10
Review Natural Killer Cells: The Secret Weapon in Dendritic Cell Vaccination Strategies Catharina H.M.J. Van Elssen 1 , Tammy Oth 1 , Wilfred T.V. Germeraad 1 , Gerard M.J. Bos 1 , and Joris Vanderlocht 2 Abstract In cancer therapy, dendritic cell (DC) vaccination is still being explored. Clinical responses, however, are diverse and there is a lack of immunologic readout systems that correspond with clinical outcome. Only in the minority of patients, T-cell responses correlate with clinical outcome, indicating that other immune cells also gain anticancer activity. We still have limited knowledge of the effect of DC vaccination on different immune effector cells. However, it has been shown that bidirectional cross-talk between natural killer (NK) cells and DCs is responsible for enhanced activation of both cell types and increases their antitumor activity. In this review, we postulate the possibility that NK cells are the secret weapons in DC vaccination and studying their behavior together with T-cell activation in vaccinated individuals might predict clinical outcome. Clin Cancer Res; 20(5); 1095–103. Ó2014 AACR. Disclosure of Potential Conicts of Interest No potential conflicts of interest were disclosed. CME Staff Planners' Disclosures The members of the planning committee have no real or apparent conflicts of interest to disclose. Learning Objectives Upon completion of this activity, the participant should have a better understanding of the potential positive role of natural killer cells in dendritic cell vaccination in cancer patients and the different tools currently available to assess natural killer cell activity in vaccinated patients. Acknowledgment of Financial or Other Support This activity does not receive commercial support. Introduction Dendritic cells (DC), the most efficient antigen-present- ing cells (APC) of the immune system, have been used in clinical vaccination strategies for cancer. Immune responses induced by DCs inhibit tumor cell growth or even eradicate tumor cells. Although many clinical DC vaccination trials have been performed, the exact immune responses induced by these DCs are not completely elucidated. In most of these trials, specific immune reactions are studied instead of investigating the effect on the whole immune system. Initial vaccination strategies focused on inducing antitumor CTLs (1). However, after understanding the function of MHC class II and the identification of Th cells, the induction of CTLs after vaccination was analyzed in the context of Th-cell polarization (2). This eventually led to development of DC differentiation and maturation cocktails that enhance Th1 responses (3). In clinical DC vaccination strategies, only a minority of patients respond to therapy by tumor regression or stable disease; in some cases, complete remission is reported (4). Clinical response rates may vary between studies, which is mainly due to differences in patient pop- ulation and different DC maturation and vaccination pro- tocols. As indicated in Table 1, when clinical responses are induced after DC vaccination, this does not always corre- spond with the induction of tumor-specific CTLs. These poor correlations could indicate that T-cell responses are analyzed at the wrong anatomic site or that other non-T-cell responses are induced. Besides T-cell responses, B-cell responses were also evalu- ated by detection of tumor-specific antibodies. Although these tumor-specific antibodies are often produced after vaccination, they correspond only in a minority of cases with clinical responses (5). It took until 2000 for other effector cells to be analyzed in the context of DC vaccination. Authors' Afliations: Departments of 1 Internal Medicine, Division of Hematology and 2 Transplantation Immunology, Maastricht University Medical Center, Maastricht, the Netherlands Corresponding Author: Catharina H.M.J. Van Elssen, Department of Internal Medicine, Division of Hematology, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht, the Netherlands. Phone: 31-433- 881-644; Fax: 31-433-884-164; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-2302 Ó2014 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 1095 on August 3, 2018. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

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Review

Natural Killer Cells: The Secret Weapon in Dendritic CellVaccination Strategies

CatharinaH.M.J. VanElssen1, TammyOth1,Wilfred T.V.Germeraad1,GerardM.J. Bos1, andJoris Vanderlocht2

AbstractIn cancer therapy, dendritic cell (DC) vaccination is still being explored. Clinical responses, however, are

diverse and there is a lack of immunologic readout systems that correspond with clinical outcome. Only in

theminority of patients, T-cell responses correlate with clinical outcome, indicating that other immune cells

also gain anticancer activity. We still have limited knowledge of the effect of DC vaccination on different

immune effector cells. However, it has been shown that bidirectional cross-talk between natural killer (NK)

cells andDCs is responsible for enhanced activation of both cell types and increases their antitumor activity.

