fas ligand expression by neoplastic t lymphocytes mediates elimination of cd8+ cytotoxic t...

Fas Ligand Expression by Neoplastic T Lymphocytes MediatesElimination of CD8� Cytotoxic T Lymphocytes in MycosisFungoides: A Potential Mechanism of TumorImmune Escape?1

Xiao Ni, Parul Hazarika, Chunlei Zhang,Rakhashandra Talpur, and Madeleine Duvic2

Department of Dermatology, University of Texas, M. D. AndersonCancer Center, Houston, Texas

ABSTRACTMycosis fungoides (MF) is the most common form of

cutaneous T-cell lymphoma (CTCL) and arises from theaccumulation and clonal proliferation of epidermotropic,CD4�/CD45RO� (helper/memory) T lymphocytes. Loss ofCD8� CTLs within MF lesions is associated with poorprognosis and disease progression. Because T-lymphocyteapoptosis is controlled mainly through the Fas/Fas ligand(FasL) pathway and tumor cells may escape immune sur-veillance by expressing FasL, triggering apoptosis of tumor-infiltrating T lymphocytes, we studied the role of this systemin MF. T-cell subsets, Fas/FasL expression, and apoptosiswere evaluated in normal and lesional skin biopsy specimensfrom 21 patients with all stages of MF and in cultured CTCLcell lines (MJ, HUT78, and HH) using immunohistochemis-try, terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL), and Western blotting. MF le-sions and paired, clinically “normal,” uninvolved skinshowed increased numbers of both TUNEL-positive epider-mal keratinocytes (n � 13; F � 31.146; P < 0.01, ANOVA)and dermal lymphocyte infiltrates (n � 13; F � 15.825, P <0.01, ANOVA) compared with the normal control skin. FasLexpression was highest in lesional epidermal keratinocytes,in CTCL tumor cell lines, and in dermal tumor lymphocytesin MF lesions compared with uninvolved skin. FasL colocal-ized with CD45RO� cells. CD8� cells in or adjacent toCD45RO� cells were positively labeled by TUNEL for ap-

optosis. In addition, CD8� cell numbers were decreased inareas in which FasL� tumor cells were abundant (2.01 �0.86%) compared with non-FasL expressing areas (13.53 �3.54%; P < 0.02). These results suggest that a potentialmechanism of tumor immune escape in MF is FasL-medi-ated apoptosis of infiltrating CD8� CTLs.

INTRODUCTIONCTCLs3 are a heterogeneous group of peripheral, extra-

nodal, non-Hodgkin’s lymphomas that arise in skin-hominglymphocytes and may progress by disseminating to lymph nodesand viscera. MF, the most indolent form of CTCL, and itsleukemic variant, the SS, are the most common CTCLs, repre-senting a disease spectrum. MF and SS originate from a clonalexpansion of epidermotropic, CD4�/CD45RO� helper/mem-ory T cells (1, 2). Between 1974 and 1984, the incidence of MFincreased by 3.2-fold, and the proportion of MF cases among alllymphomas increased from 1.6 to 2.8% (2, 3).

The stage and prognosis of MF has been defined by theskin or T-stage as reported by Bunn and Lamberg in 1979 (4).Early-stage patients (T1–2) with patches or plaques on less than10% of their skin have an excellent overall survival and prog-nosis, when they respond to skin-directed therapy (5, 6). Withthe exception of electron beam used in early patients, no cura-tive therapies exist, and progressive disease can be associatedwith morbidity, disfigurement, and death from sepsis (5–7).

One hypothesis to explain the indolent nature of MF sug-gests that clonal T cells arise and accumulate in response tochronic antigen stimulation (8). It has been suggested that an-tigens may differ from one patient to another and could beprovided by persistent infectious agents (9–11). Also in supportof chronic antigen-driven T-lymphocyte proliferation is thefinding of significant HLA Class II associations, including HLA-DRBw11 and DQB*03 with MF and DQB*502 with SS (12).Regardless of the initial cause, with disease progression, accu-mulation of malignant T cells is followed by loss of normalT-cell immunity leading to an AIDS often exacerbated by treat-ment and to death from opportunistic infections (13).

Hoppe et al. (14) reported that T1,2 MF patients tend to

Received 5/8/01; revised 6/19/01; accepted 6/21/01.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 toindicate this fact.1 Supported in part by grants from National Institute of Arthritis, Mus-culoskeletal, and Skin Diseases (NIAMS; R21-CA74117), core cancergrant to M. D. Anderson Cancer Center (CA 16672-22), clinical trialand research support from Ligand Pharmaceuticals, Inc, and CutaneousT-Cell Lymphoma Patient Research and Education Fund. This paperwas presented in part at the Society for Investigative DermatologyMeeting, May 8–11, 2001, Washington, DC.2 To whom requests for reprints should addressed, at Department ofDermatology, M. D. Anderson Cancer Center, Box 434, 1515 HolcombeBoulevard, Houston, TX 77030. Phone: (713) 745-1113; Fax: (713)745-3597; E-mail address: [email protected].

