orchestration and prognostic signiﬁcance of immune ... · w.h. fridman and c. sautes-fridman...

Biology of Human Tumors

Orchestration and Prognostic Significance ofImmune Checkpoints in the Microenvironment ofPrimary and Metastatic Renal Cell CancerNicolas A. Giraldo1,2,3, Etienne Becht1,2,3, Franck Pag�es2,3,4,5, Georgios Skliris6,VirginieVerkarre7,YannVano8,ArnaudMejean9,NicolasSaint-Aubert9, LaetitiaLacroix1,2,3,Ivo Natario1,2,3, Audrey Lupo1,2,3,10, Marco Alifano11, Diane Damotte1,2,3,10,Aurelie Cazes12, Frederic Triebel13, Gordon J. Freeman14, Marie-Caroline Dieu-Nosjean1,2,3,Stephane Oudard2,8,Wolf H. Fridman1,2,3, and Catherine Saut�es-Fridman1,2,3

Abstract

Purpose: Clear cell renal cell carcinoma (ccRCC) has showndurable responses to checkpoint blockade therapies. However,important gaps persist in the understanding of its immunemicro-environment. This study aims to investigate the expression andprognostic significance of immune checkpoints in primary andmetastatic ccRCC, in relationwithmature dendritic cells (DC) andT-cell densities.

Experimental Design: We investigated the infiltration and thelocalization of CD8þ T cells andmatureDC, and the expression ofimmune checkpoints (PD-1, LAG-3, PD-L1, and PD-L2) in rela-tion with prognosis, in 135 primary ccRCC tumors and 51 ccRCClung metastases. RNA expression data for 496 primary ccRCCsamples were used as confirmatory cohort.

Results: We identify two groups of tumors with exten-sive CD8þ T-cell infiltrates. One group, characterized byhigh expression of immune checkpoints in the absence offully functional mature DC, is associated with increasedrisk of disease progression. The second group, characteriz-ed by low expression of immune checkpoints and locali-zation of mature DC in peritumoral immune aggregates(tertiary lymphoid structures), is associated with goodprognosis.

Conclusions: The expression of the immune checkpointsand the localization of DC in the tumor microenvironmentmodulate the clinical impact of CD8þ T cells in ccRCC. ClinCancer Res; 21(13); 3031–40. �2015 AACR.

IntroductionA solid tumor is an intricate and dynamic ecosystem containing

tumor and immune cells, fibroblasts, blood, and lymphatic

vessels (1). The density and composition of the immunemicroenvironment is heterogeneous among patients and tumortypes, and it is becoming a robust tool to predict postsurgicalrecurrence and death (1, 2). In the vast majority of cancers,tumor infiltration by CD8þ memory cytotoxic T cells and Th1cells is associated with good clinical outcome (1). In addition,it has been reported that an organized local immune reaction,characterized by the presence of mature dendritic cells (DC)localized in tumor-associated tertiary lymphoid structures(TLS), is necessary to orchestrate this cytotoxic and Th1immune contexture and is associated with good clinical out-come (3–8) and response to therapeutic vaccines (9, 10).

However, several recent findings challenge this concept of theunequivocal relation of effector memory CD8þ T-cell infiltrationwith a good clinical outcome in cancer. First, in diffuse large B-celllymphoma (11), Hodgkin lymphoma (12), and clear cell renalcell carcinoma (ccRCC; ref. 13) high densities of tumor-infiltrat-ing CD8þ T cells have been associated with poor prognosis insome studies. Second, a recent publication from our groupshowed that the infiltration of lung metastases from ccRCC byCD8þ T cells was correlated with poor overall survival (OS)whereas it was a factor of good prognosis in lung metastasesfrom colorectal carcinoma (14). Finally, in non–small cell lungcancer (NSCLC) in which CD8þ T cells densities correlate withgood prognosis, a low density of TLS-DC associated with highCD8þ T-cell infiltration identifies a group of patients with highrisk of death, suggesting a functional impairment of intratumoralCD8þ T cells in this situation (4).

1INSERM, UMRS 1138, Cordeliers Research Center, Team 13 Cancer,Immune Control and Escape, Paris, France. 2Universit�e Paris Des-cartes, Sorbonne Paris Cit�e, UMRS 1138, Centre de Recherche desCordeliers, Paris, France. 3UPMC Univ Paris 06, Sorbonne Universit�es,UMRS 1138, Centre de Recherche des Cordeliers, Paris, France.4INSERM UMRS 1138, Cordeliers Research Center, Team 15 IntegrativeCancer Immunology, Paris, France. 5Department of Immunology,Hopital Europ�een Georges Pompidou, Paris, France. 6CoStim Phar-maceuticals, Boston, Massachusetts. 7Department of Pathology,Hopital Necker-Enfants Malades, Paris, France. 8Department of Med-ical Oncology, Hopital Europ�een Georges Pompidou, Paris, France.9Department of Urology, Hopital Europ�een Georges Pompidou, Paris,France. 10Department of Pathology, Hopital Cochin, Paris, France.11Department of Thoracic Surgery, Hopital Cochin, Paris, France.12Department of Pathology, Hopital Europ�een Georges Pompidou,Paris, France. 13Immutep, S.A., Orsay, France. 14Department of MedicalOncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

W.H. Fridman and C. Saut�es-Fridman contributed equally to this article.

