Dendritic Cells in Tumor-Associated Tertiary Lymphoid ... · It is generally recognized that secondary lymphoid organs, comprisinglymphnodes,spleen,andmucosal-associatedlym-phoid

Microenvironment and Immunology

Dendritic Cells in Tumor-Associated Tertiary LymphoidStructures Signal a Th1 Cytotoxic Immune Contextureand License the Positive Prognostic Value of InfiltratingCD8þ T Cells

J�er�emy Goc1,2,3, Claire Germain1,2,3, Thi Kim Duy Vo-Bourgais1,2,3, Audrey Lupo1,2,3,4, Christophe Klein2,Samantha Knockaert1,2,3, Luc de Chaisemartin1,2,3, Hanane Ouakrim1,2,3, Etienne Becht1,2,3, Marco Alifano5,Pierre Validire1,6, Romain Remark1,2,3, Scott A. Hammond8, Isabelle Cremer1,2,3, Diane Damotte1,2,3,4,Wolf-Herman Fridman1,2,3,7, Catherine Saut�es-Fridman1,2,3, and Marie-Caroline Dieu-Nosjean1,2,3

AbstractTumor-infiltrating T cells, particularly CD45ROþCD8þ memory T cells, confer a positive prognostic value in

human cancers. However, the mechanisms that promote a protective T-cell response in the tumor microenviron-ment remain unclear. In chronic inflammatory settings such as the tumor microenvironment, lymphoid neogenesiscan occur to create local lymph node–like structures known as tertiary lymphoid structures (TLS). These struc-tures can exacerbate a local immune response, such that TLS formation in tumors may help promote an efficaciousimmune contexture. However, the role of TLS in tumors has yet to be investigated carefully. In lung tumors, maturedendritic cells (DC) present in tumor-associated TLS can provide a specific marker of these structures. In this study,we evaluated the influence of TLS on the characteristics of the immune infiltrate in cohorts of prospective andretrospective human primary lung tumors (n¼ 458).We found that a high density ofmature DC correlated closely toa strong infiltration of T cells that are predominantly of the effector–memory phenotype. Moreover, mature DCdensity correlated with expression of genes related to T-cell activation, T-helper 1 (Th1) phenotype, and cytotoxicorientation. Lastly, a high density of TLS-associated DC correlated with long-term survival, which also allowed adistinction of patients with high CD8þ T-cell infiltration but a high risk of death. Taken together, our results showhow tumors infiltrated by TLS-associated mature DC generate a specific immune contexture characterized by astrong Th1 and cytotoxic orientation that confers the lowest risk of death. Furthermore, our findings highlightthe pivotal function of TLS in shaping the immune character of the tumor microenvironment, in promoting aprotective immune response mediated by T cells against cancer. Cancer Res; 74(3); 705–15. �2013 AACR.

IntroductionThe tumor microenvironment is a complex network of

different cell types comprising tumor, stromal, and immunecells, interspersed with blood and lymphatic vessels (1). Theimmune infiltrate in human tumors is heterogeneous depend-ing on the tumor type and the individual. Several reports haveidentified tumor-infiltrating T cells directed against tumor-

associated antigens, indicative of a spontaneous in situ immuneresponse in patients with cancer (2–6). Moreover, a strongassociation between T-cell density with clinical outcome hasbeen reported inmany types of humansolid cancer (7–13).Moreprecisely, memory T cells with T-helper 1 (Th1) and cytotoxicorientations seem to represent a predominant T-cell populationfor prediction of favorable clinical outcome (14). In colorectalcancer, a comparison of the T-cell infiltrate with standardpathologic criteria demonstrated the prognostic power ofimmune criteria (7, 15, 16). These findings support the propo-sition that the T-cell infiltratemay serve as a new criticalmarkerto aid in classifying cancers (17). Nevertheless, despite the well-recognized prognostic value of tumor-infiltrating lymphocytes(TIL) in some cancers, mechanisms that govern their recruit-ment and activation into the tumor remain unclear.

It is generally recognized that secondary lymphoid organs,comprising lymph nodes, spleen, andmucosal-associated lym-phoid tissues are the primary site of induction of adaptiveimmune responses (18). In addition, organized lymphoidaggregates termed tertiary lymphoid structures (TLS) candevelop locally, at sites of persistent inflammatory disorders.Lymphoid neogenesis has been described in various human

Authors' Affiliations: 1Laboratory ImmuneMicroenvironment andTumors,INSERM U872, Cordeliers Research Center; 2University Pierre et MarieCurie; 3University Paris Descartes, UMRS 872; 4Departments of Pathologyand 5Thoracic Surgery, Hotel Dieu Hospital, AP-HP; 6Department ofPathology, Institut Mutualiste Montsouris; 7Department of Immunology,European Georges Pompidou Hospital, AP-HP, Paris, France; and 8Oncol-ogy Research, MedImmune LLC, Gaithersburg, Maryland

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Marie-Caroline Dieu-Nosjean, Laboratory ImmuneMicroenvironment and Tumors, UMRS872 INSERM, Cordeliers ResearchCenter, 15 rue de l'�ecole de M�edecine, F-75270 Paris, France. Phone: 33-1-44-27-90-86; Fax: 33-1-44-27-81-17; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1342

�2013 American Association for Cancer Research.