3 The abbreviations used are: CTCL, cutaneous T-cell lymphoma; FasL,Fas ligand; L0, MF skin lesion at baseline; MF, mycosis fungoides; N0,clinically uninvolved “normal” skin from MF patient at baseline; NS,normal skin from normal control subjects; TIL, tumor-infiltrating lym-phocyte; Tdt, terminal deoxynucleotidyl transferase; TUNEL, Tdt-mediated dUTP nick end labeling; SS, Sezary syndrome; IL, interleukin;DAB, diaminobenzidine; AI, apoptosis index; TBS, Tris-buffered sa-line; HRP, horseradish peroxidase.

2682 Vol. 7, 2682–2692, September 2001 Clinical Cancer Research

have a higher proportion of CD8� cells in their skin biopsyspecimens, compared with patients with more advanced disease.Heald et al. (15, 16) also found that the loss of CD8� cells,tumor-infiltrating CTLs in MF lesions and in the peripheralblood of SS patients is associated with poor prognosis and aninability to respond to photopheresis therapy. Within each Tstage, patients with a larger proportion of CD8� cells had abetter survival rate than did patients with fewer numbers ofCD8� cells. Defects in cell-mediated immunity are reported toarise during disease progression (17, 18). The excess productionof T-helper type 2 cytokines (IL-4, 5, 6, and 10) by malignant Tcells can antagonize production of T-helper type I cytokines(IFN-� and IL-2) and interfere with cell-mediated immunity(19). However, the mechanism by which CD8� CTLs are lostwith the progression of MF has not been fully elucidated at themolecular level.

Classical mechanisms to account for tumor evasion ofthe immune system include defective antigen presentation,interference with tumor/T-cell interaction, and production ofimmunosuppressive factors (20). New evidence points toexpression of FasL as a possible mediator of tumor immuneprivilege (20). FasL (also called CD95L/APO-1 L), is atype-II integral membrane protein member of the tumor ne-crosis factor family that transduces an apoptotic death signalby binding to its receptor Fas (CD95). Fas is a type Itransmembrane protein of the tumor necrosis factor receptorfamily. The Fas/FasL system is implicated in several aspectsof immune regulation and development. It modulates T-cellselection and clonal deletion of activated T cells by thethymus, regulation of activated B-cell-specific killing ofvirally infected or transformed cells, and establishmentof “immune-privileged” sites such as testis, brain, and eyes(20, 21).

Recent data suggest that de novo expression of FasL intumors might result in the elimination of Fas� antitumor lym-phocytes, referred to as Fas counterattack, by which tumors mayfoil the immune system and become immune privileged sites.Both hematological malignancies (22) and solid tumors, such asmelanomas, have been found to induce apoptotic cell death ofTILs through Fas and FasL interactions (23). In human colo-rectal carcinomas, apoptosis in TILs was more frequent intumors that expressed FasL than in FasL-negative tumors (24).FasL expression in esophageal cancer was associated withapoptosis and depletion of TILs in vivo (25). These findingssuggest that the Fas/FasL system is an important mechanism bywhich tumors avoid attack by CTLs.

FasL-expressing tumor cells have been reported in T-cell-derived malignancies including natural killer cell lymphoma,large granular lymphocytic (LGL) lymphoma (26) and T-cellacute lymphocytic leukemia (T-ALL; Ref. 27). FasL expressionhas been studied in MF, but the results were not conclusive(28–30). This study shows that the depletion of specific CD8�CTLs by apoptosis within MF lesions is associated with FasLexpression in tumor cells. FasL expression by MF tumor cellsmight lead to the loss of CD8� CTLs, which clinically isassociated with poor prognosis. These results suggest strategiesfor monitoring therapy in the future.

MATERIALS AND METHODSTissue Specimens. Twenty-one patients with either

newly diagnosed MF or with established MF, who were offtherapy because they were being enrolled in clinical researchtrials, signed informed consent for the use of tissue for research.All clinical research was approved by the M. D. AndersonCancer Center Institutional Review Board and was conductedaccording to good clinical practice guidelines. Biopsy speci-mens were taken under local anesthesia from clinically unin-volved skin (N0) and from representative untreated skin lesion(L0) using 6-mm punch biopsies. Skin tumor (T) staging of allof the patients at baseline was conducted according to therecommendations of the European Organization for Researchand Treatment of Cancer classification for primary cutaneouslymphoma (31). Normal control skins (NS; n � 8) were ob-tained from adult breast surgeries.

Cell Lines. CTCL cell lines HH, MJ (MF), HUT78 (SS;Ref. 32) and Jurkat cell line were purchased from the AmericanType Culture Collection (Manassas, VA). Cells were grown inHEPES-buffered RPMI 1640 with 2 mmol/liter glutamine, sup-plemented with 10% fetal bovine serum, 0.25 mg/ml amphoter-icin B, 100 units penicillin G, 100 units streptomycin, and 1mmol/liter sodium pyruvate. Cytospin preparations of naıveCTCL cells lines were air-dried and fixed for 10 min with coldacetone prior to immunocytotochemistry staining.

