JCO-2011-Steidl-JCO.2010.32.8401

Molecular Pathogenesis of Hodgkin’s Lymphoma:Increasing Evidence of the Importance ofthe MicroenvironmentChristian Steidl, Joseph M. Connors, and Randy D. Gascoyne

From the British Columbia CancerAgency, University of British Columbia,Vancouver, British Columbia, Canada.

Submitted September 19, 2010;accepted January 4, 2011; publishedonline ahead of print at www.jco.org onApril 11, 2011.

Supported in part by the Michael SmithFoundation for Health Research, theLymphoma Research Foundation, and apostdoctoral fellowship from theCancer Research Society (Steven E.Drabin fund; C.S.); Project Grant No.019001 from the Terry Fox FoundationNew Frontiers Program (J.M.C.,R.D.G.); and Grant No. 178536 from theCanadian Institutes for Health Research(R.D.G.).

Authors’ disclosures of potential con-flicts of interest and author contribu-tions are found at the end of thisarticle.

Corresponding author: RandyGascoyne, MD, Department of Pathol-ogy and Experimental Therapeutics,British Columbia Cancer Agency, 675West 10th Ave, Room 5-113, Vancou-ver, BC V5Z 1L3, Canada; e-mail:[email protected].

© 2011 by American Society of ClinicalOncology

0732-183X/11/2999-1/$20.00

DOI: 10.1200/JCO.2010.32.8401

A B S T R A C T

Hodgkin’s lymphoma (HL) represents the most common subtype of malignant lymphoma in youngpeople in the Western world. Most patients can be cured with modern treatment strategies,although approximately 20% will die after relapse or progressive disease. The histologic hallmarkof the disease is the presence of the characteristic Hodgkin Reed-Sternberg (HRS) cells in classicalHL and so-called lymphocyte-predominant (LP) cells in nodular lymphocyte-predominant HL. HL isunique among all cancers because malignant cells are greatly outnumbered by reactive cells in thetumor microenvironment and make up only approximately 1% of the tumor. Expression of avariety of cytokines and chemokines by the HRS and LP cells is believed to be the driving force foran abnormal immune response, perpetuated by additional factors secreted by reactive cells in themicroenvironment that help maintain the inflammatory milieu. The malignant HRS and LP cellsmanipulate the microenvironment, permitting them to develop their malignant phenotype fully andevade host immune attack. Gene expression signatures derived from non-neoplastic cells correlatewell with response to initial and subsequent therapies, reflecting their functional relevance. Recentbiomarker studies have added texture to clinical outcome predictors, and their incorporation intoprognostic models may improve our understanding of the biologic correlates of treatment failure.Moreover, recent preclinical and clinical studies have demonstrated that the tumor microenvironmentrepresents a promising therapeutic target, raising hope that novel treatment strategies focused on theinterface between malignant and reactive cells will soon emerge.

J Clin Oncol 29. © 2011 by American Society of Clinical Oncology

INTRODUCTION

Hodgkin’s lymphoma (HL) accounts for approxi-mately 11% of all malignant lymphomas.1 The mor-phologic hallmarks of this unique lymphoma wereinitially described more than 100 years ago; it ischaracterized by the presence of Hodgkin Reed-Sternberg (HRS) cells in classical HL (cHL) and so-called lymphocyte-predominant (LP) cells innodular lymphocyte-predominant HL (NLPHL).2

Typically, malignant cells are greatly outnumberedby reactive cells in a microenvironment that in-cludes lymphocytes, macrophages, eosinophils,mast cells, plasma cells, stromal cells, fibroblasts, andother cells.3 Specifically in cHL, the frequencies of allthese cellular components, including the HRS cells,vary considerably between cHL subtypes; lympho-cyte rich, lymphocyte depleted, mixed cellularity(MC), and nodular sclerosis (NS).

Independent of advances in understanding theetiology and molecular biology of HL, the introduc-tion of polychemotherapy has reduced the numberof patients who succumb to the disease by more than

60% since the early 1970s.4 Consequently, most pa-tients are cured by modern treatment strategies. Im-portant milestones in our understanding of HLinclude first, separation of HL into cHL versusNLPHL; second, development of distinct treatmentstrategies for limited versus advanced disease; andthird, validation of the International PrognosticScore for advanced disease.5,6 Since its introductionin 1998, the International Prognostic Score has be-come the gold standard for risk assessment andstratification and been used to guide treatment de-cisions in advanced cHL. However, it can be antici-pated that progress, especially among those 20% ofpatients who are still destined to die after relapse orprogressive disease, will be tightly linked to novelbiomarker discovery and targeted therapies for re-lapsed disease.7

In this review, we will first focus on the cross-talk between malignant and reactive cells in the mi-croenvironment mediated by cytokines andchemokines expressed in HL tissue. These solublefactors mediate the specific cellular composition of

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the reactive infiltrate, promote receptor-mediated signaling, and cul-tivate immune privilege for malignant cells. We will then review theprognostic implications of the tumor microenvironment and finallydiscuss novel therapeutic approaches targeting the microenvironmentor microenvironment-dependent receptor signaling.

CROSSTALK BETWEEN CELLS: THE IMPORTANCE OFCYTOKINES AND CHEMOKINES IN HL

Cytokines and chemokines are low–molecular weight proteins with awide variety of functions that work either in a paracrine manner to

modulate the activity of surrounding cells or in an autocrine fashion todirectly affect the cells that produce them. Specifically, these autocrineand paracrine interactions lead to the particular cellular compositionfound in HL and contribute to the proliferation and antiapoptoticphenotype of HRS cells (Fig 1). Although the cytokine and chemokinemilieu in HL tissues is complex and interactive, a clear understandingof this biology will be clinically relevant in the future as therapies aredeveloped that disrupt the crosstalk between malignant cells and themicroenvironment. Hence, improved insight into the biology will becritical to introducing these novel biologic treatments into clinicalpractice. This section focuses on the involvement of cytokines in the

Eosinophil FGF1/2IL-13

IL-13R

CCL11/Eotaxin

HRS cell

Mast cell

Macrophage

T cell(Treg /TH2)

NK cell

B cell

FibroblastFibroblast

Plasma cell

Cytotoxic T cell

CCL5

CD30

CD30L

CD30L

CD30IL-5MEC

CSF1

CSF1R

TARCMDCCCL20

TNF-αLT-αTGF-βBAFF

CCR4

CD40

CD40L

TNFRfamily

CXCL16

CCR3/CCR10

CCR10

BLCIL-4

CCR3

CCR3

CX3CL1

CX3CR1

MIF

IL-10TGFβ

PDL1

T cell(TH1)