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pathologies, comprising infections, autoimmune diseases, andorgan transplant rejections (19–21). TLS exhibit strong struc-tural analogies with canonical secondary lymphoid organs andpresent features of an ongoing immune reaction site (22–24).Moreover, several studies inmousemodels have demonstratedthat TLS can induce a protective primary and secondaryimmune response independently of secondary lymphoidorgans during respiratory viral infection (25, 26). However, thepotential contribution of TLS to the promotion of an intratu-moral immune reaction and their influence on the tumorimmune contexture remain poorly investigated.

We have previously reported the presence of TLS in thetumor stroma of early-stage non–small cell lung cancer(NSCLC), composed of clusters of DC-Lampþmature dendriticcells (DC; referred as "TLSmatureDC") andT cells within T-cellarea adjacent to B-cell follicle (27). These structures aresurrounded by PNAdþ high endothelial venules (HEV), whichare specialized blood vessels mediating lymphocyte extrava-sation into canonical lymphoid organs (28). Moreover, TLSwere associated with a specific set of chemoattractant mole-cules involved in T-cell homing, suggesting their participationfor the immigration of peripheral blood T cells into the tumor(29). In addition, DC-Lampþ mature DC that home selectivelyin TLS have been associatedwith long-term survival in patientswith early-stage NSCLC supporting the involvement of TLS inthe promotion of a protective immunity (27). We hypothesizedthat TLS could represent a privileged site for the recruitmentand activation of TIL in human lung tumors and aimed toevaluate the potential influence of these structures on thetumor immune contexture.

Here, we investigated the impact of the TLS on the immunecontexture in 458 NSCLC comprising all stages of the disease.Using immunohistochemistry, flow cytometry, and quantita-tive real-time PCR, we demonstrated that TLS mature DC arestrongly associated with a specific Th1 and cytotoxic immunesignature and a long-term survival. In addition, the combina-tion of mature DC and CD8þ T-cell densities constitutes apowerful and independent prognostic factor for overall sur-vival (OS). Altogether, our data emphasize a major role for TLSin shaping the tumor immune contexture, and support theirinvolvement in the promotion of a protective immuneresponse mediated by T cells.

Patients and MethodsPatients

Fresh (n ¼ 54 patients), frozen (n ¼ 28 patients), andparaffin-embedded (n ¼ 376 patients) primary lung tumorsamples were obtained from patients with NSCLC whounderwent a complete surgical resection of their lungtumors at Institut Mutualiste Montsouris or Hotel DieuHospital (Paris, France). Three hundred seventy-six patientswith NSCLC operated between June 15, 2001, and November26, 2004, were retrieved retrospectively. Patients whoreceived neoadjuvant chemotherapy or radiotherapy wereineligible. The observation time of the cohort was theinterval between the surgery and the last contact (lastfollow-up or death of the patient). At the completion of thestudy, the minimal clinical follow-up was 90 months for the

last patient included in the cohort. The main clinical andpathologic features of the patients enrolled are presentedin Table 1 for the retrospective study and in SupplementaryTables S1 and S2 for the prospective study on fresh andfrozen tumors (20 common patients between the 2 prospec-tive cohorts), respectively. The data on long-term outcomes

Table 1. Baseline characteristics of the patientswith NSCLC enrolled in the retrospective study

Characteristics Number %

GenderMale/female 302/74 80/20

AgeMean (y) � SEM 62 � 10Range 19–83

Smoking historyCurrent 315 84Never smokers 52 14ND 9 2

Pack-years (y) � SEM 42 � 24Range 0–120

Histologic typeADC 241 64SCC 111 29Others 18 5ND 6 2

EmboliNo 141 37Yes 210 56ND 25 7

pT stageT1 85 22T2 180 48T3 84 22T4 25 7ND 2 1

pN stageN0 239 64N1 67 18N2 66 17ND 4 1

pTNM stageI 167 44II 101 27III 104 28IV 2 0.5ND 2 0.5

Vital status of patientsAlive 146 39Dead 230 61

NOTE: All parameters were evaluated among 376 patientswith NSCLC.Abbreviations: ADC, adenocarcinoma; ND, not done; pT,pathologic T stage; pN, pathologic N stage.

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Cancer Res; 74(3) February 1, 2014 Cancer Research706




were obtained retrospectively by interrogation of munici-pality registers or the family of patients. A written informedconsent was obtained from the patients before inclusion inthe prospective study. The protocol was approved by thelocal ethics committee (nos. 2008-133 and 2012-0612) inapplication with the article L.1121-1 of French law.

