30 receptor cross-linking with recombinant CD30 ligand. Clin Cancer Res. 2003;9:2744-2754. 48. Boulland ML, Meignin V, Leroy-Viard K, et al. Human interleukin-10 expression in T/natural killer-cell lymphomas: association with anaplastic large cell lymphomas and nasal natural killer-cell lymphomas. Am J Pathol. 1998;153:1229-1237. 49. Chiang AC, Massague J. Molecular basis of metastasis. N Engl J Med. 2008;359:2814-2823. 50. Weng AP, Shahsafaei A, Dorfman DM. CXCR4/CD184 immunoreactivity in T-cell non-Hodgkin lymphomas with an overall Th1- Th2+ immunophenotype. Am J Clin Pathol. 2003;119:424-430.




26

Table 1. Expression of PD-L1 among 34 human lymphoma cell lines Expression of PD-L1 and PD-L2 was measured by flow cytometry. “++” indicates ≥ 2 logs mean fluorescence intensity (MFI) above isotype control; “+” indicates < 2 logs MFI above control. Abbreviations: ALCL, anaplastic large cell lymphoma; T-ALL, precursor T acute lymphoblastic leukemia; DLBCL, diffuse large B cell lymphoma; GCB, germinal center B subtype; ABC, activated B cell subtype.

Cell line Lymphoma subtype PD-L1 PD-L2

B c

ell l

ines

Daudi Burkitt - - Raji Burkitt - - Ramos Burkitt - - BJA-B Burkitt - - MC116 Burkitt - - NU-DHL-1 DLBCL (GCB) - - OCI-Ly-2 DLBCL (GCB) - - OCI-Ly-7 DLBCL (GCB) - - OCI-Ly-19 DLBCL (GCB) - - SU-DHL-4 DLBCL (GCB) - - SU-DHL-6 DLBCL (GCB) - - SU-DHL-2 DLBCL (ABC) - - U2932 DLBCL (ABC) - - OCI-Ly-3 DLBCL (ABC) - - OCI-Ly-10 DLBCL (ABC) ++ - HBL-1 DLBCL (ABC) + - RC-K8 DLBCL (NF-κB+) + - SU-DHL-5 DLBCL - - SU-DHL-7 DLBCL - - SU-DHL-8 DLBCL - - SU-DHL-9 DLBCL - - SU-DHL-10 DLBCL - - SU-DHL-16 DLBCL - - USC-DHL-1 DLBCL - - Granta-519 Mantle cell - - JeKo-1 Mantle cell - - REC-1 Mantle cell - - BCBL-1 1° effusion - -

Cell line Lymphoma subtype PD-L1 PD-L2

T c

ell l

ines

Karpas 299 ALCL ++ - SU-DHL-1 ALCL ++ - SUP-M2 ALCL ++ - DEL ALCL + - Jurkat T-ALL + - Hut78 Sezary syndrome - -




27

Table 2. Expression of PD-L1 in primary lymphoma tissue specimens Frozen sections (A) and flow cytometry (B) on cryopreserved specimens were performed with the anti-PD-L1 antibody clone M1H1. Paraffin sections (C) were stained with clone 5H1. Frozen and paraffin sections of diffuse large B cell lymphoma were classified as germinal center origin or non-germinal center origin (D). PD-L1 expression in DLBCL occurs almost exclusively in tumors of non-germinal center origin

A Lymphoma subtype # Cases # PD-L1+ % PD-L1+

Fro

zen

Hodgkin lymphoma* 9 8 89%

Diffuse large B cell lymphoma 33 9 27% Primary mediastinal B cell 3 3 100%

B Lymphoma subtype # Cases # PD-L1+ % PD-L1+

Flo

w c

ytom

etry

Follicular lymphoma 16 0 0%

Small lymphocytic lymphoma / chronic lymphocytic leukemia

2 0 0%

Marginal zone lymphoma 3 0 0% Mantle cell lymphoma 1 0 0% Burkitt lymphoma 1 0 0%

