ﬁcientcutaneousantibacterialcompetenceincutaneous t-cell...

Biology of Human Tumors

DeficientCutaneousAntibacterial Competence inCutaneousT-Cell Lymphomas: Role of Th2-Mediated Biased Th17Function

Kerstin Wolk1,2,3, Hiroshi Mitsui4, Katrin Witte1,2, Sylke Gellrich5, Nicholas Gulati4, Daniel Humme5,Ellen Witte1,2, Melanie Gonsior2, Marc Beyer5, Marshall E. Kadin6, Hans-Dieter Volk7,8, James G. Krueger4,Wolfram Sterry5, and Robert Sabat1,2,3

AbstractPurpose: Primary cutaneous T-cell lymphomas (CTCL) are neoplastic disorders of skin-homing

T cells. Affected skin areas show morphologic similarities with alterations in other T-cell–mediated

dermatoses. Furthermore, as in atopic dermatitis but in contrast with psoriasis, patients with CTCL are

frequently afflicted by cutaneous bacterial infections that support the survival of lymphoma cells.

Our aim was to investigate the mechanisms of elevated susceptibility to cutaneous infections in patients

with CTCL.

Experimental Design: Skin samples from CTCL, psoriasis, and atopic dermatitis patients were used to

illuminate the antibacterial competence status and the presence of its modulating cytokines. For substan-

tiation of findings, 3-dimensional epidermis models, isolated and in vitro generated Th-subpopulations,

were applied. Parameters were analyzed via qPCR and IHC.

Results: CTCL lesions compared with psoriatic lesions presented an impaired upregulation of antibac-

terial proteins (ABPs), with levels even below those in atopic dermatitis. This was associated with a relative

deficiency of the ABP-inducing cytokine IL-17 and a strong presence of the ABP-downregulating cytokine

IL-13. The simultaneous presence of the Th17-cell cytokine IL-26 indicated that IL-17 deficiency in CTCL

lesions results from functional deviation of Th17 cells. Accordingly, IL-17 but not IL-26 production by Th17

cells in vitro was inhibited by IL-4Ra ligand. Levels of other ABP inducers were comparable between CTCL

and psoriasis lesions. The same was true about IL-22/TNF-a targets, including the keratinocyte hyper-

regeneration marker K16 and the matrix-degrading enzyme MMP1.

Conclusion: Our results suggest that the cutaneous bacterial infections in CTCL are caused by

impaired ABP induction as consequence of Th2-mediated biased Th17-cell function. Clin Cancer Res;

20(21); 1–10. �2014 AACR.

IntroductionPrimary cutaneous T-cell lymphomas (CTCL) are neo-

plastic lymphoproliferative disorders that are primarilylocalized in the skin. Mycosis fungoides and S�ezary syn-drome are two common CTCL forms, accounting forapproximately 67% of cases. For the United States, theirincidence has been estimated to be approximately 3 to 8cases per million inhabitants per year (1–3). Affected indi-viduals are mostly older than 50 years. The clinical presen-tation of mycosis fungoides varies over time and includesearly patches, plaques with more pronounced infiltration,and the advanced tumor stage. In S�ezary syndrome, themalignant cells are also found in the peripheral blood andlymph nodes; this condition is much more aggressive andassociated with generalized pruritic erythroderma.

In its early stage, mycosis fungoides resembles lesionsof T-cell–mediated dermatoses such as psoriasis or chron-ic atopic dermatitis, in which mediators of mixed T-cell

1Psoriasis Research and Treatment Center, University Hospital Charit�e,Berlin, Germany. 2Interdisciplinary Group of Molecular Immunopathology,Dermatology/Medical Immunology, University Hospital Charit�e, Berlin,Germany. 3Research Center Immunosciences, University Hospital Charit�e,Berlin, Germany. 4Laboratory for Investigative Dermatology, RockefellerUniversity, New York, New York. 5Department of Dermatology and Allergy,University Hospital Charit�e, Berlin, Germany. 6Department of Dermatology,Boston University, Roger Williams Medical Center, Providence, RhodeIsland. 7Institute of Medical Immunology, University Hospital Charit�e,Berlin, Germany. 8Berlin-Brandenburg Center for Regenerative Therapies,University Hospital Charit�e, Berlin, Germany.

Current address for S. Gellrich: Medical practice for Dermatology,Baumschulenstr. 74, 12437 Berlin, Germany.