Immunohistochemistry and immunofluorescenceFor each paraffin-embedded lung tumor, two observers (one

expert pathologist and one investigator trained to identify thepathologic features of NSCLC) selected the tumor sectioncontaining a representative area of tumor with adjacent lungparenchyma, and the highest density of immune cells on thehematoxylin and eosin-safran–stained tissue section. Briefly,serial 5-mm tissue sections were deparaffinized, rehydrated,and pretreated in appropriate buffer for antigen retrieval. Thesections were incubated with 5% human serum for 30 minutesbefore adding the appropriate primary antibodies followed bysecondary antibodies (see Supplementary Table S3). Enzymat-ic activity was performed as described (27). For single staining,sections were counterstained with hematoxylin. Images wereacquired using a Nanozoomer (Hamamatsu) operated withNDPview software.

Method for cell quantificationThe quantification of DC-Lampþ DC was determined as

previously described (27). CD8þ cells were enumerated in thetumor nests and the stroma of the whole tumor section withCalopix software (Tribvn), and expressed as an absolute num-ber of positive cells/mm2 of the areas of interest. Both immu-nostaining and quantification were reviewed by at least twoindependent observers (J. Goc, and R. Remark, T.K.D. Vo-Bourgais or M.-C. Dieu-Nosjean).

Flow cytometryFresh lung tumor specimens were mechanically (manual)

dissociated and digested in the presence of Cell RecoverySolution (BD Biosciences) instead of enzymes that canremove CCR7 and CD62L at the cell surface. Then, totallive mononuclear cells were isolated from the tumors, aspreviously described (29). Mononuclear cells were stainedwith multiple panels of antibodies conjugated to fluorescentdyes (see Supplementary Table S3). Briefly, after saturationwith 2% human serum, mononuclear cells were incubatedwith the primary antibodies or appropriate isotype controlsfor 30 minutes at 4�C in the dark. Cells were washed andfixed in 0.5% formaldehyde before the analysis on a LSRII orFortessa cytometer (BD Biosciences). Flow cytometry datawere analyzed with the Diva (BD Biosciences) and FlowJo(TreeStar, Inc.) softwares. The gating strategies areexplained in Supplementary Fig. S1.

RNA extraction and reverse transcriptionTotal RNA from frozen tissues was extracted with the

RNeasy Mini Kit (Qiagen) according to the manufacturer'sinstructions, and RNA quantity and quality were determinedwith 2100 Bioanalyzer (Agilent Technologies). Samples with aRNA integrity number�7 were reverse transcribed into cDNA

using the High Capacity cDNA Kit (Life Technologies) accord-ing to the manufacturer's instructions.

Quantitative PCRComplementary DNA samples were amplified using the Low

Density Array System according to themanufacturer's instruc-tions (AppliedBiosystems). The arrays (Human ImmuneArray;TaqMan) were processed on a TaqMan 2900HD (Life Tech-nologies). Four nanograms of cDNA per qRT-PCR was used.Expression levels of genes were determined using thresholdcycle (Ct) values normalized to b-actin expression as anendogenous control (DCt).

Statistical analysisWe used the Mann–Whitney test to compare the density

of infiltrating immune cells in the different tumors. Correla-tions were performed using the Spearman test. OS curveswere estimated by the Kaplan–Meier method and differ-ences between the groups of patients were calculated usingthe log-rank test. The start of follow-up for OS was the timeof surgery. Together with mature DC and CD8þ cell densi-ties, the following available clinical parameters were tested:tumor–node–metastasis (TNM) stage according to the newclassification 2009 (30), smoking history, histologic typeaccording to the classification of the WHO (31), interventiontype and emboli. With respect to immune cell densities, the"minimum P value" approach was used to determine thecutoff (Supplementary Fig. S2) for the best separation ofpatients referring to their OS outcome (outcome-orientedapproach). Optimal cutoff values are 1.964, 383, and 114 cellsper mm2 for DC-Lampþ, CD8S, and CD8T cells, respectively.Because the P values obtained might be underestimated, OSlog-rank P values were corrected using the formula proposedby Altman and colleagues (32) and validated using 100repetitions of 2-fold cross-validations. We have also ensuredthat the significance established at the optimal cutoffremained valid at the quartiles (Supplementary Table S4).A P value of less than 0.05 was considered statisticallysignificant. Parameters identified at univariate analysis aspossibly influencing the outcome (P < 0.05) were introducedin a multivariate Cox-proportional hazards regression mod-el. All analyses were performed with Prism 5 (GraphPad),Statview (Abacus Systems), and R (http://www.r-project.org/). Correlation matrix was represented with the Genesissoftware (Institute for Genomics and Bioinformatics, Gratz,Austria; ref. 33).