PD-1

IFNγ

IFNγ

Gal-1

CD7

IL-2

IL-2R

CD74

FASLCD95

APRIL

Neutrophil

IL8

IL8

CXCR1/2

HLA-G

Fig 1. Schematic of the crosstalk between malignant Hodgkin Reed-Sternberg (HRS) cells and the tumor microenvironment in classical Hodgkin’s lymphoma. In thecenter, a bi-nucleated HRS cell is shown expressing characteristic surface molecules as well as secreted cytokines and chemokines. Surrounding the HRS cells arecell types representative of the non-neoplastic cells attracted and activated by these molecules. The cells in the microenvironment themselves express a variety ofchemokines and cytokines that further shape the reactive infiltrate and provide signaling for HRS cells. Only the main activating and inhibitory functions (arrows) ofpredominantly membrane-bound (purple triangles) and secreted molecules mediated by surface receptors (pink) are illustrated; other interactions exist. IL, interleukin;FGF, fibroblast growth factor; TNF-�, tumor necrosis factor �; LT-�, lymphotoxin �; TGF-�, transforming growth factor �; BAFF, B-cell activating factor; APRIL, aproliferation-inducing ligand; BLC, B lymphocyte chemoattractant; TNFR, tumor necrosis factor receptor; FASL, Fas ligand; MEC, mucosae-associated epithelialchemokine; Treg, T regulatory cell; TH, T helper cell; TARC, thymus and activation-related chemokine; MDC, macrophage-derived chemokine; Gal-1, galectin-1; PDL,programmed cell death 1 ligand; IFN-�, interferon gamma; NK, natural killer.

Steidl, Connors, and Gascoyne

2 © 2011 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY


formation of the microenvironment and highlights how, in return,HRS cells receive signals from the microenvironment through sur-face receptors.

The clinical and pathologic features of HL reflect an abnormalimmune response that is thought to be the result of expression of avariety of cytokines and chemokines by the HRS cells, altering thecomposition and function of the cells in the surrounding microenvi-ronment and shaping the specific histopathologic appearance of thelymph node.8,9 Moreover, the reactive cells in the microenvironmentproduce specific cytokines and chemokines that help maintain andeven amplify the intense inflammatory reaction.10 Table 1 summa-rizes the most commonly described cytokines and chemokines in HLand their main functions. Of note, most of the studies in HL havefocused on NSHL and MCHL, because NLPHL, lymphocyte-rich andlymphocyte-depleted cHL account for only a minority of cases.2 Inaddition to the variability of cytokine expression in the different HLsubtypes, Epstein-Barr virus (EBV) positivity also influences the rela-tive expression of certain cytokines (eg, CXCL10, CXCL9, CCL5,and CCL3).9

COMPOSITION OF THE MICROENVIRONMENT

The most abundant cells found in HL are infiltrating CD4� T cells.58

Several studies have revealed that the main subset of these cells displaysa phenotype of T helper 2 (TH2) and T regulatory (Treg) cells, partic-ularly those in the direct vicinity of HRS cells.13,59 HRS cells secreteregulated upon activation, normal T cell expressed and secreted(CCL5), thymus and activation-related chemokine (CCL17), andmacrophage-derived chemokine (CCL22), potent chemokines thathave all been shown to attract CCR4-expressing TH2 and Treg cells inHL.32,46,47,49-52 CCL5 has also been shown to attract mast cells com-monly found in HL.46 Together with T helper 1 (TH1) cells, HRS cellsthemselves produce interferon gamma,11,15,16 an important enhancerof macrophage function. HRS cells also produce granulocyte colony-stimulating factor (CSF1) and fractalkine (CX3CL1), chemokines anddifferentiation factors of monocytes and their progenitors explainingvariable infiltration by macrophages.40,43,60,61 In general, macro-phages have been shown to be critical enhancers of tumor progression,to promote cell migration, and to suppress antitumor immunity inmany cancers, including lymphoma.62 Particularly in HL, secretion ofmacrophage migration inhibitory factor (MIF) may contribute to theproliferation of HRS cells, which frequently express the CD74 receptoron their surface20,63; CD74 has been previously implicated in signalingand accessory functions for immune cell activation.64 Interleukin (IL)-8 expression by macrophages might also contribute to neutrophilinfiltration, which has been frequently described, most commonlyin NSHL.53,54

Expression of IL-13, tumor necrosis factor (TNF) �, and fibro-blast growth factors by HRS cells has been well described,18,27,41 andthis leads to the activation and proliferation of fibroblasts, most prom-inently seen in NSHL.48,65 Fibroblasts themselves can attract eosino-phils and TH2 cells through secretion of eotaxin (CCL11).48

Furthermore, IL-5 and mucosae-associated epithelial chemokine(CCL28) expression of HRS might contribute to tissue eosino-philia.19,39 CCL28 and CXCL16 have been described as correlated withplasma cell infiltration, a variable feature in cHL.39

B cells of various maturational stages are part of the normalcellular composition of lymph nodes, but in HL, this architecture isdisturbed to varying degrees. Therefore, it remains an open question ifand how reactive B cells are specifically attracted into the microenvi-ronment of HL, or whether they represent remnants not displaced byHL. However, as part of the general inflammatory reaction, variouscytokines, including TNF-� and lymphotoxin �, with effects on B-celldifferentiation, proliferation, and chemotaxis have been shown to beexpressed, which play critical roles in the initiation of germinal centerreactions.21,33,66 In NLPHL, expression of B cell–attracting chemokine1 (CXCL13) has been demonstrated in a subset of cases.67

AUTOCRINE AND PARACRINE RECEPTOR SIGNALING BYHRS CELLS

Although the malignant HRS cells gain a survival advantage by dereg-ulated transcription factor networks and genetic lesions altering in-trinsic signaling, the fully developed proliferation and antiapoptoticphenotype of HRS cells are critically dependent on their interactionwith the microenvironment.68 In HL, the cooperation of direct genetichits in signaling pathways with microenvironment-dependent signal-ing is most evident in the activation of nuclear factor kappa B (NF�B)and Janus kinase–signal transducer and activator of transcription sig-naling (JAK-STAT; Fig 2). Many mutations and structural alterationsin the genes involved in these pathways leading to constitutive activity,including chromosomal gains, overexpression of pathway genes,69-74

and inactivating mutations of tumor suppressor genes have beendescribed.75-82 Moreover, the microenvironment provides signalingvia surface receptors, namely IL receptors,83 members of the TNFreceptor (TNFR) family,84 and receptor tyrosine kinases.85