In this review, we postulate the possibility that NK cells are the secret weapons in DC vaccination and

studying their behavior together with T-cell activation in vaccinated individuals might predict clinical

outcome. Clin Cancer Res; 20(5); 1095–103. �2014 AACR.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

CME Staff Planners' DisclosuresThe members of the planning committee have no real or apparent conflicts of interest to disclose.

Learning ObjectivesUpon completion of this activity, the participant should have a better understanding of the potential positive role of natural killer cells in

dendritic cell vaccination in cancer patients and the different tools currently available to assess natural killer cell activity in vaccinated

patients.

Acknowledgment of Financial or Other SupportThis activity does not receive commercial support.

IntroductionDendritic cells (DC), the most efficient antigen-present-

ing cells (APC) of the immune system, have been used inclinical vaccination strategies for cancer. Immune responsesinduced by DCs inhibit tumor cell growth or even eradicatetumor cells. Although many clinical DC vaccination trialshave been performed, the exact immune responses inducedby theseDCs are not completely elucidated. Inmost of thesetrials, specific immune reactions are studied instead ofinvestigating the effect on thewhole immune system. Initialvaccination strategies focused on inducing antitumor CTLs(1). However, after understanding the function of MHC

class II and the identification of Th cells, the induction ofCTLs after vaccinationwas analyzed in the context of Th-cellpolarization (2). This eventually led to development of DCdifferentiation and maturation cocktails that enhance Th1responses (3). In clinical DC vaccination strategies, only aminority of patients respond to therapy by tumor regressionor stable disease; in some cases, complete remission isreported (4). Clinical response rates may vary betweenstudies, which is mainly due to differences in patient pop-ulation and different DC maturation and vaccination pro-tocols. As indicated in Table 1, when clinical responses areinduced after DC vaccination, this does not always corre-spond with the induction of tumor-specific CTLs. Thesepoor correlations could indicate that T-cell responses areanalyzed at thewrong anatomic site or that other non-T-cellresponses are induced.

Besides T-cell responses, B-cell responses were also evalu-ated by detection of tumor-specific antibodies. Althoughthese tumor-specific antibodies are often produced aftervaccination, they correspond only in a minority of caseswith clinical responses (5). It took until 2000 for othereffector cells to be analyzed in the context of DC vaccination.

Authors' Affiliations: Departments of 1Internal Medicine, Division ofHematology and 2Transplantation Immunology, Maastricht UniversityMedical Center, Maastricht, the Netherlands

Corresponding Author: Catharina H.M.J. Van Elssen, Department ofInternal Medicine, Division of Hematology, Maastricht University MedicalCentre, POBox616, 6200MDMaastricht, theNetherlands.Phone: 31-433-881-644; Fax: 31-433-884-164; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2302

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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Tab

le1.

Tan

dNKce

llac

tivationin

clinical

DC

vacc

inationtrials

Disea

seVac

cina

tion

Clin

ical

resp

ons

eSpec

ificTce

llsReferen

ce

AML

WT1

mRNA–elec

trop

orated

DCs

5/10

patie

nts(2/10with

long

-lastingresp

onse

s)2/5Patients.

Bes

tT-ce

llresp

onse

sin

bes

tclinical

resp

onde

rs(67)

Melan

oma

Wild

-typ

ean

dmod

ifies

gp10

0pep

tide-pulse

dDCs

9/27

patie

nts(1/27co

mplete

resp

onse

s)3/27

Patients.

Bes

tT-ce

llresp

onse

sin

thebes

tclinical

resp

onse

s(68)

Breas

tca

ncer

MUC1ge

ne–tran

sfec

tedDCs

1/10

patie

nts

4/10

patie

nts

(69)

Ren

alce

llca

ncer

DC

vacc

inationwith

tumor

celllysa

teor

tumor

celllinelysa

te10

/27patients(2/27co

mplete

resp

onse

s)5/6Patients.