Corresponding Author: Robert Sabat, Psoriasis Research and Treat-ment Center; Interdisciplinary Group of Molecular Immunopathology,Dermatology/Medical Immunology; Research Center Immunosciences;University Hospital Charit�e, Charit�eplatz 1, D-10117 Berlin, Germany.Phone: 49-30-450-518625; Fax: 49-30-450-518964; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-0707

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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infiltrates, by altering skin cell biology, and cause changesof skin architecture, including acanthosis and hyperker-atosis (4, 5). Only in later stages, the mycosis fungoidesdiagnosis is facilitated by the profound intraepider-mal accumulations of lymphoma cells in the form ofso-called Pautrier’s microabsesses and by the occurrenceof atypical nuclei of the lymphoma cells (2, 3). Theetiology of mycosis fungoides/S�ezary syndrome remainsunknown. It has been hypothesized that mycosis fun-goides cells are derived from skin-infiltrated, chronicallyactivated Th cells (6, 7). In fact, they are generally CLAþ

a/b TCRþ, CD4þ, CD45ROþ, and CD26�/dim. However,the antigen(s) that are responsible for their primary stimu-lation has not been found, yet.

According to the altered skin architecture, already early-stage mycosis fungoides lesions show an impaired skinbarrier function. This, in turn, is associated with bacterialskin infections, for example, with Staphylococcus aureus(8–10). Those infections support survival and expansionof lymphoma cells (11). In line with this, antibiotictreatment has been shown to lead to clinical improvementof lesions (8–10). Moreover, if those local infectionsspread systemically in connection with the impaired sys-temic T-cell repertoire in S�ezary syndrome, they maybecome life threatening (12, 13). Importantly, animpaired skin barrier function is also found in patientswith psoriasis and atopic dermatitis (14, 15). However,only atopic dermatitis skin shows an increased incidenceof bacterial infections, which is mainly due to the limitedupregulation of antibacterial proteins (ABP) by the kera-tinocytes of these patients (16). In contrast, in psoriasis,the antimicrobial competence of the disturbed skin isincreased to a level that almost perfectly prevents cutane-ous infection (16, 17).

ABP expression in the skin is known to be upregu-lated by specific cytokines, including those producedpreferentially by monocytes/macrophages and keratino-

cytes (TNF-a and IL-1b) and by T cells (IL-17A, IFN-g ,and IL-22; refs. 18–22). Moreover, mediators of Th2 cellsincluding IL-4 and IL-13 reduce keratinocyte ABP levels(16, 23, 24). Previous studies demonstrated that therelative deficiency of the Th17-cell cytokine IL-17 andthe overproduction of Th2-cell cytokines are respon-sible for the limited cutaneous ABP production in atopicdermatitis.

In this study, we aimed to shed light on the mechanismsunderlying the increased bacterial infection risk of the skinin patients with mycosis fungoides.

Materials and MethodsPatients

For analysis of mRNA expression, skin samples wereobtained from control donors, psoriasis vulgaris patients(65.2% moderate to severe disease), patients with mycosisfungoides (5 with patch stage mycosis fungoides, 2 withplaque stagemycosis fungoides, 3 with tumor stagemycosisfungoides), and patients with atopic dermatitis (100%moderate to severe disease). Of the patients with mycosisfungoides (age: from 52 to 89 years, 80% male), 8 hadno therapy at the time of study, one got topical steroids andone had undergone PUVA therapy. For IHC analysis, skinbiopsies were obtained from patients with mycosis fun-goides, patients with psoriasis, and healthy volunteers(3 from each group). Samples were approved by the clini-cal institutional review boards of the Charit�e UniversityHospital (Berlin, Germany) or the Rockefeller University(New York, NY) and informed consent was obtained fromeach subject.

Cell cultureUnderdeveloped EpiDerm-201 reconstituted human

epidermis (RHE; MatTek Corp.) was cultured as describedpreviously (25) and stimulated or not (control) with acytokine mix containing recombinant human IL-22(1 ng/mL), IL-17A (1 ng/mL), IFN-g (0.1 ng/mL), IL-20(5 ng/mL), and TNF-a (0.1 ng/mL) or with the mixlacking one of the T-cell cytokines for 72 hours.