Prominent receptor family members involved in activation of thecanonical NF�B pathway, including CD30 (TNFR superfamily [SF],member 8), CD40 (TNFRSF5), receptor activator of NF�B(TNFRSF11A), and TNFR1 (TNFRSF1A), have been shown to beexpressed on the surface of HRS cells.29,45,86,87 These receptors can beactivated by membrane-bound or secreted ligands such as receptoractivator of NF�B ligand (TNFSF11, autocrine29), CD30L (TNFSF8,mast cells,30 eosinophils31), CD40L (TNFSF5, TH2/Treg86), TNF-�(autocrine, macrophages and lymphocytes33,34), and lymphotoxin �(autocrine21). Furthermore, in EBV-positive HL, the expression ofviral oncogene LMP1 leads to protein aggregates in the cellular mem-brane mimicking an active CD40 receptor that similarly activatesNF�B signaling.88 Alternative activation (so-called noncanonical acti-vation) of the NF�B pathway can be achieved via the receptors trans-membrane activator and calcium modulator and cyclophilin ligandinteractor (TNFRSF13B) and B-cell maturation antigen (TNFRSF17)after engagement by their ligands a proliferation-inducing ligand(TNFSF13) and B-cell activating factor (TNFSF13B).28,89 Notably, theseligands are expressed not only by HRS cells but also by a variety of cellsin the microenvironment, including macrophages, neutrophils, mastcells, and plasma cells, suggesting a prominent paracrine network.28

The constitutive activity of the Janus kinase–STAT pathway isexemplified by autocrine and paracrine signaling of IL-13, whichengages the IL-13 receptor and leads to HRS cell proliferation andsurvival by phosphorylated STAT6.18,90 Additionally, STAT3 activa-tion through IL-21 and STAT5 activation have been described inHL.91,92 Overall, expression of a large number of ILs that display

Microenvironment in Hodgkin’s Lymphoma

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Table 1. Cytokines and Chemokines Involved in the Pathogenesis of Hodgkin’s Lymphoma

NameAlternative

Name Biological Function/Function in Hodgkin’s LymphomaReference

No.

TH1 cytokinesIL-2 Lymphokine T-cell growth factor 11, 12IL-12 — Growth factor for activated T and NK cells 13, 14IFN-� — Macrophage activation, CXCL10 induction 11, 15-17

TH2 cytokinesIL-4 BCGF1 B-cell activation 12, 16IL-5 EDF Eosinophil differentiation 12, 18, 19IL-6 IFNB2 B- and plasma cell activation and maturation 12, 20-22IL-9 P40 Mast cell activation 23, 24IL-10 CSIF Negative regulation of TH1 cytokines 16, 25, 26IL-13 — B-cell maturation and activation, fibroblast stimulation, CCL17 and

CCL22 induction18, 27

TNF receptor ligandsTNFSF13B BAFF B- and HRS cell proliferation and differentiation 28TNFSF11 RANKL Augmentation of T-cell activation by dendritic cells, HRS cell stimulation 29TNFSF13 APRIL B- and HRS cell proliferation and differentiation 19TNFSF8 CD30L T-cell stimulation 30, 31CD40LG TNFSF5 B- and HRS cell proliferation 32LTA TNFSF1 B-cell growth factor, collagen synthesis 21TNF-� TNFSF2 CCL11 production enhancement through fibroblasts, collagen synthesis 33, 34

Other cytokinesIL-1A IL-1� Proinflammatory cytokine, fibrosis and sclerosis induction 33, 35IL-1B IL-1� Fibrosis and sclerosis induction 33, 35TGF-� — B- and T-cell suppression, fibroblast proliferation, associated with

nodular sclerosis subtype26, 36

IL-3 Multi-CSF Growth factor for HRS cells 37, 38IL-7 — Growth factor for B and T cells 39MIF — Blocks cytotoxic T lymphocyte response 40VEGF — Endothelial cell growth, angiogenesis 41, 42CSF1 M-CSF Monocyte growth and differentiation 20, 43, 44CSF2 GM-CSF Granulocyte, macrophage, and eosinophil differentiation 12, 22, 45

ChemokinesCC chemokines (�-chemokines)

CCL1 I-309 Monocyte chemotaxis 32, 39CCL2 MCP-1 Monocyte and basophil chemotaxis 33CCL5 RANTES T-cell, eosinophil, and mast cell recruitment 16, 46, 47CCL11 Eotaxin Eosinophil accumulation, produced by fibroblasts (TNF-� induced) 16, 39, 48CCL13 MCP-4 Monocyte chemotactic protein 14

32CCL17 TARC Induction of TH2 response and regulatory T cells 49-51CCL19 MIP-3� Promotes B- and T-cell migration 32CCL20 MIP-3� Lymphocyte recruitment 32CCL22 MDC TH2 response and regulatory T cell induction 16, 32, 39, 52CCL28 MEC Plasma cell and eosinophil recruitment 39

CXC chemokines (�-chemokines)CXCL8 IL-8 Neutrophil recruitment 53-56CXCL9 MIG Reactive infiltrate in EBV-positive disease 9, 16CXCL10 IP-10 Reactive infiltrate in EBV-positive disease 16, 32, 40, 50CXCL12 SDF1 Monocyte migration stimulation 57CXCL13 BLC B lymphocyte migration 32, 57CXCL16 SRPSOX Recruitment of plasma cells 39

CX3C chemokinesCX3CL1 Fractalkine Chemotactic for T cells, NK cells and monocytes 40

Abbreviations: TH, T helper cell; IL, interleukin; NK, natural killer; IFN-�, interferon gamma; TNF, tumor necrosis factor; TNFSF, TNF superfamily; BAFF, B-cellactivating factor; HRS, Hodgkin Reed-Sternberg; RANKL, receptor activator of nuclear factor kappa B ligand; APRIL, a proliferation-inducing ligand; LTA, lymphotoxinalpha; GM-CSF, granulocyte macrophage colony-stimulating factor; MIF, macrophage migration inhibitory factor; VEGF, vascular endothelial growth factor; MCP,monocyte chemotactic protein; TARC, thymus and activation-related chemokine; MDC, macrophage-derived chemokine; MEC, mucosae-associated epithelialchemokine; MIG, monokine induced by gamma interferon; EBV, Epstein-Barr virus; IP-10, 10KDa interferon gamma-induced protein; SDF, stromal cell–derivedfactor; BLC, B lymphocyte chemoattractant; SRPSOX, scavenger receptor for phospatidylserine and oxidised low-density lipoprotein.




