In vivo depletion of DC impairs the anti-tumor effectof agonistic anti-CD137 mAb

Oihana Murillo�1, Juan Dubrot�1, Asıs Palazon1, Ainhoa Arina1,

Arantza Azpilikueta1, Carlos Alfaro1, Sarai Solano1, Marıa C. Ochoa1,

Carmen Berasain1, Izaskun Gabari1, Jose. L. Perez-Gracia2,

Pedro Berraondo1, Sandra Hervas-Stubbs��1 and Ignacio Melero��1,2

1 Gene Therapy Unit, Centro de Investigacion Medica Aplicada, Universidad de Navarra,

Pamplona, Spain2 Clinica Universitaria, Universidad de Navarra, Pamplona, Spain

Anti-CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor

models and are undergoing clinical trials for cancer. The anti-tumor effect involves co-

stimulation of tumor-specific CD81 T cells. Whether antigen cross-presenting DC are required

for the efficacy of anti-CD137 mAb treatment has never been examined. Here we show that

the administration of anti-CD137 mAb eradicates EG7-OVA tumors by a strictly CD8b1 T-cell-

dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to deter-

mine the identity of the draining lymph node cell type responsible for tumor antigen cross-

presentation revealed that CD11c1 cells, most likely DC, are the main players in this tumor

model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor-

draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates

in anti-OVA CTL induction. Using CD11c diphtheria toxin receptor-green fluorescent protein-

C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we

confirmed the involvement of DC in tumor antigen cross-presentation in CTL induction

against OVA257–264 epitope and in the antitumor efficacy induced by anti-CD137 mAb.

Key words: CD137 (4-1BB) . Cross-priming . CTL . DC . Tumor immunity

Supporting Information available online

Introduction

CD137, also known as 4-1BB, is a TNF-receptor family co-

stimulatory molecule originally identified on activated T cells [1].

Its natural ligand CD137L is found on APC, including B cells,

macrophages, and DC [2]. In the presence of TCR engagement,

signaling through CD137 leads to increased T-cell proliferation,

cytokine production, and prolonged CD81 T-cell survival in vitro

[2]. Administration of agonistic anti-CD137 mAb either alone or

following peptide vaccination is capable of inducing the eradica-

tion of established tumors in several transplantable tumor models

[3, 4]. This anti-tumor effect is dependent on CD81 T cells that

show increases in tumor-specific cytotoxic activity and associated

IFN-g production [3, 4]. Recent studies have demonstrated that

the activity of anti-CD137 mAb treatment is also dependent on

their ability to enhance the survival of CD81 CTL, and on the

prevention/reversal of established anergy [5–7]. In addition,

antigen-independent anti-CD137 mAb effects have been

described for memory T cells, and CD137 seems to be an

�These authors contributed equally to this work.��These authors share equal senior authorship.

Correspondence: Professor Ignacio Meleroe-mail: imelero@unav.es

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.200838958 Eur. J. Immunol. 2009. 39: 2424–2436Oihana Murillo et al.2424

important player in the differentiation of memory cells [8–10].

CD137 can also be expressed by activated NK cells [11], myeloid

leukocytes [12] including DC [13, 14], and tumor endothelial

cells [15]. However, the role of CD137 molecules expressed on

these non-T cells during tumor immunotherapy remains unde-

fined [13, 16].

As naıve CTL do not express CD137 on their surface, susceptibility

to CD137 triggering depends on prior up-regulation of this receptor

[5]. This requires antigen recognition through the TCR-CD3 complex

[5]. Two mechanisms have been proposed for tumor antigen

presentation to naıve CD81 T cells. Tumor cells reaching lymph

nodes may directly present peptides derived from tumor antigens via

their MHC class I molecules to naıve CD81 T cells [17]. Alternatively,

tumor antigens may be presented to CD81 T cells by professional APC

in a process referred to as cross-presentation [18]. Cross-presentation

involves the uptake and processing of exogenous antigens that reach

proteasomal degradation and the MHC class I presentation pathway

[19]. It is still unresolved whether cross-presentation or direct

presentation is the relevant mechanism governing tumor immunity

[20, 21]. Although some studies have suggested that the two

processes act as redundant non-mutually exclusive mechanisms [22],

other authors have proposed cross-presentation to be the major

mechanism for the activation of naıve CD81 T cells [23].

Experimentation in BM chimeric mice with differential

expression of antigen-presenting molecules and in humans

vaccinated with allogeneic tumor cells has demonstrated that the

ability to cross-present cell-associated antigens is restricted to

BM-derived APC [24, 25]. Several groups have investigated the

identity of the BM-derived APC involved in MHC class I-restricted

presentation of exogenous antigens. In the past few years, a large

body of literature has arisen that assigns this function to DC

[26–30]. In particular, CD8a-expressing DC seem to play a

crucial role in cross-presentation of cell-associated antigens to

CD81 T cells in vivo [27–30]. Such CD8a1 DC might capture

antigens directly from living or dead tumor cells or indirectly

from other intermediary DC [31, 32]. Cross-presentation to naıve

CD81 T cells is believed to take place mainly in tumor-draining

lymph nodes (TDLN). Upon contact with antigen-presenting DC,

CD81 T cells up-regulate CD137 expression and become

susceptible to agonistic anti-CD137 mAb activity [13]. Based on

this scheme, it is conceivable that DC-mediated cross-priming to

CD81 T cells would be a requirement for efficacy of anti-CD137

treatment. In the present study we have attempted to determine

whether DC-mediated antigen cross-presentation is a requisite for

the therapeutic immune-mediated activity of anti-CD137 mAb.