Bes

tT-ce

llresp

onse

sin

bes

tclinical

resp

onders

(70)

Hep

atoc

ellularca

rcinom

aDC

vacc

inationwith

tumor

celllysa

te7/25

patie

nts

5/10

Patients,

noco

rrelationwith

clinical

resp

onse

s(71)

Melan

oma

Tumor

antig

enco

cktail–load

edDCs

11/36patients

6/10

Patients,

noco

rrelationwith

clinical

resp

onse

s(72)

Multip

lemye

loma

Idiotype-pulse

dDCs

5/9patients

4/5Patientsha

daclinical

resp

onse

(73)

Colorec

talc

ance

rTu

mor

lysa

te—load

edDCs

8/24

patie

ntsaredisea

sefree

15/24Patients(significa

ntly

bettersu

rvival

whe

nsp

ecificT-ce

llresp

onse

s)(74)

Disea

seAna

lysisofNK

cells

resp

ons

esNK

cellresp

ons

esan

dclinical

resp

ons

esReferen

ce

AML

>40%

ofHLA

-DRþ

NKce

llspos

tvac

cina

tion

4/5Clinical

resp

onders

(67)

0/5Non

resp

onders

CEA-pos

itive

tumors

(Colorec

tal,lung

,urach

al)

Increa

sein

totalN

Kce

llnu

mbers

4/5Clinical

resp

onders

(75)

Increa

sein

cytotoxicac

tivity

(K56

2an

dRaji)

0/4Non

resp

onders

Increa

sedNKG2D

andNKp46

expression

Non

-Hod

gkin

lympho

ma

Increa

sein

totalN

Kce

llnu

mbers

5/6Clinical

resp

onders

(76)

1/4Non

resp

onders

Adva

nced

canc

er(m

yeloma,

anal,ren

al)

Increa

sein

totalN

Kce

llnu

mbers

Increa

sein

NKp30

,NKp46

,and

NKp44

expression

0/2Clinical

resp

onders

0/3Non

resp

onders

(77)

Metas

tatic

gastrointestinal

canc

erIncrea

sein

NKce

llcy

totoxicity

(K56

2)2/3Clinical

resp

onders

(78)

3/14

Non

resp

onders

Abbreviation:

AML,

acutemye

loge

nous

leuk

emia.

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In mice, DC-induced natural killer (NK) cell activation wasproven to be an absolute prerequisite for induction of Th1polarization (6). Human in vitro data demonstrate that NKcells and DCs cooperate in pathologic conditions (7). Thistriggered the evaluation of NK cell responses in clinical DCvaccination studies (Table 1). The immunostimulatory effectof NK–DC interaction is based on a bidirectional cross-talkby which both cell types become activated. For DC vaccina-tion techniques, activation of both cell types in vitro has beendescribed, and their effect on other immune and tumor cellshas been suggested to be beneficial for antitumor responses(8, 9). We postulate that NK cells are the secret weapons inDC vaccination. Here, we review the different effects of NK–DCinteractionand their impact inDC-vaccination strategies.

NK cell–dependent DC responsesNK cells hold the capacity to control and enhance DC-

mediated antitumor immune responses, by inducing mat-uration of Th1-polarizing DCs, providing DCs with anti-genic material for presentation and killing of inappropri-ately matured DCs.

NK cell–induced DC maturationNK cells mediate immunoregulatory "helper" functions.

These cells were phenotyped as CD83þCCR7þCD56dim NKcells that possess DC-activating capacities (9), which aremediated by both NK cell–derived soluble factors, mainlyIFN-g and TNF-a (10, 11), as well as ligation of surfacereceptors (Fig. 1A). NK–DC interaction results in the devel-opment of stable, type-1 polarized DCs that produce highamounts of proinflammatory cytokines and, thereby, areable to enhance Th1 and CTL-mediated immunity againstintracellular pathogens and cancer (Fig. 1A; refs. 10, 12). Inthe absence of NK cells, Th polarization is biased towardTh2 polarization, without induction of CTLs (13). NK cell–derived IFN-g induces upregulation of the major Th1 tran-scription factor T-bet and inhibition of the Th2 transcrip-tion factor GATA-3 (6). These "helper" NK cells gained theirfunction after activation by interleukin (IL)-12 and IL-18(9). Other proinflammatory cytokines (IL-2, IL-15, type-IIFNs: IFN-a/b), NK cell–sensitive tumor cell lines, andopsonizing tumor-specific antibodies can synergize in a

