Immunological study on CD3 defective cutaneous T cell lymphoma cells from a patient with Sézary syndrome

Immunological study on CD3 defective cutaneous T cell lymphoma cells from apatient with Sezary syndrome

S. SANO, Y. MATSUI, S. ITAMI & K. YOSHIKAWA Department of Dermatology, Osaka University Medical School,Suita, Osaka, Japan

(Accepted for publication 23 April 1998)

SUMMARY

Here we investigated the nature of cutaneous T cell lymphoma (CTCL) cells lacking surface CD3. Alarge number of CD3¹CD4 T cells were found in the peripheral blood and lesional skin of a patient withSezary syndrome, which is a variant of CTCL. Southern blot analysis revealed that a clonalrearrangement of T cell receptor (TCR) genes was detected in the separated CD3¹CD4 cells, whereasCD3þCD4 cells showed no clonal rearrangement, indicating that the CD3¹CD4 cells represented CTCLcells. However, the CTCL cells expressed TCR with a particular Vb apart from CD3. The CTCL cellsshowed significant responses to staphylococcal enterotoxins (SEs)in vitro, although they hardlyresponded to phytohaemagglutinin,Mycobacterium tuberculosisantigen, and alloantigen. Theyrequired antigen-presenting cells (APC) to respond to SEB. Blocking analyses with MoAbs revealedthat they recognized SEB through TCR depending on HLA-DR and intercellular adhesion molecule-1(ICAM-1). Taken collectively, these results indicate that the CTCL cells lacking surface CD3 couldproliferate in response to bacterial superantigens, whereas the responses to conventional antigens weregenerally suppressed. These results also implied that CTCL could be exacerbated by bacterial infection.

Keywords staphylococcal enterotoxins skin lymphoma CD3

INTRODUCTION

Cutaneous T cell lymphoma (CTCL) is a malignant proliferation ofT lymphocytes, which initially involves the skin as plaque-stagedermatosis and later involves lymph nodes, and finally othervisceral organs. Se´zary syndrome is the leukaemic variant ofCTCL characterized by generalized erythroderma, and atypical Tlymphocytes, namely Se´zary cells, in peripheral blood as well asskin infiltrate [1]. CTCL cells are mostly of a mature T helperphenotype (CD3þCD4þ TCR a/bþ) [2], which is shared by themajority of lymphocytes in a wide variety of inflammatory skinlesions. However, it has been reported that certain cases of Tlymphoma show loss of surface TCR and/or CD3 [3–7], and such aphenotypic aberration could be of considerable help for thediagnosis of T lymphoma, since anomalous phenotypes were notobserved in any of non-lymphomatous infiltrates [8,9].

The TCR and CD3 form a functional unit in antigen recogni-tion and signal transduction, and their association and co-expression appear to be essential for normal T cells to exertimmunological function [10]. Thus abnormalities in the expressionof TCR/CD3 should induce unresponsiveness of T cells to antigen

stimuli [11]. In the present study, we focused on a patient withSezary syndrome, since the malignant cells showed phenotypicalaberrancy, of which we took advantage to separate them from non-lymphomatous T cells, and successfully established a CTCL line.The imunological nature of the CTCL with TCR/CD3 discordancewas examinedin vitro.

PATIENT AND METHODS

Patient dscriptionA 55-year-old Japanese man who was born and lived in Osakadeveloped pruritic erythematous patches on the legs in 1992.Thereafter the eruption rapidly spread over the entire body todevelop erythroderma, which was accompanied by alopecia andenlargement of lymph nodes in the axillae and groins. Histologicalexaminations of the lesional skin and the involved lymph nodesshowed morphologic features indicative of CTCL. In 1993 he wasdiagnosed as Se´zary syndrome because of the presence of circulat-ing atypical lymphocytes, known as Se´zary cells. The patient wasthen treated with combined systemic chemotherapy for lymphoma,and ultraviolet irradiation, which resulted in partial remission, butgradual relapse of the eruption and enlargement of lymph nodesfollowed in 1994. Haematological data disclosed that a total

Clin Exp Immunol 1998;113:190–197

190 q 1998 Blackwell Science

Correspondence: Shigetoshi Sano, Department of Dermatology, OsakaUniversity Medical School, 2-2 Yamadaoka, Suita, Osaka, Japan 565.

leucocyte count was in normal range with 10–20% of atypicallymphocytes (cytological Se´zary cells), and with moderate eosino-philia. Serum anti-HTLV-I antibody was negative. Other labora-tory findings showed normal values, and there was no visceralinvolvement detected.