Peripheral blood mononuclear cells (PBMC) were sep-arated from citrated venous blood of healthy volunteersand patients with S�ezary syndrome by standard densitygradient centrifugation using LeucoSep tubes (Greiner)and Ficoll–Paque Plus (Amersham and Biochrom). Th1,Th2, and Th17 were isolated from PBMC by flow-cyto-metric sorting as described below and were stimulated viaCD3/CD28 for 2 days. Naive CD4þ T cells were isolatedfrom PBMC of healthy donors by the MACS system usingthe Naive CD4þ T-cell isolation kit II (Miltenyi Biotec;mean purity 95.8 �0.44%). Cells were stimulated for 8days with Dynabeads Human T-activator CD3/CD28beads (Invitrogen; 1 bead/T cell) in the presence of eitherIL-12 and anti-IL-4 mAb (Th1), IL-4 and anti-IFN-g mAb(Th2), IL-1b, IL-6, IL-23, TGF-b1.2, anti-IL-4 mAb, andanti-IFN-g mAb (Th17), IL-6, TNF-a, anti-IL-4 mAb, anti-IFN-g mAb, anti-TGF-b1.2 mAb, and 6-formylindolo[3,2-

Translational RelevanceCutaneous bacterial infections, cutaneous T-cell lym-

phoma (CTCL) patients frequently suffer from, supportlymphoma cell survival, and may be life-threateningwhen the T-cell receptor repertoire becomes limited. Inthe context of the increased skin infection risk, wedemonstrated a strongly limited upregulation of anti-bacterial proteins (ABP) in CTCL lesions, which wasassociated with specific deficiency of IL-17. The presenceof other ABP-inducing cytokines did not compensate theIL-17 absence, demonstrating that synergistic action ofseveral mediators is necessary for effective cutaneousantibacterial defense. IL-17 deficiency seems to becaused by specific inhibition of its production in Th17cells by IL-4Ra ligands. Our results suggest that thera-peutic IL-4Ra antagonizing (e.g., by blocking antibo-dies) may improve the prognosis of patients with CTCL.

Wolk et al.

Clin Cancer Res; 20(21) November 1, 2014 Clinical Cancer ResearchOF2




b] carbazole (Enzo Life Sciences; Th22), or IL-10, TGF-b1.2, anti-IL-4 mAb, anti-IFN-g mAb, and all-trans-reti-noid acid (Sigma-Aldrich; regulatory Th cells), as previ-ously described (26). In further experiments, Th17-celldifferentiation was carried out in the absence of IL-1b,IL-6, IL-23, or TGF-b or in the presence of 10 ng/mL IL-10.Finally, Th17 cells, generated during 8 days, were stim-ulated for another day with anti-CD3/anti-CD28 Ab-coated Dynabeads in the presence and absence of 10ng/mL IFN-g or 20 ng/mL IL-4. Th1, Th2, and Th17cellsisolated from PBMC of healthy donors by FACS cellsorting (see below) were left unstimulated or were stim-ulated with anti-CD3/anti-CD28 Ab-coated Dynabeads(Invitrogen; 1 bead/T cell) for 2 days.If not indicated otherwise, antibodies and recombinant

cytokines were purchased from R&D Systems.

Flow cytometryCell sorting was done starting with PBMC by means of

FACSAria. Fluorescence-labeled Ab clones against the fol-lowing molecules were used: CD4 (SK3; BD Biosciences),CXCR3 (49801; R&D Systems), CCR4 (255410; R&D Sys-tems), CCR6 (11A9; BD Biosciences), andCCR10 (314305;R&D Systems). Th cells were defined as CD4þ CXCR3þ

CCR6� cells (Th1), CD4þ CCR6� CCR4þ cells (Th2), andCD4þ CCR6þ CCR4þ CCR10� cells (Th17).To assess the purity of total and na€�ve CD4þ T-cell iso-

lations, cells were stained with fluorescence-labeled Abclones against the following molecules: CD3 (SK7), CD56(NCAM16.2), CD45RA (HI100), CD45RO (UCHL1; allfrom BD Bioscience), CD4 (13B8.2), CD8 (B9.11), CD14(RMO52), CD19 (J4.119), CD16 (3G8; all from BeckmannCoulter).

Quantitative gene expression analysisSnap-frozen skin biopsies and surgically excised skin

areas were homogenized during thawing in lysing solutionfrom the Invisorb RNA Kit II (Invitek/Stratec molecularGmbH) by means of the Omni labor homogenizer(S€udlabor). Isolation of total cellular RNA from homoge-nized cells and tissues was performed using Invisorb RNAKit II (Invitek/Stratec molecular GmbH). Reverse transcrip-tion of mRNA was performed as described previously (27).qPCR on reverse transcribed mRNA (qPCR) was performedin triplicate assays using universal master mix (AppliedBiosystems) or the Maxima Probe/ROX qPCR Master Mix(Thermo Fisher Scientific/Fermentas), sequence systemswith double-labeled fluorescent probes and comparableamplification efficiency, and either the Stepone plus or theABI Prism 7700 Sequence Detection System (both fromApplied Biosystems). Oligonucleotide sequences used forexpression analysis ofb-defensin2,b-defensin3, IL22, IL26,and matching hypoxanthine phosphoribosyl-transferase1 (HPRT) were published previously (18, 28). All otherdetection systems were purchased from Applied Biosys-tems. Expression levels were calculated relative to those ofHPRT, which was analyzed in parallel with the matchinganalysis system.