receptor-specific binding similar to that seen with IL-13 has beenreported in HL8,93 (Table 1), including ligands binding to IL-2 recep-tor complexes94,95 and the IL-3,37,38 IL-6,12,96 and IL-7 receptors.97

Recently, receptor tyrosine kinase signaling has been reported toplay an important role in the pathogenesis of HL. In particular, the

surface receptors mesenchymal-epithelial transition factor (c-MET),tyrosine receptor kinase A (TRKA), and discoidin domain receptortyrosine kinase 2 (DDR2) are expressed and mediate survival signalingin HRS cells through paracrine signaling.98-102 The respective ligandsof these receptors are thought to be produced by cells of the

RANK

CD40 TNFR1/2

CD30 BCMA TACI

DDR2

collagen type I

TRKA

NGFBAFFAPRIL

CD40LCD30LRANKL

c-MET

HGF

Canonical NFκB Alternative NFκB Tyrosine receptor kinases

IL-2RIL-3RIL-6RIL-7RIL-13R

STAT-3

STAT-6

IL-4, IL13

JAK-STAT signaling

IL-6, IL-10IL-2, IL-7, IL-9, IL-15IL-3, IL-5, GM-CSF

STAT-5

TNF-αLT-α

NFκB complex

I

c M

RIP

IKKβ

NEMOIKKα

TRAF

IκB complex

MAP3K14

IKKαIKKβ

Transcriptional regulation:

Proteasomal

- Inflammation- Cell proliferation

- Survival- Differentiation

degradation

TRAF

STAT

STAT

STAT dimers/oligomers

STAT STAT

JAK1JAK2JAK3TYK2

STATSTAT

Apoptosis, cell cycle control,protein synthesis, DNA damage repair

IKK complex

IκBαIκBε

p50 RELAp52 RELB

p100 RELB

P

P

PP

Januskinase

P

P

RAS

RAF

MEK

ERK

SHP2

SOS

GAB1

PDK

AKT

p21MDM2 mTOR

PI3KGRB2

BAD

Fig 2. Pathway activation in Hodgkin Reed-Sternberg cells through signaling from the microenvironment. Soluble and membrane-bound signaling molecules producedby reactive cells (paracrine activation) activate the Janus kinase–signal transducer and activator of transcription signaling (JAK-STAT) and canonical and alternativenuclear factor kappa B (NF�B) pathways and receptor tyrosine kinases. For the JAK-STAT signaling pathway, the most commonly expressed interleukins (ILs), ILreceptors, and activated STATs are shown. For the NF�B pathway, only the principal activation cascades are shown, and inhibitor of � kinase (I�K) complex activationby other kinases are described. Downstream signaling of receptor tyrosine kinases is shown using the example of tyrosine kinase receptor A (TRKA) and illustratingthe Ras and Akt pathways; other signal transduction pathways exist. GM-CSF, granulocyte macrophage colony-stimulating factor; RANKL, receptor activator of NF�Bligand; TNF-�, tumor necrosis factor �; LT-�, lymphotoxin �; APRIL, a proliferation-inducing ligand; BAFF, B-cell activating factor; HGF, hepatocyte growth factor; NGF,nerve growth factor; TACI, transmembrane activator and CAML interactor; BCMA, B-cell maturation antigen; c-MET, mesenchymal-epithelial transition factor; DDR,discoidin domain receptor tyrosine kinase; TYK2, tyrosine kinase 2; RIP, receptor-interacting protein; TRAF, TNF receptor–associated factor 1; MAP3K, mitogen-activated protein 3 kinase; SHP2, Src homology 2 domain–containing tyrosine phosphatase; SOS, son of sevenless homolog 1 (Drosophila); GRB2, growth factorreceptor–bound protein 2; PI3K, phosphoinositide 3-kinase; ERK, extracellular signal-regulated kinase; MEK, MAP-ERK kinase; PDK, 3-phosphoinositide–dependantprotein kinase; BAD, BCL2-associated agonist of cell death; MDM, mouse double minute; mTOR, mechanistic target of rapamycin.




microenvironment. Hepatocyte growth factor (HGF), a specific li-gand of c-MET, is expressed by CD21� follicular dendritic cells.99

Collagen type I, the high-affinity ligand of TRKA, is expressed byfibroblasts, and nerve growth factor (NGF) –producing granulocyteswere found abundantly in the direct vicinity of HRS cells.100 Further-more, autocrine activation loops have been suggested for the platelet-derived growth factor receptor (PDGFRA) and EPH receptor B1(EPHB1). In summary, cytokine and chemokine expression have beenextensively studied in HL, and specific ligand-receptor interactionshelp explain the composition of the tumor environment and down-stream receptor signaling critically influencing the phenotype of ma-lignant cells.

IMMUNE ESCAPE BY HRS CELLS

As discussed, HRS cells are dependent on the extensive inflammatorymicroenvironment and use receptor signaling pathways to maintaintheir high proliferative capacity and antiapoptotic phenotype. How-ever, HRS cells also appear to orchestrate their microenvironment inmany ways to evade host immune attack. As evidenced in HIV-associated HL, malignant cells can thrive in an environment in whichhost immunity is globally impaired.103 Moreover, in recent years,specific mechanisms have been uncovered detailing how HRS cellsinduce a favorable microenvironment with suppressed antitumor im-mune activity.

Major advances have been made that characterize the pheno-types and functions of specific T-cell subsets,104 including immuno-suppressive Treg cells, which are abundant in tissue biopsies ofHL.105,106 Specifically, it has been shown that HRS cells producechemokines such as CCL5, CCL17, and CCL22, which attract TH2and Treg cells49,52,107 and secrete IL-7, an IL capable of inducingdifferentiation of CD4�naive T cells toward FOXP3�Treg cells.97,108

Treg and HRS cells themselves produce IL-10 and transforminggrowth factor � (TGF-�), which exert inhibitory effects on T-celleffector functions, especially on cytotoxic T lymphocytes(CTLs).8,25,105,109 In addition, prostaglandin E2 (PGE2) has been im-plicated as a contributor to immunosuppression and pathogenesis ofHL110,111 similar to cancer progression of solid tumors.112 PGE2 leadsto decreased CD4� T-cell activation through inhibition of leukocyte-specific protein tyrosine kinase (LCK),113 suggesting that PGE2 con-tributes to the CD4� T-cell impairment observed in HL. The cellsproducing PGE2 have yet to be determined.