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IL-12

IL-18

IL-2

IL-15

IFN-α/β

IL-12

Th1

Th1

NK

NK

NK

NK

NK

IDC

IDC

DC

DC

DC

IL-2

Activation

Activation

Apoptotic

bodies

CD94/NKG2A

Granzymes

Perforins

A

B

C

NKp30

NKp30

NKp30

HLA-E

FcR

TNF-α/IFN-γ

IFN-γ

Figure 1. NK cell–dependent DC activation. NK cells can enhance DCfunction by three different mechanisms. A, NK cells can becomeactivated to produce IFN-g and TFN-a by a two-signal mechanism,including activation by cytokines (IL-12, IL-18, IL-15, IL-2, IFN-a, andIFN-b), by induction of antibody-dependent cell-mediated cytotoxicity(ADCC) via antibodies or by ligation of activating receptors (NKp30,DNAM-1). NK cell–derived cytokines enhance maturation of IL-12-producing DCs, which effectively induce Th1 polarization. In addition,NK cell–derived IFN-g directly enhances Th1 polarization. B,because of NK cell–mediated tumor cell lysis, tumor antigens becomeavailable for DCs to engulf and present. Activated NK cells alsoupregulate the expression of MHC class II and become weak antigenpresenters themselves. C, NK cells kill iDCs because of low HLA-Eexpression. The ligation of NKp30 enhances this NK cell function.By elimination of iDCs, NK cells prevent T cells from becomingtolerogenic due to contact with inadequately matured DCs.

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two-signal mechanism (12, 14, 15). Whether these "help-er" NK cells also naturally occur in vivo and whether theyindeed represent a distinct subpopulation is stillunknown. Reports on the IFN-g–secreting NK cell sub-population are controversial, and both CD56bright andCD56dim subsets have been shown to produce IFN-g afterNK–DC interaction (8, 9). NK cells display a high plas-ticity in phenotype and upon stimulation can easily up-and downregulate surface receptor expression. However,after they have acquired the expression of killer immu-noglobulin-like receptors (KIR) and perforin, the expres-sion of CD56 and CD16 is stable (16).

Facilitation of antigen presentationNK cells have the natural capacity to kill virally infected or

malignantly transformed cells (17). This function is not onlyof direct use for tumor elimination, but also facilitates uptakeandcross-presentationof tumor-relatedantigenicmaterialbyDCs (refs. 18, 19; Fig. 1B). In addition, activated NK cells areinduced toexpressMHCclass II and costimulatorymolecules(like CD80, CD83, and CD86) and display APC-like prop-erties, with unique pathways for antigen uptake and presen-tation to T cells (Fig. 1B; refs. 20, 21). TheseNKcellswithDC-like qualities have been described in humans and mice andhave been reported as a new immune cell subpopulationreferred to as NKDCs as well as IKDCs (IFN-producing killerdendritic cells; refs. 22–24). Developmentally,mouse IKDCsare absent from recombinase activating gene-2-null, com-mon g-chain-null (RAG2�/�IL2rg�/�) mice, which lack NKcells but not DCs (25, 26). Therefore, although the nomen-clature is confusing, it is assumed that these different subsetsare most likely to resemble activated NK cells (22).

NK cell–dependent lysis of immature DCsNK cells also have the capacity to eliminate autologous

and allogeneic immature DCs (iDC; ref. 10). NKp30 hasbeen shown to play a major role in iDC lysis and coop-erates with DNAX accessory molecule-1 (DNAM-1;ref. 27; Fig. 1C). Because both mature as well as immatureDCs express ligands for NKp30 and DNAM-1 (27), anadditional mechanism must be involved. Analysis of NKcell clones has revealed that killing of iDCs was confinedto NK cells that lack expression of inhibitory KIRs specificfor self-HLA class I alleles, but do express the HLA-E–specific CD94/NKG2A inhibitory receptor (28). The iDCbecomes susceptible to NK cell–mediated cytotoxicitybecause of low expression of HLA-E irrespective of expres-sion of other HLA molecules. In contrast, mature DCsexpress higher amounts of HLA-E and are, therefore, keptuntouched (28, 29). iDCs have been implicated in tol-erance and induction of regulatory T cells (30). Thus, itcan be hypothesized that by elimination of iDCs, NK cellsensure the activation of adaptive immune responses bypreventing inadequately matured DCs from interactingwith T cells (31, 32). Although in apparent paradox, NKcells can induce DC maturation as well as lysis of iDCs,depending on the relative numbers of each cell type (10).

DC-induced NK cell responsesNot only are NK cells capable of DC activation, but also

reciprocally, DCs stimulate NK cells by soluble as well ascontact-dependent activators, thereby enhancing their cyto-kine production, proliferation, survival, and cytotoxicity.