Results

Anti-CD137 mAb efficacy on tumors is criticallydependent on CD8b1 T cells but not on CD41 orNK cells

EG7-OVA thymoma transfectants combine the features of a

relatively immunogenic tumor and the expression of a surrogate

tumor antigen to follow the CTL immune response [33]. CTL

recognizing the OVA257–264 epitope are traceable in vivo by a

number of techniques. To evaluate the therapeutic activity of anti-

CD137 treatment in the EG7-OVA tumor model, C57BL/6 mice

received a s.c. injection of 5�105 EG7-OVA viable cells in their right

flanks on day 0. On days 7, 9, 11, and 13, after tumors had

developed, tumor-bearing mice were i.p. injected with either anti-

CD137 mAb or control rat IgG. We found that anti-CD137 treatment

resulted in profound inhibition of tumor growth. Consistently more

than 50% of mice were completely cured more than 3 months after

the initiation of treatment. It is worth noting that, as observed with

other transplantable tumors, some of the lesions lethally grew after

transient reduction or stabilization, indicating a tumor-escape

mechanism. By contrast, the injection of control rat IgG did not

induce any therapeutic effect (Fig. 1A and B).

To identify the cell types responsible for the therapeutic anti-

tumor activity in this tumor model, we carried out in vivo

depletion of leukocyte subsets prior to initiation of anti-CD137

treatment. As shown in Fig. 1B, depletion of CD8b1 T cells

completely abrogated the therapeutic effect of anti-CD137 mAb.

The anti-CD8b mAb used for depletion recognizes T cells only,

and not subpopulations of DC that express CD8aa dimers. NK cell

depletion had no significant effect. Interestingly, CD41 T cell

depletion clearly improved efficacy, in agreement with a recent

publication implying regulatory T cells that are depleted under

these conditions [34]. Rechallenge of mice that had already

rejected EG7 tumors after anti-CD137 mAb treatment with EL-4

tumor cells resulted in rejection or control of EL-4 tumor prolif-

eration (Fig. 1C). However, no such control of B16 melanoma

cell proliferation was observed (data not shown), indicating

priming to the EL-4 endogenous antigens in addition to the

artificial OVA antigens expressed by the EG7-OVA thymoma

transfectant.

Having observed that anti-CD137 mAb-induced EG7-OVA

tumor rejection by a purely CD81 T-cell-dependent mechanism,

subsequent studies focused on the requirements for antigen

presentation to this lymphocyte subset.

Rejection of EG7-OVA tumors elicited by anti-CD137mAb correlates with increased CTL activity

We evaluated the effect of anti-CD137 mAb on the generation of

tumor-specific CTL responses in EG7-OVA-bearing mice by

determining the percentage of OVA-tetramer1 (OVA-Tetr1) cells

and the OVA-specific CTL in vivo activity. Surprisingly, the

percentages of OVA-Tetr1 cells detected in the spleen of mice

treated with anti-CD137 mAb were comparable to that of control

tumor-bearing mice (Fig. 2A). In addition, OVA-Tetr1 cells up-

regulated the activation marker CD44 and down-regulated the

lymph-node-homing receptor CD62L in mice treated either with

anti-CD137 mAb or with control rat IgG (Supporting Information

Fig. 1). This indicates that EG7 tumor growth is sufficient to

expand antigen-specific CD81 T cells in spite of lethal tumor

progression.

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The expansion of OVA-specific CD81 T cells indicates that

OVA antigen presentation is not a limiting factor for tumor

rejection in the EG7 model. However, although the control IgG-

treated mice showed weak OVA-specific cytolytic activity in vivo

(o10% of specific lysis), the anti-CD137-treated mice showed

relevant CTL responses against the immunodominat surrogate

tumor antigen (around 40% of specific lysis) (Fig. 2B). Accord-

ingly, in this tumor model, anti-CD137 mAb mainly acts to

promote efficient CTL effector function.

In vivo OVA257–264 epitope presentation in EG7-OVAtumor-bearing mice mainly occurs in TDLN

Together with adoptive transfer of TCR transgenic cells [35], the

EG7-OVA tumor model allows the delineation of mechanisms

involved in tumor control or rejection. To assess if presentation of

tumor-derived OVA to CD81 T cells occurs in the TDLN [36, 37],

CFSE-labeled OVA-specific OT-I CD81 T cells were adoptively

transferred into EG7-OVA-tumor-bearing mice and OT-I prolif-

eration was analyzed 3 days later. OT-I CD81 T cells were mainly

primed in TDLN (Supporting Information Fig. 2). The slight

proliferation of OT-I CD81 T cells in spleen and contralateral

lymph nodes CLN was probably caused by the recirculation of

activated cells rather than a local antigen-induced expansion.

Therefore, in EG7-OVA model, tumor-specific CTL are selectively

activated in the TDLN.

Both cross-priming and direct priming have been postulated

as mechanisms to induce tumor-protective CTL immunity [20, 24,

38, 39]. Direct priming by EG7-OVA tumor cells should result in

migration of tumor cells to the TDLN, where encounters with

naıve T cells might take place. To study whether OVA-specific

CTL could be primed directly by tumor cells in the TDLN or by

professional APC, we first analyzed whether tumor cells could be

found in the secondary lymphoid organs of tumor-bearing

animals. In total 8–10 days after s.c. injection of EG7-OVA cells,

Figure 1. Anti-CD137 mAb induces the eradication of EG7-OVA tumors via a CD81 T cell-dependent mechanism. (A) C57BL/6 mice (seven per group)were injected with 5� 105 EG7-OVA cells on day 0, and on days 7, 9, 11, and 13 were treated i.p. with anti-CD137 mAb or the control rat IgG at 100 mgper dose. (B) Tumor-bearing mice (seven per group) were treated with depleting anti-CD4, anti-CD8b, or anti-NK1.1 mAb prior to anti-CD137treatment. A total of 200 mg per dose of each depleting mAb was administered. Anti-CD4 and anti-CD8b mAb were i.p. injected every other day forfour times beginning 3 days before treatment onset, and then every 5th day for the remainder of the experiment. NK1.1-specific mAb wasadministered i.v. for five consecutive days, beginning 3 days before treatment onset, and thereafter administration continued in a 3-day-interval.(C) Mice cured of EG7-OVA tumors with anti-CD137 mAb were rechallenged (four per group) 4–6 wk later with the untransfected EL-4 cell line(5� 105 per mouse). A naıve group of four mice was used as a negative control. Data are representative of at least two independent experiments.Fraction of surviving tumor-free mice is provided in each graph. Data were compared by t-test. p-Values ofo0.05 were considered significant.