IHCIHC analysis was performed on frozen sections of

human mycosis fungoides, psoriasis vulgaris, and normalskin samples (n¼ 3 for each). Antibodies used in this studywere anti-DEFB2 (Perpro Tech, goat polyclonal, diluted1/20), anti-Lipocalin 2 (Abcam,mousemonoclonal, cloneHYB211-01, diluted 1/100), and anti-MMP1 (Abcam,mouse monoclonal, clone SB12e, diluted 1/100). Sectionswere incubated with the primary antibody overnight at4�C. The staining signal was amplified using avidin–biotincomplex (Vector Laboratories) for 30 minutes at roomtemperature and developed using chromogen 3-amino-9-ethylcarbazole. Themicroscope usedwas aNikonEclipse50i with a Nikon DS-Fi1 camera.

Statistical analysisData are presented as the mean � SEM. For further

analyses, SPSS 14.0 software (SPSS Inc.) was used. Com-parison of results between mycosis fungoides patients andcontrol patients/participants were analyzed using theMann–Whitney U test (two tailed). Correlations were ana-lyzed on the basis of the Spearman correlation coefficient.Results on paired in vitro treated cell cultures were testedusing the Wilcoxon matched-pairs signed-rank test (twotailed).

ResultsCTCL skin lesions show a relative deficiency in ABPproduction

Similar to patients suffering from atopic dermatitis butin contrast with patients with psoriasis, patients withCTCL have an increased susceptibility to cutaneous bac-terial infections. We hypothesized that, like in patientswith atopic dermatitis, an attenuated increase of ABPsmay be responsible. To investigate this hypothesis,mRNA expression of different ABPs including lipocalin2, b-defensins, RNase 7, LL37, and S100 proteins wasquantified by qPCR in skin lesions of patients withmycosis fungoides (carrying patch stage, plaque stage,and tumor stage lesions). For comparison, analyses ofhealthy skin from control donors and lesional skin frompatients suffering from psoriasis and from atopic derma-titis were included. As demonstrated in Fig. 1A, theexpression of most ABPs was upregulated in the patientgroups compared with healthy skin. However, as it is thecase for atopic dermatitis, the upregulation in CTCL wasstrongly limited, with significantly lower levels of allABPs in CTCL compared with those observed in psoriaticskin. A clear difference between CTCL and psoriasiswas also found upon IHC analyses of skin sectionsperformed for lipocalin-2 and b-defensin 2 (Fig. 1B). Incase of b-defensin 2 and 3, LL37, S100A7, and S100A9,the expression in CTCL was even lower than that inatopic dermatitis lesions (Fig. 1A). The relative deficiencyin ABP expression may therefore account for theincreased susceptibility to bacterial infections in patientswith CTCL.

Cutaneous Antimicrobial Defense and Th17-Cell Function in CTCL

www.aacrjournals.org Clin Cancer Res; 20(21) November 1, 2014 OF3




CTCL lesions show reduced upregulation of IL-17Aexpression and high levels of IL-13

We then analyzed the levels of cytokines knownto induce (TNF-a, IL-1b, IL-17A, IFN-g , IL-22) or down-regulate (IL-13) epidermal ABP expression. As demon-strated by qPCR data, most of the analyzed cytokineswere expressed at similar levels in CTCL comparedwith psoriatic lesions (Fig. 2A). However, two were not:IL-17A was found having a significantly lower expres-sion, whereas IL-13 expression was higher comparedwith psoriasis. Hence, the cytokine expression patternin CTCL highly resembled that found in atopic derma-titis lesions (Fig. 2A).

The limited cutaneous IL-17A production may accountfor the low keratinocyte ABP levels in CTCL

We then asked how the differential cytokine expressionpatterns in CTCL compared with psoriasis lesions arerelated to the limited ABP expression. We first investigat-ed the statistical relationship between lesional cytokineand ABP expression in both patient groups. As demon-strated by Table 1, the positive statistical correlationbetween IL-17A and most of the analyzed ABP expres-sions indeed supported the idea of a causal relationshipof deficient upregulation of IL-17A expression and lowABP expression in CTCL versus psoriasis. No correlationwas found between IL-13 and ABP expression (Table 1).