Induction of cytokine production and proliferationDCs activate NK cells to produce cytokines (mainly TNF-

a and IFN-g), which are mediated by a DC-derived two-signalmechanismanddepend onboth soluble and contact-dependent factors (Fig. 2). Depending on their maturationstimuli (33, 34), DCs upregulate the expression of NK cell–activating surface molecules and start producing NK cell–stimulating cytokines.

Among the DC-derived soluble factors, IL-12, IL-18, IL-2,and IL-15 have been shown highly efficient in NK cellactivation (8, 35, 36). We and others have shown that NKcell–activating capacity is confined to DCs triggered bypattern recognition receptor signaling (8, 37). IL-15 actsboth as a soluble and a contact-dependent factor (38), asDCs and DC exosomes can bind IL-15 to their IL-15Ra,thereby presenting IL-15 in trans. The effect of IL-15 onNK cell proliferation, survival, and cytokine secretion ismuch stronger when presented in trans than in its solubleform (39).

In addition to these soluble factors, NK–DC cross-talk isenhanced when both cell types are in contact with eachother, suggesting that contact-dependent factors areinvolved (8). These factors include surface moleculesexpressed on DCs that ligate with activating NK cell recep-tors. One of these NK cell receptors is NKG2D, which is a C-type lectin coactivation receptor that binds to stress-induc-ible members of the polymorphic MHC class I–relatedchain A/B (MICA/MICB) family (40, 41). The NK cell–activating receptors NKp30 and NKp46 are also involvedinNK–DC cross-talk (42–44).Moreover, 2B4, which ligateswith CD48, enhances NK–DC cross-talk (45). Its function,however, is still promiscuous as the 2B4 pathway can beboth activating as well as inhibitory (46, 47). Which path-way is induced depends on the NK cell maturation statusand localization (48). All the previously mentioned surfacemolecules are stress-dependent factors that can be upregu-lated during DC maturation, thereby mediating dangersignals to the surrounding tissue.

Induction of NK cell–mediated cytotoxicityThe cytotoxic activity of NK cells is based on a delicate

balance of inhibitory and activating signals and dependson the expression of MHC class I and activating ligandson the target cell and the expression of the respectivereceptors by NK cells (49, 50). NK cell cytotoxicity can beenhanced by DC-dependent mechanisms (Fig. 2; refs. 51,52), including DC-derived IL-12 (8), IL-2, and IL-15 (53)as well as contact-dependent factors like NKG2D, NKp46,and NKp30 (43). The NK cell–activating mechanism usedby DCs has been shown to relate to the stimuli used in DCmaturation (54).

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Common playground for NK cells and DCs?For reciprocal NK–DC activation, it is obligatory for the

two cell types to meet. It has been proposed that NK–DCinteraction takes place at the site of inflammation as well asin the lymph nodes (7). During an inflammatory response,a burst of immunoactive molecules is secreted, includingchemokines, resulting in immune cell recruitment. ForNK–DC cross-talk specifically, it is necessary that one cell typeproduces chemokines, important for recruitment of theother cell type. We have shown that upon maturation,Toll-like receptor (TLR)–triggered DCs produce large quan-tities of many different chemokines (55, 56). Among these,there are NK cell–recruiting chemokines produced, likeCCL5, CXCL10, and CCL19. Different chemokines andchemokine receptors have been shown to be involved inDC-mediatedNK cell recruitment (6, 8). Therefore, it seemsthat there are multiple, nonredundant mechanisms torecruit NK cells to the site of inflammation. Which of thesemechanisms is useddepends on theDCmaturation triggers.Because maturing DCs migrate into the draining lymph

nodes, chemokine production by mature DCs is possiblyalso responsible for the recruitment of NK cells into thelymph node. It has been shown in CXCR3�/�mice that Th1polarization was inhibited, due to impaired recruitment ofNK cells into the lymph nodes (6). In humans, however,additional NK cell–recruiting mechanisms have been pro-

posed.NK cells are able to upregulate their CCR7 expressionand subsequently their responsiveness to the lymph node–homing chemokine CCL19 upon activation with IL-18 andafter NK–DC cross-talk (8, 9). This would represent anadditional mechanism bywhich DCs induce NK cell migra-tion into the draining lymph nodes (Fig. 3).