Another important mechanism by which HRS cells evade hostimmune response is by overexpression of surface molecules maintain-ing peripheral tolerance. For example, the overexpression of Fas ligand(CD95L) that has been described in virtually all HRS cells of NS andMC subtypes114-116 induces apoptosis in tumor-specific CTLs.117

Similarly, galectin-1 (LGLAS1) was found to be overexpressed on thesurface of HRS cells in HL, correlating with a reduced CD8� infiltra-tion at the tumor site.118 In cocultures of activated T cells with HL celllines, galectin-1 induced secretion of TH2 cytokines and expansion ofTreg cells, whereas knockdown of galectin-1 increased overall T-cellviability and restored the TH1/TH2 balance.119 Furthermore, overex-pression of ligands of the receptor molecule programmed cell death 1(PD1), PD1 ligand 1 (PDL1; CD274) itself, and PDL2 (CD273) hasbeen described in HL.120,121 PD1 belongs to the CD28 costimulatoryreceptor superfamily expressed on CTLs and can modify T-cell activ-

ity by providing a second signal to T cells via the PD1 receptor, inconjunction with signaling through the T-cell receptor.122 In the set-ting of HL, PD1 ligand overexpression has been shown to contributeto the T-cell exhaustion observed in infiltrating T cells. A recent studyestablished a link between copy number gains of chromosome 9pwhere PDL1 and PDL2 reside and overexpression of these moleculesin NSHL.121

Loss of HLA class I and II molecules on the surface of HRS cellsoccurs in a substantial proportion of cases and is correlated with latentEBV infection. HRS cell escape from immune surveillance has beensuggested as a functional consequence of this finding,123-125 becausethe reduced immunogenicity of HRS cells could impair activation ofCTLs (major histocompatibility complex [MHC] class I recognition)and CD4� T cells (MHC class II recognition). However, the under-lying causes for this downregulation of HLA molecules and the exactfunctional consequences are largely unexplored in HL. In particular,no correlation between chromosomal loss and underexpression ofHLA molecules has been found in two HL cell lines.126 Interestingly,certain HLA types are correlated with genetic susceptibility to HL—EBV-positive HL cases in particular—suggesting that certain poly-morphisms might effect antigen presentation to CTLs.127-129

Furthermore, expression of the nonclassical MHC class I gene HLA-G,the ligand for inhibitory receptors found on natural killer (NK) cells,subsets of T cells, and other immune cells may lead to evasion ofNK-cell and CTL-mediated immune responses and has been impli-cated in the pathogenesis of HL.130 Such surface expression has beenidentified by immunohistochemistry (IHC) in more than half of cHLcases, and a strong correlation with absence of classical MHC class Imolecule expression and EBV-negative status has been seen.

In summary, it is now well established that the phenotypicchanges that characterize HRS cells allow for efficient escape from thehost immune attack through various mechanisms, including release ofchemokines shaping an immunosuppressive microenvironment aswell as the expression of surface molecules gaining immune privilegethrough the reduction of HRS cell immunogenicity. However, most ofthe underlying genetic mechanisms remain to be discovered.

BIOMARKERS IN HL: CORRELATIONS TO TREATMENT OUTCOME

As the impact of the microenvironment on the specific pathobiologyof HL becomes increasingly clear, more and more work is focused oncorrelation of biomarkers with treatment outcome. Although treat-ment regimens have been remarkably stable in the last two decades,specific aspects of patient selection, varying histologies, and treat-ments might influence cross-comparability and in part explain differ-ing conclusions from these studies. In general, although a plethora ofbiomarkers associated with clinical outcome have been described,none of these factors has influenced clinical practice to date. The mainreason for this unsuccessful clinical translation lies in the lack ofreproducibility between independent patient cohorts and prognosticimplications that are not of significant magnitude to justify a change inclinical management. For individual biomarkers, reproducibility ofIHC scoring has been suggested as a reason for inconclusive results,and thus more robust multigene predictors have been reported basedon expression profiling.131,132 Table 2 summarizes some of thebiomarkers for which outcome correlations have been described, sep-arated into studies using IHC and gene expression profiling. The focus




of these studies has been cHL, because there are no biologic markersyet identified that can predict treatment outcome or transformation toan aggressive lymphoma in patients with NLPHL.

HISTOPATHOLOGY

The first biomarker studies in cHL used classical histopathologic ap-proaches to describe the specific cellular composition of subtypes andinvestigate correlations with treatment outcome. Although in initialreports, the rare lymphocyte-depleted subtype and grade 2 nodularsclerosis were reported to be associated with poor response to initialtherapy and decreased survival,149 there is now overwhelming evi-dence that none of the cHL subtypes are correlated with survival usingmodern treatment modalities.133,150-152 Similarly, the number of HRScells is not prognostically relevant. Tissue eosinophilia is still a subjectof debate; a large study investigating diagnostic biopsies found a strong

correlation between the presence of eosinophils and freedom fromtreatment failure and overall survival (OS),31 in agreement with areport showing prolonged disease-free survival in patients with heavyeosinophilic infiltration in NSHL.153 However, several other studiescould not confirm this finding.150,154,155 Mast cell infiltration has alsobeen linked to poor prognosis in cHL,156 and a recent study usingc-kit/CD117 IHC was able to link the number of mast cells to unfa-vorable outcome after primary therapy157; however, these data haveyet to be validated. These factors affecting HRS cells and the microen-vironment as assessed by routine histopathology are of interest. How-ever, none has yet had an impact on clinical practice.

MACROPHAGES

Some of the first biomarker studies in the modern treatment era wereconducted in the 1980s and suggested that increased numbers of

Table 2. Biomarkers With Outcome Correlations Described in the Literature

Immunohistochemistry

Marker/Signature Expressed on/Staining Pattern Function Outcome CorrelationReference

No.