Isolation of peripheral blood mononuclear cells and skin-infiltrating lymphoid cellsPeripheral blood mononuclear cells (PBMC) were isolated fromheparinized whole blood by Ficoll–Paque (Pharmacia, Uppsala,Sweden) density gradient centrifugation. Skin-infiltrating lym-phoid cells (SILC) were collected as follows. With the informedconsent of the patient, lesional skin was biopsied, and which wascut into pieces, then immersed in RPMI 1640 (Life Technologies,Grand Island, NY) supplemented with 10% heat inactivatedfetal calf serum (FCS), 2 mM L-glutamine, 50mM 2-mercaptoethanol(2-ME), and antibiotics. After 4 days culture at 378C in 5% CO2,migrating cells into the medium were collected, and used as SILC.

Monoclonal antibodiesMoAbs used in the current study included: anti-CD3 (Leu-4;Becton Dickinson, Mountain View, CA), UCHT1 (Coulter, Hia-leah, FL), OKT3 (Ortho, Tokyo, Japan), HIT3a (PharMingen,San Diego, CA), and NUT3 (Nichirei, Tokyo, Japan), anti-CD2(Leu-5b; Becton Dickinson), anti-CD4 (Leu-3a; Becton Dickin-son), anti-CD8 (Leu-2a; Becton Dickinson), anti-CD16 (Leu-11c;Becton Dickinson), anti-HLA-DR (Becton Dickinson), anti-CD25(IL-2R; Becton Dickinson), anti-CD1a (Leu-6; Becton Dickinson),anti-CD14 (Leu-M3; Becton Dickinson), anti-intercellular adhe-sion molecule-1 (ICAM-1; R&D Systems, Abingdon, UK), anti-TCR a-, b-, andd-chain (aF1, bF1, and TCRd1, respectively; TCell Diagnostics, Cambridge, MA), and a screening panel for anti-TCR variable chains (Diversi-T; T Cell Diagnostics) includinganti-Vb5.2þ 5.3 (1C1), anti-Vb5.3 (W112), anti-Vb5.1 (LC4),anti-Vb6.7 (OT145), anti-Vb8 (16G8), anti-Vb12.1 (S511), andanti-Va2 (F1). Phenotypic analyses of cells was carried out bystaining with two-colour immunofluorescence of FITC- and PE-conjugated MoAbs. When unconjugated MoAbs were used, cellswere subsequently stained with FITC-conjugated rat anti-mouseimmunoglobulin (Dako, Glostrup, Denmark). Isotype-matchedmouse immunoglobulin (Coulter) was used as a negative control.Ten thousand cells per sample were analysed on a FACSortanalyser (Becton Dickinson). For blocking experiments, MoAbswere dialysed to remove sodium azide, and then added to theculture.

Separation of CD3þCD4 and CD3¹CD4 cellsPBMC or SILC were stained with anti-CD4 and anti-CD3, fol-lowed by sorting CD3þCD4þ and CD3¹CD4þ subpopulationsusing EPICS ELITE (Coulter). Purity of the phenotype sortedwas>99%. In some experiments the CD3¹CD4 cells were isolatedby magnetic immunoselection (Dynal, Oslo, Norway) by sortingCD4 cells followed by depleting CD3þ cells. On the other hand,the CD3þCD4 cells were obtained by sequential sortings of CD3þ

and CD4þ cells.

Establishment of CD3¹ CTCL cloneAfter isolation of CD3¹CD4þ cells from the patient’s PBMC, theywere stimulated for 2 days with 1mg/ml of SEB in the presence of30 Gy-irradiated autologous PBMC. They were then washed twice

with medium, and cultured in the presence of recombinant IL-2(50 U/ml; Shionogi, Osaka, Japan). After 3 days culture, viablecells were collected using Ficoll–Paque gradient, and maintainedin the presence of 20 U/ml of IL-2. The phenotype of the lined cellswas confirmed to be unchanged through the experiments.