Role of NK cells in DC vaccinationThe reciprocal effects of NK–DC interaction provide a

strong rationale for the combineduseofNKcells andDCs inimmunotherapy. The use of allogeneic NK cells has shownpromising results in cancer immunotherapy (57), and thesecells have been proven to be the main effector cells inhaploidentical stem cell transplantation in acute myeloge-nous leukemia (AML; ref. 58). However, in these therapies,NK cells were always used as lonely killers. With our recentunderstanding of the NK–DC interaction, one could arguethat DCs used in vaccination strategies should be in vitromatured in the presence of NK cells (14). From anotherpoint of view, there is also a rationale for the in vivo use ofDCs that are able to optimally activate and recruit NK cells(8, 37). We and others have shown that for optimal NK cellactivation and recruitment, DC maturation protocolsshould contain TLR agonists, whereas PGE2, another DC-activating molecule produced during inflammation, has anegative effect on NK–DC cross-talk (37, 59). Despite the

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© 2014 American Association for Cancer Research

Th1

NK

NK

NK

NK

NK

NK

DC

DC

DC

TLR

Proliferation

Cytokine secretion

Cytotoxicity

Contact independent

IL-2, IL-12, IL-15, IL-18,

IFN-α, IFN-β

Contact dependent

NKp46, NKp30, NKG2D,

2B4, CD27, IL-15 in trans

PathogenGranzymes

Perforins

TNF-α, IFN-γ

Figure 2. DC-induced NK cellactivation. DCs can affect NK cellfunction by augmentation ofcytotoxicity, cytokine secretion(IFN-g and TNF-a), andproliferation. This depends oncontact-dependent (ligation ofNKp46, NKp30, NKG2D, 2B4, andCD27 and presentation of IL-15in trans) as well as soluble factors(IL-2, IL- 12, IL-15, IL-18, IFN-a, andIFN-b). IFN-g secretion by NK cellsis, in turn, responsible for DCmaturation and Th1 polarization,whereas augmentation of NK cellcytotoxicity contributes to tumorcell lysis. Which cytokines orcontact-dependent factors areinvolved depends on the triggersinvolved in DC activation (mostlikely TLR ligands).

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reports on the negative effects of PGE2-mediated DC mat-uration, most clinical trials use PGE2-matured DCs. Therationale behind this is the in vitro capacity of these DCs tomigrate (60). However, in in vivo studies, these DCsmigratejust as poorly as non-PGE2–matured DCs (61). The lack ofmigratory capacity of TLR-matured DCs’ combination withtheir NK cell–recruiting capability opens the possibility forthe combination of NK–DC therapy. In these therapies, NKcells and DCs can either be simultaneously injected or onlyDCs are injected to recruit NK cells.

For the simultaneous injection of NK cells and DCs,intratumoral injection seems an attractive strategy. NK cellslyse tumor cells, thereby making antigenic material avail-able for DCs to take up and present. In addition, NK cellsinduce maturation of bystander DCs and lyse inappropri-ately matured iDCs. Reciprocally, mature DCs can activateNK cells to proliferate, secrete cytokines, and augment NKcell cytotoxicity. Another option is the injection of NK cellsandDCs in the tumor-draining lymph nodes where the Th1polarizing capacity of the NK cell can be used to its fullest inthis T cell–rich environment. Subcutaneous systemic injec-tion of DCs together with NK cells seems a less attractivestrategy. Because of the lack of migration of DCs, both celltypes will possibly not home into tissue in the direct tumorvicinity and, thereby, will not stimulate the appropriateeffector cells.

In addition to the simultaneous injection of both celltypes, we propose a different option for NK–DC interactionin DC vaccination: the development of a tertiary lymphoidstructure. In this strategy, only DCs are administered andthis option is based on the capacity of TLR-matured DCs toproduce chemokines and, thereby, selectively recruit effec-tor cells (55). Because of the lack of in vivo DC migration,DCs could be injected in tumor-draining lymph nodes orinto the tumor vicinity, where they can recruit and interactwith NK cells. DC-mediated chemokine production is alsoattractive for recruitment of CTLs and Th1 cells (62–64).Wehypothesize that by recruitment of all these effector cells byDCs, the same interactions that take place in lymph nodescan be induced extranodally in a tertiary lymphoid struc-ture. Whether the immunosuppressive effect of the tumormicroenvironment has detrimental effects on effector cellinduction by DCs remains to be elucidated.