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secondary lymphoid organs were examined. PCR amplification of

the DNA-encoding OVA unequivocally revealed that tumor

cells had reached TDLN, but not other lymph nodes or the spleen

(Fig. 3A).

Next, we investigated whether these tumor cells, after

reaching the TDLN were also capable of presenting tumor

antigens in vivo. To this end, we took advantage of the

H2Kb�/� mouse model. In this experimental system, mouse

cells lacking the MHC class I Kb molecule are incapable of

presenting the OVA immunodominant tumor antigen to

OT-I CD81 T cells. Hence, in this experimental system, tumor

antigen presentation depends primarily on presentation by

tumor cells. Unfortunately, s.c. injection of EG7-OVA cells into

H2Kb�/� mice led to the transient growth and subsequent

regression of tumors in all mice because H-2Kb molecule on

EG7-OVA cells acts as a strong major alloantigen for H-2Kb�/�

mice (data not shown).

In contrast, in H2Kb�/�-C57BL/6 BM chimeric mice

(generated through reconstitution of lethally irradiated recipient

mice (C57BL/6) with H2Kb-/- BM) EG7-OVA tumors grafted as in

normal mice (data not shown) because chimeric mice are

tolerant to this MHC class I molecule. Therefore, we generated

H2Kb�/�-C57BL/6 BM chimeras. After complete BM recon-

stitution and verification that their DC did not express H2-Kb

(Fig. 3B), mice were injected with EG7-OVA tumor cells by s.c.

injection. When palpable tumors had developed, CFSE-labeled

OT-I CD81 T cells were adoptively transferred and antigen

presentation (indicated by OT-I proliferation) was analyzed. As

shown in Fig. 3C, the surrogate tumor antigen OVA induced

about fourfold less proliferation of adoptively transferred OT-I

cells in the TDLN of H2Kb�/�-WT BM chimeras compared with

tumor-bearing WT C57BL/6 controls. Therefore, antigen

presentation is chiefly performed by host cells as distinct from the

transplanted tumor cells. However, there is a direct antigen-

presenting activity mediated by tumor cells, which reach

lymphoid tissue in small quantities that account for measurable

direct priming.

Furthermore, reduction in priming efficiency of the OVA

immunodominant antigen in the H2Kb�/�-WT BM chimeras

upon anti-CD137 mAb treatment was substantiated by sequential

follow-up of H2-Kb-OVA peptide tetramer-specific CD81 T cells

(Fig. 3D) and by in vivo killing assays (Fig. 3E). In Fig. 3D, a

tendency to accumulate OVA Tetr1CD81 T cells in peripheral

blood can be observed on day 14 in the WT mice, but is not seen

in the chimeric mice. More importantly, the level of CTL specific

activity attained in vivo is much lower in the chimeric mice

lacking H-2Kb in their hematopoietic compartment compared

with the WT control. H2Kb�/�WT BM chimeras reject EG7 upon

treatment with anti-CD137 mAb (Supporting Information Fig. 3),

indicating that EL-4 endogenous tumor antigens can be primed in

these mice in agreement with the rechallenge experiment shown

in Fig. 1C.

Figure 2. EG7-OVA therapy with anti-CD137 mAb leads to enhanced cytolytic activity. C57BL/6 mice were injected with 5�105 EG7-OVA cells andtreated with anti-CD137 mAb (12 mice) or the control rat IgG (11 mice) as in Fig. 1. When first tumor rejections were observed (8–10 days after theinitiation of Ab treatment), spleen cells were harvested and analyzed for (A) frequency of OVA257–264-specific CD81 T cells (OVA-Tetr1) and (B) anti-tumor CTL response as determined by in vivo killing assay (see the Materials and methods). A naıve group of two mice was used as a negative control.Pooled data from two independent experiments are shown. Data were compared by t-test. p-Values ofo0.05 were considered significant.

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Figure 3. EG7-OVA cells reach TDLN but antigen presentation by BM-derived H2Kb1/1 cells accounts for most to the antigen presentation to OVA-specific T cells. (A) C57BL/6 mice (five per group) were injected s.c. with 5� 105 EG7-OVA cells and 8–10 days later, when the tumors were palpable, theTDLN, contralateral lymph nodes, spleen, and the tumors were harvested and analyzed for the presence of OVA-coding DNA by real-time PCR. Mouse-genomic DNA for SOCS3 was used as control. (B) Flow cytometry analysis of spleen cells of WT, H2Kb�/�, and H2Kb�/�-C57BL/6 BM chimeric miceindicating H2Kb expression by CD11c1 DC. Open histograms represent either BM chimeric cells (gray lines) or H2Kb�/� cells (black lines) and filledhistograms represent WT cells. (C) WT and H2Kb�/�-C57BL/6 BM chimeric mice (12 mice per group) were injected s.c. with 5� 105 EG7-OVA cells and,8–10 days later, were adoptively transferred with CFSE-labeled OT-I CD81 T cells (106 cells per mouse). Tumor Ag presentation was assessed in TDLN 3days later. A naıve group of three mice was used as a negative control. Pooled data from two independent experiments are shown. Data were comparedby t-test. p-Values ofo0.05 were considered significant. (D) WT or H2Kb�/�-C57BL/6 BM chimeric mice (six per group) were injected with 5� 105 EG7-OVA cells and treated with anti-CD137 mAb or the control rat IgG as described before. At 6, 10, 14, and 17 days after treatment onset, blood wascollected and analyzed for (D) frequency of OVA257–264-specific CD81 T cells (OVA-Tetr1) and (E) anti-tumor CTL response as determined by in vivokilling assay. Data are representative of at least two independent experiments and compared by t-test. p-Values ofo0.05 were considered significant.