Southern blot analysisDNA from cells was extracted by cell lysis, proteinase K digestion,phenol extraction, and ethanol precipitation. Cellular DNA wasdigested with appropriate restriction enzymes, sized in 0·8%agarose gels, and transferred to a nylon membrane. Biotinylatedprobes for Jb1 and Jb2 (Oncor, Gaithersburg, MD) were hybrid-ized and visualized by streptavidin-conjugated enzyme reactionwith a substrate.

Immunohistochemical stainingSections (5mm) were immunostained with the antibodies using theavidin-biotin-peroxidase complex (ABC) technique (VectastainABC kit; Vector Labs, Burlingame, CA). Briefly, the stainingconsisted of a first-stage incubation with one of the MoAbs. Afterwashing, biotinylated goat anti-mouse immunoglobulin wasapplied prior to a third stage of peroxidase-conjugated streptavidin.The sections were then incubated in diaminobenzidine, followedby counterstaining with methyl green.

mRNA extraction and reverse transcriptase-polymerase chainreactionmRNA was extracted from cells using a Quick Prep kit (PharmaciaBiotech, Tokyo, Japan) according to the manufacturer’s instruc-tions. mRNA (0·1mg) was reverse transcribed into cDNAusing random 9mers (Takara Biochemicals, Tokyo, Japan)as primer in a total volume of 20ml, and prepared cDNAwas amplified together with primers in a total volume of 80-mlreaction using reagents from Takara Biochemicals. Primerswere designed according to cDNA sequences (GenBanks) ofCD3 chains and b-actin as follows: CD3-g: sense, 50-CTCATCCTGGCTATCATTCTTCTTCAAGG-30, anti-sense, 50-GGTTTCCTTGAAGGTGGCTGTACTGG-30; CD3-d, sense, 50-CTGGACCTGGGAAAACGCATC-30, anti-sense, 50-GTACTGAGCATCATCTCGATC-30; CD3-«, sense, 50-GACATGCCCTCAGTATCCTGGATC-30, anti-sense, 50-GCAGTGTTCTCCAGAGGGTCAGATG-30; CD3-y, sense, 50-CCTCAGCCTCTGCCTCCCAGCCTCT-30, anti-sense, 50-CTGGGCGTCTGCAGGTCTGGCCTTT-30; b-actin, sense, 50-GTGGGGCGCCCCAGGCACCA-30, anti-sense, 50-CTCCTTAATGTCACGCACGATTTC-30. Thirtycycles of amplification were performed with denaturation at 958Cfor 1 min, annealing at 568C for 1 min, and extension at 728C for2 min. DNA products were analysed on 2% agarose gels, stainedwith ethidium bromide.

Intracellular stainingViable cells were incubated in PBS containing 0·2% Tween-20(Sigma, St Louis, MO) for 1 h at room temperature. Permeability ofthe plasma membrane was confirmed by a dye exclusion test withtrypan blue (Life Technologies), followed by staining with MoAbsas described.

Proliferation assayUsed stimulants included phytohaemagglutinin (PHA; 2mg/ml;Sigma), staphylococcal enterotoxins (SEA, SEB, SEC1, SED,SEE and TSST-1, 1mg/ml unless otherwise indicated; Toxin

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q 1998 Blackwell Science Ltd,Clinical and Experimental Immunology, 113:190–197

Technology, Sarasota, FL), andMycobacterium tuberculosis(H37Ra; Difco, Detroit, MI) whose antigen was prepared as follows:100 mg of heat-killedM. tuberculosis(Mtb) was sonicated in 10 mldistilled water and soluble fraction was used as an antigen at 1:30 ofthe final concentration. In the presence of antigen or mitogen, 1·5–3×104 responder cells per well were cultured in a flat-bottomedmicrotitre plate (Corning, Corning, NY) together with 30 Gy-irradiated autologous or allogeneic PBMC (1×105/well) as antigen-presenting cells (APC) in 100ml of the culture medium describedabove. Unless otherwise indicated, cells were cultured for 72 hpulsed with3H-thymidine (0·5mCi/well; Amersham, Aylesbury,UK) for the last 8 h, and then the cells were harvested onto glassfilters and counted radioactivity incorporated with ab-liquidscintillation counter. In some experiments, 0·5mg/ml of SEB wasprepulsed to allogeneic PBMC 24 h before they were intensivelywashed, irradiated (30 Gy), and used as SEB-prepulsed APC.Secondary mixed lymphocyte reaction (MLR) assays were carriedout as follows: cells were cultured with irradiated (30 Gy) allo-geneic PBMC for 3 days, and cultured for another 3 days with10 U/ml of IL-2, then viable cells were collected after removal ofdead cells through Ficoll–Paque gradient. Then cells (2×104/well)were cultured with irradiated PBMC (105) from the same allo-geneic donor and were assessed for proliferative responses asdescribed. All assays were performed in triplicate.