ResultsMature DCdensity is associatedwith early-differentiatedand intermediate effector–memory CD8þ T-cellinfiltration in human lung tumors

We performed large-scale flow cytometry analyses on 54freshly resected human NSCLC to characterize the immuneinfiltrate according to the density of DC-Lampþ mature DC(Fig. 1). We observed a significant higher percentage of totalCD3þ, CD3þCD4þ, and CD3þCD8þ T cells, a nonsignificanttrend for CD19þ B cells and no difference for CD3�CD56þ

Mature DC Coordinate Intratumoral Immune Reaction

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natural killer (NK) cells (Fig. 1A) among total livemononuclearcells from the tumor between patients with a high density ofDC-Lampþ DC (DC-LampHi patients) versus patients with alow density of DC-Lampþ DC (DC-LampLo patients). DC-LampHi tumors had a significantly greater amount ofCD62LþCD4þ and CD62LþCD8þ T cells than DC-LampLo

tumors (Fig. 1B, left), in accordance with the selective local-ization of CD62Lþ T cells inside the TLS (Supplementary Fig.S3; ref. 29). We also observed a significant and concomitantincrease of antigen-experienced CD62L�CD4þ and CD62L�

CD8þ T cells, which represent the majority of TIL among totalmononuclear cells (Supplementary Fig. S4A), between DC-LampHi versus DC-LampLo tumors (Fig. 1B, right). Interesting-ly, a positive correlation was observed between the proportionof CD62Lþ and CD62L� T-cell subsets among total live mono-nuclear cells in the tumors (Supplementary Fig. S4B).

As compared with DC-LampLo tumors, DC-LampHi tumorswere more infiltrated by activated CD38þ or CD69þ CD8þ Tcells (Fig. 1C) and by the fourmain subpopulations of effector–memory CD8þ T cells (CD45RA�CCR7�CD27þ or �CD28þ or �;Fig. 1D) whereas no difference was seen for terminally differ-entiated effector–memory T cells (also called TEM-RA), whichwere detected at a very low frequency.

Altogether, these results demonstrate that DC-LampHi

tumors have higher numbers of TLS T cells, as well as a higher

number of activated and effector–memory non-TLS T cells,than DC-LampLo tumors.

Density of mature DC signals a coordinated in situ Th1,cytotoxic, and activated T-cell immune reaction

Because the density of mature DC that home selectively inTLS is associated with an increased number of TIL, we nextevaluated the impact of TLS on the functional characteristicsof the immune cell infiltrate with a focus on T lymphocytes.Gene expression levels related to the main immune popula-tions, TLS, Th1 and T-helper 2 (Th2) orientations, CD8þ T-cellcytotoxicity, T-cell activation, immunosuppression, inflam-mation, and angiogenesis were assessed in whole frozentumors from 14 patients with DC-LampHi tumors, and com-pared with 14 patients with DC-LampLo tumors (Fig. 2A,Supplementary Fig. S5, and Supplementary Tables S2 andS5). As a control, genes related to the TLS cluster (CCL19,CCR7, CD28, CD62-L, and lymphotoxin-a) were significantlyoverexpressed among DC-LampHi versus DC-LampLo tumors(P ¼ 0.0006). DC-LampHi tumors and not DC-LampLo tumorswere also associated with the overexpression of sets of genesclustered into specific groups: immune cell populations (P ¼0.0011), Th1 polarization (P ¼ 0.0002), CD8þ T-cell cytotox-icity (P ¼ 0.0013), and T-cell activation (P ¼ 0.0005). Most ofthese molecules were detected at the protein level by

Figure 1. Phenotypic analysis ofthe immune cell infiltrate accordingto the high and low density ofmature DC. Large-scale flowcytometry analysis of immune cellpopulations stratified by thedensity of mature DC in 54 freshlung tumors (34 DC-LampLo

tumors vs. 20 DC-LampHi

tumors). Density of matureDC was evaluated byimmunohistochemistry. Thepercentage of the different celltypes among total livemononuclear cells from thetumors of DC-LampLo (whitecircles) and DC-LampHi (blackcircles) is shown. A, percentage oftotal CD3þ T cells, CD4þ andCD8þ

T-cell subsets, CD19þ B cells, andCD56þCD3� NK cells in groups ofDC-LampHi tumors versus DC-LampLo tumors. B, proportion ofCD3þ CD4þ and CD3þ CD8þ T-cell subsets expressing eitherCD62Lþ or CD62L� T cells inDC-LampHi versus DC-LampLo

tumors. C and D, proportion ofCD3þ CD8þ T cells expressing theactivationmarkersCD38andCD69(C) with effector–memoryphenotype (D) among groups ofDC-LampHi versus DC-LampLo

tumors. �, P < 0.05; ��, P < 0.01;Mann–Whitney U test. NA, notapplicable.

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immunohistochemistry and/or by flow cytometry on T cells:immune cells (Figs. 1 and 3; ref. 1), TLS signature (Fig. 1;ref. 29), Th1 orientation (Supplementary Fig. S6; ref. 27),chemokine receptors (29), and T-cell cytotoxicity and activa-tion (Supplementary Fig. S6).