TUNEL. After proteinase K treatment at 20 �g/ml for 30min, endogenous peroxidase activity was blocked with 0.3%hydrogen peroxide in methanol for 60 min. Sections weretreated with permeabilization solution (0.1% Triton X-100 in0.1% sodium citrate) for 2 min on ice. The labeling reaction wasperformed in buffer with TdT and fluorescein-dUTP accordingto the instructions (In situ cell death detection kit-POD; Roche,Mannheim, Germany). Slides were coverslipped and incubatedat 37°C for 60 min in a humidity chamber before incubationwith sheep antifluorescein antibody Fab fragment conjugatedwith HRP at 37°C for 30 min. TUNEL-positive color develop-ment (brown) was obtained by incubating the sections withDAB substrate solution for 15 min. DNase I-treated specimens(1.0 �g/ml for 10 min at room temperature) served as positivecontrols, and TdT was omitted from negative control slides.

Immunohistochemical Detection of FasL in SkinLesions and Cytospin Preparations. Six to eight 5-�m con-secutive sections from each specimen were stained using im-munohistochemistry methods described by Mullauer et al. (33)with slight modifications. After deparaffinization and rehydra-tion, sections were microwaved in 0.01 M citrate buffer for 10min at high power for antigen retrieval. Endogenous peroxidasewas quenched with 0.3% hydrogen peroxide in methanol for 30min. After blocking with 1% normal horse serum-0.1 M PBS(pH 7.2) for 30 min, sections were incubated at 37°C for 60 min,and then overnight at 4°C with 1:100 dilution of mouse mono-clonal antibody (clone 33). The antibody was raised against apeptide comprising the COOH-terminal amino acid 116–277 ofhuman FasL (Transduction Laboratories, Lexington, KY) andused in 1% normal horse serum-0.1 M PBS (pH 7.2). Sectionswere rinsed in 0.1 M PBS (pH 7.2), and bound antibody wasdetected with a three-step biotin-avidin method (ABC-Elite kit;Vector Laboratories, Burlingame, CA). DAB was used as a

2683Clinical Cancer Research

chromogen substrate. Tissues were counterstained with hema-toxylin. The lymph nodes from a patient with B-cell lymphomawas included as a positive control. Omission of the primaryantibody was the negative control. A rabbit polyclonal antihu-man FasL IgG (NH2-terminal amino acid, 2–19; Santa CruzBiotechnology, Santa Cruz, CA) and immune peptide were usedto confirm the results. Staining of cytospins was by the sameprotocol without antigen retrieval, using 0.1% Saponin (w/v%)-0.1 M PBS (pH 7.2).

Colocalization of FasL and CD45RO or CD8/FasL byDoublestaining. CD45RO or CD8 staining was performedfirst and was followed by FasL doublestaining to identifyFasL� T-cell subsets within MF lesions. Staining was per-formed at room temperature according to the manufacturer’sinstructions (Dako Envision Doublestain System; Dako, Carpin-teria, CA) with minor modifications. Briefly, after microwaveantigen retrieval and endogenous peroxidase blocking, mouseantihuman CD45RO monoclonal IgG (1:20; UCHL; Dako A/S,Denmark) or mouse antihuman CD8 monoclonal IgG (1:20;clone C8/144B; Dako, A/S) or negative control reagent wasadded to the slides and incubated for 30 min. The slides werenext incubated with labeled polymer-HRP for 30 min and thenwith DAB substrate for 10 min. Sections were blocked for 3 minand then incubated with mouse monoclonal antihuman FasL-specific IgG1 at 1:100 in blocking buffer for 30 min and withpolymer-alkaline phosphatase for 30 min. FasL-positive colorwas detected after application of alkaline phosphatase substratesolution (Vector Blue; Vector Laboratories) for 10 min. Whenviewed under light microscopy, FasL single-positive cellsstained blue. CD45RO or CD8/FasL dual-positive cells ap-peared with blue cytoplasmic/cell surface and brown cell-surface staining.

Detection of TUNEL Staining in CD45RO or CD8� TCells by Doublestaining. CD45RO or CD8 staining was per-formed first on MF sections followed by TUNEL detection toidentify cells undergoing apoptosis. Briefly, doublestain-block-ing solution (Dako Envision Doublestain system) was appliedfor 3 min, followed by primary antibody (anti-CD45RO oranti-CD8) as above for 30 min, and labeled polymer-AP for 30min (Dako Envision Doublestain system). Sections were thenincubated for 10 min with Vector Blue substrate solution.TUNEL was performed in the same sections as above, but with3-amino-9-ethylcarbazole (AEC) color development. Whenviewed under light microscopy, CD45RO or CD8 single-posi-tive cells stained blue, and CD45RO or CD8/TUNEL dual-positive cells showed red nucleus staining and blue cytoplasmic/cell surface staining.

Semiquantitative Analysis of ImmmunohistochemistryStaining and Cell Number. To quantify TUNEL-positivecells, FasL staining, and CD45RO� or CD8� cells, stained sec-tions were observed under light microscopy at �400. The numberof TUNEL-positive cells per 500 epidermal keratinocytes and 500dermal infiltrating cells within each of five areas per section werecounted. AIs for keratinocytes and infiltrating cells were expressedas the percentage of TUNEL-positive cells per 500 total keratino-cytes and dermal infiltrating cells counted (34, 35).