RESULTS

Detection of CD3 defective CD4 lymphocytes in the peripheralblood and lesional skin of a patient with Se´zary syndromeTo examine immunophenotypes of the PBMC and SILC, weperformed flow cytometry analysis. Two-colour analysis revealedthat a large number of CD4 lymphocytes were lacking CD3 (Leu-4) in both PBMC and SILC. The CD3¹CD4 cells were moreabundant in SILC (88% of all the CD4 cells) than in PBMC (68%)(Fig. 1). Using other MoAbs against CD3 (UCHT1, OKT3, HIT3aand NUT3), the percentage of CD3¹CD4 cells was identical (datanot shown). Further phenotypic analysis disclosed that theseaberrant CD3¹CD4 cells were CD2þCD8¹CD5þCD1a¹

CD14¹CD16¹ TCRd1¹ lymphocytes.

Identification of CTCL and its phenotypeThe cell sorter allowed us to purify CD3¹CD4 cells and CD3þCD4

cells from the patient’s PBMC with>99% purity. To determineclonality in each CD4 subpopulation, we performed Southern blotanalysis on the TCRb genes. In isolated CD3¹CD4 cells, discreterearranged bands were detected after hybridization of EcoRI,BamHI, and HindIII digests with the Jb2 probe (Fig. 2), but nomonoclonal rearranged bands were detected in CD3þCD4 cells.Also, discrete rearranged bands with the Jb1 probe were observedin the CD3¹CD4 cells (data not shown), while CD3þCD4 cellsagain showed germ-line configuration. This finding suggeststhat both alleles of TCRb gene in the CD3¹CD4 cellsunderwent monoclonal rearrangement with VbDb1Jb1 andVbDb2Jb2 configuration.

Thus, we identified the CD3¹CD4 cells as CTCL, whereas theCD3þCD4 cells were non-lymphomatous. It should be noted thatthe identical rearranged bands were detected in the CD3¹CD4 cellsderived from the lesional skin (data not shown) to those fromPBMC. Also, the malignant nature of the CD3¹CD4 lymphocyteswas clinically evaluated in that anti-lymphoma chemotherapy forthe patient selectively decreased their number in the peripheralblood, being accompanied by clinical improvement in skin rashand in swelling of lymph nodes. Furthermore, to examine whetherthe CD3¹CD4 lymphoma cells express the surface T cell antigenreceptor (TCR) chains, we utilized a panel of MoAbs directed toclonotypic Vb region products. As shown in Fig. 3a, most of theCD3¹CD4 cells isolated from PBMC or SILC were stained by1C1, a MoAb to Vb5.2þ 5.3, whereas they were not stained withanti-Vb5.3, indicating that they expressed Vb5.2. In contrast,CD3þCD4 non-lymphomatous cells carried no predominant Vb

region segments. To ensure that the CTCL expressed TCRab

chain, we performed immunohistochemical staining of the lesional

192 S. Sanoet al.


Fig. 1.Flow cytometric analysis on the peripheral blood mononuclear cells(PBMC) and skin-infiltrating lymphoid cells (SILC) from the patient withSezary syndrome. The PBMC and SILC were stained with PE-conjugatedanti-CD3 (Leu-4) and FITC-conjugated anti-CD4 (Leu-3) MoAbs, andanalysed on FACSort. The percentages of cells in quadrants are indicated.

Fig. 2. Southern blot analysis on the CD3¹CD4 cells isolated from thepatient’s peripheral blood mononuclear cells (PBMC). After digestion withEcoRI (E), BamHI (B), and HindIII (H), DNA was hybridized with Jb2probe. Germ-line control digests (G) are shown. From CD3¹CD4 cells,rearranged discrete bands (open triangles) are shown in each digest togetherwith germ-line bands (closed triangles), which were presumably fromanother allele where rearrangement of the Jb1 gene occurred (data notshown). On the other bands, from CD3þCD4 cells, digests were hybridizedin germ-line configuration except for a smear (circle) in BamHI digest,which were referred to polyclonal rearrangement of the Jb2 gene. Size ofthe fragments is given in kilobase pairs.

skin with MoAbs against common region ina- and b-chain ofTCR. The infiltrating cells in the dermis of the lesion werepositive foraF1, bF1 (Fig. 3b), indicating that CTCL expressedTCR ab. Taken collectively, the CD3¹CD4 cells representedCTCL expressing TCR with a particular Vb.