Corresponding Author: Catherine Saut�es-Fridman, Centre de Recherche desCordeliers, InsermUMRS 1138, 15 rue de l'�ecole demedicine, Paris 75006, France.Phone: 33-1-44-27-91-03; Fax: 33-1-40-51-04-20; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-2926

�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 3031

on November 29, 2020. © 2015 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 16, 2015; DOI: 10.1158/1078-0432.CCR-14-2926

http://clincancerres.aacrjournals.org/

Immune checkpoints on infiltrating T cells are key regulators ofimmune escape in cancer. In primary ccRCC, several studies haveshown that the expression of the inhibitory receptor PD-1 on theimmune cells (15, 16) or its ligand PD-L1 on tumor cells (17–19)is associated with a poor clinical outcome. Interestingly, antibo-dies that block the PD-1 axis have yielded a 20% to 30% responserate in metastatic ccRCC (20, 21) that seems to be related to theexpression of PD-L1 by the tumor cells (20, 22). Another inhib-itory molecule that has gained recent attention is the lymphocyteactivation gene-3 (LAG-3), which is coexpressed with PD-1 onCD8þ tumor-infiltrating lymphocytes in melanoma (23), andtogether with PD-1 synergistically regulates T-cell function (24).We expected immune checkpoints high ccRCC patients to have aworse prognosis than the immune checkpoint low.

In these newly described scenarios in which CD8þ T-cellinfiltration correlates with poor prognosis, it is important todefine the combination of immune-based biomarkers that willpredict patients' prognosis and further guide immunotherapeuticapproaches. This study aims to investigate the expression andprognostic significance of immune checkpoint receptors andpaired ligands on primary and metastatic ccRCC in relation withTLS-DC and T-cell densities.

Materials and MethodsPatients

A cohort of 135 primary ccRCC human tumors and another of51 ccRCC lung metastases were collected. The primary casesderived fromspecimensof radical nephrectomyoperatedbetween1999 and 2003 at the hospital Necker-Enfants Malades (Paris,France). The ccRCC lung metastases cohort resected at the HotelDieu hospital (Paris, France) or Hopital Europ�een George Pom-pidou (HEGP, Paris, France) between 1992 and 2010 was alreadydescribed in ref. (14). This research was conducted according tothe recommendations outlined in the Helsinki declaration andapproved by the medical ethics boards of all participating institu-tions, and with the agreement of the Ile-de-France II ethicscommittee (no. 2012-0612). The demographic characteristics ofthe cohorts are depicted in Supplementary Table S1 (14).

In addition, expression data for 496 primary ccRCC sampleswith complete follow-up were downloaded from The CancerGenome Atlas' (TCGA) KIRC study, using version 2 of the nor-malized RNA sequencing data. Corresponding clinical data(updated on 2013-04-06) were downloaded from (25).

Clinic and pathologic featuresThe original histologic diagnosis was confirmed on archival

hematoxylin and eosin–stained slides, and histopathologic fea-tures such as histologic subtype (26), tumor size, regional lymphnode invasion, distant metastases at surgery, Fuhrman nucleargrade (27), and sarcomato€�d features were collected. All tumorswere pathologically staged according to the TNM (tumor–node–metastasis) classification (28). The duration of follow-up wascalculated from the date of the surgery (nephrectomy or metas-tasectomy) to the date of cancer progression, last follow-up ordeath.

Immunohistochemical and immunofluorescence stainingSerial 5-mm formalin-fixed paraffin-embedded (FFPE) tissue

sections from primary and metastatic ccRCC were stained usingautostainerPlus Link 48 (Dako). Antigen retrieval and deparaffi-nization were carried out on a PT-Link (Dako) using the EnVisionFLEX Target Retrieval Solutions (Dako). The antibodies used inthis study for IHC and immunofluorescence (IF) are listed inSupplementary Table S2. IF-stained slides were scanned aftersecondary antibody incubation and mounting. For the IHCstaining, peroxidase activity was detected using 3-amino-9-ethyl-carbazole substrate or Novared and alkaline phosphatase usingalkaline phosphatase substrate III (Vector Laboratories).

Tests of the specificity and sensitivity of PD-1, PD-L1, PD-L2and LAG-3 monoclonal antibodies for IHC experiments wereperformed using generated FFPE cell pellets from transfected300.19 cells (for PD-1, PD-L1, and PD-L2; ref. 29) and CHO cells(for hLAG-3), whereas parental untransfected cells served asnegative controls (Supplementary Fig. S1; Costim Pharmaceuti-cals). Multiple organs (n ¼ 32), human TMA (FDA999a, USBiomax), and malignant cancer tissues (n ¼ 10) from differentoncology indications were also tested using the abovementionedprotocols. Normal human FFPE tonsil sections for PD-1, LAG-3,PD-L2, and normal placenta for PD-L1 were used as positivecontrols (Supplementary Fig. S1).