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CD11c1 cells from the TDLN cross-present tumorantigens to CD81 T cells

These results indicate that the predominant cell responsible for

antigen presentation to naıve CTL is of host origin and derived

from the BM. APC have the ability to take up cell-associated

antigens and present them in the context of MHC class I

molecules to CD81 T cells in a process referred to as cross-

presentation [40]. A series of reports have indicated that cross-

priming is a specific property of DC [19, 40, 41]. Here, we sought

to investigate the involvement of DC in tumor antigen cross-

presentation in the EG7-OVA tumor model. To this end, the TDLN

and spleens of mice-bearing EG7-OVA tumors were harvested and

CD11c1 cells were purified (purity480% of CD11c1 I-Ab1). The

CD11c1 population was then incubated with CFSE-labeled OT-I

CD81 T cells, and proliferation of the latter (indicated by CFSE

dilution) was analyzed 3 days later. As shown in Fig. 4A, CD11c1

cells from TDLN induced OT-I CD81 T-cell proliferation, whereas

CD11c1 cells from spleen had no significant effect. These findings

suggest that CD11c1 cells from TDLN, most likely DC, are

involved in cross-presentation of the surrogate tumor antigen to

CD81 T cells. However, similar experiments stimulating OT-I

CD81 T cells with TDLN B2201 cells (mainly B lymphocytes) or

CD11c�CD11b1 myeloid/macrophage populations consistently

rendered negative results (data not shown), indicating CD11c1

cells to be the main population responsible for cross-presentation.

Interestingly, the fraction lacking CD11c1B2201CD11b1 cells

retained some in vitro priming activity for OT-I CD81 T cells

(data not shown). This was attributable to tumor cells in the

lymph node. OT1 CD81 T cells did not proliferate in the presence

of DC similarly purified from pooled lymph nodes of tumor naıve

mice that were used as specificity controls (Fig. 4B), whereas the

TCR-transgenic lymphocytes proliferated when the OVA cognate

peptide was exogenously loaded onto the DC.

In vivo depletion of DC impaired tumor-antigencross-presentation

To study the involvement of DC in tumor antigen cross-

presentation to CD81 T cells in vivo, we took advantage of a

mouse model that allows for ablation of this cell subset [42].

These mice carry a transgene encoding a simian diphtheria toxin

receptor and the green fluorescent protein (DTR-GFP fusion

protein), both under control of the murine CD11c promoter. As a

result, diphtheria toxin (DTx) treatment induces a transient and

profound depletion of CD11chigh cells in vivo, although not of

CD11cint cells containing certain NK cell subsets and plasmacy-

toid DC (data not shown). However, CD11cDTR-GFP transgenic

mice do not tolerate repeated DTx injections because of liver

toxicity presumably caused by leaky expression of the transgene

in this organ (data not shown). Therefore, the time window for

DC depletion is rather limited. In contrast, CD11cDTR-GFP-

C57BL/6 BM chimeras, generated through reconstitution of

lethally irradiated recipient mice (C57BL/6) with CD11cDTR-

GFP BM cells, can be treated with DTx for prolonged periods

of time without adverse side effects [43]. We generated

CD11cDTR-GFP-C57BL/6 BM chimeric mice, and, after

complete BM reconstitution, we verified that that all CD11chigh

were fluorescent (Fig. 5A), and confirmed depletion after

toxin administration (Fig. 5B). Importantly, the CD8a1 DC,

whose function in cross-priming has been established [41],

expressed the transgenes (Fig. 5A) and became depleted

(data not shown). To assess the involvement of DC in tumor

antigen cross-presentation, EG7-OVA tumors were established

by s.c. injection in these chimeric mice. Tumor-bearing mice

were divided randomly into two groups and were treated

with either DTx or the control vehicle as described in the

Materials and methods. At the time when palpable tumor

had developed (8–10 days after tumor inoculation) CFSE-labeled

OT-I CD81 T cells were i.v. injected as a probe to monitor antigen

presentation into tumor-bearing mice. Depletion of DC drastically

reduced OT-I CD81 T cell in vivo proliferation (Fig. 5B and C),

although OT-I CD81 T cell proliferation was not completely

abrogated.

Figure 4. Tumor antigens are presented to CD81 T cells in the TDLN byCD11c1 cells. (A) C57BL/6 mice were injected s.c. with 5�105 EG7-OVAcells. Upon first appearance of palpable tumor, TDLN of 10–15 tumor-bearing mice were pooled and ground to prepare cell suspensions.CD11c1 were magnetically sorted from TDLN and spleens and co-cultured with CFSE-labeled OT-I CD81 T cells at a ratio of 2:1 (DC:OT-I).Ag presentation, indicated by OT-I CD81 T-cell proliferation, wasdetermined 3 days later. All profiles obtained were gated on CFSE1

Va21CD81 cells. Data are representative of four independent experi-ments. (B) As a control, MACS-sorted CD11c1 cells from lymph nodesremoved from naıve C57BL/6 mice were exposed to OT-I cells loadedwith OVA peptide or a control irrelevant peptide as indicated. OT-I cellsdo not dilute CFSE unless DC had been in vitro loaded with theantigenic.