In contrast, genes involved in Th2 polarization, immuno-suppression, inflammation (excepting CSF-2), and angiogene-sis were not differentially expressed between the two groups ofpatients (Fig. 2A). We constructed a correlation matrix anddemonstrated that the expression of all genes overexpressed

Figure 2. Gene expression levelsrelated to immune populations,TLS, Th-orientation, cytotoxicity,T-cell activation,immunosuppression,inflammation, and angiogenesisaccording to the high and lowdensity of mature DC. Geneexpression levels were assessedby qRT-PCR and determined usingthreshold cycle (Ct) valuesnormalized (DCt) to the actinhousekeeping gene (n ¼ 28 frozenlung tumor samples). The heatmaprepresentation of clusters of genesrelated to immune populations,TLS, Th1 and Th2 orientation,cytotoxicity, T-cell activation,immunosuppression,inflammation, and angiogenesis isshown in A. Genes are plotted fromthe minimal level of expression(blue) to the maximal level(red). Density of mature DCwas evaluated byimmunohistochemistry.Comparison of gene expressionlevels between DC-LampHi versusDC-LampLo patient groups wasperformed using the Mann–Whitney test. A correlation matrixwas constructed with the samegenes. Each square inB representsa Spearman correlation coefficient(blue, negative correlation; red,positive correlation).






among DC-LampHi tumors (immune cell subsets: CD3, CD4,CD8, CD20; TLS: CCL19, CCR7, CD28, CD62L, and LTA; Th1orientation: CXCR3, IFN-g , interleukin (IL)-2, IL-12B, IL-15,T-bet, and TNF-a; cytotoxicity: FAS-L, GNLY, GZMB, and PRF1;and T-cell activation: CCL5, CCR2, CCR4, CCR5, CD40L, CD86,CTLA-4, HLA-DRa, and ICOS) was also significantly correlat-ed (correlation coefficient in Fig. 2B, and P value in Supple-mentary Fig. S7A and S7B).

Altogether, these results demonstrate that mature DC den-sity correlates with a specific intratumoral immune contexturecharacterized by the overexpression and coordination of genesrelated to T-cell activation, Th1 orientation, and cytotoxiceffector functions.

High density of mature DC predicts high levels of CD8þ

T-cell infiltration in lung tumorsA correlation between CD8þ T-cell infiltration and a favor-

able clinical outcomewas previously reported inmany types ofhuman solid cancer (8, 9, 13–15). As we observed a closeassociation betweenmature DC density with cytotoxic effectorfunction, we further investigated the relationship betweenmature DC and CD8þ T-cell infiltration. Because CD8þ T cellsare expected to establish a contact with tumor cells to exerttheir cytolytic function, we discriminated CD8þ T cells presentin the tumor nests and in the stroma in the following analysis.

In a retrospective series of 376 patients with NSCLC (Table1), we quantified stromal CD8þ T cells (CD8S), tumor nest

Figure 3. Analysis of the distributionof CD8T and CD8S positive cells inDC-LampHi versus DC-LampLo

patients. Immunostainings wereperformed on 376 paraffin-embedded tumor samples. Noticethat CD8þ cells (red cellssurrounded by a green line) werecounted selectively in thestroma (A) and pan-cytokeratinsþ

tumor nests (B, blue cells stainedwith AE1/AE3 antibodies).TLS are detected byimmunohistochemistry in NSCLCcounterstained with hematoxylinand eosin (HES; C). TLS arecomposed by T-cell–rich areas thatcomprised DC-Lampþ DC (D; red)in contact with CD3þ T cells (blue).E, TLS are surrounded by PNAdþ

HEV (red). He, hematoxylin. F, theCD3þ T-cell zone (blue) is adjacentto CD20þ B-cell follicles (red).Lower magnification ofimmunohistochemistry stainings isshown in Supplementary Fig. S8.Quantification of positive cells wasperformed on the whole section. G,distribution of the density of CD8T(left) andCD8S (right) T cells amonggroups of DC-LampLo (white bars)versus DC-LampHi tumors (graybars). Statistical significance wascalculated by the Mann–Whitneytest. H, DC-LampHi and DC-LampLo patients stratified by highor low density of CD8S and CD8Tcells.