Immunohistochemical quantificationStained slides were scannedwith aNanozoomer (Hamamatsu)

and analyzed with Calopix software (Tribvn). For quantificationpurposes, tissues were divided into Invasive Margin (IM) andTumor Center (TC) as previously described (30), and the densityof positive cells was calculated in the whole tumor region (IM andTC). Because of the small size (or absence) of TC region in themajority of the metastases, the analysis on this cohort was donenot discriminating between the two regions. The percentages oftumor cells stained positive for PD-L1 and PD-L2 were quantifiedby two independent reviewers (A. Lupo and N.A. Giraldo) with-out prior knowledge of patient outcome, and the tumors above5% tumor cell expression were considered as positive in accor-dance with studies in other types of cancer (20, 31).

Statistical analysisComparisons among the demographic andpathologic features,

immunemarker densities, andPD-L1 andPD-L2 expressionswere

Translational Relevance

Clear cell renal cell carcinoma (ccRCC) is an enigma of thetumor microenvironment studies because, in contrast withmost malignancies, high densities of CD8þ T cells correlatewith poor clinical outcome. We characterize the microenvi-ronment of ccRCC primary tumors and lung metastases asso-ciated with an extensive CD8þ T-cell infiltrate. In one group ofpatients, a T-cell exhausted microenvironment characterizedby high expression of immune checkpoints, and the absence offully functional mature dendritic cells (DC) was found andcorrelated with poor prognosis. In the second group, lowexpression of immune checkpoints and DC localized in peri-tumoral immune aggregates were found and conversely asso-ciatedwith goodprognosis.Our data provide novel prognosticbiomarkers highlighting the central role of PD-L2 and LAG-3in the immunomodulation of ccRCC. The combination ofthese immune profiles could guide the selection of patientswho are likely to respond to checkpoint blockade therapies.

Giraldo et al.

Clin Cancer Res; 21(13) July 1, 2015 Clinical Cancer Research3032




evaluated by using c2, Fisher exact, and Wilcoxon rank-sum tests.Association of variables to prognosis was assessed using theKaplan–Meier method, univariate and multivariate Cox regres-sion analyses. To segregate patients in two groups based onnumerical variables (cell density or gene expression), Log-rankP values of each possible cutoff were computed. The cutoff thatminimized the P value of a log-rank test for DFSwas retained, andthe corresponding P value was corrected using the method pub-lished by Altman and colleagues (32). These cutoffs were laterused to segregate patients into two groups, and their associatedKaplan–Meier curves are displayed throughout the figures. Tofurther confirm and validate the prognosis association of the celldensities, we determined themedian and third quartile cutoff andcalculated the corresponding univariate Cox-regression P values(Supplementary Table S3). Only those variables univariatelyassociated with prognosis were included in the multivariate Coxregression analysis. The duration of follow-up was calculatedfrom the date of nephrectomy or metastasectomy to the date ofdeath or last follow-up.

ResultsTumor infiltration by CD8þ T lymphocytes and expression ofTh1-associated genes correlate with poor prognosis in ccRCC

The density of CD8þ T cells in the IMof the primary tumorswasheterogeneous in the cohort of 135 primary ccRCC. On the basisof the Optimal P value cutoff for DFS (OPv; 630 cells/mm2) wefound that the CD8High (n ¼ 41/135, 30%) group had a shorterdisease-free survival (DFS, P ¼ 0.0001, Fig. 1A) and OS (P ¼0.001, Fig. 1A). The density of CD8þ T cells in the TC had noprognostic impact (Supplementary Table S4). The density ofCD8þ T cells in the IM correlated with the Fuhrman grade

(Supplementary Fig. S2), but not with any other pathologicvariables, including TNM (data not shown).

An independent cohort of lung metastases of ccRCC, inwhich the negative impact of high densities of CD8þT cells onOS had been previously described using semiquantitativecounting techniques (14), was reanalyzed using a quantitativeapproach on 51 cases. The OPv for CD8þ T cells density was490 cells/mm2, and the CD8High (n ¼ 14/51, 27%) groupdisplayed a shorter OS (P¼ 0.001, Fig. 1B). This result confirmsour previous observations (14).

From TCGA public database of 496 primary ccRCC, we ana-lyzed the expressionof 844 immune-related genes, fromwhichweextracted data concerning seven genes expressed in a Th1- andCD8þ T-cell–oriented response according to Galon and collea-gues (30). We found that the expression of most of the genesassociated with this cell signature correlated with poor prognosis:CD8A P¼ 0.04, TBX21 P¼ 0.03, IRF1 P¼ 0.01, GZMB P¼ 4.4�10�5, and IFNG P¼ 3.17� 10�7 that displayed the lowest P value(Fig. 1C). On the basis of theOPv for IFNG, patients that had highquantities of intratumoralmRNA for this gene displayed a shorterOS (P ¼ 0.006, Fig. 1D).

Simultaneous expression of immune checkpoints in primaryand metastatic ccRCC identifies patients with poor clinicaloutcomePrimary tumors. To investigate the impact of immune checkpointmolecules on the negative prognostic impact of the CD8High

group, we analyzed the protein expression of PD-1, LAG-3, PD-L1, and PD-L2 molecules in this group of tumors (we couldanalyze 40/41 tumors) and a randomly matched group of thesame size (n ¼ 40/94) coming from the CD8Low.