C Lymphoma subtype # Cases # PD-L1+ % PD-L1+

Par

affi

n Anaplastic large cell lymphoma 5 4 80%

Follicular lymphoma 7 0 0% Diffuse large B cell lymphoma 30 6 20%

D PD-L1 detection method n GCB non-GCB

P-value + - + -

Frozen (M1H1 antibody) 33 1

(5%) 18

(95%) 8

(57%) 6

(42%) 0.0004

Paraffin (5H1 antibody) 30

0 (0%)

11 (100%)

6 (32%)

13 (68%)

0.0613

*Staining in Hodgkin and Reed-Sternberg cells.




28

Figure legends

Figure 1. High-level PD-L1 expression among ALCL but not 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 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.

Figure 2. PD-L1 blockade enhances the activation of T cells co-cultured with allogeneic

ALCL cells. (A) Irradiated Karpas 299 ALCL cells (PD-L1+), were stimulated for 1 week with

T cells from 2 healthy donors, 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 (lower panels), or antibody was

replenished and cells pulsed with 3[H]-thymidine overnight to measure T cell proliferation (upper

panels). 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 allo-specific proliferation (upper panel) or IFN-

γ secretion (lower panel). (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 panels), or cells were pulsed with 3[H]-thymidine overnight to measure T cell

proliferation (upper panels). Proliferation data are represented as mean ± SD of quadruplicate

cultures. P values shown are for anti-PD-L1 antibody versus isotype control antibody.

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 two rows show results for the primary tumor (ascites), with far left panel displaying forward




29

and side scatter plot 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 grey.

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 PHA 0.5 or 1.0 μg/ml, 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.

Figure 4. PD-L1 expressed in primary B cell lymphoma is biologically active but readily

lost during in vitro culture. (A) PBMC from a patient with aggressive mature B cell lymphoma

were analyzed by flow cytometry. Malignant cells co-express 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 PBMC 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

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 co-express 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 based on 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) versus 20% human plasma (MFI = 465).




Karpas 299 (ALCL) Jurkat (T-ALL)A

Figure 1

SU-DHL-1 (ALCL) SUP-M2 (ALCL)

HBL-1 (DLBCL) OCI-Ly-10 (DLBCL)Ramos (Burkitt) SU-DHL-6 (DLBCL)

PD-L1 PD-L1 PD-L1 PD-L1

PD-L1 PD-L1 PD-L1 PD-L1

BRamos: No stimulation Ramos: CpG + IL-4 stimulation

PD-L1 PD-L2 PD-L2PD-L1

Research.

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2:1 1:1 E alone0

Effector : Target ratio2:1 1:1 E alone

0

50

Effector : Target ratio2:1 1:1 E alone

05000

Effector : Target ratio

2500

5000

7500

10000

12500

15000

17500

Ave

rage

CPM

5000

10000

15000

20000

Ave

rage

CPM

10000

20000

30000

40000

Ave

rage

CPM

P = 0.007P = 0.965

P = 0.010

P = 0.204

P = 0.063

P = 0.019

4:1 2:10

2500

Effector : Target Ratio4:1 2:1

0

Effector : Target Ratio4:1 2:1

0


500600700800

/ml)

1000

1500

/ml)

30354045

/ml)

4:1 2:10

100200300400500


IFN

- γ (p

g/

4:1 2:10

500


IFN

- γ (p

g/

4:1 2:105

10152025


IFN

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300

400

500

600

N-γ

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Primary RS-27 tumor

PHA 0 PHA 0.50

100

200

Cell conditions

IFN

C DRS-27 cell line

MFI = 465

OCI-Ly-10, 20% human plasma

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Weaned gradually from human serum

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Published OnlineFirst May 3, 2011.Clin Cancer Res David J Andorsky, Reiko Yamada, Jonathan W Said, et al. tumor-associated T cellsnon-Hodgkin lymphomas and inhibits the activity of Programmed death ligand 1 (PD-L1) is expressed by

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