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CD8þ T cells (CD8T), and mature DC-Lampþ DC (Fig. 3A, B,and D). As previously observed in early-stage lung tumors (27),we confirmed that mature DC home selectively in the T-cell–rich areas of TLS (Fig. 3C andD) adjacent to PNAdþ vessels andB-cell follicles (Fig. 3E and F) in all stage lung tumors.In accordance with the results above, we observed a higher

density of bothCD8T andCD8S cells (Fig. 3G) amongDC-LampHi

versus DC-LampLo tumors (mean¼ 254 vs. 138 CD8T/mm2, P¼0.0003; mean¼ 843 vs. 553 CD8S/mm2, P < 0.0001, respectively).Consequently, substratification of DC-LampHi and DC-LampLo

patients according toCD8S andCD8T cell densities revealed that84% of DC-LampHi patients were CD8Hi in at least one region,and in particular 55% were high in both regions (Fig. 3H). Theseproportions were reduced in DC-LampLo patients with 61% ofCD8Hi in at least one region, and only 33% in both regions.Interestingly, patients with CD8S

Lo/CD8THi tumors were rare in

both DC-Lamp groups, in accordance with the trafficking ofinfiltrating T cells from the stroma to the tumor nests. Themaindifferences between DC-LampHi versus DC-LampLo patientsconcerned the percentage of CD8S

HiCD8THi and CD8S

LoCD8TLo

patients whereas the percentages of mix groups (CD8SHiCD8T

Lo

and CD8SLoCD8T

Hi) were quite unchanged. There were nodistinguishable clinical characteristics [except for gender, his-tologic type, and pathologic TNM (pTNM) stage] between thepatients with DC-LampHi versus DC-LampLo tumors (Supple-mentary Table S6). The gender and histologic type are alsocorrelated, as most females were diagnosed with a squamouscell carcinoma (SCC; adenocarcinoma, SCC, and other types:85%, 11%, and 4% of females, and 59%, 34%, and 7% of males,respectively, P ¼ 0.0026). This observation may, in part, explainthe differential distribution of gender and histologic type amongthe groups ofDC-LampHi versusDC-LampLo patients. UsingCoxmultivariate regression analyses, we demonstrate that pTNMstage and DC-Lamp density were two independent prognosticfactors (Supplementary Table S7).Altogether, these results demonstrate that a high density of

mature DC is closely related to a strong CD8þ T-cellinfiltration.

Density of TLS DC allows the identification of CD8Hi andCD8Lo patients with high risk of deathBecause we observed that high densities of CD8þ T cells

were detected in both groups of DC-LampHi and DC-LampLo

patients, we next evaluated the prognostic value of eachvariable alone and in combination (Fig. 4).The Kaplan–Meier curves indicate that the densities of

matureDC (P¼ 9.1� 10�05; Fig. 4A), CD8S cells (P¼ 0.0019; Fig.4B), and CD8T cells (P ¼ 0.0228; Fig. 4C) were correlated withlonger OS.Because the presence of mature DC and CD8þ cells in the

tumors positively influence the outcome of patients with lungcancer, we stratified the patients into four groups according tothe high or lowdensity of eachmarker (DC-LampHi/CD8Hi, DC-LampHi/CD8Lo, DC-LampLo/CD8Hi, and DC-LampLo/CD8Lo; Fig. 4D). We observed that the group of patients withDC-LampHi tumors regardless of the density of CD8S cells hadthe lowest risk of death (P ¼ 3.4 � 10�07, median OS were 92months for DC-LampHi/CD8S

Hi patients and 100 months for

DC-LampHi/CD8SLo patients), as was observed for DC-LampHi

patients (Fig. 4A). Interestingly, only the DC-LampHi patientspresent an improved survival as compared with the wholecohort. In contrast, patients with a low density of both den-dritic cells (DC) and CD8S cells were at highest risk of death(median OS was 22 months) as compared with each immunemarker alone (median OS: DC-LampLo¼ 36 months, CD8S

Lo¼40 months). Patients with DC-LampLo/CD8S

Hi tumors were atan intermediate risk of death (median OS¼ 41 months). Sameresults were obtained when the analysis was performed on thecombination of DC-Lamp with CD8T cells (data not shown).Additional analyses with 100 repetitions of 2-fold cross-valida-tions confirmed the high and significant prognostic value ofDC-Lamp/CD8S score (cross-validated 99/100 tests, medianP value ¼ 4.7 � 10�04). Using Cox multivariate regressionanalyses (Table 2), the pTNM stage and DC-Lamp/CD8S scorewere the only criteria significantly and independently associ-atedwith OS (HR¼ 1.70 and 0.71, and P¼ 2.83� 10�07 and 4.50� 10�07, respectively).

All together, these data demonstrate that DC-Lamp aloneis a good marker for the identification of patients with afavorable outcome whereas the combination of CD8 withDC-Lamp allows the identification of patients with thehighest risk of death. Finally, the DC-Lamp/CD8S score andpTNM stage constitute two independent and powerful prog-nostic factors.

DiscussionThemajor clinical impact of TIL has emphasized the need to

better identify the mechanisms underlying their recruitmentand activation (14). Based on the structural analogywith lymphnodes, we hypothesized that lung tumor TLS could play a keyrole in the promotion of a local immune reaction. The ability ofTLS to promote an efficient and protective T-cell response inmany chronic inflammatory contexts (19–21) provided therationale for this study.