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Figure 1. CTCL skin lesions show adeficient induction of ABPexpression. A, biopsies of healthyskin from control participants(n ¼ 9) and of skin lesions frompatients with psoriasis (n ¼ 13),CTCL (n¼ 9), and atopic dermatitis(n ¼ 10) were analyzed for ABPmRNA expression by qPCR.Data are given as mean � SEMrelative to HPRT expression.Significances compared withthe CTCL group are indicated.�, P < 0.05; ��, P < 0.01;��, P � 0.001. B, biopsies ofhealthy skin from controlparticipants (n ¼ 3) and of skinlesions from patients with CTCL(n ¼ 3) and psoriasis (n ¼ 3) wereanalyzed for ABP expression byIHC. Representative pictures(magnification: 200-fold) arepresented.

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We additionally performed in vitro studies with 3-dimen-sional epidermis models composed of stratified humankeratinocytes. Using a mix of different cytokines imitatingthe inflammatory condition in psoriasis (includingIL-17A, IL-22, IFN-g , TNF-a, IL-20), strongly induced ABPexpressions were found after 3 days of stimulation (Fig.2B, here shown for lipocalin-2). In parallel, the mix withone of the T-cell cytokines omitted at each time wastested. Omitting IL-17A had the clearest limiting effecton the induction of lipocalin 2. These data indicate thatIL-17A deficiency limits the cutaneous upregulation ofABPs even in the presence of other ABP inducers anddemonstrate the importance of this observation for thecutaneous situation in CTCL.

IL-22/TNF-a-dependent epidermal functions aresimilarly altered in CTLC and psoriasis

Next, we asked whether, apart from the antimicrobialcompetence, CTCL differs from psoriasis regarding furtherkeratinocyte alterations. Using qPCR, we analyzed respec-tive skin samples for a marker of the keratinocyte hyper-proliferation/regeneration (K16) as well as for the matrix-degrading enzyme MMP1. Both parameters are essentiallyregulated by IL-22, especially in combination with TNF-a,but not by IL-17A (29, 30). In line with the comparableexpression of IL-22 and TNFa in psoriasis versus CTCL (Fig.2A), the expression of K16 and MMP1 was similar in thesediseases (Fig. 3A). Comparable epidermal MMP1 levelswere also confirmed by IHC analysis (Fig. 3B).

Figure 2. The lesional expression ofselected ABP-regulating cytokinesdiffers between patients with CTCLand psoriasis patients. A, biopsiesof healthy skin from controlparticipants (n ¼ 9) and of skinlesions from patients with psoriasis(n ¼ 13), CTCL (n ¼ 9), and atopicdermatitis (n ¼ 10) were analyzedfor cytokine mRNA expression byqPCR. Data are given as mean �SEM relative to HPRT expression.Significances compared with theCTCL group are indicated. �, P <0.05; ��, P < 0.01; ��, P� 0.001. B,epidermis models were stimulatedor not (control) for 72 hours with acytokine mix described in theMaterial and Methods section orwith the same mix but lacking oneof the T-cell cytokines as indicated.Lipocalin-2 mRNA expression wasanalyzed relative to HPRTexpression by qPCR, and data fromthree experiments are given asmean � SEM as the percentage ofthe nonstimulated group.






CTCL lesions do not demonstrate a general Th17cytokine deficiency

IL-17A is mainly produced by Th17 cells. Apart fromIL-17A, these cells have been described to produce IL-17Fand IL-26. Analyzing cytokine expressions by qPCRrevealed, similar to IL-17A, only low IL-17F expressionin CTCL lesions, with mean levels being approximately 50times lower than in psoriasis lesions (Fig. 4A). Surpris-ingly however, IL-26 expression was much higher inCTCL versus healthy skin and did not differ betweenpsoriasis and CTCL.

To rule out that Th cells other than of the Th17 lineageare main IL-26 producers, we investigated Th-cell popula-tions that were either isolated from the peripheral bloodby flow-cytometric sorting based on their chemokineexpression patterns or were generated from naive T cells

in vitro (see Materials and Methods section). As demon-strated by qPCR data shown in Fig. 4B and C, activatedTh17 cells (stimulated via CD3 and CD28) were indeedthe strongest producers not only of IL-17F, but also of IL-26. These data suggest that the production of individualcytokines is differently regulated in Th17 cells, explainingthe selective relative IL-17A/F deficiency in CTCL.