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Figure 1.CD8þ T cells and CD8/Th1 gene signature are associatedwith poor prognosis in ccRCC. OS andDFS according to the presence of a high or low density of CD8þ T cellsin the IM of primary ccRCC (A) and ccRCC lung metastases (B). The P values by univariate Cox regression analysis for OS on seven Th1-related genes aredisplayed, P ¼ 0.05 dotted black line, HR >1.0 gray columns (C). OS according to IFNG gene expression in primary ccRCC (D).

The Suppressive and Dysfunctional Immune Contexture in RCC

www.aacrjournals.org Clin Cancer Res; 21(13) July 1, 2015 3033




On the basis of the OPv cutoff, 15 tumors of 80 wereconsidered as PD-1High in the IM and they displayed a shorterDFS (P ¼ 0.0005, Fig. 2A) and OS (P ¼ 0.03, Fig. 2A). Thedensity of PD-1þ cells in the TC was not significantly associatedwith prognosis (Supplementary Table S4). The Fuhrman gradewas associated with the PD-1þ cell density in the IM (Supple-mentary Fig. S2).

Of the 80 patients, 9 were considered as LAG-3High (subdividedby theOPv cutoff) in the IM and they displayed a shorter DFS (P¼0.02, Fig. 2A), and did not reach significance for the OS (P ¼0.07, Fig. 2A). The density of LAG-3þ cells in the TC was notsignificantly associated with patients DFS or OS (SupplementaryTable S4). Representative pictures of the IHC staining of highlyand poorly PD-1– and LAG-3–infiltrated lesions are shown inSupplementary Fig. S3A and S3B, respectively.

On the basis of the 5% cutoff generally used in clinical trialswith anti-checkpoint antibodies (20), 22 of 80 (27%) and 27 of80 (34%) patients were PD-L1þ and PD-L2þ, respectively(Supplementary Fig. S3C and S3D); among them, 9 tumorswere double positive. Patients with PD-L1þ tumors had ashorter OS than those with less than 5% positive tumor cells(P ¼ 0.02, Fig. 2A); a trend toward shorter DFS was also found(P ¼ 0.06, Fig. 2A). Univariately, patients with PD-L1þ tumorswere 2.9 times more likely to die in the 5 years post-nephrec-tomy than patients with less than 5% PD-L1þ tumor cells [OSrisk ratio, 2.87; 95% confidence interval (CI), 1.2–7.1; P¼ 0.02,

Supplementary Table S4]. Patients with PD-L2þ tumors dis-played a shorter OS compared with those with less than 5%positive tumor cells (P ¼ 0.005, Fig. 2A), but no impact on theDFS was found (P ¼ 0.13, Fig. 2A). Univariately, patients withPD-L2þ tumors were 3.4 times more likely to die than thosewith negative tumor cells (risk ratio, 3.4; 95% CI, 1.4–8.5; P ¼0.005, Supplementary Table S4).

Patients with tumors exhibiting both high densities of PD-1þ

lymphocytes in the IM and PD-L1þ and/or PD-L2þ tumor cells(n ¼ 11) had the worst prognosis, as assessed by DFS and OS(DFS, P ¼ 1.3 � 10�5; OS, P ¼ 0.005; Fig. 2B). Patients that metthese criteria had 6.1 times more risk to progress after resectionthan patients who did not (risk ratio, 6.08; 95% CI, 2.4–15.0;P < 0.001, Supplementary Table S4). Strikingly, 91% of thepatients having a PD1High infiltrate and tumor cells expressingPD-L1 and/or PD-L2 progressed in the subsequent 5 years ofsurgery versus 36% in the negative group (complete 5-yearsfollow-up n ¼ 70, Fisher exact test P ¼ 0.001, Fig. 2C). In theTCGA public datasets of 496 primary ccRCC, the gene expres-sion of PD-1 (PDCD1), LAG-3, and PD-L2 (PDCD1LG) wasassociated with shorter OS (P ¼ 0.03, P ¼ 0.0001, and P ¼0.0003, respectively) whereas PD-L1 (CD274) was not (P ¼0.67; Fig. 2D).

It has been reported that IFNg can induce PD-L1 expression ontumor cells (27). We found a significant positive correlationbetween the gene expression of IFNG with PD-L1 (R ¼ 0.13,

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Figure 2.Expression of immune checkpoints correlates with unfavorable clinical outcome for patients with primary ccRCC. OS and DFS according to the presence of a high orlow density of PD-1þ and LAG-3þ cells, and the expression of PD-L1 or PD-L2 by >5% of the tumor cells in primary ccRCC (A). OS and DFS (B) and pie chart(C) representing the percentage of patients that had progressed after 5 years of surgery according to the expression of PD-L1 and/or PD-L2 on tumor cellsrelated with high densities of PD-1þ lymphocytes. The P values by univariate Cox regression analysis for OS on four genes are displayed, P¼ 0.05 dotted gray line,HR >1.0 gray columns (D).