Immunohistochemical staining of FasL (either brown or blue)was analyzed by two semiquantitative methods. The intensity ofstaining was graded as negative (�), weakly positive (�), moder-

ately positive (��), or strongly positive (��) for the epidermalkeratinocytes and the dermal infiltrating cells. Secondly, the num-ber of positive-staining cells in each section was scored as 0 for�5% positive cells, 1 for 5–25% positive cells, 2 for 25–50%positive cells, and 3 for �50% positive cells.

The number of CD8� infiltrating cells was determined by themethod described by Bennett et al. (25) modified as follows. Theanalysis was performed by two blinded, board-certified dermatol-ogists (X. N., M. D.) with expertise in cutaneous biology research.Areas of MF lesions that had high-FasL-expression versus non-FasL-expression were first identified in CD8/FasL-doublestainedsections. Areas stained with strong and moderate intensity of FasL(�� ) were defined as FasL-expressing areas, and thosewith negative or weak intensity (� �) were defined as non-FasL-expressing areas. One thousand cells from each specimenwere evaluated twice. The number of CD8� infiltrating cells inFasL-expressing and in non-FasL-expressing areas were countedtwice for each specimen. The data were expressed as the averagepercentage of CD8� cells per 1000 total nuclei counted.

ELISA of FasL. Soluble and cellular (membrane-bound)FasL in supernatants and lysates of CTCL cell lines (HH, MJ,and HUT78) and Jurkat T cell lines were measured with asandwich ELISA kit (FasL ELISA; Calbiochem, Cambridge,MA) according to the manufacturer’s instructions. The data areshown as the mean of duplicate determinations.

Western Blot Analysis of FasL. Cytoplasmic proteinsfrom MJ, Hut 78, and HH cells (15 �g for each sample) andprotein from the control human endothelial lysate provided bythe company (Transduction Laboratories, Lexington, KY) werefractionated on 12% SDS-PAGE and transferred to nitrocellu-lose membrane by electroblotting. After being blocked with 3%nonfat dry milk in TBS, the membranes were incubated withmouse monoclonal antihuman FasL-specific IgG1 (1:1000)overnight at 4°C in 3% powdered milk in TBS. Membraneswere washed with TBS supplemented with 0.05% Tween 20 andincubated with 1:5000 HRP-conjugated goat antimouse poly-clonal (Jackson ImmunoResearch, West Grove, PA) for 1 h atroom temperature. After a washing with TBS, antibody bindingwas visualized using enhanced chemiluminescence (ECL detec-tion kit; Amersham, Buckinghamshire, United Kingdom) asdescribed previously (36). The equivalent loading of proteins ineach well was confirmed by Ponceau staining and actin control.

Statistical Analysis. The sign test and Fisher’s exacttests were used to assess the difference in FasL expressionbetween MF lesions and paired uninvolved patient skin. Spear-man’s rank correlation coefficient was used to test the correla-tion between FasL expression and MF stages. The difference inthe average CD8� T-cell number between FasL-expressing andnon-FasL-expressing areas was compared using the Wilcoxonsigned-rank test for matched pairs. Other data expressed asmean SD were tested by Student’s t test or ANOVA andconsidered significant at P � 0.05.

RESULTSPatient Demographic Data. The demographics and

stages of the 21 patients (10 male and 11 female) are shown inTable 1. The median age of the subjects was 67 years old (range,40–84). Clinical staging of lymphoma was done on all patients

2684 Correlation between FasL� and CD8� T-Cell Loss in MF

at baseline, and representative patients from each stage wereincluded as follows: Stage IA (n � 4), Stage IB (n � 4), StageIIA (n � 1), Stage IIB (n � 4), Stage III (n � 2), Stage IVA(n � 4), and Stage IVB (n � 2). Representative skin lesions ofeach patient’s skin T stage (Table 1) were selected for study asfollows: T1, patch or plaque covering �10%; T2, patch orplaque covering �10%; T3, tumor stage; and T4, erythroderma.

Increased AI Is Present in Baseline Untreated MFLesions and in Clinically Uninvolved Skin. It has been sug-gested that FasL, expressed by several neoplastic cell lines andsome tumors in vivo, is able to trigger the apoptotic process inactivated T lymphocytes (23, 25). This system may constitute akey element of the immunological escape mechanism used bymany types of neoplasms. To see whether this mechanism wasimportant in MF, we first examined the amount of baselineapoptosis by TUNEL in situ in 13 untreated MF lesions pairedwith the same patient’s uninvolved skin. MF lesions and paired,clinically “normal” uninvolved skin both contained increasednumbers of TUNEL-positive epidermal keratinocytes (n � 13;F � 31.146; P � 0.01, ANOVA) and dermal lymphocyteinfiltrates (n � 13; F � 15.825; P � 0.01, ANOVA) comparedwith normal control skins (n � 8). Representative sections areshown in Fig. 1. Surprisingly, clinically uninvolved skin fromMF patients had higher AIs than did MF skin lesions, whichsupports the systemic nature of this disease (Fig. 2).