We therefore looked for conditions that may cause adifferential regulation of IL-17 versus IL-26 productionby Th17 cells. The generation and maintenance of Th17cells have been described to depend on IL-1b, IL-6, IL-23,and TGF-b. When omitting one of these cytokines duringthe in vitro Th17-cell generation period, we found thatIL-17F expression was highly dependent on the presenceof TGF-b, whereas IL-26 was not (Fig. 5A). We thereforeanalyzed TGF-b expression in the patients’ skin using

Table 1. IL-17 is an important regulator of the keratinocyte ABP expression

TNF-a IL-1b IL-13 IFN-g IL-17A IL-22

LCN2 0.14 (0.548) 0.63 (0.004) �0.40 (0.101) 0.09 (0.699) 0.81 (0.000) 0.39 (0.087)b-defensin 2 �0.30 (0.915) 0.57 (0.011) �0.43 (0.078) 0.18 (0.454) 0.95 (0.000) 0.33 (0.161)b-defensin 3 0.15 (0.547) 0.67 (0.002) �0.45 (0.056) 0.13 (0.578) 0.86 (0.000) 0.28 (0.241)RNase 7 0.31 (0.201) 0.35 (0.168) �0.10 (0.721) �0.12 (0.622) 0.41 (0.086) 0.38 (0.104)LL37 �0.10 (0.679) 0.76 (0.000) �0.21 (0.399) �0.12 (0.600) 0.50 (0.028) 0.18 (0.455)S100A7 0.04 (0.881) 0.25 (0.297) �0.16 (0.521) 0.02 (0.920) 0.72 (0.000) 0.44 (0.058)S100A8 0.04 (0.849) 0.28 (0.247) �0.21 (0.399) 0.25 (0.271) 0.81 (0.000) 0.33 (0.149)S100A9 �0.06 (0.811) 0.30 (0.223) �0.15 (0.554) �0.03 (0.884) 0.68 (0.001) 0.42 (0.062)

NOTE: Messenger RNA expression data were statistically correlated with ABP mRNA expression data obtained by qPCR analysis oflesional skin samples from CTCL (n ¼ 9) and psoriasis (n ¼ 13) patients. Correlation coefficients rs and, in parentheses, P values ascalculated by means of the Spearman correlation test are indicated and, in case of significant relationships, are italicized.

Figure 3. The expression ofparameters known to be regulatedby IL-22/TNF-a are similarlyregulated in CTLC and psoriasis.A, biopsies of healthy skin fromcontrol participants (n ¼ 9) and ofskin lesions from patients withpsoriasis (n¼ 13) and CTCL (n¼ 9)were analyzed for K16 and MMP1mRNA expression by qPCR. Dataare given as mean � SEM relativeto HPRT expression. Significancescompared with the CTCL groupare indicated. ��, P � 0.01. B,biopsies of healthy skin fromcontrol participants (n ¼ 3) and ofskin lesions from patients withCTCL (n ¼ 3) and psoriasis (n ¼ 3)were analyzed for MMP1expression by IHC. Representativepictures (magnification: 200-fold)are presented.

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qPCR. However, no TGF-b deficiency was found in CTCLlesions compared with psoriatic lesions that wouldexplain the differential expression of IL-17 versus IL-26in these samples (Fig. 5B). Another candidate of a reg-ulator of differential Th17-cytokine expressions in CTCLis IL-10, whose expression was more highly expressed inCTCL compared with psoriatic skin (Fig. 5C). However,when performing Th17-cell differentiation, the presenceof IL-10 had no influence on the IL-17F or IL-26 expres-sion of these cells (Fig. 5D). Further mediators, whoseexpression highly differs between CTCL versus psoriaticlesions, are IL-13 (Fig. 2A) and IL-4 (31, 32). Bothcytokines share the IL-4Ra subunit and have been shownto inhibit the IL-17A production by Th17 cells (26, 33).When exposing in vitro generated human Th17 cells toIL-4 and, as comparison, with IFN-g , IL-4 indeed inhib-ited IL-17 expression but had no influence on IL-26expression of these cells (Fig. 5E). The differential pres-ence of IL-4Ra ligands in CTCL versus psoriasis skin maytherefore account for the differential expression amongTh17-cell cytokines in these patients.