CD3 mRNA expression and intracellular CD3 in the CTCLBy reverse transcriptase-polymerase chain reaction (RT-PCR)analysis, mRNAs for CD3-g, «, d, y were detectable in theCD3¹ CTCL line as well as CD3þCD4 cells (data not shown).Moreover, after permeabilization of cellular membrane, anti-CD3stained all the CTCL cells (data not shown). This finding suggested

that they expressed CD3 molecule in the cytoplasm, but not in thecell surface.

CD3¹ CTCL responded to staphylococcal enterotoxinsTo study the immunological character of the CTCL, we culturedthemin vitro with various stimuli including a T cell mitogen PHA,Mtb antigen, staphylococcal enterotoxins, and allogeneic cells. Asshown in Exp. 1 of Table 1, either fractionated CD4 subpopulationfrom the patient’s PBMC showed unresponsiveness to PHA andMtb. This result was in favour of previous descriptions that thecell-mediated immune systems are impaired in patients withCTCL, in particular Se´zary syndrome [2,12]. In contrast, PBMC



Fig. 3. (a) Reactivities of isolated CD3þCD4 cells and CD3¹CD4 cells with a panel of MoAbs against Vb products. The sorted cells werecultured for 2 days in order to remove antibodies on the cell surface, and then stained by PE-conjugated anti-CD3 together with FITC-conjugated MoAb as indicated. Note that most CD3¹CD4þ cells, regardless of derivation, were stained by 1C1, an antibody to Vb5.2þ 5.3,but not by anti-Vb5.3, indicating that they expressed VP5.2. (b) TCRab expression of cutaneous T cell lymphoma (CTCL) in the skin.Primary antibodies used in immunohistochemical staining included mouse IgG as a control (left,×200),aFl (middle,×200), andbF1 (right,×400). Infiltrating cells were mostly TCRabþ, and they infiltrated in upper dermis (middle), and perifollicular (right).

from control donors responded well to PHA or Mtb. However,comparable proliferation to SEB was demonstrated in the CTCL(CD3¹CD4 cells) as well as CD3þCD4 cells and PBMC from thecontrols. In secondary allogeneic MLR assay, the differencebetween the two CD4 subpopulations was conspicuous in thatthe CTCL showed marginal anti-allogeneic response, whereasCD3þCD4 cells appeared to be intact (Exp. 2 of Table 1). Variousstaphylococcal enterotoxins were tested for stimulating activity onthe CD3¹CTCL cells (Fig. 4). They responded significantly toSEA, SEB, SEC1, SEE and TSST-1 in a dose-dependent manner,but marginally to SED. SEB appeared to be greatest stimulus.

Response of the CTCL cells to SEB was TCR-mediated andrequired APCTo investigate whether the CTCL recognized SEB through TCR,we performed a blocking experiment using the specific MoAb toTCR Vb. A result shown in Fig. 5a demonstrated that the MoAb tospecific Vb (closed bar) completely inhibited the proliferativeresponse to SEB at a concentration of>6mg/ml. This resultindicated that the CTCL recognized SEB through the particularVb. Furthermore, to examine if they required APC for antigenrecognition, experiments were performed in the presence orabsence of APC (Table 2). The CTCL grew well to SEB whenco-cultured with APC, while they showed no response to SEBwithout APC. Even stronger proliferation was observed when co-cultured with SEB-prepulsed APC. These data indicate that theCTCL cells recognized SEB associated with APC, but they didnot respond to cell-unbound SEB. This was also true forCD3þCD4 cells (data not shown). Finally, the SEB response ofthe CTCL was significantly blocked by inclusion of antibodies toHLA-DR or ICAM-1 (Fig. 5b), illustrating that they recognized

194 S. Sanoet al.


Table 1. Proliferative responses of isolated CD3þCD4 cells and CD3¹CD4 cells to a variety of stimuli for Tcells

3H-TdR incorporation, ct/min (s.e.m.)