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Another site at which anti-CD137 may mediate an anti-tumor

effect is on DC themselves. Indeed, splenic CD11cbright cells dimly

express surface CD137 in a fashion that is up-regulated

by treatment with endotoxin (Supporting Information Fig. 4).

We did not detect increases of costimulatory molecules or

maturation markers on DC upon treatment with agonistic

anti-CD137 mAb, whereas LPS readily does so in the same

12 h period of time. A recent publication from the Kwon group

[44] also shows lack of enhancing effects on DC maturation,

whereas these authors describe inhibition of DC apoptosis by

agonist anti-CD137 mAb.

DC ablation reduces the therapeutic activity of anti-CD137 treatment

Because of the features of this model, we sought to examine the

impact of DC ablation on the therapeutic activity of anti-CD137

mAb. To this end, EG7-OVA tumor cells were s.c. inoculated

into CD11cDTR-GFP-C57BL/6 BM chimeras. These mice were

injected i.p. with DTx or the control vehicle prior to anti-CD137

treatment. Specifically, DTx was administered every other day

beginning 3 days before tumor cell inoculation, and administra-

tion continued in 2-day intervals afterwards. Depletion of DC

markedly impaired (445% reduction), but failed to completely

abrogate, the in vivo anti-OVA CTL activity in anti-CD137-treated

mice (Fig. 6A). Similarly, DC ablation reduced but did not

completely abolish the effect of agonistic anti-CD137 mAb on

tumor growth (Fig. 6B). When highly purified WT CD81 T cells

and NK cells were adoptively injected into the CD11cDTR-GFP-

C57BL/6 chimeric mice to rule out effects attributable to the

depletion of these subsets that also express CD11c under certain

circumstances [45] similar results were obtained (Supporting

Information Fig. 5).

As a whole, the results advocate an important role

of DC-mediated MHC class I presentation in anti-CD137 immu-

notherapy. However, such presentation is not an absolute

requirement, since presentation by tumor cells or other yet

unidentified host hematopoietic cells may also participate in this

model.

Figure 5. Ablation of DC markedly impaired but failed to abrogate anti-OVA257–264 CTL priming. (A) Phenotypic characterization of CD11cDTR-GFP-C57BL/6 BM chimeric mice. Flow cytometric analysis of spleen cells of WT, CD11cDTR-GFP transgenic, and CD11cDTR-GFP-C57BL/6 BMchimeric mice indicating GFP expression by CD11chigh DC. Histograms show GFP expression in CD8a1 and CD8a� DC subsets. (B) Analysis of DTxsensitivity of the DC compartment in CD11cDTR-GFP-C57BL/6 BM chimeric mice. Flow cytometric analysis of spleen and LN cells 24 h afterinjection of DTx or the vehicle control. The percentages of CD11c1GFP1 DC are indicated. Data are representative of two independent experiments.(C) Assessment of the involvement of DC in tumor Ag presentation in vivo. CD11cDTR-GFP-C57BL/6 BM chimeric mice were injected s.c. with5�105 EG7-OVA cells and treated with DTx (16 mice) or the vehicle control (15 mice) as described in the Materials and methods. Upon firstappearance of palpable tumors, mice were adoptively transferred with CFSE-labeled OT-I CD81 T cells (106 cells per mouse). Tumor Agpresentation, indicated by OT-I proliferation, was analyzed in the TDLN 3 days later. A naıve group of three mice was used as a negative control.Pooled data from two independent experiments are shown. Student’s t-test was used to determine p-values. p-Values ofo0.05 were consideredsignificant.

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Discussion

This study provides evidence for an involvement of DC in the

augmentation of anti-tumor CTL immunity by agonist anti-

CD137 mAb. Surprisingly, the role of DC was not as crucial as

anticipated, probably as a result of antigen presentation by tumor

cells whose performance as APC might be augmented by artificial

T-cell costimulation via CD137.

Conceivably, anti-CD137 mAb are not beneficial for initiating a

CTL response, but rather act on CTL that have already been acti-

vated by tumor antigens [13, 46]. Antigen stimulation is considered

necessary to induce CD137 expression on naıve CTL, and thereby

provide targets for the immunotherapeutic Ab [5]. The dominant

mechanism of tumor antigen presentation to CTL in tumor-bearing

hosts has been argued to be mediated by professional APC (most

likely DC) that cross-present tumor-derived antigen after uptake and

Figure 6. Assessment of the anti-tumor CTL response induced by anti-CD137 mAb in DC depleted mice. (A) CD11cDTR-GFP-C57BL/6 BM chimericmice (6 per group) were treated as indicated and used to comparatively determine the effect of DTx depletions in OVA-specific in vivo killing activityin the spleen. The activity in naıve mice is included as a control. Data are representative of two independent experiments. (B) Evaluation of thetherapeutic activity of anti-CD137 mAb in EG7-OVA tumor-bearing mice. DTx or control vehicle was administered every other day beginning 3 daysbefore tumor cell inoculation and administration continued at 2-day interval afterward until day 30. Treatment with anti-CD137 mAb was given ondays 8, 10, and 12 (100 mg i.p. per dose) after tumor cell inoculation while control animal received control rat IgG. In this experiment, 5 or 10 mice pergroup were included as indicated in the graphs. Fraction of surviving tumor-free mice is shown in each graph.

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processing tumor cells or tumor cell debris [23]. Based on this

notion, it has been proposed that antigen cross-presentation by DC

is a prerequisite for the efficacy of anti-CD137 treatment [3].