Giraldo et al.





P ¼ 0.004) and with PD-L2 (R ¼ 0.42, P ¼ 2.2�10�16) in theTCGA cohort (Fig. 3A). In addition, a significant positive corre-lationwas foundbetween the densities of PD-1þ andCD8þ T cells(R ¼ 0.31, P ¼ 0.004), LAG-3þ and CD8þ T cells (R ¼ 0.42, P ¼0.001) and PD-1þ and LAG-3þ cells (R¼ 0.55, P < 0.0001) in theIM of the 80 primary ccRCC (Fig. 3B and 3C). An even strongercorrelation was found at the gene-expression level between PD-1and LAG-3 (R ¼ 0.81, P ¼ 0.002), PD-1 and CD8A (R ¼ 0.95, P <0.001), and LAG-3 and CD8A (R ¼ 0.96, P < 0.001) in the TCGAcohort of 496 primary tumors (Fig. 3B and C). In accordancewith all these observations, we also detected the presence oftriple-positive CD8þ/PD-1þ/LAG-3þ cells in 6 of 7 cases from theCD8High group of primary tumors by IF on paraffin sections(representative pictures are displayed in Fig. 3D).

Metastases. Metastatic ccRCC has been one cancer in whichantibodies inhibiting the PD-1 axis have induced remarkabletumor regression in some patients (20), highlighting the neces-sity to define prognostic and predictive immune-based bio-markers in this disease. Of the 51 patients with ccRCC lungmetastasis, 13 were considered as PD-1High, and the latterdisplayed a shorter OS (P ¼ 0.008, Fig. 4A). Seven of 51 wereconsidered as LAG-3High, and again they displayed a shorter OS(P ¼ 0.048, Fig. 4A).

On the basis of a 5% cutoff, 5 of 51 (10%), and 15 of 51 (29%)metastases were PD-L1þ or PD-L2þ, respectively; of them, 3 were

double positive. Whereas patients with PD-L1þ metastases didnot have a significantlyworse clinical outcome (P¼0.12, Fig. 4A),the expression of PD-L2 on tumor cells was associated with ashorter OS (P¼ 0.03, Fig. 4A). Univariately, patients with PD-L2þ

metastasis were 2.2 times more likely to die compared withpatients with PD-L2- ones (risk ratio, 2.17; 95% CI, 1.03–4.35;P ¼ 0.04, Supplementary Table S4).

Patients with high densities of PD-1þ lymphocytes and PD-L1þ

and/or PD-L2þ tumor cells in their ccRCC lung metastases (n ¼12)had theworst prognosis as assessedbyOS (P¼0.003, Fig. 4B).The patients that met these criteria had 3.1 times more risk to dieafter metastasectomy than patients who did not (risk ratio, 3.1;95% CI, 1.28–6.66; P ¼ 0.003, Supplementary Table S4). Strik-ingly, 100% of the patients having a PD1High infiltrate andmetastases simultaneously expressing one or both of its ligands(PD-L1 or PD-L2) died in the subsequent 5 years after surgeryversus 57% in the negative group (Fisher tests, P value ¼ 0.004,Fig. 4C).

A significant and strong correlation was found between den-sities of PD-1þ and CD8þ T cells (R¼ 0.51, P¼ 0.0001), LAG-3þ

and CD8þ T cells (R ¼ 0.4, P ¼ 0.004; Fig. 4D) and PD-1þ andLAG-3þ cells (R ¼ 0.71, P < 0.0001).

In conclusion, the combined analysis of the expressionof PD-1,PD-L1, and PD-L2 identified a group of patientswith a high risk ofprogression anddeath inprimary and in an independent cohort ofmetastatic ccRCC.

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Figure 3.Expressionof immune checkpoints correlateswithCD8þT cells infiltration inprimary ccRCC. Dot plot of thegene expressionofPD-L1 (red dots) andPD-L2 (blue dots;y-axis) against IFNG (x-axis; A). Dot plot of the Log10 cell density and gene expression of PD-1 (red dots) and LAG-3 (blue dots; y-axis) against CD8 (x-axis;B). Dot plot of the log10 cell density and gene expression of PD-1 (y-axis) against LAG-3 (x-axis; C). Pearson r value and the number of samples for each correlation aredisplayed. IF staining on 1 paraffin-embedded ccRCC showing the colocalization of CD8 (green), PD-1 (red), and LAG-3 (white) proteins in lymphocytes (D);nuclei are stained with DAPI.






Opposite correlations of TLS-DC and NTLS-DC with prognosisand their association with immune checkpoints

It has been shown that TLS-DC orchestrate intratumoral CD8þ

cytotoxic T cells and Th1 responses in NSCLC (3, 4). In theprimary ccRCC cohort (n ¼ 135), DC-Lampþ cells were detectedwithin TLS (TLS-DC) in the IM. They coexpressed CD83 and highamounts of MHC Class II, and were localized in the vicinity ofPNAdþ high endothelial venules (HEV; Fig. 5A) as described inother tumor types (3). There was a trend where high densities ofTLS-DC were associated with longer DFS (OPv ¼ 0.09), but notwith OS (Supplementary Table S4). TLS-DC densities in the TCwere not associatedwithprognosis. Interestingly, highdensities ofTLS-DC (based on the OPv cutoff) in the IM identified a group ofpatients with good prognosis among the CD8High group for bothDFS (P ¼ 0.001, Fig. 5A) and OS (P ¼ 0.03, Fig. 5A).