Patient Normal control

CD3þCD4þ cells CD3¹CD4þ cells(CD4 control) (CTCL) Donor 1 Donor 2

Exp.1*Nil 80 (29) 87 (7) 177 (449) 402 (61)PHA 359 (66) 42 (11) 16362 (1149) 23684 (3341)Mtb 32 (6) 39 (5) 4142 (370) 5583 (95)SEB 58593 (2544) 22230 (888) 68235 (119) 69604 (1813)Exp.2†Nil 255 (37) 339 (51) 314 (76) NDAllo PBMC 31697 (3336) 2062 (198) 18208 (540) ND

* The proliferative reactivities of cell sorter (EPICS ELITE)-isolated CD4 subpopulations from the patient’speripheral blood mononuclear cells (PBMC) were investigated using irradiated autologous PBMC as antigen-presenting cells (APC).

† Allogeneic PBMC was from a donor Se´zary syndrome (HLA-DRw15, DRw8, and DRw52), andviceversa, the patient’s PBMC (DR4 and DR6) were used for stimulating T cells of a donor Se´zary syndrome.Assays were performed as described in Patient and Methods. Results shown are representative of three separateexperiments and are means of triplicate values with s.e.m. shown in parentheses.

PHA, Phytohaemagglutinin; Mtb,Mycobacterium tuberculosis;ND, Not done.

20 000

SEA

SEB

SEC

SED

SEE

TSST-1

15 000

10 000

5000

0

ng/ml

Pro

life

rati

on

(ct/

min

)

10001001010.10.01

Fig. 4. CD3¹ cutaneous T cell lymphoma (CTCL) cells proliferate inresponse to various staphylococcal enterotoxins.In vitro proliferation assayon CD3¹ CTCL cells was performed as described in Patient and Methods.Concentration of the toxins used in culture is indicated in the figure. Asymbol represents each toxin depicted in the figure. S.e.m. in the valueswas <15%, and is omitted for simplicity. Medium control gave11946 166 ct/min.

SEB with HLA-DR, and required an adhesion molecule ICAM-1. Taking all data together, it is likely that CD3¹ CTCLrecognized staphylococcal superantigens in the same fashionas do conventional CD4 cells [13].

DISCUSSION

We studied a patient with Se´zary syndrome, a variant of CTCL[1]. A number of aberrant T-helper cell populations(CD3¹CD4þ TCRþ) were found in the peripheral blood andlesional skin. The finding that the cells with aberrant phenotypewere more abundant in the skin than peripheral blood (Fig. 1)suggests that they propagated in the skin, and ‘spilled over’ into thecirculation [14]. Results of Southern blot analysis (Fig. 2) clearlyindicated that the aberrant CD4 cells represented CTCL, but theadmixed CD4 cells bearing CD3 were non-lymphomatous. Thus,taking advantage of the absence of surface CD3 to discriminateCTCL, comparative studies were performed in the present study.

The lack of surface CD3 in the CTCL cells is strange. Theabsence of the surface CD3 was confirmed by five differentMoAbs, and therefore it is unlikely that they were lacking asingle epitope of CD3 for a specific antibody. However, mRNAsfor CD3-g, d, «, y were expressed in the CTCL (data not shown). Inaddition, intracellular CD3 was stained in the CTCL cells afterpermeabilization of plasma membrane (data not shown). Thisresult may account for a defect in cytosomal transport of CD3molecule to plasma membrane because of incomplete assembly ofthe TCR–CD3 complex in the cytoplasm [6,15–17], since thesemolecules should be assembled in the cytoplasm to be expressedon the cell surface [6,11]. Therefore, TCR–CD3 discordanceregarding surface expression observed in the present study isextremely peculiar, because the defect in surface expression ofthe TCR and CD3 proteins is likely to be mostly coincidental[6,11,18]. Nevertheless, it is evident that they expressed clonallydistributed TCR by the following results: (i) Southern blot analysisof CD3-defective cells using Jb probes revealed they had mono-clonally rearranged TCRb gene (Fig. 2); (ii) most of themwere stained with a MoAb against a particular Vb chain inFACS analysis (Fig. 3a), and it blocked their proliferativeresponse (Fig. 5a); (iii) immunohistochemical examination of thelesional skin revealed that they were positive forbF1 andaF1(Fig. 3b), which are antibodies specific for common framework of



4000(a)

(b)