Discussion

The skin forms the major outer barrier of organismsagainst infections (15, 34). Among the different mechan-isms to achieve this protection, the production of ABPs suchas b-defensins, RNase-7, S100 proteins, lipocalin-2, andcathelicidin/LL37 by keratinocytes is of high importance(17, 35). ABPs directly inhibit the growth of microbes(17, 35). b-defensins, for example, are very small, highlypositively charged proteins that kill microbes by destabiliz-ing their membranes. S100A7 acts through zinc sequestra-tion and also through permeabilization of the bacterialmembrane. SomeABPs are constitutively produced, thoughhigh ABP levels are attained by the presence of cytokines(18–22). Theupregulation of cutaneousABP levels seems tobe especially important in situations with disturbed skinbarrier function. This is the case not only after wounding,but also in chronic-inflammatory skin diseases. In psoriasis,the cutaneous ABP production is increased to a dimensionthat effectively prevents cutaneous infection (16, 17). Thisseems to be achieved by the synergistic action of different

Figure 4. The cutaneous expressionof IL-26, another Th17-cell cytokine,is not deficient in patients withCTCL. A, biopsies of healthy skinfrom control participants (n¼ 9) andof skin lesions from patients withpsoriasis (n ¼ 13) and CTCL (n ¼ 9)were analyzed for cytokine mRNAexpression by qPCR. Data aregiven as mean � SEM relative toHPRT expression. Significancescompared with the CTCL group areindicated. ��, P � 0.001. B, CD4þ

CXCR3þ CCR6� cells (Th1), CD4þ

CCR6� CCR4þ cells (Th2), andCD4þ CCR6þ CCR4þCCR10� cells(Th17), isolated from peripheralblood, were stimulated via CD3/CD28 for 2 days. Cytokineexpression was analyzed by qPCR.Mean data�SEM from 3 donors aregiven as percentage of maximumexpression. C, naïve CD4þ T cellswere stimulated for 8 days via CD3and CD28 in the presence of Th-subset-polarizing conditions (seeMaterials and Methods section) togenerate Th1, Th2, Th17, Treg, andTr1-cells. Cytokine expression wasanalyzed by qPCR. Mean data�SEM of five experiments are givenas percentage of Th17-cellexpression. Significancescompared with the Th17 cells areindicated. �, P < 0.05.






mediators of the local cytokinemilieu, including the Th-cellcytokines IL-17 and IL-22 and also proinflammatory cyto-kines derived from activated dendritic cells/macrophagesand tissue cells (e.g., TNF-a, IL-24, IL-20; ref. 36). Incontrast, relative IL-17 deficiency and overproduction ofABP-downregulating Th2 cytokines do not allow effectiveABP induction in atopic dermatitis (16, 23, 24, 37),whereasdeficient production of IL-22 and its downstreammediatorIL-20 prevents sufficient ABP upregulation in Hidradenitissuppurativa (acne inversa; ref. 36). In addition to atopicdermatitis and Hidradenitis suppurativa lesions, CTCLlesions are known for their increased susceptibility towardbacterial infections.

Inour current study,wedemonstrate that, comparedwithpsoriatic lesions, upregulation of ABP production in CTCLlesions was highly impaired. Low levels of IL-17A andIL-17F in CTCL lesions may be responsible for this defi-ciency. Apart from our observation of very low IL-17A and

IL-17F expression in CTCL lesions, which is in line withprevious reports (31, 38, 39), we demonstrate that theseexpressions are significantly lower compared with those inpsoriatic skin, and that IL-17 absence had a strong negativeimpact of the ABP induction evenwhen other inducers werepresent. The presence of IL-4Ra ligands (IL-13 and IL-4)might further contribute to the relative ABP deficiency inthese lesions. Apart from their known direct ABP-down-regulating capacity, they seem to limit Th17-cell IL-17production. The situation in CTCL lesions therefore resem-bles that in lesional skin of patients with atopic dermatitis.Importantly, the lacking significant upregulation of IL-17Aand IL-17F in CTCL skin seems to be caused by the inhi-bition of their production rather than the absence of Th17cells. This was indicated by high expression of IL-26, anoth-er Th17-cell cytokine, in CTCL lesions. Importantly, incontrast with IL-17A/F, IL-26 production by Th17 cells wasnot affected by IL-4Ra ligands.

Figure 5. Possible mechanisms underlying the cutaneous deficiency in the upregulation of IL-17 in patients with CTCL. A, Th17 cells were generated asdescribed in Fig. 4C or with one of the polarizing cytokines omitted as indicated. Cytokine mRNA expressions were analyzed by qPCR. Mean data�SEM of five experiments are given as percentage of the Th17 full polarization group. Significances compared with the Th17 full polarizationgroup are indicated. �, P < 0.05. B, biopsies of skin lesions from patients with psoriasis (n ¼ 13) and CTCL (n ¼ 9) were analyzed for TGF-b mRNAexpression by qPCR. Data are given as mean � SEM relative to HPRT expression. No significance was detected between both patient groups.C, biopsies of skin lesions from patients with psoriasis (n ¼ 13) and CTCL (n ¼ 9) were analyzed for IL-10 mRNA expression by qPCR. Data are given asthe mean � SEM relative to HPRT expression. Significance compared with the CTCL group is indicated. �, P < 0.05. D, Th17 cells weregenerated as described in Fig. 4C in the presence or absence of IL-10. Cytokine mRNA expressions were analyzed by qPCR. Mean data �SEM offive experiments are given as percentage of the Th17 group without IL-10. E, Th17 cells generated as described in Fig. 4C were treated or not(control) with IFN-g or IL-4 from day 8 to day 9. Cytokine mRNA expression was analyzed by qPCR. Mean data �SEM of three experiments are givenas percentage of the control group.