However, in contrast with NSCLC where most of the DC-Lampþ cells are localized within TLS (4), in ccRCC a largepercentage (79% � 20%) of DC-Lampþ cells with DC morphol-ogy was found isolated and outside TLS (NTLS-DC, Fig. 5B). Theycolocalized with CD34þ blood vessels, but not with PNAdþHEV,expressed low amounts of MHC class II and were CD83 negative(Fig. 5B). The majority of NTLS-DC was localized in the IM(71�14%) and their high densities (based on the OPv cut-off)were associated with poor clinical outcome in primary ccRCCpatients (DFS P ¼ 0.006, OS P ¼ 5.1�10�5, Fig. 5B).

The opposite influences of TLS-DC andNTLS-DC on prognosisprompted us to explore their relationships with the expression ofPD-1 and its ligands in the CD8High group of patients. A negativecorrelationwas foundbetween thedensities of TLS-DCandPD-1þ

cells (r ¼ �0.23, P ¼ 0.04, Fig. 5C). Tumors containing PD-L1þ

and/or PD-L2þ tumor cells exhibited less TLS-DC (P ¼ 0.014;Supplementary Fig. S4), but similar densities of CD8þ T cells (P¼0.96). In contrast, the density of NTLS-DC was associated withthe tumor expression of PD-L1 (OR, 6.54; 95% CI, 1.23–45.45;

P¼0.012) andPD-L2 (OR, 2.7; 95%CI, 1.2–18.1;P¼0.04).Mostof the patients that were PD-1High and PD-L1þ and/or L2þ (8 of11) were also CD8High and TLS-DCLo in primary ccRCC (OR,15.02; 95% CI, 1.66–72.7; P¼ 0.004), and the presence of one orboth patterns correlated with shorter DFS (P < 0.0001) and OS(P ¼ 0.001, Fig. 5D).

Identification of a group of patients with worst prognosis inprimary and metastatic ccRCC

To define the independent prognostic significance of the pre-viously mentioned immune profiles (CD8High and TLS-DCLow orPD-1High and PD-L1þand/orL2þ), we included them in a multi-variate Cox regression analysis with the other significant prog-nostic clinical variables (TNM and Furhman grade). The strongestindependent poor prognostic factors in primary ccRCC for DFSwere to have a CD8High and TLS-DCLow (P ¼ 0.001, Table 1) orPD-1High and PD-L1þand/orPD-L2þ (P ¼ 0.03, Table 1) immuneprofile. For the metastatic cohort, we found that the strongestindependent worst prognostic variable for OS was being CD8High

(P¼ 0.004, Table 1) or having a tumorwith PD-1High and PD-L1þ

and/or PD-L2þ immune profile (P ¼ 0.02, Table 1).

DiscussionCompared with other neoplasia, the immune microenviron-

ment in ccRCC has not yet been studied in detail. However, it is ofparamount importance to understand its regulation in view of theparadoxical correlation of CD8þ T-cell infiltration with poorprognosis (13, 14) and the recent advances of immunotherapywith anti-checkpoint antibodies (anti PD-1 and anti PD-L1;refs. 20, 21).

ccRCC has been described as a proinflammatory neoplasiawhere tumor cells produce several cytokines (such as VEGF, IL6and TGFb; refs. 33–35) that may lead to the recruitment and

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Giraldo et al.





activation of polyclonal CD8þ T cells (36–39). Our data suggestthat these recruited CD8þ T cells could only be locally educatedwhen high densities of TLS-DC are present, and in only in thesecases their density correlates with favorable prognosis. ccRCC isthe first tumor type where a large proportion of DC-Lampþ cellsoutside TLS structures have been observed (NTLS-DC). Interest-ingly, we found that these cells do not express activation andcostimulatory markers, and they are probably recruited directlyfrom the blood into the tumor stroma—contrary to the usual paththat DC-Lampþ cells follow in other type of tumors (5, 40).Accordingly, several studies have shown that the ccRCC micro-environment can induce a dysfunctional DCmaturation, a down-regulation of costimulatory molecules and tolerogenicity

(34, 41, 42), whereas DC in the TLS are likely to be protectedfrom these effects (43). Altogether, our results suggest that theparticular proinflammatory milieu initiated by tumor cellsinduces the recruitment of CD8þ T cells that—due to the lownumber of fully functional mature DC present in specialized T-cell–priming sites, or the presence of DC with suppressive phe-notype—are not able to mount an effective antitumor immuneresponse, but rather express exhaustion/inhibition molecules.

This work reinforces the concept that T-cell exhaustion/inhi-bition (44) plays an important role in ccRCC pathogenesis. It hasbeen described that the density of PD-1þ cells (15, 16) and thetumor expression of PD-L1 (17–19) in primary ccRCC are asso-ciated with a poor clinical outcome. In this study, we confirm

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Figure 5.Characteristics of TLS-DC and NTLS-DC and their relationships with prognosis and immune checkpoints. IHC photomicrographs of DC-Lamp(Red)/CD3(Blue)illustrating the presence of mature DC in TLS (A, white arrows) or outside TLS (B, black arrows); DC-Lamp(Red)/PNAd(Blue) and DC-Lamp(Red)/CD34(Blue) illustrating TLS-DC near HEV (A) and NTLS-DC near endothelial cells (B) in primary ccRCC. IF staining showing the colocalization of CD3 (green), DC-Lamp(red) with HLA-DR or CD83 (white) expression in TLS-DC (A), but not in NTLS-DC (B); nuclei are stained with DAPI. OS and DFS according to the presenceof a high- or low-density TLS-DC in the CD8High group (A, bottom) or NTLS-DC in the entire cohort (B, bottom). Dot plot of the PD-1þ against TLS-DCþ celldensities (blue dots; C); Pearson r value is displayed. OS and DFS according to the presence of a CD8High and TLS-DCLow and/or PD-1High and PD-L1 and/or L2þ

immune profiles (D).






these findings and extend them to ccRCC lung metastases. Thisinformation is highly relevant because metastatic ccRCC-treatedpatients have shown one of the highest objective durableresponse rates to PD-1 blockade (approximately 30%; ref. 20)and many efforts are being dedicated to define theranostic toolsin this pathology. Furthermore, we describe for the first time theprognostic significance of PD-L2. This molecule seems to beexpressed in a higher proportion of tumors than PD-L1, and upto 30% of them might express it solely according to our results.This might be of clinical relevance because—although in twodifferent nonrandomized cohorts—the anti–PD-L1 treatmentalone seems to have a lower response rate than the anti–PD-1(12% and 27%, respectively; refs. 20, 21). Furthermore, thereare PD-L1–negative tumors that respond to anti–PD-1 treat-ment (22), suggesting that indeed there are other moleculesbeside PD-L1 implicated in the PD-1 inhibition axis of ccRCC.Few publications have reported PD-L2 mRNA or proteinexpression in other tumors, including primary mediastinallarge B-cell lymphoma (29), NSCLC (45), ovarian (46), andesophageal (47), where it has shown a limited impact ofpatients' prognosis.

To our knowledge, this is the first report on the poor prognosticimpact associated with high densities of LAG-3þ cells in humantumors. Furthermore, we provide clear evidence that the expres-sion of PD-1 and LAG-3 is highly correlated in ccRCC. Somestudies on mouse models have shown a synergistic effect of theinhibition of both pathways in boosting antitumor immuneresponse (24). Therefore, our data support the rationale of dualblockade of these molecules in ccRCC.

Although recent works have emphasized that PD-L1 is prefer-entially upregulated by IFNg (31, 48), whereas PD-L2 is regulatedby IL4 (49), the weak association of PD-L1 mRNA with IFNGmight suggest an important role for posttranscriptional regulationof PD-L1 expression in ccRCC as for other aggressive tumors (50).Furthermore, it is highly suggestive that IFNg can also induce PD-L2 upregulation in tumors, as in immune cells (51), and supportsthe rationale to use therapeutic antibodies targeting this ligand.Taken together, our results suggest that the expression of thesemolecules is related with a chronic inflammatory and highlysuppressive process that is unselectively recruiting CD8þ andNTLS-DC cells from the circulation, and overall is associated witha poor prognosis.

Another characteristic of ccRCC is the lack of prognostic sig-nificance of the immune cells in the TC. Although CD8þ, PD-1þ,LAG-3þ, and NTLS-DC densities in the IM of the primary tumorswere associated with poor prognosis, they had no prognosticsignificancewhen present in the TC. In colorectal carcinoma, both

regions are important to define the best immune score, whichcorrelates with patient's longer survival (30).Whether this dichot-omy between ccRCC and colorectal carcinoma reflects differenttumor tissue organization or relates to other factors of the tumormicroenvironment remains to be clarified.

Recent unsupervised gene clustering of stage IV primaryccRCC showed that tumors with high inflammatory immuneinfiltrate (approximately 15%) also have a high expression ofPDCD1 (PD-1) and its ligands, and correlate with the worstprognosis (52). Indeed, this inflammatory/proangiogenic pro-file (probably originated from the ccRCC tumor cells) found inccRCCprimary tumors and in ccRCC lungmetastases (14), is notfound in colorectal carcinoma lung metastases (14), highlysupporting that it may contribute to local immunosuppressionprocess, the absence of fully mature DC, and high expression ofimmune checkpoints (53).

In summary, we identify a subset of primary and metastaticccRCC patients characterized by (i) an extensive CD8þ T-cellinfiltrate, (ii) expression of immune checkpoints, and (iii) theabsence of fully functional DC, which is associated with poorclinical outcome. Our results highlight novel independent prog-nostic factors in ccRCC based on the concomitant quantificationof densities of DC, CD8þ, PD-1þ, and LAG-3þ lymphocytes inaddition to PD-L1/PD-L2 expression by tumor cells. Theseimmune profiles should guide the selection of suitable patientsto receive immunotherapies and need to be further validated inlarger and independent cohorts. Because this choice depends onboth the extent of CD8þ T-cell infiltration and thematuration andlocalization of DC, it invites revision of the idea that intratumoralCD8þ T cells are always associated with favorable prognosis inhuman tumors.

Disclosure of Potential Conflicts of InterestY. Vano reports receiving other research grants from Novartis and is a

consultant/advisory board member for Novartis and Pfizer. G.J. Freeman hasownership interest (including patents) inAmplimmune, Boehringer-Ingelheim,Bristol-Myers Squibb, EMD Serono, Merck, Novartis, and Roche and is aconsultant/advisory board member for Novartis and Roche. S.M. Oudardreports receiving speakers bureau honoraria from and is a consultant/advisory board member for Janssen, Novartis, Pfizer, Roche, and Sanofi.W-H. Fridman is a consultant/advisory board member for Costim. No poten-tial conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design:N.A. Giraldo, G.J. Freeman, S. Oudard, W.H. Fridman,C. Saut�es-FridmanDevelopment of methodology: N.A. Giraldo, G. Skliris, L. Lacroix, G.J.Freeman, M.-C. Dieu-Nosjean, S. Oudard

Table 1. Multivariate Cox regression analysis for DFS and OS of the pathologic and immune variables in primary and metastatic ccRCC

Multivariate Cox regression analysis DFS Multivariate Cox regression analysis OSPrimary ccRCC HR (95% CI)(lower–upper) P HR (95% CI)(lower–upper) P

CD8high TLS-DClow (yes vs. no) 3.19 (1.42–3.83) 0.001 1.57 (0.45–1.82) 0.41PD-L1 or L2 5%–100% þ PD-1high (yes vs. no) 1.88 (1.56–2.89) 0.03 2.22 (0.80–1.16) 0.24Fuhrman grade 1.90 (0.99–2.00) 0.06 2.68 (0.99–2.16) 0.32TNM stage (I–IV) 1.59 (0.18–2.33) 0.12 2.74 (1.01–3.05) 0.002

Multivariate Cox regression analysis OSMetastatic ccRCC HR (95% CI)(lower–upper) PCD8 group (high vs. low) 2.86 (1.21–3.11) 0.004PD-L1 or L2 5%–100% þ PD-1high (yes vs. no) 1.41 (1.19–2.48) 0.02Lymph node invasion (yes vs. no) 0.72 (0.35–1.42) 0.47

Giraldo et al.





Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N.A. Giraldo, F. Pag�es, V. Verkarre, Y. Vano,N. Saint-Aubert, L. Lacroix, I. Natario, A. Lupo, M. Alifano, D. Damotte, A.Cazes, S. OudardAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):N.A. Giraldo, E. Becht, A. Lupo, M.-C. Dieu-Nosjean,S. Oudard, W.H. Fridman, C. Saut�es-FridmanWriting, review, and/or revision of the manuscript: N.A. Giraldo, E. Becht,F. Pag�es, G. Skliris, Y. Vano, A. Mejean, F. Triebel, G.J. Freeman, M.-C. Dieu-Nosjean, S. Oudard, W.H. Fridman, C. Saut�es-FridmanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N.A. Giraldo, F. Pag�es, V. Verkarre, A. Mejean,L. Lacroix, D. Damotte, F. Triebel, S. Oudard, W.H. Fridman, C. Saut�es-FridmanStudy supervision: N.A. Giraldo, W.H. Fridman, C. Saut�es-Fridman

AcknowledgmentsThe authors thank Romain Remark for his help in the case/block selection

and collection of clinical data from the metastatic cohort, Jose Balcaceres(Association pour la recherche sur les Th�erapeutiques en Canc�erologie) for

his support in the collection of the clinical data of the primary cohort,Patricia Bonjour for cutting the paraffin-embedded blocks, Dr. Marc Riquetfor the case selection of the metastatic cohort and all members of the teams13 and 15 in the Cordeliers Research Center for their valuable discussions.

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

la Recherche M�edicale," the University Paris-Descartes, the University Pierreet Marie Curie, the Institut National du Cancer (2011-1-PLBIO-06-INSERM6-1 and PLBIO09-088-IDF-KROEMER), CARPEM (CAncer Research forPErsonalized Medicine), Labex Immuno-Oncology (LAXE62_9UMS872FRIDMAN), Universidad de los Andes School of Medicine (to N.A. Giraldo),Colciencias (to N.A. Giraldo), and NIH (P50CA101942; to G.J. Freeman).

The costs of publication of this articlewere defrayed inpart 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 November 14, 2014; revised January 9, 2015; accepted January 27,2015; published OnlineFirst February 16, 2015.

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Giraldo et al.




2015;21:3031-3040. Published OnlineFirst February 16, 2015.Clin Cancer Res Nicolas A. Giraldo, Etienne Becht, Franck Pagès, et al. Cancerin the Microenvironment of Primary and Metastatic Renal Cell Orchestration and Prognostic Significance of Immune Checkpoints

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