MF Lesions Contain Higher FasL Expression Than NS.FasL-positive cells were studied in 21 MF patients’ skin lesionsand clinically normal skin at all stages of the disease (Tables 1,

2, and 3). Granular staining was observed in both the cytoplasmand cell membrane of positive cells. Representative sectionsdemonstrating the pattern and degree of FasL staining in thenormal skin, the uninvolved skin, and paired lesional skin areshown in Fig. 3, A, B, and C–D, respectively. The strongestimmunoreactivity for FasL was found in lymphocytes withcerebriform nuclei within lesions (Fig. 3D). Low FasL stainingwithin the epidermal keratinocytes of normal uninvolved skinfrom MF patients was similar to that seen in NS from controlsubjects (Fig. 3, A and B). Eighty-one % (17 of 21) MF patients’lesions showed weak to strong (� to ��) staining of FasL inepidermal keratinocytes, and the number of positive lesions wassimilar across all four stages (Table 2). FasL� infiltratingdermal lymphocytes were detected in only one-half of the StageI (four of eight) or II (three of five) lesions but were seen in fiveof six Stage IV lesions (Table 2), but the differences were notstatistically significant by Fisher’s exact test (P � 0.05). Wealso analyzed FasL staining in patients classified by T stage asshown in Table 3 with similar results. In T3 and T4 lesions,FasL-positive lymphocytes were detected in four of five andthree of five patients, respectively. Staining of FasL was moreintense in dermal lymphocytic infiltrates of all stages of MFlesions than it was in dermal lymphocytes found in paired,clinically uninvolved skin (10 of 15; sign test, �2 � 8.1; P �0.01) and in control NS.

FasL Expression Is Higher in CD45RO� T Cells Thanin CD8� Lymphocytes in MF Lesions. To determine whichcells in MF lesions were expressing FasL, we studied 10 rep-resentative MF lesions (patients 2, 3, 8, 10, 13, 15, 17, 19, 20,and 21). Double-labeling methods were used to first detecteither CD45RO or CD8 followed by FasL. These patients wereselected because their lesions contained FasL� dermal lympho-cytes (shown in Table 1). All of the 10 lesions containedinfiltrates of lymphocytes staining for both CD8 and CD45RO.CD45RO� infiltrating cells were located in groups or clustersaround dermal vessels and in the epidermis. Most CD45RO�-infiltrating cells stained blue for FasL, with intensity varyingfrom � to ��. The highest immunoreactivity for FasL wasobserved in CD45RO� atypical dermal and epidermal lympho-cytes with cerebriform nuclei as shown in Fig. 4, A and B.CD8�-infiltrating cells also showed weak (�) to moderate(��) blue counter-staining for FasL, but it was less intense thanthat seen in the CD45RO� tumor cells (Fig. 4, C and D).

FasL Is Expressed by CTCL Tumor Cell Lines in Vitro.To confirm that MF tumor cells express FasL, we also examinedFasL expression in three well-established CTCL lines (HH, MJ,and Hut78). Cytospin preps from all three of the cell linesshowed homogeneous, strong immunoreactivity to FasL anti-body (Fig. 5, A–C). The FasL bands by Western blot analysiswere stronger in three CTCL cell lines than in human endothe-lial cells as a positive control, shown in Fig. 5D. We usedELISA to determine further the relative amount of cellular(membrane-bound) versus soluble FasL in CTCL lines and inJurkat control T cells. The level of expression of cellular FasLwas higher than soluble FasL in all three of the CTCL cell lines(n � 3; t � 1.976; P � 0.05, paired t test) as shown in Fig. 6.HUT78 had the highest expression of both forms of FasL,especially the membrane-bound form of FasL. Cellular FasL

Table 1 FasL expression in MF lesion epidermal keratinocytes anddermal infiltrates

Patient no. Age/Sex Stagea

Keratinocyte Infiltrating cell

N0 L0 N0 L0

1 43/F IA, T1 NAb ��2c NA �02d 67/M IA, T1 �1 ��2 �0 �23d 84/F IA, T1 ��2 ��3 �0 ��24 72/M IA, T1 �3 �2 �2 �25 53/F IB, T2 NA ��2 NA �06 71/M IB, T2 NA ��2 NA �07 40/F IB, T2 ��3 �2 �0 �08d 70/F IB, T2 �1 ��2 �1 �19 65/M IIA, T1 �2 �2 �0 �0

10d 66/M IIB, T3 NA �0 NA �111 78/M IIB, T3 NA �0 NA �012 69/M IIB, T3 ��3 �2 �0 �213d 54/F IIB, T3 ��2 �1 �0 ��314 76/F III, T4 NA �2 NA �015d 68/F III, T4 ��2 ��3 �1 ��216 63/F IVA, T1 �0 �0 �0 �117d 67/F IVA, T1 ��2 �2 �2 ��218 75/M IVA/SS, T4 �0 �1 �0 �019d 65/F IVA, T4 �0 ��2 �0 ��220d 66/M IVB, T4 ��1 ��3 �0 ��221d 57/M IVB, T3 �1 �0 �0 ��2a T stage: T1, patches and plaques �10% body surface area; T2,

patches and plaques �10% body surface area; T3, tumors; T4, general-ized erythroderma.

b NA, not available.c Intensity (�, �, ��, ��) and frequency (0, 1, 2, 3) of

immunohistochemical staining.d Selected for study of FasL versus CD8� and CD45RO dou-

blestaining.


was higher in all three of the CTCL lines than in the controlJurkat T cells.