OVA was chosen as tumor surrogate antigen model because

specific immune responses can be monitored in vivo and in vitro.

The similarity of such protein to a real tumor antigenic sequence

is debatable, but allows demonstration of proof-of-concept [4, 47,

48]. In the case of EG7 cells, it must be taken into account that

OVA is secreted to the cellular milieu [49] and that tumor

exosomes may also participate in antigen transfer [50].

Our experiments required an immunogenic tumor sensitive to

anti-CD137 mAb treatment as monotherapy. Administration of

agonistic anti-CD137 mAb induced the eradication of well-estab-

lished EG7-OVA-derived tumors by a CD81 T-cell-dependent

mechanism that correlated with an enhanced in vivo CTL activity

against the immunodominant tumor antigen. These findings are in

agreement with previous studies that pinpoint CD81 T cells as the

most consistent protagonist of the immune rejection process induced

by anti-CD137 mAb [13]. Importantly, in this model NK cells do not

play any necessary role [11] and the presence of CD41 cells seem to

decrease therapeutic efficacy, as described previously [34].

It seems EG7-OVA tumor growth is sufficient to expand and

activate antigen-specific CD81 T cells, but, despite this fact,

tumor rejection of established tumors does not take place. This

finding is in line with other publications that report the existence

of primed CD81 T cells in mice with progressing tumors. These T

cells have been described as ‘‘poised’’ T cells, ready for action, not

deleted or anergized, but also unfit to migrate or exert anti-

tumoral effector functions [23, 51]. Alternatively, multifaceted

suppressive mechanisms could be restraining the effector func-

tions of such potentially tumoricidal CD81 T cells [52]. In vivo

treatment with anti-CD137 mAb in this system would enhance

effector functions acting directly or indirectly as a signal-three-

type co-stimulation. Signal-three-stimuli are those that are

required for the acquisition of effector functions by CTL [48].

Using H2Kb�/�-C57BL/6 BM chimeric mice, we have shown

that tumor cells themselves, which can be traced by sensitive PCR

techniques to lymph node, prime CD81 T cells in TDLN of tumor-

bearing mice even in the absence of DC expressing the antigen-

presenting molecule. However, cells derived from the BM are

primarily responsible for antigen presentation to OVA-specific CTL.

Tumor cells may also contribute to this function. These observa-

tions can also be explained by the superior efficiency of profes-

sional APC compared with tumor cells to communicate with CTL

since most tumors lack the co-stimulatory functions required for

CTL induction. It has been proposed that antigen presentation by

tumor cells is inefficient, and therefore cannot elicit protective

anti-tumor CTL immunity [38, 53]. Zinkernagel’s group has chal-

lenged this view proposing that CTL priming is critically dependent

on tumor cells reaching lymphoid tissue [17]. ‘‘Peaceful’’ coex-

istence between DC cross-priming and direct presentation is taking

place in our model as proposed previously [21], although the

involvement of BM-derived cells seems to predominate.

DC are considered the principal cross-presenting cells [19, 40,

41]. In our hands TDLN B cells and macrophages do not cross-

present OVA to sensitive OT-I CD81 T cells. The fraction lacking

CD11c1, B2201, and CD11b1 cells retained in vitro priming

activity for OT-I CD81 T cells that could be mediated by tumor

cells. Currently we are exploring the potential involvement of

plasmacytoid DC in the residual presentation because these are

not depleted in the CD11cDTR-GFP transgenic system (data not

shown). The function of plasmacytoid DC in antigen cross-

presentation to CD81 T cells has been recently reported [54].

The role of DC in tumor immunotherapy based on anti-CD137

mAb was found to be more dispensable than anticipated. Removal

of DC clearly reduces the level of CTL stimulation achieved, but

residual mechanisms under artificial costimulation through CD137

mAb are still capable of inducing tumor rejection with certain delay

in a number of cases. The decreased efficacy following DTx deple-

tion is apparently DC-specific, since transfer of DTR-negative T and

NK lymphocytes did not rectify loss of CTL activity (Supporting

Information Fig. 5). We have not explored in detail the possibilities

that activated DC express surface CD137 (Supporting Information

Fig. 4) [14, 44] and that depletion with DTx could result in the

removal of a potential target of the injected therapeutic Ab.

We cannot definitively rule out that a small fraction of DC

could be surviving DTX chronic treatment or that radio-resistant

host Langerhans cells could play a role [55] in generating an

immune response in the chimeras. Additionally, resistance to the

DTx may occur when exposure is extended [56]. Nonetheless, we

confirm a drastic reduction of DC throughout our depletion

experiments. A new transgenic mouse with less leaky expression

of the DTR has been recently described that permits repeated

depletions without need to generate the BM chimeras [56].

Indirect evidence for the involvement of DC in CD137-targe-

ted therapies has been provided previously [57, 58]. These

reports show that artificially eliciting activation/maturation of

DC in vivo synergizes with anti-CD137 mAb. Such synergistic

effects were precisely optimal when apoptosis of tumor cells is

induced. Apoptosis under certain conditions releases antigens

which thereby become available for cross-presentation [59, 60].

Anti-CD137 mAb acting directly on splenic DC have been shown

to protect these cells from apoptosis and may act by extending

their antigen-presenting function [44].

The optimal performance of DC will be presumably more

critical for therapeutic success in less intrinsically immunogenic

tumor models, such as those in which anti-CD137 immunostimu-

latory mAb needs to be combined with tumor vaccines or cytokine

therapy to achieve efficacy [4, 61–64]. Overall our data show in

the immunogenic EG7-OVA model that DC-mediated cross-prim-

ing is important for the outcome of CD137-targeted therapy.