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12

20 000

15 000

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5000

0Control Ab HLA-DR ICAM-1

SEBSEBNil SEB SEB

Antibody added

Antigen

Fig. 5. (a) Anti-TCR Vb blocked the proliferative response of CD3¹

cutaneous T cell lymphoma (CTCL) cells to staphylococcal enterotoxinB (SEB). Proliferative response of the CTCL to SEB (0·03 pg/ml)was completely abrogated by anti-clonotypic Vb MoAb (1C1, B) ata concentration of> 6mg/ml, whereas control mouse IgG had amarginal affect (A). 3H-TdR uptake of the cells in medium only and SEBwithout antibody was 4216 35 ct/min and 34656 210 ct/min, respectively.(b) Effect of anti-HLA-DR and anti-intercellular adhesion molecule-1(ICAM-1) on the proliferative responses of CD3¹ CTCL cells to SEB.The proliferative responses to SEB (1mg/ml) of either PBMC (B)- orskin-infiltrating lymphoid cells (SILC) (A)-derived CD3¹ CTCLcells were significantly blocked by anti-HLA-DR (1:50) or anti-ICAM-1(2mg/ml).

Table 2. The cutaneous T cell lymphoma (CTCL) requiredantigen-presenting cells (APC) for responding to staphylococcal

enterotoxin B (SEB)

3H-TdR incorporation‡,APC* SEB† ct/min (s.e.m.)

– – 112 (11)– þ 114 (15)þ – 246 (16)þ þ 3834 (418)SEB-prepulsed§ – 8206 (706)

* Irradiated (30 Gy) allogeneic peripheral blood mononuc-lear cells (PBMC; 5×104/well) were used as APC.

† 0·1mg/ml.‡ The CTCL lined cells were cultured, and pulsed with3H-

thymidine for the final 6 h of total 48 h culture. Results shown arerepresentative of three separate experiments and are means oftriplicate values with s.e.m. expressed in parentheses.

§ Allogeneic PBMC was prepulsed with 0·5mg/ml of SEBfor 24 h and washed intensively, and then irradiated beforeculturing with the CTCL cells.

TCRb- anda-chain, respectively; (iv) they expressed Cb2, but noCb1 mRNA in RT-PCR analysis (not shown). Like our case,previous reports have demonstrated many cases of T lineagemalignancy with TCR–CD3 aberrance [3–7]. T cells in retro-viral-related diseases such as AIDS [19] or adult T cell leukaemia[20] reportedly underwent a surface CD3 defect. In most cases withT lineage malignancy of childhood, CD3 antigen was absent fromthe cell surface but was detected in the cytoplasm, and it wasassumed that they arose from immature thymocytes [7]. Further-more, it is intriguing that a high frequency of surface CD3-defective CD4 cells has been observed in a patient with ataxiateleangiectasia [15,21], characterized by abnormalities of T cellfunction and development of T cell lymphoma.

Very intriguing was the fact that the CD3¹ CTCL cells couldrespond to SEB as well as control CD3þCD4 cells and normaldonors’ PBMC, although PHA, Mtb or alloantigen gave the CTCLcells a marginal stimulation (Table 1). Moreover, their response toSEB required APC, and was HLA-DR- and ICAM-1-dependent(Table 2 and Fig. 5b), indicating that the CTCL cells recognizedstaphylococcal toxins in the same way as do normal helper T cells[13]. On the other hand, it seems unlikely that SEB directlystimulated the CTCL cells via HLA-DR as previously described[22], for the following reasons: (i) the CTCL cells from freshPBMC marginally expressed HLA-DR (<7%), which was up-regulated to 50% after activation to become a line. Proliferativeresponse to SEB was, however, equally demonstrated in the freshlyseparated and lined CTCL (Tables 1 and 2); (ii) HLA-DR trigger-ing by a specific antibody alone or in combination with otherstimuli did not induce proliferation of the CTCL cells (data notshown), while Spertiniet al. have demonstrated significant prolif-eration of DRþ T cells by stimulation with anti-DR together withanti-CD3 [22]; (iii) SEB could not stimulate the CTCL cells in theabsence of APC (Table 2). If they responded to SEB through theirDR, they could have been activated by SEB without help fromprofessional APC. Moreover, we have shown even stronger stimu-lation by SEB-prepulsed APC than by soluble SEB in the presenceof APC (Table 2). Since SEB the binding site to HLA-DR had beenpreoccupied by APC when prepulsed, it is unlikely that the DRþ

CTCL cells, if any, could bind SEB through their DR under theseconditions; (iv) anti-TCR Vb specific for the CTCL inhibitedthe proliferative response to SEB in a dose-dependent manner(Fig. 5a). This result was suggestive of definitive involvement ofTCR in the recognition of superantigens. So far, we do not knowhow the signal through TCR was introduced in the CTCL cellswhen the surface CD3 was absent. Further biochemical analysis ofthe surface proteins is needed to elucidate this point.