NK responses in DC vaccinationThe evidence on reciprocal interactions of NK cells and

DCs indicates that there is a rationale for monitoring NKactivity after immunotherapeutic approaches, includingDCvaccination. Notably, there is no consensus on assays to beroutinely applied for NK cell monitoring, and as shownin Table 1, the definition of NK cell responsiveness differsbetween studies. Here, we propose a combination of assays

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© 2014 American Association for Cancer Research

Th1

NK

NK

NK

NK

NK

NK

DC

DC

DC

Lymph node

D

C

B

A

NK–DC cross-talkNK–DC cross-talk

CXCR3

CCR5

CCR7↑CCR7↑

Chemokines

Tumor cells

Chemokines

Figure3. WheredoNKcells andDCsinteract? Tissue-resident DCsmature upon inflammatory stimuliproduced by the tumor and itssurrounding tissue. Uponmaturation, these DCs upregulateMHC, costimulatory molecules,and CCR7. In addition, these DCsstart to produce cytokines andchemokines. These chemokinesare responsible for NK cellrecruitment (either CCR5 or CXCR3dependent). A, blood-derived NKcells are then recruited into thetumor environment and interactwith DCs, resulting in reciprocalactivation of both cell types. Uponmaturation, DCs upregulate theirCCR7 expression and migrate intothe draining lymph nodes. B, whilemigrating, DCs still producechemokines and, thereby, recruitNK cells into the lymph nodes. C, inaddition, iKDC interaction inducesthe upregulation of CCR7expression by NK cells, whichconstitutes a secondarymechanism by which NK cellsmigrate into the draining lymphnode. D, because of migration ofboth cell types into the lymph nodeand also the presence of lymphnode–residing NK cells, NK–DCcross-talk can take place in thelymph node.

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that should be applied as a means to standardize thedefinition of NK cell responsiveness.The different assays include both phenotypic and func-

tional characterizations. Phenotypic characterization is usu-ally performed by flow cytometry. The surface markersCD69 and CD25 allow discrimination between resting,recently activated, and activated NK cells. CD69 expressionis transiently present on NK cells after activation and is,therefore, considered a surface marker of recently activatedNK cells, whereas CD25 has a more sustained expressionprofile upon activation (65). Upon activation, NK cells alsoinduce the expression of HLA-DR (22). In addition, theseactivation markers are often combined markers for theidentification of different NK cell subsets, such as CD16 incombination with CD56, KIRs, and natural cytotoxicityreceptors (e.g., NKp30 and NKp46).To analyze NK cell functionality, intracellular staining

measures their content of granzymes and perforins,which is considered a benchmark for cytotoxic ability.NK cells that have recently degranulated are identified bytheir surface expression of CD107a (66). The secretedcytokine quantity can be measured by ELISA, and thepercentage of cytokine-secreting cells is determined byintracellular staining or ELISPOT. The ultimate readoutfor assessing NK cell function after immunotherapy isstudying whether NK cells increase their cytotoxicity totumor cells. The K562 tumor cell line is MHC class I–negative and is, therefore, readily killed by NK cells. The

Raji cell line is a more attractive cell line because it is notsusceptible to NK-mediated lysis unless the NK cells arepreactivated. If patients’ tumor cells are accessible, it ispossible to assess whether the immunotherapeutic inter-vention has resulted in increased cytotoxicity to thetumor. An additional advantage of the accessibility oftumor cells is that these cells and their microenviron-ment can be studied following immunotherapy for pro-duction of NK cell–suppressive factors such as prosta-glandins or TGF-b.

ConclusionsIn this review, we postulate that NK cells might be the

secret weapons in DC-vaccination strategies. NK cells canbidirectionally interact with mature DCs and, thereby,enhance both antitumor T-cell responses as well as NK cellresponses. ThisNK–DC interactionmight occur at the site ofthe tumor or in the lymph node. Until now, only limited,albeit promising human in vivo proof has been shown thatNK cell activation after DC vaccination correlates withclinical outcome. To learn more about this NK–DC inter-action in vivo, it is important for monitoring of NK cellactivation to be incorporated into clinical DC vaccinationtrials. This process may lead to a system by which clinicaloutcome can be predicted.

Received September 23, 2013; revised December 17, 2013; acceptedJanuary 5, 2014; published online March 3, 2014.

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2014;20:1095-1103. Clin Cancer Res   Catharina H.M.J. Van Elssen, Tammy Oth, Wilfred T.V. Germeraad, et al.   Vaccination StrategiesNatural Killer Cells: The Secret Weapon in Dendritic Cell

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