High FasL Expression by MF Lesion Tumor Cells IsAssociated with Low Numbers of CD8� Infiltrating Cells.To evaluate whether FasL expression by MF tumor cells influ-enced or was associated with CD8� CTLs in MF lesions, wecounted the number of CD8� infiltrating cells in predeterminedFasL-expressing versus non-FasL-expressing areas in lesionsfrom seven MF patients. Three of 10 patient samples used abovewere excluded because they had either strong staining (patients3 and 13) or weak staining (patient 8) of FasL over the entireinfiltrate. As shown in Fig. 4, C and D, we observed that theCD8� infiltrating lymphocytes were most commonly distrib-

uted at the periphery of FasL�-expressing tumor cell aggre-gates, and only a few single CD8� lymphocytes were foundinside areas of CD45RO� cell infiltrates. CD8� infiltrating cellnumbers were lowest in areas in which FasL-expressing lym-phocytes were most abundant compared with areas that did notcontain CD45�RO cells in the same MF lesion sections. Asshown in Table 4, mean numbers of CD8� lymphocytes weresignificantly less (a 7.41-fold decrease) in areas of high expres-sion of FasL on CD45RO� cells as compared with areas inwhich the expression of FasL was low (P � 0.02; n � 7; range,

Fig. 1 Increased apoptotic cells in clinically un-involved skin specimens and MF lesions. TUNELwas used to detect apoptotic cells in paraffin-embedded sections of skin from a representativeMF lesion and paired clinically uninvolved skin(patient 2). TUNEL-positive cells were seldomfound in normal control skin (A) but was found inMF clinically uninvolved skin in both epidermalkeratinocytes and dermal infiltrating cells (B); andin MF lesional skin as well as within both epider-mal keratinocytes (C) and dermal infiltrating cells(D). �200.

Fig. 2 The AI in control NS and in MF clinically uninvolved skinspecimens and lesions. TUNEL-positive cells were counted in controlNS (NS; n � 8), MF clinically uninvolved skin (N0; n � 13), and MFlesion (L0; n � 13). AIs for keratinocytes and lymphocytes wereexpressed as the percentage of TUNEL-positive cells per 500 totalkeratinocytes and lymphocytes counted.

Table 2 FasL expressiona in MF patients’ skin by stage of disease

FasL Stage IA,B Stage II Stage III Stage IV All stages

MF L0

Keratinocytes 8/8 3/5 2/2 4/6 17/21Lymphocytes 4/8 3/5 1/2 5/6 13/21

MF N0


a Expression was graded as positive if the staining was � ��in sections viewed.

Table 3 FasL expressiona in MF patients’ skin by T stage

FasL T1 T2 T3 T4 All T stages

MF L0


MF N0

Keratinocytes 5/6 2/2 3/3 3/4 13/15Lymphocytes 2/6 1/2 1/3 3/4 7/15a T1, patch or plaque �10% body surface area; T2, patch or plaque

�10% body surface area; T3, tumor; T4, generalized erythroderma.


4.77- to 12.33-fold; Wilcoxon’s signed-rank test for matchedpairs).

Colocalization of TUNEL and CD8� in MF DermalLymphocytes. To further investigate the status of CD8� T-cell infiltrates observed in the areas of high versus low FasL-expression in MF lesions, we performed CD8/TUNEL dou-blestaining. The majority of CD8� T cells were labeled by theTUNEL, regardless of whether they were in FasL-expressing ornon-FasL-expressing areas of the MF lesions (Fig. 7, C and D).In fact, the absolute numbers of CD8�TUNEL� cells withinareas of high FasL expression in MF lesions were too low forquantitative analysis or comparison. CD45RO/TUNEL dou-blestaining also showed some of CD45RO� cells labeled by theTUNEL (Fig. 7, A and B).

DISCUSSIONWe demonstrate that MF tumor cells in skin lesions from

all stages of the disease express FasL, and confirm tumorexpression of membrane-bound FasL in CTCL lines. Both

lymphocytes and keratinocytes appear to have a high-apo-ptotic rate in normal and lesional MF skin biopsy specimens,based on TUNEL staining. The high rate of keratinocyteapoptosis in MF normal and lesional skin compared withcontrol NS could explain why epidermal atrophy is commonin MF patients’ skin. The high rate of apoptosis in normaluninvolved skin appears to be unique for MF, because normalapoptosis was found in the uninvolved skin of patients withhyperproliferative skin diseases including Darier’s disease,pityriasis rubra pilaris, and psoriasis (37). The most impor-tant finding was an inverse correlation between the distribu-tion of FasL� and CD8� cells within MF lesions. Areas ofdermal infiltrates that were high-FasL-expressing areas con-tained significantly fewer CD8� T cells than did areas lack-ing high FasL-expression. In addition, CD8� T cells in theseareas of high FasL were undergoing apoptosis, as determinedby a TUNEL assay. The significance of these data are thatMF CD45RO� tumor cells may escape immune recognitionand elimination by FasL-induced apoptosis of CD8� TILs.Seven of 10 patients that were selected for high-Fas-L-expressing lesions, had late-stage disease, suggesting thatthis mechanism may play important roles in advanced MFpatients. They support the previous correlation between lossof CD8� TILs and disease progression, stage, and responseto immunotherapy, i.e., photopheresis (15, 16).