By combining immunohistochemical and bio-molecularanalyses, we demonstrated that a high density of TLS matureDC is associated with a specific immune orientation char-acterized by the overexpression and coordination of genesrelated to T-cell activation, Th1 polarization, and cytotoxiceffector functions. By contrast, immune genes related to Th2polarization, immunosuppression, inflammation, and angio-genesis were not differentially expressed between groups ofpatients with high or low densities of TLS mature DC. Inagreement with these results, previous reports have alsodemonstrated that TLS correlated with an increased numberof T-betþ lymphocytes or high levels of cytotoxic geneexpression in NSCLC and colorectal cancers, respectively(27,34). This specific Th1 and cytotoxic orientation related tomature DC density suggests a major influence of TLS matureDC in the shaping of the tumor immune contexture.

The significant increase of T-cell proportion, comprisingCD62Lþ T cells among the group of DC-LampHi versus DC-LampLo tumors, is in agreement with the selective presence ofCD62Lþ T cells located close to PNAdþHEV (and vice versa) inTLS (29). Interestingly, a crucial role for DC in the maturation






of HEV (35) and maintenance of TLS (22, 23) has been dem-onstrated in mice. In addition, close correlations betweenthe density of HEV and lymphocyte infiltrate have beenreported in melanoma (36, 37), human breast cancer (38), and

in themethylcholanthrene-induced fibrosarcomamousemod-el (39), which argue for functional features of HEV in tumormicroenvironment. Altogether, these data strongly supportthat intratumoral HEVmediate recruitment of CD62Lþ T cells

Figure4. Overall survival for patientswithNSCLCaccording to thepresenceof a high or lowdensity ofDC-LampþmatureDC (A), CD8Scells (B), andCD8T cells(C), or both DC-Lampþ DC and CD8S cells (D). OS curves for patients with NSCLC were estimated by the Kaplan–Meier method, and differencesbetween groups of patients were evaluated using the log-rank test. Tables show the number of events, censored and at-risk patients according to thecell density group. Tables show the 24-month, 60-month, and 120-month OS rates (%) according to the group of patients, respectively.

Goc et al.





from the blood into TLS, which could represent a majorgateway for T cells into the tumor.The density of TLS mature DC is also associated with a

strong infiltration of experienced CD62L� T cells that arepredominantly of the effector–memory phenotype. Theseexperienced T cells may derive, in part, from TLS CD62Lþ Tcells, which have undergone a local activation and differenti-ation. Studies inmousemodels indicated that TLS could play akey role in the induction of a local immune response. Devel-opment of TLS was associated with the generation of specificCD8þ T cells and viral clearance in mice lacking secondarylymphoid organs (19, 20). A recent study also demonstratedthat the activation, expansion, and differentiation of na€�veCD8þ T cells into effector cells could occur directly in thetumors of mice with B16 melanoma and devoid of secondarylymphoid organs (40). These data highlight that extranodalactivation of TIL is possible and can generate an immuneresponse against the tumor independently of a responseinitiated in canonical lymphoid organs. Furthermore, de novopriming of tumor-infiltrating CD8þ na€�ve T cells has beendescribed in TLS induced by modified vaccine virus Ankarademonstrating that these structures represent a major site forT-cell priming (23). Strikingly, induction of lymphoid neogen-esis in tumors manipulated to express LIGHT (lymphotoxins,inducible expression, competes with HSV glycoprotein D forHVEM, expressed by T cells) or lymphotoxin-a generates amassive infiltration of na€�ve T cells, followed by T-cell activa-tion, expansion, and tumors rejection (41, 42). Altogether, thesedata underline that TLS may serve as an important site forpriming TIL during the generation of a local immune response.Three major studies have previously reported strong asso-

ciations between CD8þ T-cell infiltrate with the outcome ofpatients with lung cancer (9, 43, 44). In agreement with thesedata and similar results in many other types of cancers (8, 13–15), we found a strong correlation between CD8þ cell densityand improved clinical outcome in patients with NSCLC. Com-pared with our first study performed in patients with early-stage NSCLC (27), we confirmed that mature DC density is stilla favorable prognostic biomarker for patient survival (cohort ofpatients with stages I to IVNSCLC). Interestingly, patients withDC-LampHi/CD8Lo tumors were scarce, arguing for a causallink between TLS and CD8þ cells density in which TLS would

be an active site for CD8þ T-cell proliferation. More impor-tantly among CD8Hi patients, those with a concomitant DC-LampHi density had a significant clinical benefit as comparedwith patients with no or few TLS. Thus, DC-Lamp represents anew marker allowing the identification of CD8Hi patients withelevated risk of death, and the combination of both variablesallows the identification of a worst-risk group (DC-LampLo/CD8Lo patients).