Materials and methods

Mice and cell lines

C57BL/6 WT mice (5–6 wk old) were purchased from Harlan

Laboratories (Barcelona, Spain). H2-Kb�/� mice (C57BL/6Ji-Kbtm1

Eur. J. Immunol. 2009. 39: 2424–2436Oihana Murillo et al.2432

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

N12) were obtained from Taconic Farms (Germantown, NY, USA).

OT-I TCR transgenic mice (C57BL/6-Tg (TcraTcrb) 1100Mjb/J),

harboring OVA-specific CD81 T cells, and CD11cDTR-GFP trans-

genic mice (B6.FVB-Tg Itgax-DTR/GFP 57Lan/J) were purchased

from the Jackson Laboratory (Bar Harbor, ME, USA) and bred in

our animal facility under specific pathogen-free conditions. All

animal procedures were conducted under institutional guidelines

(study approval number 003/02) that comply with national laws

and policies.

The murine thymoma cell line EG7-OVA was obtained from

the American Type Culture Collection (ATCC, Manassas, VA

CRL-2113) and maintained in complete RPMI medium (RPMI

1640 with Glutamax (Gibco, Invitrogen, Carlsbad, CA, USA)

containing 10% heat-inactivated FBS (Sigma-Aldrich, Dorset,

UK), 100 IU/mL penicillin and 100mg/mL streptomycin

(Biowhittaker, Walkersville, MD, USA) and 5�10�5 mol/L

2-mercaptoethanol (Gibco)).

Generation of BM chimeric Mice

BM chimeric mice were generated by irradiation of recipient mice

(C57BL/6) with a single-lethal dose of 800 cGy. Recipient mice

were then reconstituted with 2�5�106 donor BM cells. For

generation of H2-Kb�/�-C57BL/6 BM chimeric mice, T cells

were removed from donor BM cells suspension by using anti-CD4,

anti-CD8, and anti-Thy-1-coupled magnetic beads according to

the manufacturer’s protocol (Miltenyi Biotec, Gladbach,

Germany). BM chimeric mice were kept on antibiotic-containing

drinking water for 15 days and allowed to reconstitute for

8–10 wk before use.

Reagents

The hybridoma cell lines anti-CD4 (GK 1.5), anti-CD8b (H3S-

17-2), and anti-NK1.1 (PK136), were obtained from ATCC; the anti-

CD137 (2A) hybridoma was a kind gift from Dr. L. Chen (Johns

Hopkins. Baltimore, MD, USA) [4]. The mAb produced by these

hybridomas were purified from respective culture supernatant by

affinity chromatography in sepharose protein G columns according

to manufacturer’s instructions (GE Healthcare Bio-sciences AB,

Uppsala, Sweden). IgG from Rat serum was obtained from Sigma-

Aldrich and was used as control Ab. DTx was purchased from

Sigma-Aldrich and was used for in vivo DC depletion.

In vivo tumor growth and depletion of lymphocytesubsets

For the EG7-OVA tumor model, C57BL/6 mice received

a s.c. injection of 5�105 EG7-OVA viable cells on day 0

and on days 7, 9, 11, and 13 were treated i.p. with either anti-

CD137 mAb or with control rat IgG at 100 mg per dose. Tumor

diameters were measured by electronic caliper every 2–4 days,

and tumor size was determined by multiplying perpendicular

diameters.

For in vivo leukocyte subset depletion, tumor-bearing mice

were injected with depleting anti-CD4, anti-CD8b, or anti-NK1.1

mAb prior to anti-CD137 treatment. A total of 200mg per dose of

each depleting mAb were administered. Anti-CD4 and anti-CD8bmAb were i.p. injected every other day 4 times beginning 3 days

before treatment onset, and then every 5th day for the remainder

of the experiment. NK1.1-specific mAb was administered i.v. for

five consecutive days, beginning 3 days before treatment onset,

and thereafter administration continued in 3-day intervals. For

systemic DC depletion, mice were injected i.p. with DTx at 500 ng

per dose. DTx was injected every other day beginning 3 days

before tumor cells inoculation.

OVA-PCR

C57BL/6 mice received an s.c. injection of 5� 105 EG7-OVA

viable cells in their right flanks. At the time when tumors had

developed (8–10 days after tumor cell inoculation), total DNA

from tumor, spleen, and lymph node tissue was isolated by

using the DNeasy tissue Kit (Qiagen, Madrid, Spain). OVA

DNA was amplified by real-time PCR using the following

pair of specific primers: 50-GCAAACCTGTGCAGATGATG-30

and 50-CCATCTTCATGCGAGGTAAG-30. Real-time PCR was

performed using an iCycler (Bio-Rad, Hercules, CA, USA) and

the iQ SYBR Green Supermix (Bio-Rad, El Prat, Spain). The

amplification of mouse genomic DNA for SOCS3 using the

primers 50-TACCAGCTGGTGGTGAACGC-30 and 50-GCGGCATG-

TAGTGGTGCACC-30 was used as control. To monitor the

specificity, final PCR products were analyzed by melting curves

and electrophoresis. OVA DNA values were represented by the

formula: 2DCt � 102, where DCt represents the difference in

threshold cycle between the control and target gene.

Isolation of mononuclear cells from spleen and lymphnodes

At the indicated time points, spleens and TDLN from tumor-

bearing mice or naıve mice were harvested. They were incubated

in Collagenase D and DNase I (Roche, Basel, Switzerland) for

15 min at 371C, and then they were pressed between two semi-

frosted microscopic slides. Dissociated cells from spleens and

TDLN were passed through a 70 mm nylon mesh filter (BD Falcon,

BD Bioscience, San Jose, CA, USA) and washed. Spleen cells were

also treated with ammonium chloride lysing buffer and washed

before further use.