CTCL represents a unique tumor model because both theneoplastic and the reactive cells are T lymphocytes. In the caseof MF studied here, the tumor cells are clonal, expressing onetype of variable regions of the � chain (Vb) of the T-cellreceptor, CD4 and CD45RO (memory) protein. These cells areepidermotropic, based on the expression of various moleculesincluding chemokines and their receptors, especially IFN-induc-ible protein-10 (IP-10) and T cell chemokine receptor CXCR3(38), and CTL-associated antigen-4 (CTLA-4; Ref. 30). Al-though in several human cancer models (22–26) tumor cellsexpressing FasL can destroy TILs by the Fas/FasL pathway, thispossibility has not been previously investigated in CTCLs. Wehave shown for the first time that tumor cells expressingCD45RO coexpress FasL in MF lesions, and that three CTCLcell lines express membrane FasL in vitro. We hypothesize thatFasL-bearing neoplastic T cells may be capable of killingCD8� cytotoxic T cells via Fas/FasL-mediated apoptosis in MFlesions, based on our finding of high apoptotic labeling withinlesional CD8� cells.

Fas/FasL-mediated elimination of T cells after repeatedantigenic activation involves both CD4� and CD8� T lympho-cytes (39, 40). It is possible that malignant CD4� T lympho-cytes could also mediate apoptosis via a suicide or fratricidemechanism, as in HIV infection (41, 42). Although viral inte-gration of human T-lymphotropic virus type-1 (HTLV-1) taxfragments has been detected in CTCL cell lines, a retroviralcause for CTCL has not been proved (10). Regardless of themechanism, our data support coexpression of CD45RO andFasL by atypical T cells. The high AI of neoplastic T cells in MFlesions (as judged by TUNEL) could explain the indolent courseof the disease. MF clearly differs from other variants of CTCLsthat are more aggressive and lack epidermotropism as a patho-logical feature. It is possible that apoptotic rates at presentationmay be used to help determine the prognosis of CTCLs. Some

Fig. 3 FasL expression is higher in MF lesions than in paired clinicallyuninvolved skin and NS controls. Immunohistochemical analysis wasperformed with FasL monoclonal antibody (1:100). Normal control skin(A), MF patient 17 paired clinically uninvolved skin (B) and lesionalepidermis (C), and dermal infiltrating cells (D). Arrow, FasL� granularcytoplasmic/cell surface staining. �400.


CTCLs are reported to have reduced apoptosis rates (43). Therecent study by Zoi-Toli et al. (28) demonstrated the loss of Fasexpression in aggressive types of CTCLs but not in indolenttypes of CTCLs.

Tateyama et al. (44) reported that CD4� T lymphocytesare primed to express FasL by CD4 cross-linking and to con-tribute to CD8� T-cell apoptosis via Fas/FasL death-signalingpathway. Piazza also verified that activated CD4� helper T-

Fig. 4 High FasL expression colocalizes with CD45RO� tumor cells and is also associated with low numbers of CD8� infiltrating lymphocytes.Immuno-alkaline phosphatase staining (blue) using a FasL-specific mouse monoclonal IgG1 (1:100) was used to identify FasL-expressing andnon-FasL-expressing infiltrating cells in paraffin-embedded MF sections from patient 19. CD45RO or CD8 were detected using immunoperoxidasestaining (brown) on the same sections. A, B, (A, �100; B, �400) CD45RO (brown)/FasL (blue) doublestaining showed that the majority of infiltratesin epidermis and dermis had CD45RO� cell surface staining (brown; A) and CD45RO� cells costained strong FasL� cytoplasmic/cell surfacestaining (blue); C, D, (C, �100; D, �400). Consecutive sections costained with CD8 (brown) and FasL (blue). In FasL� areas (blue, few CD8� cellsare present (C) and CD8� cells also showed weak FasL� cytoplasmic/cell surface staining (D).

Fig. 5 FasL protein is highly expressed in CTCL cell lines. Immunocytochemical staining of cytospin preps for cell lines MJ (A), HUT78 (B), andHH (C). Western blot analysis (D) using FasL monoclonal antibody (1:1000) for the control human endothelial cell and CTCL cell lines. Cytoplasmprotein (15 �g) from MJ, HUT78, and HH cells was electropheresed on 12% SDS-PAGE and transfered by Western blot using enhancedchemiluminescence (ECL) detection. Scale bar, 20 �m (�100, A, B, and C). KD, Mr in thousands.


cells were able to mediate apoptotic events in Fas-positiveCD8� T cells (45). In the present study, we studied the possi-bility that expression of FasL by MF tumor cells could decreaseCD8� T cells by quantitating CD8� T-cell number in FasL-expressing versus non-FasL-expressing areas of MF lesions.There was a 7-fold reduction in CD8-positive CTLs in areas inwhich FasL� tumor cells were abundant compared with non-FasL-expressing areas in identical sections. Direction interac-tions between CD4� and CD8� T cells are necessary to me-diate apoptotic signal in CD8� T cells (45). We have shownthat FasL expressed by CTCL cell lines is mainly the mem-brane-bound form. These findings suggest that MF tumor cellsin vivo express FasL, which in turn enhances apoptosis ofCD8� T cells, resulting in their depletion over time.