TLS could potentiate an antitumor CD8þ T-cell response inmultiple ways. We previously reported that TLS T cells con-sisted primarily of CD4þ T cells (27, 29). Interestingly, a majorrole of CD4þ T-cell help for the recruitment, activation, andeffector functions of na€�ve CD8þ T cells was demonstrated in amodel of pancreatic tumors (45). This is consistent with thestrong coordination between mature DC infiltration, Th1polarization, T-cell activation, and cytotoxic orientation thatwe report in this study.

We speculate that TLS mature DC could present tumor-associated antigens that will promote continuous generationof specific T cells directly in the tumor. As a result, TLS couldinduce a local immune reaction that would be more adaptableto the shifting expression of tumor-associated antigens duringtumor progression. A major challenge will now be to charac-terize and to compare the specificity of experienced T cellsaccording to DC density to evaluate a potential associationbetween TLS and clonal diversity of TIL.

Altogether, our results suggest that TLS represent a privi-leged area for T-cell recruitment and activation in the primarysite of lung tumor, which could play amajor role in the shapingof a tumor immune contexturewith survival benefit. Lymphoidneogenesis could represent a major phenomenon to promotea protective immune response in lung or other types of cancer,which may provide new opportunities to improve immuno-therapy strategies.

Disclosure of Potential Conflicts of InterestS.A. Hammond is an employee of MedImmune LLC/AstraZeneca and has

ownership interest in AstraZeneca. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: J. Goc, L. de Chaisemartin, S.A. Hammond, C. Saut�es-Fridman, M.-C. Dieu-Nosjean

Table 2. Multivariate Cox proportional hazards analysis for overall survival in patients with NSCLC

Variable PHA test HR 95% CI P

Intervention type (lobectomy vs. pneumonectomy) 0.200 1.5 0.98–2.23 0.0629Emboli (no vs. yes) 0.489 1.35 0.95–1.90 0.0901pTNM stage 2009 0.171 1.70 1.39–2.08 2.83e�07DC-Lamp/CD8S densities 0.278 0.71 0.62–0.81 4.50e�07

NOTE: All parameters were evaluated among 325 patients with NSCLC. Patients were stratified into four groups according to thehigh/low densities of mature DC and stromal CD8þ cells (DC-LampHi/CD8Hi, DC-LampLo/CD8Hi, DC-LampHiCD8Lo, and DC-LampLo

CD8Lo). All categorical variableswere transformed intodiscretenumeric variablesbefore theywereadded into theCoxmodel. PHA test,P < 0.05, violates the proportional hazards assumption.Abbreviations: CI, confidence interval; PHA, proportional hazards assumption.






Development of methodology: J. Goc, T.K.D. Vo-Bourgais, C. Klein, L. deChaisemartin, D. Damotte, M.-C. Dieu-NosjeanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Goc, T.K.D. Vo-Bourgais, A. Lupo, S. Knockaert,H. Ouakrim, M. Alifano, P. Validire, R. Remark, D. DamotteAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Goc, T.K.D. Vo-Bourgais, L. de Chaisemartin,E. Becht, S.A. Hammond, M.-C. Dieu-NosjeanWriting, review, and/or revision of the manuscript: J. Goc, C. Germain,H. Ouakrim, S.A. Hammond, I. Cremer, W.-H. Fridman, C. Saut�es-Fridman, M.-C.Dieu-NosjeanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Goc, C. Germain, T.K.D. Vo-Bourgais,H. Ouakrim, P. Validire, D. Damotte, M.-C. Dieu-NosjeanStudy supervision: M.-C. Dieu-Nosjean

AcknowledgmentsThe authors thank Patricia Bonjour for technical assistance and Martine

Bovet for help in clinical data collection. They also thank Estelle Devevre and

Helene Fohrer-Ting from the "Centre d'Imagerie Cellulaire et de Cytom�etrie"(Cordeliers Research Center, Paris) for excellent technical assistance and sup-port in flow cytometry.

Grant SupportThis work was supported by the "Institut National de la Sant�e et de la

Recherche M�edicale," the "Fondation ARC pour la recherche sur le cancer," the"Canc�eropole Ile-de-France," Universit�e Paris-Descartes, Universit�e Pierre etMarie Curie, Institut National du Cancer (2011-1-PLBIO-06-INSERM 6-1,PLBIO09-088-IDF-KROEMER), CARPEM (Cancer Research for PersonalizedMedicine), Labex Immuno-Oncology (LAXE62_9UMRS872 FRIDMAN), andMed-Immune LLC.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 9, 2013; revised November 7, 2013; accepted November 23, 2013;published OnlineFirst December 23, 2013.

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2014;74:705-715. Published OnlineFirst December 23, 2013.Cancer Res Jérémy Goc, Claire Germain, Thi Kim Duy Vo-Bourgais, et al.

T Cells+Positive Prognostic Value of Infiltrating CD8Signal a Th1 Cytotoxic Immune Contexture and License the Dendritic Cells in Tumor-Associated Tertiary Lymphoid Structures

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