Granzyme B Cytotoxic T cells Target cell lysis Adverse (PFS, OS) 133-137TIA-1 Cytotoxic T cells Target cell lysis Adverse (EFS, OS) 136,138FOXP3 Regulatory T cells Transcriptional regulation Favorable (EFS) 137-139CD20 Background B cells B-cell differentiation Favorable (OS, EFS) 132,140BCL11A Background B cells, plasmacytoid dendritic

cellsTranscriptional regulation Favorable (OS, EFS) 140

HLA-DR HRS cells Antigen presentationCD68, PNA Macrophages Scavenger receptor Favorable (FFS) 123ALDH1A1 Macrophages Oxidative

pathway/metabolismAdverse (PFS, DSS) 132,141

STAT1 Macrophages Transcriptional activation Adverse (DSS) 142EBV-encoded small RNAs HRS cells Activation of NF�B Adverse in elderly patients,

favorable in youngpatients (FFS, OS)

143-147

MMP11 HRS cells, macrophages, endothelial cells,extracellular

Tissue remodeling Adverse (PFS) 132

Gene Expression Profiling

Main Gene Components Outcome Correlation Reference No.

Angiogenic signature ADH1B, CD93, SRPX, PLA2G2A, GPR126 Adverse (primary treatment failure) 132Adipocyte signature GLUL, MGST1, COL1A2, FABP4 Adverse (primary treatment failure) 132Fibroblast function/extracellular

matrix remodelingAdverse: MMP2, MMP3, TIMP1, COL1A1,

COL4A1, COL4A2, COL5A1, COL18A1,COL16A1, MFAP2, THBS1/2, FN1,EDNRA, ITGB5, LAMA4; favorable:TIMP4, SPON1, LAMB1, TACR1, CCL26

Discordant: adverse/favorable(primary treatment outcome)

142,148

B-cell signature BCL11A, BANK1, STAP1, BLNK, FCER2,CD24, CCL21

Favorable (primary treatmentoutcome)

132,140

Cytotoxic T-cell signature CD3D, CD8B1, CTSL, CD26, SH2D1A,IFI16, RGS13, CR2, ELL3, CCDC23,PPM1L, TRA@, PIK3CA

Adverse (primary treatmentoutcome)

131,132,142

Plasmacytoid dendritic cells ITM2A, SRPX, CTSB, APP Adverse (primary treatmentoutcome)

132

Macrophage signature ALDH1A1, LYZ, STAT1, ITGA4, CCL13,MS4A4A, CCL23, VCAN, HSP90AB3P,HSP90AB1, CTSB, CFL1, JMJD6,MAPK7, IKBKG, RAB7A, RXRA,MAPK13

Adverse (primary treatmentoutcome)

131,132,142

Abbreviations: PFS, progression-free survival; OS, overall survival; TIA-1, T-cell–restricted intracellular antigen 1; EFS, event-free survival; FOXP3, forkhead boxprotein 3; BCL11A, B-cell lymphoma/leukemia 11A; HRS, Hodgkin Reed-Sternberg; FFS, failure-free survival; PNA, peanut agglutinin; DSS, disease-specific survival;ALDH1A1, aldehyde dehydrogenase 1A1; STAT, signal transducer and activator of transcription signaling; EBV, Epstein-Barr virus; NF�B, nuclear factor kappa B;MMP11, matrix metalloproteinase 11.




macrophages, as measured using peanut agglutinin–binding cells,were associated with more frequent constitutional symptoms andrelapse.141,158 The association of macrophages with adverse treatmentoutcome has now been confirmed in an increasing number of patientcohorts. In a gene expression profiling study investigating the mi-croenvironment, the authors found several genes expressed by mac-rophages, including STAT1 and ALDH1A1, to be significantlycorrelated with disease-specific survival when investigated by IHC.142

The importance of these markers was also confirmed in an indepen-dent patient cohort using quantitative reverse-transcriptase polymer-ase chain reaction from archival paraffin material.131 However, it wasnot confirmed in a second study by the same group, in which theauthors found a correlation of the macrophage activation genes LYZand STAT1 with favorable outcome.159 Most recently, tumor tissuesignatures of monocytes and tumor-associated macrophages werefound by gene expression profiling in patients with therapy-refractorydisease or relapse when compared with patients who sustained long-term remissions.132 Most importantly, in independent validation ex-periments, the authors demonstrated that the enumeration of CD68�macrophages in lymph node biopsies was a strong and independentpredictor of disease-specific survival. These data are encouraging; inthis study, it could be demonstrated that the number of CD68�macrophages was also significantly associated with the outcome ofsecondary treatments such as autologous stem-cell transplanta-tion. Overall, the adverse prognostic impact of tumor-associatedmacrophages in cHL biopsies can now be considered as firmlyestablished in patients treated with doxorubicin, bleomycin, vin-blastine, and dacarbazine (ABVD) or ABVD-like chemotherapyregimens. The correlation of tumor-associated macrophages withadverse clinical course and tumor progression has also been re-ported in follicular lymphoma160 and many other solid tumors inwhich macrophages stimulate tumor growth, angiogenesis, tumorcell migration, and invasion.62

T-CELL SUBSETS

Another biomarker in the microenvironment of HL was described byOudejans et al,134 who found that an increased number of activatedCTLs in tissue biopsies of patients was associated with unfavorableclinical outcome. The authors used granzyme B (GrB) as an IHCmarker and found GrB-expressing cells in the vast majority of patients,but they showed that patients with more than 15% GrB� cells hadsignificantly shorter progression-free survival and OS. In a subsequentstudy of 83 patients with cHL using GrB IHC, this same factor wasfound to correlate with OS and to be independent of both age andstage in multivariate analysis.135 Several subsequent studies by inde-pendent groups validated these initial studies of CTLs as useful predic-tors of prognosis in cHL but also identified other T-cell subsetsassociated with treatment outcome.133,135-138,157,161 Despite this ear-lier work, the most useful T-cell marker combination for determiningprognosis using IHC is still a subject of debate. In a large series of 257patients with cHL represented on a tissue microarray, the number ofFOXP3� Treg cells correlated with prolonged event- and disease-freesurvival, and the authors were able to validate the negative prognosticimpact of CTLs.138 However, in this study, TIA-1—another cytotoxicgranule marker—was used to enumerate CTLs, and the number ofGrB-activated CTLs was not significantly associated with poor prog-

nosis. Accordingly, high ratios of FOXP3�/TIA-1� T cells138 wereassociated with favorable event- and disease-free survival and OS,whereas high ratios of FOXP3�/GrB� cells137 were shown to beassociated with favorable failure-free survival and OS. The correlationof high numbers of intratumoral FOXP3� regulatory cells with OSand failure-free survival was validated in a subsequent study.139 Incontrast, high numbers of regulatory T cells have mostly been reportedto be associated with unfavorable outcome in various solid tumors.162

The reason for these unexpected findings remains unclear. In anattempt to link T-cell infiltration to the specific biology of HRS cells,the prognostically favorable low number of cytotoxic cells (CD8� Tlymphocytes, CD57� NK cells, and GrB� cells) was linked to over-expression of antiapoptotic proteins in HRS cells.163 Interestingly,recent work has shown that lack of HLA class II cell-surface expressionon HRS cells was frequently observed, and this finding was associatedwith EBV-negative disease status, HLA class I loss, and adverse out-come in multivariate analysis.123 However, because loss of HLA class IIlikely leads to diminished activation of TH1 cells and thereby a re-duced cytotoxic antitumor response, the exact functional mechanismsremain to be clarified linking increased numbers of intratumoralCTLs with unfavorable outcome.