Wolk et al.





Bacterial infections may be pathogenetically importantfor CTCL as they may initiate and/or trigger the diseasecourse. It has been suggested that malignant T-cell trans-formation arises upon persistent stimulation of T cells (7)and that even transformed cells do not necessarily showhigh autonomous proliferation but require external stim-ulation (40, 41). This is in line with observed increasingmonoclonality of T-cell populations arising with progres-sion of CTCL only. A role of bacteria in these processes isalso in line with the clinical improvement of lesions due toantibiotic treatment of patients (8, 9). Premalignant/malig-nant cell stimulationbybacteriamayoccur either directly bybacterial superantigens (9, 10) or indirectly via inductionof cytokines, in macrophages and dendritic cells (42),nonmalignant T cells, or tissue cells (43–46), which serveas growth factors for CTCL cells. Furthermore, followingpresentation viaMHCclass II bymacrophages anddendriticcells, microbial antigens matching the specificity of thetransformed T cells might play an important role in theactivationof respectiveCTCL cells, as recently demonstratedfor a subset of human B-cell lymphomas (47). In addition,in the final stages of CTCL, when the T-cell repertoire inthe body becomes restricted to that of the tumor cells,local infections may be origin of fatal systemic infections(12, 13).Besides IL-17A/F, CTCL lesions show a cocktail of other

different T-cell cytokines. It will be interesting to determinefor each of these cytokineswhether it ismainly derived fromthe lymphoma cells themselves or from infiltrating reactiveimmune cells especially present in the dermis of the CTCLlesions (48, 49). These cytokines may be responsible forspecific epidermal alterations observed in CTCL lesions. Infact, the lesions often show tissue cell reactions, which arealso known from nonmalign chronic-inflammatory skindiseases. In this study, we additionally demonstrate a pso-riasis/atopic dermatitis-like increased expression of thehyper-regeneration marker K16 and the matrix-degrading

enzymeMMP1 inCTCL lesions. The fact that the expressionof both parameters is particularly regulated by IL-22/TNF-a(50) might explain the similar levels of these parameters inthe three investigated diseases. Although K16 is a parameterhistologically associated with acanthosis, MMP1 may beimportant for the infiltration of lymphoma and normalimmune cells into the epidermis.

In summary, we demonstrated a relative deficiency in theantibacterial defense of CTCL lesions that might be due tothe inhibition of Th17-cell IL-17A/F production caused byhigh abundance of IL-4Ra ligands.

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

Authors' ContributionsConception and design: R. Sabat, J.G. KruegerDevelopment of methodology: K. Wolk, K. Witte, J.G. Krueger, R. SabatAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.):H.Mitsui, K.Witte, S. Gellrich, N. Gulati, E.Witte,M. Beyer, W. SterryAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): K. Wolk, J.G. Krueger, W. Sterry, R. SabatWriting, review, and/or revision of the manuscript: K. Wolk, H. Mitsui,S. Gellrich, D. Humme, M. Gonsior, M. Beyer, M.E. Kadin, H.-D. Volk,J.G. Krueger, R. SabatAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Mitsui, W. Sterry, R. SabatStudy supervision: K. Wolk, H.-D. Volk, W. Sterry, R. SabatOther (editing and discussion of the manuscript): E. WitteOther (partial funding of research): J.G. Krueger

AcknowledgmentsThe authors thank Annette Buss and Brigitte Ketel for steady and excellent

technical assistance.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMarch 24, 2014; revised July 25, 2014; accepted August 18, 2014;published OnlineFirst September 11, 2014.

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Wolk et al.




Published OnlineFirst September 11, 2014.Clin Cancer Res Kerstin Wolk, Hiroshi Mitsui, Katrin Witte, et al. FunctionT-Cell Lymphomas: Role of Th2-Mediated Biased Th17 Deficient Cutaneous Antibacterial Competence in Cutaneous

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