Loss of cellular immunity and increased immunosuppres-sion characterize disease progression in CTCL patients (17, 18).MF patients with earlier-skin-stage (T1,2) disease tended to havea higher proportion of CD8� cells in their skin biopsy speci-mens, compared with patients with more advanced diseasestages (14). We also found that a higher percentage of MFinfiltrates expressed FasL in later stages of MF than in earlystages of the disease. Hence, loss of CD8� CTLs may occur viaFasL mechanisms, leading to disease progression. Nevertheless,CD8� cells undergoing apoptosis were also found in areas thatlacked high FasL expression, which suggested that there may beother mechansims by which TILs are lost in CTCLs. Of interest,a longitudinal study by Terheyden et al. (46), analyzing primarymelanomas and subsequent metastases, showed that highernumbers were positive for FasL among metastatic melanomathan among primary melanomas.

On the other hand, it has been demonstrated that most TILsare not functional. This has been attributed to inhibition bytumor-derived immunosuppressive factors, such as transforminggrowth factor �, gangliosides, nitric oxide, IL-10, and FasL(47). transforming growth factor � inhibits the expression ofpore-forming protein gene in human peripheral blood CD8� T

lymphocytes (48). The development of immunodeficiency inMF has been explained by a shift toward Th2 cytokine produc-tion observed in the peripheral blood and skin lesions of patientswith CTCL (17, 19, 49). Functional and molecular assays havedemonstrated a cytokine profile in CTCL patterns similar to thatproduced by the Th2-type helper T cells (50). We also suggestthat FasL may serve as yet another immunosuppressive factor, apotent mediator of immunlogical tolerance and privilege in thecontext of MF. Additional studies are now under way to confirmand to elucidate this mechanism.

FasL also plays a role in mediating immune privilegedsites, in contributing to immunological tolerance in the periph-ery, in down-sizing immune responses, and in supporting al-lograft survival (50). All of these involve FasL-mediated apo-ptotic depletion of leukocytes. A recent study reports that FasLgene transfer prolongs rat renal allograft survival and down-regulates antiapoptotic Bcl-2-associated athanogene (Bag-1) inparallel with enhanced Th2-type cytokine expression (51). Thisrole is contradicted by the finding that, in certain cases, murineallografts, genetically manipulated to express FasL, caused neu-trophil infiltration and allograft destruction (52). Neutrophilicinfiltrates are never a feature of MF lesions. Of interest, adeno-virus-mediated overexpression of FasL in mouse ankle jointsameliorated collagen-induced arthritis, thus providing powerfulevidence for an anti-inflammatory role for FasL in vivo (53).Why FasL transfection could have such contradictory effects inexperimental models is not yet clear (54).

It has been reported that cyclosporin A (CsA) treatmentinduces an inhibition of FasL mRNA expression in T cells(55) as well as inhibiting the ability of CD4� T cells to killCD8� target cells (45). Cyclosporin A administration hasbeen associated both with progression to more aggressivelymphomas (56) and with remission of MF (57). Thus, un-derstanding the significance of the Fas counterattack as amechanism of immune evasion in CTCL should facilitatedevelopment of more effective therapy for CTCL that doesnot further contribute to the destruction of the remainingtumor cytotoxicity. Fas sensitization of tumor cells or pro-tection of CD8� T cells from FasL could represent promis-ing targets for future therapeutic strategies.

Fig. 6 Cellular FasL protein is highly expressed in CTCL cell lines.Soluble and cellular (membrane-bound) FasL in supernatants and ly-sates of CTCL cell lines (HH, MJ, and HUT78) and Jurkat T cell lineswere measured by a sandwich ELISA. The level of expression ofcellular FasL was higher than of soluble FasL in all three of the CTCLcell lines (n � 3; t, 1.976; P � 0.05 paired t test). HUT78 had thehighest expression of both forms of FasL, especially the membranebound-form of FasL. Cellular FasL was higher in all three of the CTCLlines than in control Jurkat T cells.

Table 4 Reduction of CD8� TILs in FasL-expressing versus non-FasL-expressing areas of MF lesions

Patient no.

FasL-expressingareaa

(CD8� TILs %)(1)

Non-FasL-expressingareab

(CD8� TILs %)(2) (3) � (2)/(1)

2 2.20 10.5 4.7710 1.28 15.7 12.3315 1.54 12.5 8.1417 2.52 13.0 5.1619 1.43 10.0 6.9920 1.41 12.5 8.9021 3.66 20.33 5.55

Mean SD 2.01 0.86 13.53 3.54 7.41 2.67a Areas stained with strong and moderate intensity of FasL

(��) were defined as FasL-expressing.b Areas stained with negative or weak intensity (��) were

defined as non-FasL-expressing areas.


ACKNOWLEDGMENTSWe thank Leonhard Muellauer (Department of Clinical Pathology,

University of Vienna, Allgemeines Krankenhaus-General Hospital) forhelpful suggestions and for providing FasL staining protocol; RachelMacMillen for technical assistance and Drs. Hongbing Sheng andBreuer-McHam for helping with statistical analysis.

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