Furthermore, signatures of cytotoxic T cells have been identifiedusing gene expression profiling, suggesting that overexpression ofcertain T-cell activation genes such as CD8B1, CD3D, CTSL, CD26,and SH2D1A might be correlated with inferior outcome.131,132,142

Additional biomarker studies investigating the prognostic impact ofT-cell subsets together with tumor-associated macrophages are antic-ipated to address the question of whether combinations of IHC mark-ers with well-established clinical risk factors might further improve thepredictive value of a single biomarker alone.

BACKGROUND B CELLS

Recently, the number of CD20� background B cells was reported tobe significantly associated with treatment outcome in two indepen-dent studies.132,140 Both studies found an overrepresentation of signa-tures of benign B cells in diagnostic biopsies of patients whose primarytreatments were successful and were able to demonstrate using CD20IHC significant correlations with OS and disease-specific survival,respectively. Although in one study the prognostic impact of thenumber of CD20� background B cells on OS was independent ofclinical parameters,140 the second study found a strong correlation ofCD20� cells with clinical stage.72 Interestingly, another marker ex-pressed on B cells—BCL11A—was an even better predictor for OS,possibly because of an association of BCL11A� plasmacytoid den-dritic cells with favorable outcome.

FOLLICULAR DENDRITIC CELLS

The presence and distribution of follicular dendritic cells (FDCs)have been studied in both cHL and NLPHL using IHC.164,165 Thesestudies reveal the close spatial relationship of HRS and LP cells with theFDC network. In one study of cHL, the authors reported a correlationbetween the absence of FDCs and unfavorable outcome.166 In afollow-up study, the association with outcome was validated, but onlyin the context of certain patterns of CD21� FDC staining.167 The




longest OS was observed in patients with well-defined follicle-likestructures compared with visible but largely destroyed FDC mesh-works (intermediate survival) and absence of FDCs (poor survival).These data suggest that not only the number of FDCs but also the levelof destruction of normal lymph node architecture might be an impor-tant variable for outcome prediction.

EBV ASSOCIATION

EBV infection of HRS cells occurs in up to 60% of patients in somestudies but varies with geographic location, age, sex, clinical stage,and histologic type.168 Recent gene expression profiling data haveshed more light on immune-related cells in EBV-positive versusEBV-negative disease, specifically identifying a TH1/antiviral re-sponse in EBV-positive patients.140 Furthermore, an increasednumber of cytotoxic T cells has been linked to EBV positivity.134

The impact of EBV infection on outcome is controversial, withsome studies showing no impact150,169 and others favorable143,170 orunfavorable outcomes.144,145 These discrepancies might result in partfrom patient selection and confounding differences in demographicfactors, because there is an increasing body of work supporting anassociation between EBV positivity and adverse outcome in olderadult patients144-146 and a favorable effect in younger, specificallypediatric, patients.143,144,147 Additional studies might clarify the clini-cal value of these findings; the apparent age dependence has hamperedthe translation of EBV status into a widely used biomarker.

SERUM MARKERS

It has been shown that certain cytokines, chemokines, and othersoluble factors are detectable in the sera of patients with HL, andmultiple studies have focused on the negative prognostic impact ofthese molecules.17,171,172 In general, increased levels of these serummarkers can be interpreted as surrogates for active crosstalk betweenHRS cells and their microenvironment. Characteristically, many ofthose markers, including sCD30, serum IL-2 receptor, solubleintercellular adhesion molecule (sCD54), and vascular cell adhe-sion molecule 1 (sCD106), have shown correlations to one an-other, and elevated levels have been reported to be associated withadvanced tumor stage and unfavorable prognosis.173-176 Althoughall these markers have shown prognostic significance, their inde-pendent value in comparison with the established clinical surro-gate markers for tumor burden and disease activity has not beenconvincingly demonstrated.

GENOME-WIDE STUDIES

Gene expression profiling studies have contributed substantially toimproved understanding of the disease with respect to the inherentphenotypic features of malignant HRS cells177,178 and the specificcomposition of the microenvironment.132,140,142,148 Using the un-biased approach of transcriptome-wide gene expression profiling,gene signatures have been identified that correlate with failure orsuccess after primary treatments. Many of these signatures can berelated to cellular compartments represented in whole-tissue biop-sies. Some of these signatures have been subsequently validated by

IHC, including tissue-associated macrophages, background reac-tive B cells, and CTLs132,140,142; however, confirmation is stillneeded for others (Table 2). Of note, the prognostic implications ofangiogenesis, adipoctyes, and dendritic cells have been studied inother lymphomas.179-181

Proteomic studies have recently been performed in HL. Twoapproaches have been chosen thus far, one using total cell lysates,the other studying the secretome of HRS cells.40,182,183 These stud-ies have demonstrated the potential value for developing noveldiagnostic tools by identifying specific profiles linked to certainlymphoma entities and have helped to characterize secreted pro-teins that are critically involved in the crosstalk of HRS cells withtheir microenvironment.40 However, no outcome correlationshave been described to date.

Only one study has investigated correlations of a micro-RNAprofile, which was previously found in HL,184 with outcome. Theauthors found that miR-135a expression was associated with a higherprobability of relapse and shorter disease-free survival.185

THERAPEUTIC APPROACHES TARGETING THETUMOR MICROENVIRONMENT

Although HL is overall a successfully treated disease, treatment failurein a substantial proportion of patients and treatment-related early andlate sequelae of chemotherapy and radiotherapy underscore the needfor novel therapeutic approaches, especially for relapsed HL. No newdrugs have been approved for HL in the past two decades186; however,many agents are being tested in preclinical studies and currently en-rolling active trials. Table 3 lists a selection of novel therapeutic agentsin HL that are thought to target reactive cells in the microenvironmentor surface receptors on the malignant cells or to interfere with down-stream receptor signaling.