Flow cytometry

Single-cell suspensions from spleen and TDLN were washed in

PBS supplemented with 5% FBS, and subsequently pre-treated

Eur. J. Immunol. 2009. 39: 2424–2436 Cellular immune response 2433

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

with Fc-Block (anti-CD16/32, eBioscience, San Diego, CA) to

reduce the non-specific staining. Afterwards, cells were incubated

with the staining Ab. Ab to the following mouse antigens

conjugated to FITC, PE, or allophycocyanin were used: CD44,

CD62L, H2Kb, CD3, CD8, CD11c, and Va2. These specific Ab and

their corresponding isotype controls were obtained from BD

Pharmingen (San Agustin de Guadalix, Spain). For tetramer

staining, single-cell suspension from spleen were incubated with

PE-conjugated Kb-SIINFEKL tetramers (Beckmann Coulter,

Madrid, Spain), washed, and subsequently stained with both

anti-CD8 FITC and anti-CD3 APC Ab. Surface marker expression

was assayed using a FACSCalibur (BD Biosciences) flow

cytometer, and data analysis carried out using Cell Quest Pro

(BD) software.

In vivo analysis of tumor Ag cross-presentation

OVA-specific T-cell proliferation was assessed using CFSE-labeled

OT-I T cells as described elsewhere [23]. Briefly, single-cell

suspensions were prepared from the spleens of OT-I TCR

transgenic mice. OVA-specific CD81 T cells (CD81 T OT-I cells)

were purified using a negative selection kit (mouse CD8a1 T-cell

isolation kit, Miltenyi Biotec) and the AutoMACS cell separation

system (Miltenyi Biotec). Purified lymphocytes were labeled with

2.5 mM CFSE (Sigma) for 15 min, at room temperature. CFSE-

labeled cells were adoptively transferred by i.v. injection into

recipient mice (106 cells). In total 72 h after transfer, axillary and

inguinal TDLN were collected from individual mice for analysis.

Proliferation of CD81 OT-I T cells, indicated by loss of CFSE

staining, was determined by flow cytometric analysis (FACSCa-

libur; BD Biosciences).

DC purification and co-culture with CFSE-labeled OT-ICD81 T cells

DC were positively selected from the TDLN and spleens of tumor-

bearing mice using magnetized Ab for CD11c (mouse CD11c

microbeads, Miltenyi Biotec) and the AutoMACS cell separation

system (Miltenyi Biotec), following manufacturer’s instructions.

Similarly, CD81 OT-I T cells were purified from the spleens of

OT-I TCR transgenic mice by AutoMACS cell separation. In both

cases the cell purity was 480%.

CFSE-labeled 5� 104 CD81 OT-I T cells were incubated with

1� 105 CD11c1 cells in U-bottom 96-well plates for 96 h. Cell

proliferation was analyzed by CFSE dilution (FACSCalibur; BD

Biosciences).

In vivo killing assay

Splenocytes from naıve C57BL/6 mice were divided into two

samples. One sample was pulsed with the OVA257–264 peptide

(NeoMPS, Strasbourg, France) (10 mg/mL) for 30 min at 371C in

5% CO2, washed extensively, and subsequently labeled with high

concentration 1.25mM of CFSE (Sigma). The non-pulsed control

sample was labeled with 0.125 mM of CFSE. Then, both CFSEhigh-

and CFSElow-labeled cells were mixed at a 1:1 ratio and injected

i.v. (5� 106 cells of each population) into tumor-bearing mice. In

total 24 h after transfer spleens were harvested and specific

cytotoxicity was analyzed by flow cytometry. Specific cytotoxicity

was calculated as follows: 100–[100� (%CFSEhigh tumor-bearing

mice/%CFSElow tumor-bearing mice)/(%CFSEhigh naıve mice/

%CFSElow naıve mice)].

Statistical analysis

Prism software (Graph Pad Software) was used to analyze tumor

growth, frequency of OVA-Tetr1 cells, in vivo killing assay data,

and cell proliferation and to determine statistical significance of

the differences between groups by applying unpaired Student’s

t-tests or U-Mann–Whitney tests. p-values ofo0.05 were consid-

ered significant.

Acknowledgements: Professor Lieping Chen is acknowledged for

critical discussion and for kindly providing the 2A hybridoma. The

authors are grateful to Elena Ciordia and Eneko Elizalde for animal

care and husbandry. Financial support was from from MEC

(SAF2005-03131 and SAF2008-03294), Departamento de

Educacion del Gobierno de Navarra, Departamento de Salud del

Gobierno de Navarra (Beca Ortiz de Landazuri). Redes tematicas

de investigacion cooperativa RETIC (RD06/0020/0065), Fondo de

investigacion sanitaria (PI060932), European commission VII

famework program (ENCITE) and SUDOE (IMMUNONET).

Fundacion Mutua Madrilena, and ‘‘UTE for project FIMA’’.

Sandra Hervas-Stubbs receives a Ramon y Cajal contract from

Ministerio de Educacion y Ciencia.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: DTR: diphtheria toxin receptor � DTx: diphtheria toxin �GFP: green fluorescent protein � TDLN: tumor-draining lymph nodes

Full correspondence: Professor Ignacio Melero, CIMA. Avenida de Pio XII,

55, 31008 Pamplona, Spain

Fax: 134-948-194717

e-mail: imelero@unav.es

Supporting Information for this article is available at

www.wiley-vch.de/contents/jc_2040/2009/38958_s.pdf

Received: 12/10/2008

Revised: 25/5/2009

Accepted: 12/6/2009

Eur. J. Immunol. 2009. 39: 2424–2436Oihana Murillo et al.2436

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu