The Prostate 68:1^10 (2008)

Dominance of CD4+ Lymphocytic InfiltratesWithDisturbed EffectorCell Characteristics in theTumor

Microenvironmentof ProstateCarcinomaKathleen Ebelt,1,2* Gregor Babaryka,3 Ainhoa M. Figel,1 Heike Pohla,1,4

Alexander Buchner,5 Christian G. Stief,5 Wolfgang Eisenmenger,6

Thomas Kirchner,3 Dolores J. Schendel,1,2 and Elfriede Noessner1

1Institute ofMolecular Immunology, GSF-National ResearchCenter for EnvironmentandHealth,Munich,Germany2Clinical CooperationGroup‘‘ImmuneMonitoring’’,GSF-National ResearchCenter for EnvironmentandHealth,

Munich,Germany3Institute of Pathology, Ludwig-Maximilians-University,Munich,Germany

4Laboratory forTumor Immunology, Ludwig-Maximilians-University,Munich,Germany5DepartmentofUrology,University-Hospital Grosshadern,Clinicofthe Ludwig-Maximilians-University,

Munich,Germany6Institute of LegalMedicine, Ludwig-Maximilians-University,Munich,Germany

BACKGROUND. Prostate cancer is the most common cancer of men in the Western world.Despite the over-expression of tumor-associated antigens, like PSA or PSMA, immune activationis inefficient. The goal of this investigationwas to assess in situ characteristics of prostate cancer-infiltrating lymphocytes and to determine their activation status and effector function.METHODS. We compared 17 carcinoma containing tissues, four benign prostatic hyperplasiatissues and eight healthy prostate tissues regarding lymphocyte subset composition, locore-gional distribution, and functional status using immunohistological staining of cryopreservedtissues. For determination of lymphocyte subsets, serial sections were stained with CD3, CD4,and CD8 antibodies. Activation status and effector function were studied using CD69,interferon-gamma (IFNg), perforin, and CD3 zeta chain antibodies. T-cell-receptor repertoire(TCR) analysis was made to determine the complexity of infiltrating lymphocytes.RESULTS. CD3þ, CD4þ, and CD69þ T lymphocytes were prominent in tissues derived frompatients with prostate carcinoma. CD8þ lymphocytes were significantly less than CD4þ

lymphocytes. IFNg and perforin were downregulated on infiltrating lymphocytes compared tocells of healthy prostate tissue. Very few lymphocytes were detected within cancerous lesionswhereas surrounding tissues showed extensive lymphocyte cluster formation. The TCRrepertoire of infiltrating lymphocytes was broad and similar to that of healthy prostate tissue,giving no evidence for specific lymphocyte recruitment.CONCLUSIONS. In the prostate cancer microenvironment, CD4þ T lymphocytes dominatedwhile CD8þT cellswere sparse. The lymphocytes exhibited signs of disturbed effector function.Consequently, the immune response against autologous tumor cells is likely to be inefficient incontrolling tumor growth. Prostate 68: 1–10, 2008. # 2007 Wiley-Liss, Inc.

KEY WORDS: tumor-infiltrating lymphocytes; IFN-gamma; perforin; T-cell-receptorrepertoire

Grant sponsor: The Deutsche Forschungsgemeinschaft; Grantnumbers: SFB 455, SFBTR 36.

*Correspondence to: Kathleen Ebelt, MD, GSF-National ResearchCenter for Environment and Health, Institute of Molecular Immu-nology, Marchioninistr. 25, 81377 Munich, Germany.E-mail: kathleen.ebelt@gsf.de

Received 5 April 2007; Accepted 6 August 2007DOI 10.1002/pros.20661Published online 18 October 2007 in Wiley InterScience(www.interscience.wiley.com).

� 2007Wiley-Liss, Inc.

INTRODUCTION

Prostate cancer is the most common cancer of menin the Western world and follows lung cancer andintestinal cancer as the third most frequent cause ofcancer-related death in men. Among all malignanturological tumors, prostate cancer is the leading causeof death [1,2]. Despite its high incidence, the prognosisfor patients with prostate cancer is excellent whenthe carcinoma is detected at a stage when curativetreatments, like prostatectomy or radiation therapy,can be implemented. However, when the carcinomahas reached an advanced or hormone refractory status,curative treatments are no longer available and theprognosis for these patients declines dramatically.Therefore, there is an interest to develop immune-based therapies to avoid tumor relapse.

Although there are a number of different targetantigens with potency to activate immune responsesin vitro it appears that an efficient immune responsecannot be easily activated against prostate carcinomasin vivo [3]. The reasons for immune escape in cancerpatients despite expression of tumor-specific antigensare currently unclear. An important aim for futureimmunotherapy developments for prostate carcinomais to understand and counter-act immune escapemechanisms that hinder effective anti-tumor immunity.Recently, improved knowledge regarding the require-ments for induction of effective immune responses hasinspired attempts to develop new therapeutic strategiesto boost anti-tumor immunity [4–7].

The infiltration of lymphocytes into the tumormicroenvironment seems to play an important role inanti-tumor immunity [8]. For a number of solid tumorsinfiltration of lymphocytes into malignant tumors hasbeen described [9], however, little is known abouttheir actual role in controlling cancer progression andtheir significance for the survival of patients withcancer. Some studies suggest a survival advantagefor patients with tumors containing high numbers oftumor-infiltrating lymphocytes [10]. Obviously, for themajority of cases, lymphocyte infiltration in the tumorenvironment is not sufficient for tumor eradication.A multitude of mechanisms appear to contribute toescape from immune responses [8,11,12].

In this study, we investigated the compositionand activation status of infiltrating lymphocyteswithin the prostate cancer tumor environment andbenign prostatic hyperplasia (BPH) in comparison tohealthy prostate tissue. In addition, we utilized T-cell-receptor (TCR) analysis to investigate whether theinfiltrating lymphocytes were progeny of one or afew T lymphocyte clones or represented a complexmixture of different T cells with a heterogeneous TCRrepertoire.

A comparative analysis of lymphocyte infiltrationof normal versus cancerous tissue is particularlyimportant in prostate carcinoma due to the specialgrowth pattern, whereby areas of normal tissueare interspersed with infiltrative carcinoma tissue.Thus, within one sample, carcinoma areas existalongside non-malignant tissue. On the basis of thisbiological distinction, it is possible to compare the non-transformed tumor environment within view of thecarcinoma tissue.

PATIENTS,MATERIALS, ANDMETHODS

Tissue Samples

Tissues were collected from 17 untreated patientswho underwent radical curative prostatectomy in theDepartment of Urology of the Ludwig-Maximilians-University Hospital in Munich. All prostate cancersamples showed histopathological evidence of carci-nomawith a differentiation grade according toGleasonof 6–9 (Table I). Four tissues with BPH were collectedfrom patients who underwent surgical adenomaresection (Table II).

Furthermore, prostate tissue from eight healthy menwas obtained during autopsy (Table III). These tissuesamples were available through the Institute of LegalMedicineof theLudwig-Maximilians-University,Munich.Autopsy tissues were collected by a procedure securinganonymity. The Ethics Committee of the Ludwig-Maximilians-University, Munich, consented to this tissuecollection. All normal and cancer tissue samples were

The Prostate DOI 10.1002/pros

TABLE I. Characteristics of Investigated ProstateCarcinoma Patients

Patient Age (years)Gleasonscore

Tumor stage(TNM of UICC 2002)

1 78 6 pT2c2 61 6 pT2a3 66 8 pT2b4 67 7 pT3b5 75 8 pT2a6 66 6 pT2a7 63 7 pT2c8 66 7 pT3a9 63 6 pT2c

10 65 8 pT3a11 70 7 pT3a12 59 8 pT3b13 64 9 pT3b14 69 6 pT2c15 59 7 pT2c16 69 6 pT2c17 61 6 pT2c

2 Ebelt et al.

frozen immediately in liquidnitrogen and stored at�808Cprior to further investigations.

Pathological Evaluation of ProstateTissue

All prostate tissue samples were evaluated by anexperienced pathologist for the existence of infiltrativecarcinoma or for the absence of pathological findingsin the cases of the autopsy-derived tissue samples.For pathological investigation, 5 mm cryosections wereprepared and stained with haematoxylin eosin.

Immunohistological Analysis

Tissue samples were embedded in Tissue Tek1

compound (Sakura; Zoeterwoude, The Netherlands)for preparation of cryosections. Immunohistochemis-trywas performed using the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. Serial 5 mmcryosections were fixed in 100% ice-cold acetone.After fixation and three washing steps with phos-phate-buffered saline (PBS) (Invitrogen, Karlsruhe,Germany), a blocking step using 2% bovine albumin(Sigma–Aldrich, St. Louis, USA) was employed beforeapplying the primary antibody, diluted in 8% human,type AB, male serum (Cambrex, Verviers, Belgium) inPBS, with subsequent incubation for 1 hr at roomtemperature. After three washing steps with PBS, two

consecutive antibody incubation steps followed, firstusing polyclonal rabbit anti-mouse immunoglobulin(dilution 1:20) and then applying monoclonal mouseAPAAP (dilution 1:40) (both antibodies from DAKO,Glostrup, Denmark). For visualization of antibodybinding, a developing solution consisting of 40 mglevamisole, 50 mg naphtol AS phosphate disodiumsalt, 600 ml dimethylformamide (all Sigma–Aldrich),40 mg sodium nitrite (Merck, Darmstadt, Germany)and 200 ml New Fuchsin (Sigma–Aldrich) dissolvedin tris(hydroxymethyl)amino-methane (Merck) wasprepared. Nuclear counterstain was performed withMayer’s hemalum solution (Merck).

Antibodies

The following antibodies were used: mouse anti-human CD3 (clone UCHT-1; DAKO) and anti-humanCD4 (clone MT 310), anti-human CD8 (clone C8/144B), anti-human CD69 (clone FN50), anti-humanCD3 zeta chain (clone 1D4), anti-human interferon-gamma (IFNg) (clone B27), and anti-human perforin(clone delta G9) (all BD Pharmingen, Heidelberg,Germany). All antibodies were diluted in 8% human,type AB, male serum (Cambrex).

TCRAnalysis

Total RNA was extracted from 10 to 20 cryosectionsof 5 mm with the help of RNeasy Mini Kit (Qiagen,Hilden, Germany) following the commercial productprotocol. RNA integrity was confirmed by diethylpy-rocarbonate (DEPC) water containing agarose gelelectrophoresis. c-DNA synthesis of rearranged TCRgene segments was made using the TCR primers CaST(a Chain: 50-CAC TGA AGA TCC ATC ATC TG-30) orTCR b-chain primer CbST (b Chain: 50-TAG AGGATG GTG GCA GAC AG-30) with the commercialTranscriptor First Strand c-DNA Synthesis Kit fromRoche (Penzberg, Germany) and an instrumentsetup of 558C for 30 min following the commercialprotocol.

Semiquantitative TCR repertoire analysis was per-formed as previously described [13,14]. The PCR wasperformed with a commercial Taq polymerase kit(GE Healthcare, Buckinghamshire, UK). The dNTPswere purchased from Invitrogen. c-DNA (1.5 ml)reaction was used for 35 cycles of a 40-ml PCRreaction using 2 U Taq polymerase with the suppliedbuffer. Standard PCRwas performed for 30 sec at 948C,30 sec at 588C, and 1 min at 728C, using a Biometrainstrument (Biometra, Gottingen, Germany). To visual-ize the TCR repertoire, each amplification productwas applied to a 2% melting ultra-pure agarose gel(Invitrogen).

The Prostate DOI 10.1002/pros

TABLE II. Characteristics of PatientsWith BenignProstaticHyperplasia (BPH)

Patient Age (years) Pathology

1 71 BPH2 64 BPH3 69 BPH4 63 BPH

TABLE III. Characteristics ofHealthy ProstateTissueDonor

DonorAge

(years) Cause of death Pathology

1 24 Accident None2 31 Aneurysma bleeding None3 34 Suicide None4 47 Accident None5 51 Head injury None6 46 Head injury None7 33 Natural dead None8 38 Suicide None

Quiescent Lymphocytes in Prostate Cancer 3

RESULTS

Lymphocyte Subpopulations inTissuesContainingProstate Carcinoma,Benign ProstaticHyperplasia,

andHealthy ProstateGlandTissue

We investigated the T cell infiltrates in sampletissues with respect to CD4 and CD8 composition andin situ distribution in tissues containing areas ofprostate cancer versus non-malignant regions in thesame tissue section. The results were compared tothe findings in healthy prostate gland tissue andBPH tissue. In the samples derived from prostatecarcinoma patients, CD3þ lymphocytes formed prom-inent clusters adjacent to carcinoma or adenoma areas,which appeared to be separated from the lymphocyticinfiltration (Fig. 1A,D). Directly within the carcinomaareas only a very low number of CD3þ lymphocyteswas found,without any evidence of focal accumulationor cluster formation (Fig. 1G).

The pattern shown is representative of tumorsamples obtained from 17 patients. Patient 6 is shownas a representative example in all figures.

A similar pattern, showing massive lymphocyteclustering outside the carcinoma-infiltrated tissueand only scattered intratumoral lymphocytes, wasobserved for CD4þ lymphocytes (Fig. 1B,E,H). TheCD3þ and CD4þ lymphocyte clusters were preferen-tially localized in the non-malignant tissue surround-ing the carcinoma. The numbers of CD8þ lymphocyteswere significantly less than CD3þ or CD4þ lympho-cytes, but showed the same distribution pattern,clustered in the same peritumoral areas with theCD3þ and CD4þ lymphocytes (Fig. 1C,F). Only a fewindividual CD8þ lymphocytes were found dispersedwithin the carcinoma areas (Fig. 1I). Thus, the peritu-moral areas apparently presented an environment thatattracted T lymphocytes, with a significant preferencefor CD4þ T lymphocytes.

Healthy prostate gland tissue, without any patho-logical features, also containedCD3þ, CD4þ, andCD8þ

lymphocytes (Fig. 1J,K,L). However, positively stainedcells were sporadic, in particular CD8þ cells. Theirdistribution was dispersed without cluster formationand showed preferential localization to the interstitialstroma. Some lymphocytes were also found withinthe adenoid structures in the prostate gland. Onerepresentative example (donor 2) of eight tissues isshown.

BPH tissue also had CD3þ, CD4þ, and CD8þ

lymphocytes. These lymphocytes were dispersed,similar to the healthy tissue. Sometimes small lympho-cyte cluster formations were observed (Fig. 1M,N,O).The clusterswere significantly smaller than those of thecarcinoma tissue. More CD8þ cells were present in

comparison to prostate carcinoma containing tissuesand healthy prostate tissue samples. BPH patient 4 isshown as a representative example.

Tissue-Inf|ltrating Lymphocytes Expressthe CD69ActivationMarker

To assess the activation status of infiltratinglymphocytes, we stained cryosections with antibodydetecting the CD69 molecule. A substantial number ofCD69þ cells was found in the lymphocyte clusters andmost lymphocytes in the carcinoma area were alsoCD69þ (Fig. 2A,D,G). CD69þ cells were also detectableas individually dispersed cells in tissue of the BPH(data not shown) and in the healthy prostate glandsamples (Fig. 2J). In the healthy tissue the numbersclosely matched those of CD3þ cells. Thus, tissue-infiltrating lymphocytes had a phenotype indicatingthat theywere apparently activated. The comparison ofhealthy tissue versus malignant tissue revealed thatcarcinoma-infiltrated prostate tissue contained farmore CD69þ cells than tissue of a healthy gland orBPH tissue.

Lymphocytes of CancerousTissue donotSecrete IFNg

To assess whether infiltrating lymphocytes wereable to achieve effector function, we stained for IFNg asa marker of cell function. In malignant tissues, IFNgþ

cells were rare events in the lymphocyte clusters andcarcinoma areas. In fact, in most samples virtually noIFNgþ cells could be detected (Fig. 2B,E,H). In contrast,IFNgþ cells were readily detectable in healthy prostatetissue (Fig. 2K). In the BPH tissue, IFNgþ cellswere alsorare events or absent (data not shown). In this situationthe absence of IFNg may be related to the chronicinflammatory status of the infiltrating T cells. Thus, inthe cancer-infiltrated tissue samples, the infiltratinglymphocytes were either not stimulated or, alterna-tively, their capacity to secrete cytokine was inhibited.

Lymphocytes of CancerousTissue donotExpress Perforin

Perforin expression by CD8þ and some CD4þ

lymphocytes is associated with their capacity to exertcytolytic activity. In our samples, perforin-expressingcells were very rare. If they were found, they had apreferential localization in the peritumoral non-tumor-ous glandular structures. Perforin-expressing cellswere absent in both the carcinoma areas and thelymphocyte cluster formations (Fig. 2C,F,I). In contrast,perforin-expressing cells were readily detectable in thehealthy prostate gland tissue, despite the sparsepresence of CD3þ and CD8þ lymphocytes. As seen in

The Prostate DOI 10.1002/pros

4 Ebelt et al.

The Prostate DOI 10.1002/pros

Fig. 1. Immunohistological StainingofProstateTissue: Serial 5mmthickcryosectionsprostate carcinoma tissueswere stained (APAAPmethod)with anti-human CD3 (dilution 1:5,000) (A,D,G); anti-human CD4 (dilution 1:1,000) (B,E,H); anti-human CD8 (dilution 1:700) (C,F,I)antibodies. Views in A^C of lower magnification (magnification 25�) reveal the tissue morphology with close proximity of benignadenoma and malignant carcinoma and lymphocyte clusters. The carcinoma area on the right, one small adenoma on the left anda lymphocyte cluster formation between these two regions can be distinguished. D^F (magnification 100�) show representative areas ofthe lymphocyte cluster formation. G^I depict the carcinoma area (magnification 200�). These patterns of locoregional distributionand lymphocyte subset composition are representative of all 17 tissues derived from prostate carcinoma patients (patient 6 is shown).Immunohistological staining (APAAP method) of healthy prostate gland tissue, using anti-human CD3 (dilution 1:3,000) (J); anti-humanCD4 (dilution 1:1,000) (K); and anti-human CD8 (dilution 1:700) (L) antibodies on serial 5 mm cryosections of one healthy prostategland tissue (healthy prostate donor 2), J,K (magnification100�), and L (magnification 200�) is shown. Immunohistological staining (APAAPmethod) of benign prostatic hyperplasia tissue, using anti-human CD3 (dilution 1:3,000) (M); anti-human CD4 (dilution 1:1,000) (N),and anti-human CD8 (1:700) (O) antibodies on serial 5 mm cryosections of benign prostatic hyperplasia tissue (BPH patient 4) is shown(allinmagnification100�).

Quiescent Lymphocytes in Prostate Cancer 5

the tumor samples, perforin-expressing cells preferen-tially localized to the glandular structures in healthytissues. An interstitial localization of perforin-express-ing cells was unusual (Fig. 2L).

In the BPH tissue perforin-expressing cellswere rarein accordance with the chronic inflammatory status ofthe infiltrate (data not shown).

Tissue-Inf|ltrating LymphocytesAre Polyclonal

To gain insight into whether the tissue-residentlymphocyte populations represented the progeny of afew infiltrating lymphocytes which then underwent

antigen-driven or local environmental expansion or,instead, represented unselected cellular infiltrates, weanalyzed thedistribution of TCRa andTCRb families inthe prostate cancer containing tissues and the healthyprostate tissues.

The TCR repertoire analysis included assessmentof 25 TCRVa and 32 TCRVb gene families, therebycovering most of the known TCRVa and TCRVbfamilies. This TCR analysis revealed that all 25 TCRVaand 32 TCRVb families were present in all the investi-gated tissues. Figure 3 shows representative examplesof the TCRVb1 to Vb15 repertoire of one prostatecarcinoma tissue and of one healthy prostate tissue.

The Prostate DOI 10.1002/pros

Fig. 2. ImmunohistologicalStainingofProstateTissue: Serial 5mmthickcryosectionsprostate carcinoma tissueswere stained (APAAPmethod)with anti-human CD69 (dilution1:20) (A,D,G); anti-human IFNg (dilution1:50) (B,E,H) and anti-human perforin (dilution1:100) (C,F,I) anti-bodies.Views in A^C of lowermagnification (magnification 25�) reveal the tissuemorphology with close proximity of benign adenoma andmalignant carcinoma and lymphocyte clusters.The carcinoma area on the right, one small adenoma on the left and a lymphocyte clusterformation between these two regions can be distinguished.D^F (magnification 200�) showrepresentative areas of the lymphocyte clusterformation.G^Idepictthecarcinomaarea(magnification200�).Thesepatternsoflocoregionaldistributionandlymphocytesubsetcompositionare representative of all 17 tissues derived from prostate carcinoma patients (patient 6 is shown). Immunohistological staining (APAAPmethod)ofhealthyprostateglandtissueusinganti-humanCD69(dilution1:20) (J); anti-humanIFNg (dilution1:50) (K); andanti-humanperforin(dilution1:100)(L)antibodiesonserial5mmcryosectionsofonehealthyprostateglandtissue(allinmagnification200�).InsetinK:IFNgþcellsatamagnificationof630�withoilimmersion;arrowsinL:perforin-expressingcells.The figures showhealthyprostateglandofdonor2.

6 Ebelt et al.

A similar polyclonality was observed for the TCRVafamilies (data not shown). The results presented inFigure 3 were representative for all patients andhealthy control tissues. Thus, we found no evidencefor preferential infiltration or expansion of selectedT lymphocytes within prostate tissues.

The infiltrating T cells were also stained for the CD3zeta chain and found to be positive (data not shown).The number anddistribution of zeta chain positive cellswas similar to the number of CD3þ cells. Comparablefindings were made in healthy prostate gland tissue.

DISCUSSION

The goal of this investigation was to define thelymphocyte subsets, their activation status andtheir locoregional distribution within prostate tissuesobtained from patients with prostate carcinoma com-pared to healthy prostate tissue. These various charac-teristics can provide some information about hostimmune system–tumor interactions and insight intopotential immune evasion mechanisms. This informa-tion is required for the better design of anti-tumorimmunotherapies.

Analysis of infiltrating lymphocytes in the tumorenvironment has been described for various malignantsolid tumors [9,15]. However, very few studieshave addressed the lymphocyte infiltration of prostatecancer tissues. The prostate gland tissue tends toinflammation [16]. Therefore, to discern cancer-relatedlymphocytic infiltration characteristics, a comparisonwith normal and non-malignant tissue is essential.One study revealed predominant infiltration of CD3þ

lymphocytes in the prostate gland without detecting a

difference between hyperplasia or carcinoma-inflictedprostate tissue, suggesting that there were no dif-ferences between benign and malignant disease of theprostate gland [17]. The comparison between malig-nant and BPH tissue however is problematic, becausepatients with BPH are pretreated (i.e., with alpha-receptor-antagonists or 5-alpha-reductase-inhibitors)and no certain knowledge is available about theinfluence of used substances on the immune response.In addition, the clinical problems of patients withBPH are chronic and associated with inflammatoryprocesses of the prostate and the urinary tract. Onthe basis of the clinical symptoms of these patients alymphocytic infiltration is expected. Because of theinflammatory condition of BPH and the pretreatmentstatus of the patients, untreated healthy prostate tissueis the better comparison to prostate cancer tissuesfor the evaluation of cancer-associated immune mod-ulation.

We observed a significant difference betweencancer-inflicted and healthy prostate tissue, regardingboth the overall numbers of infiltrating lymphocytesand their anatomical distribution. Whereas lympho-cytes of tumor tissue formed large peritumoral clusters,lymphocytes of healthy tissue occurred as single cells,detectable over the full tissue section, without anynoteworthy cluster formation.With respect to lympho-cyte subset composition, we observed a dominance ofCD4þ cells over CD8þ cells, with an overall similarlocoregional distribution and peritumoral clustering.

Infiltration of tumor tissue with lymphocytesmay have relevance for survival prognosis of cancerpatients, as indicated in a study of colorectal cancerwhere patients with a low density of CD3þ lymphocyte

The Prostate DOI 10.1002/pros

Fig. 3. TCRRepertoireb-Chainof InfiltratingLymphocytes.TCRVb familyrepresentationofprostatecancer tissue(A) andhealthyprostategland (B). Outer lanes represent molecular standards (1kb ladder, Biorad). Numbers above each lane indicate the family-specific TCRprimer in the Arden nomenclature [38]. The amplification product of slower electrophoresis mobility that is identical in every lane andwhich served as an internal PCR control represents theTCR constant region amplicon.The bands of higher mobility, which are variable inlength in different lanes, correspond to the specificTCRVb family indicated by the primer number.The figures show prostate cancer tissueofpatient6andhealthyprostateglandofdonor2.

Quiescent Lymphocytes in Prostate Cancer 7

infiltration had a poor survival prognosis [18]. Suppor-tive of this interpretation are results of another studywhich showed that the number of tumor-infiltratinglymphocytes was higher in earlier stage versus laterstage human colorectal cancer [19].

Prostate cancer is a malignant disease with excellentclinical prognosis regarding 5-year-survival times forthose patients capable of undergoing curative treat-ments, like prostatectomy or radiation therapy, and aslong as the tumor cells remain sensitive to hormoneablative therapy. The strong infiltration of CD3þ cellsmight be related to the slow progression and betterprognosis of this kind of cancer. A strong lymphocyticinfiltration may also contribute to the generally smalltumor size of prostate cancer. A similar associationwasmade for small cell lung cancer, where high numbers ofintratumoral T cells and CD8þ lymphocytes wereassociated with significantly smaller tumor size [20].

All our prostate carcinoma tissue samples camefrom curatively operated patients who received noadditional treatment. The 5-year-survival is the rule forthese prostate cancer patients and therefore notenough time has passed for our patients to experienceprogress. All patients have an excellent clinicalprognosis after surgery and the deficiency of CD8þ

infiltrating lymphocytes is evidently of no disadvant-age if curative treatments are achieved. However, thepaucity of CD8þ lymphocytes and their peritumoralclustering without strong intratumoral infiltration,alongside with an apparent lack of functional activity,may be relevant to disease progression in the absence ofcurative treatment. Certainly a multitude of mecha-nisms are at work to render tumor-infiltrating lympho-cytes insufficient to control tumor growth. A pivotalrole of CD8þ lymphocytes in immune responsesagainst tumors and an association with prognosis ofpatients with cancer disease is debated. For example,ovarian cancer patients with a low representation ofCD8þ lymphocytes had lower median survival timesthan patients with a high infiltration of CD8þ cells [10].A similar finding was reported for esophageal carci-nomas [21].

Several of our findings may be relevant to theapparent inefficiency of the tissue-resident lympho-cytes to control tumor growth. CD69, a generalmarker for T cell activation, was strongly expressed inperitumoral and intratumoral lymphocytes. In con-trast, IFNg and perforin, which are markers forlymphocyte effector function were notably absent intumor tissue while being readily detected in thehealthy prostate gland. This discrepancy suggests thatlymphocytes within tumor-inflicted tissue are acti-vated but not functionally active or perhaps evensuppressed in effector capacity. Alternatively, the lackof IFNg, which is a Th-1-associated cytokine, in tumor

tissue, might indicate that the CD4þ cells belong to anon-IFNg-producing Th population, such as the Th2 orTh17 lineage [22]. The paucity of perforin relative to theCD8þ lymphocyte number may be explained eitherby a loss of perforin expression within the tissue or bythe infiltration of the tissue with non-terminally dif-ferentiated CD8þ T cells, as suggested by studies inmelanoma patients [23]. Regardless of the reason, thelack of perforin and IFNg expression has high impactfor tumor immune escape, as these two moleculesare significant mediators of tumor destruction [24].Perforin release by cytotoxic lymphocytes can mediatedirect tumor cell destruction, while IFNg can controltumor cell growth either through direct cytostaticproperties or indirectly through action on tumorstroma and tumor vascularization [25].

Lower perforin expressionwas previously observedin tumor-infiltrating lymphocytes of colorectal cancercompared to antigen-experienced peripheral bloodcells [26]. Kondo et al. [27] described the influence ofadequate IFNg expression on patient survival in renalcell carcinoma. Enhanced IFNg mRNA expressionof CD4þ and CD8þ tumor-infiltrating lymphocyteswas seen in renal cell carcinoma in comparison toautologous peripheral blood cells from patients [28]. Incontrast, no significant difference in IFNg formalignantovarian cancer and normal tissuewas found.However,it was observed that patients with increased IFNgexpression had significantly longer survival times [29].In contrast to our observations, which indicate a lackof IFNg within the prostate carcinoma microenviron-ment, Elsasser-Beile found increased IFNg mRNA inCD3þ tumor-infiltrating lymphocytes of prostate carci-noma compared to those of benign hyperplasia ofprostate [30]. These apparently contrasting resultscould be due to the application of different methods,immunohistology, and PCR, respectively.

The lack of effector molecules, despite CD69þ

expression as an activation marker, might point toanother function of CD69 which is discussed recently.Apparently CD69þ expression can also indicate anegative immunoregulatory cell function comparablewith the known function of regulatory T cells. In mice,the gene forCD69 is localized in a region carrying genesinvolved in several autoimmune diseases [31]. CD69-deficient mice were shown to develop an exacerbatedform of chronic autoimmune arthritis in comparisonto CD69-competent mice [32]. Furthermore, in somemouse models an enhanced anti-tumor immunity wasseen in CD69-deficient mice [33]. The high prevalenceof CD69þ lymphocytes observed in the microenviron-ment of prostate cancermay thereby be an indicator forthis newly discussed function of CD69þ lymphocytes.Thus, it is conceivable that the high expression ofCD69þ in the microenvironment of prostate cancer is

The Prostate DOI 10.1002/pros

8 Ebelt et al.

not an indicator of activation of lymphocytes capable ofreacting against the cancer, but rather may indicate adownregulation of lymphocyte function and inductionof self-tolerance against autologous tumor cells.

In our analysis the expression of the CD3 zeta chainin lymphocytes was not apparently downregulated intumor samples or in healthy tissue. Decreased expres-sion of CD3 zeta chain in human cancer was reviewedby Whiteside [34]. Interestingly, patients with cervicalcancer after successful chemotherapy showed a resto-ration of zeta chain-expressing T cells [35]. Therefore, adecrease of CD3 zeta chain-expression in malignantcancer processes is reversible.

In renal cell carcinoma, clonal expansion of T cellsexpressing specific TCRs within tumors was reportedin some patients and shown to be associated withtumor-associated T cell responses [14]. After cytokinetherapy with IL-2 and IFNa a restricted TCR repertoirewas detectable in melanoma metastases [36]. Both,renal cell carcinoma and melanoma are known asimmunogenic tumors and immunological treatmentsare available for these patients. The TCR repertoire ofprostate cancer-infiltrating lymphocytes was polyclo-nal and similar to that of healthy prostate gland tissue.This broad polyclonality suggests that the lymphocyticinfiltrate represents an unspecific inflammatory infil-tration of lymphocytes into the tumor environment,without evidence for antigen-specificity or expansionof lymphocytes in situ. The accumulation ofT cells in aninflammatory milieu may expose them to regulatorymechanisms designed to dampen inflammation, there-by contributing to a general failure of the cellularinfiltrates to mediate effective anti-tumor activity.

CONCLUSION

Collectively, our findings reveal that prostate tissueis predominantly infiltrated by lymphocytes of theCD4þ type. A barrier appears to separate carcinomaareas from peritumoral non-malignant prostate tissueand adenoma regions. Most of the lymphocytesclustered within the peritumoral normal tissue, withonly a limited intratumoral distribution. Lymphocytesassembled around the carcinoma areas without show-ing effector characteristics, such as perforin and IFNgexpression. It seems that instigation of inflammatoryreactions is possible in the tumor environment butinfiltrating lymphocytes are hindered in mediatingeffective tumor control. The high expression of CD69þ

lymphocytes in this tumor environment may representthe recruitment and activation of cellswith a regulatoryfunction rather than an anti-tumor effector function.

Bronte and co-workers described that the immuneresponse in prostate cancer could induce an immunesuppression in vitro [37]. In accordance with our

findings, the immune response within themicroenviron-ment apparently was not able to induce an efficientimmune response against autologous carcinoma cells.

To our knowledge, this is the first investigationof IFNg and perforin expression in situ by tumor-infiltrating lymphocytes in the tumor environment ofprostate cancer as compared to healthy prostate glandtissue. We found an impressive difference betweeninfiltrating lymphocytes in healthy and malignanttissue. An inflammatory response may slow cancerprogressionbut it is ultimatelynot able to control tumorgrowth. Therapeutic intervention to allow infiltrationof perforin-expressing, functionally competent lym-phocytes may help to control tumor progression andspread of metastatic disease.

REFERENCES

1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ.Cancer statistics, 2006. CA Cancer J Clin 2006;56:106–130.

2. Hsing AW, Tsao L, Devesa SS. International trends and patternsof prostate cancer incidence and mortality. Int J Cancer (PredOncol) 2000;85:60–67.

3. Pawelec G. Tumour escape: Antitumour effectors too much of agood thing? Cancer Immunol Immunother 2004;53:262–274.

4. Tarassoff CP, Arlen PM, Gulley JL. Therapeutic vaccines forprostate cancer. Oncologist 2006;11:451–462.

5. Karnes RJ, Whelan CM, Kwon ED. Immunotherapy for prostatecancer. Curr Pharm Des 2006;12(7):807–817.

6. Fong L, Small EJ. Immunotherapy for prostate cancer. Curr UrolRep 2006;7(3):239–246.

7. Shariat SF, Sadeghi F, Slawin KM. Vaccine-based immunother-apy for prostate cancer. Rev Urol 2000;2(4):222–227.

8. Rosenberg SA. Progress in human tumour immunology andimmunotherapy. Nature 2001;411:380–384.

9. Balch CM, Riley LB, Bae YJ, Salmeron MA, Platsoucas CD,Eschenbach von A, Itoh K. Patterns of human tumor-infiltratinglymphocytes in 120 human cancers. Arch Surg 1990;125(2):200–205.

10. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F,Jungbluth AA, Frosina D, Gnjatic S, Ambrosone C, Kepner J,Odunsi T, Ritter G, Lele S, ChenYT, OhtaniH, Old LJ, Odunsi K.Intraepithelial, CD8þ tumor-infiltrating lymphocytes and a highCD8þ/regulatory T cell ratio are associated with favourableprognosis in ovarian cancer. Proc Natl Acad Sci U S A 2005;102(51):18538–18543.

11. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S. Escape ofhuman solid tumors from T-cell-recognition: Molecular mech-anisms and functional significance. Adv Immunol 2000;74:181–273.

12. Radoja S, Frey AB. Cancer-induced defective cytotoxic Tlymphocyte effector function: Another mechanism how anti-genic tumors escape immune-mediated killing. Mol Med 2000;6:465–479.

13. SteinleA, Reinhardt C, Jantzer P, Schendel DJ. In vivo expansionof HLA-B35 alloreactive T cells sharing homologous T cellreceptors: Evidence for maintenance of an oligoclonallydominated allospecificity by persistent stimulation with anautologousMHC/peptide complex. J ExpMed 1995;181(2):503–513.

The Prostate DOI 10.1002/pros

Quiescent Lymphocytes in Prostate Cancer 9

14. Jantzer P, Schendel DJ. Human renal cell carcinoma antigen-specific CTLs: Antigen-driven selection and long-term persis-tence in vivo. Cancer Res 1998;58:3078–3086.

15. TsutaK, IshiiG,KimE, ShionoS,NishiwakiY,EndohY,KodamaT, Nagai K, Ochiai A. Primary lung adenocarcinoma withmassive lymphocyte infiltration. Am JClin Pathol 2005;123:547–552.

16. Blumenfeld W, Tucci S, Narayan P. Incidental lymphocyticprostatitis. Selective involvement with nonmalignant glands.Am J Surg Pathol 1992;16(10):975–981.

17. McClinton S, Miller ID, Eremin O. An immunohistochemicalcharacterisation of the inflammatory cell infiltrate in benign andmalignant prostatic disease. Br J Cancer 1990;61:400–403.

18. Galon J, Costes A, Sanchez-Gabo F, Kirilovsky A, Mlecnik B,Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P,Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z,Fridman WH, Pages F. Type, density and location of immunecells within human colorectal tumours predict clinical outcome.Science 2006;313(5795):1960–1964.

19. Xie ZJ, Jia LM, He YC, Gao JT. Morphological observation oftumor infiltrating lymphocytes in human rectal cancer. World JGastroenterol 2006;12(11):1757–1760.

20. Eerola AK, Soini Y, Paakko P. A high number of tumor-infiltrating lymphocytes are associated with a small tumor size,low tumor stage and a favourable prognosis in operated smallcell lung carcinoma. Clin Cancer Res 2000;6(5):1875–1881.

21. Schumacher K, Haensch W, Roefzaad C, Schlag PM. Prognosticsignificance of activated CD8þ T cell infiltrations withinesophageal carcinomas. Cancer Res 2001;61:3932–3936.

22. Harrington LE, Mangan PR,Weaver CT. Expanding the effectorCD4 T-cell repertoire: The Th17 lineage. Curr Opin Immunol2006;18(3):349–356.

23. Mortarini R, Piris A, Maurichi A, Molla A, Bersani I, Bono A,Bartoli C, SantinamiM, Lombardo C, Ravagnani F, Cascinelli N,ParmianiG,AnichiniA. Lackof terminally differentiated tumor-specificCD8þTcells at tumor site in spite of antitumor immunityto self-antigens in human metastatic melanoma. Cancer Res2003;63:2535–2545.

24. Voskoboinik I, Trapani JA. Adressing the mysteries of perforinfunction. Immunol Cell Biol 2006;84(1):66–71.

25. Blankenstein T, Qin Z. The role of interferon-gamma in tumortransplantation, immunity and inhibition of chemical carcino-genesis. Curr Opin Immunol 2003;15:148–154.

26. Ye SW,WangY, Valmori D, AyyoubM,Han Y, Xu XL, ZhaoAL,QuL,Gnjatic S, RitterG,OldLJ, Gu J. Ex-vivo analysis of CD8þTcells infiltrating colorectal tumors identifies a major effector-memory subset with low perforin content. J Clin Immunol 2006;26(5):447–456.

27. Kondo T, Nakazawa H, Ito F, Hashimoto Y, Osaka Y,FutusuyamaK, TomaH, Tanabe K. Favorable prognosis of renal

cell carcinoma with increased expression of chemokinesassociated with a Th1-type immune response. Cancer Sci 2006;97(8):780–786.

28. Elsasser-Beile U, Rindsfuser M, Grussenmeyer T, Schultze-SeemannW,WetterauerU. Enhanced expression of IFN-gammamRNA in CD4þ or CD8þ tumour infiltrating lymphocytescompared to peripheral lymphocytes in patients with renal cellcancer. Br J Cancer 2000;83(5):637–641.

29. Marth C, Fiegl H, Zeimet AG, Muller-Holzner E, Deibl M,DopplerW, Daxenbichler G. Interferon-gamma expression is anindependent prognostic factor in ovarian cancer. Am J ObstetGynecol 2004;191(5):1598–1605.

30. Elsasser-Beile U, Przytulski B, Gierschner D, Grussenmeyer T,KatzenwadelA, LeiberC,DeckartA,WetterauerU.Comparisonof the activation status of tumor infiltrating and peripherallymphocytes of patients with adenocarcinomas and benignhyperplasia of the prostate. Prostate 2000;45(1):1–7.

31. Sancho D, Gomez M, Sanchez-Madrid F. CD69 is an immuno-regulatory molecule induced following activation. TrendsImmunol 2005;26(3):136–140.

32. Sancho D, Gomez M, Viedma F, Esplugues E, Gordon-AlonsoM, Garcia-Lopez MA, Fuente de la H, Martinez-A C, LauzuricaP, Sanchez-Madrid F. CD69 downregulates autoimmune reac-tivity through active transforming growth factor-b productionin collagen-induced arthritis. J Clin Invest 2003;112(6):872–882.

33. Esplugues E, Sancho D, Vega-Ramos J, Martinez-A C, Syrbe U,Hamann A, Engel P, Sanchez-Madrid F, Lauzurica P. Enhancedantitumor immunity in mice deficient in CD69. J Exp Med 2003;197(9):1093–1106.

34. Whiteside TL. Down-regulation of z-chain expression in T cells:A biomarker of prognosis in cancer? Cancer Immunol Immun-other 2004;53:865–878.

35. Ferradina G, Ranelletti FO, Legge F, Salutari V, Martinelli E,Fattorossi A, Lorusso D, Zannoni G, Vellone V, Paglia A,Scambia G. Celecoxib up-regulates the expression of the zChainof T cell receptor complex in tumor-infiltrating lymphocytes inhuman cervical cancer. Clin Cancer Res 2006;12(7):2055–2060.

36. Willhauck M, Scheibenbogen C, Pawlita M, Mohler T, Thiel E,Keilholz U. Restricted T-Cell Receptor repertoire in melanomametastases regressing after cytokine therapy. Cancer Res 2003;63:3483–3485.

37. Bronte V, Kasic T, Gri G, Gallana K, Borsellino G, Marigo I,Battistini L, Iafrate M, Prayer-Galetti T, Pagano F, Viola A.Boosting antitumor responses of T lymphocytes infiltratinghuman prostate cancers. J Exp Med 2005;201(8):1257–1268.

38. Arden B, Clark SP, Kabelitz D, Mak TW. Human T-cell receptorvariable gene segment families. Immunogenetics 1995;42(6):455–500.

The Prostate DOI 10.1002/pros

10 Ebelt et al.