The concept that the tumor microenvironment might be apromising therapeutic target has been reinforced by reports in re-lapsed HL about the efficacy of the pyrimidine analog gemcitabine,210

a cytotoxic agent that has been reported to specifically target Tregcells.211 Furthermore, the results of a study combining the anti-CD20monoclonal antibody rituximab with gemcitabine in relapsed HL areencouraging and suggest that a combined treatment targeting reactiveB and T cells in the microenvironment might be an effective ap-proach.188 Trials targeting CD20� B cells using radioimmunoconju-gates have been initiated.191 However, it remains controversial if theefficacy of anti-CD20 immunotherapy results from the direct killing ofHRS cells that do occasionally express CD20212 or from the depletionof HRS cell–supporting reactive B cells in the microenvironment.187

Of note, two gene expression profiling studies have observed a corre-lation between increased numbers of background B cells and favorableprognosis, a finding that needs further clarification in the context ofrituximab treatment.132,140 Support for a direct effect on HRS cells alsocomes from a recent study suggesting that HRS progenitor cells mightexpress CD20 on their surface and could be eradicated by ritux-imab.213 Unequivocally, treatment effects in NLPHL have been attrib-uted to a direct effect on the CD20-expressing LP cells.189,190 Otherantibodies with a predominant effect on the microenvironment thatare currently being tested in clinical trials including patients with HLare represented by alemtuzumab (anti CD52)193 and galiximab (anti-CD80).192 Furthermore, immunotherapy with EBV-specific cytotoxic




effector cells has shown promising results, providing proof of princi-ple for adoptive immunotherapy with ex vivo expanded viral antigen-specific T cells.208 Remarkably, in a subsequent study, five of sixpatients with relapsed HL had a tumor response; of these five, fourachieved complete remissions that were sustained for more than9 months.209

Because of the increased expression of members of the TNFreceptor family and the dependence of malignant cells on TNFreceptor downstream signaling, these molecules are consideredideal targets for specific agents in HL. TNFRSF8 (CD30) in partic-ular has been the target of a number of preclinical and clinicalstudies, of which the anti-CD30 compounds SGN-30 andMDX060 can be considered representative but disappointing inphase I/II clinical trials194,214 because of limited efficacy in relapseddisease. However, using an alternative strategy of conjugating ananti-CD30 antibody with the cytotoxic antimicrotubule agentmonomethyl auristatin E (ie, SGN-35 or brentuximab vedotin),195

much higher partial and complete remission rates were achieved.196 Itis noteworthy that in a heavily pretreated patient cohort, tumorreductions were documented in more than 80% of patients. As aresult, additional clinical trials with brentuximab vedotin as a

single agent and in combination with ABVD are currently recruit-ing. An anti-CD40 (TNFRSF5) antibody (HCD122198) trial and astudy of the combination bortezomib with agonistic anti–TNF-related apoptosis-inducing ligand receptor 2 (TNFRSF10B) com-pound AMG655197 are enrolling patients with HL; however, resultsare still pending. A number of novel drugs have downstream re-ceptor signaling as their primary targets, of which everolimus(mammalian target of rapamycin, phosphoinositide 3-kinasepathway),199 bortezomib (TNFR signaling, NF�B pathway),200

and vorinostat (STAT6, IL signaling)204 have shown promisingpreclinical results. Of these, however, bortezomib as a single agentin relapsed HL had low clinical efficacy.202 Combination therapiesare now being tested.

In summary, therapies targeting cells in the microenvironmentor disrupting microenvironment-dependent signaling in the malig-nant cells seem to be effective and show promise in patients withrelapsed HL. Current clinical trials are also focusing on combinationtherapies with classical chemotherapy agents. Randomized trials com-paring these novel agents with the current standard of care for first-and second-line therapies will ultimately determine their significancein the overall landscape of HL treatment.

Table 3. Selected Therapeutic Agents in Hodgkin’s Lymphoma and Their Presumed Targets

Drug Main Target Reference No./Clinical Trial No.

Cellular targets in microenvironmentRituximab CD20� B cells 187-190

CD20� HRS cells (LP cells)131�I�tositumomab CD20� B cells NCT00484874

CD20� HRS cells (LP cells)90�Y�ibritumomab tiuxetan CD20� B cells 191

CD20� HRS cells (LP cells)Galiximab CD80� B cells, follicular dendritic cells, HRS cells 192

NCT00516217Alemtuzumab CD52� cells in microenvironment 193

NCT01030900Receptor signaling on HRS cells

MDX-060 CD30� HRS cells 194NCT00284804

SGN-35 CD30� HRS cells 195,196NCT00947856, NCT01100502, NCT01060904

AMG655 TNFRSF10B (TRAIL-R2) on HRS cells 197NCT00791011

HCD122 CD40� HRS cells, TH2/Treg signaling 198NCT00670592

Downstream signalingEverolimus TNFR signaling: PI3K, mTOR 199

NCT00967044Bortezomib NF�B signaling: inhibition of degradation of I�B 200-202

NCT00439361Vorinostat Histone modification, JAK-STAT signaling (pSTAT6) 203,204Panobinostat Histone modification 205

NCT00742027Other

Lenalidomide Immunomodulation, anti-angiogenic 206,207NCT00540007

Cytotoxic T lymphocytes EBV-positive disease: viral LMP2 208,209

NOTE. US Food and Drug Administration clinical trail numbers according to http://clinicaltrials.gov.Abbreviations: HRS, Hodgkin Reed-Sternberg; LP, lymphocyte predominant; TNFRSF, tumor necrosis factor receptor superfamily; TH, T helper cell; Treg, T

regulatory cell; PI3K, phosphoinositide 3-kinase; mTOR, mechanistic target of rapamycin; NF�B, nuclear factor kappa B; I�B, inhibitory kappa B; JAK-STAT, Januskinase–signal transducer and activator of transcription signaling; EBV, Epstein-Barr virus; LMP2, latent membrane protein 2.




AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTSOF INTEREST

The author(s) indicated no potential conflicts of interest.

AUTHOR CONTRIBUTIONS

Manuscript writing: All authorsFinal approval of manuscript: All authors

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Acknowledgment

We thank Sibrand Poppema, MD, PhD, for his many contributions to the field and for reviewing this manuscript prior to submission.




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