Exposure of peripheral T cells to bacterial toxins results inprofound activation and propagation of T cells of appropriate Vb

specificity [23–25]. A large body of studies has been accumulatedregarding bacterial superantigen-related human diseases [25],including skin disorders such as atopic dermatitis [26,27], psoriasis[27–29], and CTCL [12,30]. Here, all but one staphylococcal toxintested gave significant responses of the CTCL cells expressingVb5.2 (Fig. 4). However, T cells positive for Vb5 have beenreported to be well stimulated by SEA, SEC1, SED, marginally bySEB, but not by SEE. The responses to the toxins of the CTCL cellswas therefore likely to be non-specific. It is possible that theyrecognized a shared antigenic epitope of the toxins whose aminoacid sequences are homologous [31,32] in some different way fromthat of normal T cells. Although a causative relationship betweenthe TCR–CD3 aberrance and the developmental mechanism of T

lymphoma remains to be elucidated, it is feasible that an anom-alous signal could be delivered through impaired TCR–CD3molecules to induce malignant transformation by repeated stimu-lation with ubiquitous antigens such as bacterial superantigens,which otherwise induce clonal elimination [32]. Thus, we believethat CTCL could be a suitable model to investigate the mechanismsby which T lymphoma grows in response to bacteria, since the skinprovides a particular milieu for T cell immunity.

REFERENCES

1 Edelson RL. Cutaneous T cell lymphoma: mycosis fungoides, Se´zarysyndrome, and other variants. J Am Acad Dermatol 1980;12:89–106.

2 Heald P, Edelson RL. Immunology of cutaneous T-cell lymphoma.J Natl Cancer Inst 1991;83:400–4.

3 Weiss LM, Picker LJ, Grogan TMet al. Absence of clonal beta andgamma T-cell receptor gene rearrangements in a subset of peripheral Tcell lymphomas. Am J Pathol 1988;130:436–42.

4 Ng CS, Chan JKC, Hui PKet al. Application of a T cell receptorantibodybF1 for immunophenotypic analysis of malignant lymphomas.Am J Pathol 1988;132:365–71.

5 Michie SA, Abel EA, Hoppe RTet al. Expression of T-cell receptorantigens in mycosis fungoides and inflammatory skin lesions. J InvestDermatol 1989;93:116–20.

6 Fischer MB, Hauber 1, Fodinger Met al. Defective TCR surfaceexpression associated with impaired TCR beta-chain assembly in apatient with cutaneous T-cell lymphoma. J Invest Dermatol 1995;104:537–40.

7 Link MB, Stewart RA, Warnke RAet al.Discordance between surfaceand cytoplasmic expression of the Leu-4 (T3) antigen in thymocytesand in blast cells from childhood T lymphoblastic malignancies. J ClinInvest 1985;76:248–53.

8 Haynes BF, Hensley LL, Jegasothy BM. Phenotypic characterization ofskin-infiltrating T cells in cutaneous T cell lymphoma: comparison withbenign cutaneous T-cell infiltrates. Blood 1982;60:463–73.

9 Willemze R, De Graaff-Reitsma CB, Cnossen Jet al. Characterizationof T-cell subpopulations in skin and peripheral blood of patients withcutaneous T-cell lymphomas and benign inflammatory dermatoses. JInvest Dermatol 1983;80:60–66.

10 Clevers H, Alarcon B, Wileman Tet al. The T cell receptor/CD3complex: a dynamic protein ensemble. Ann Rev Immunol 1988;6:629–62.

11 Aranaiz-Villena A, Timon M, Rodriguez-Gallego Cet al.Human T-cellactivation deficiencies. Immunol Today 1992;13:259–65.

12 Tokura Y, Heald PW, Yan SLet al. Stimulation of cutaneous T-celllymphoma cells with superantigenic staphylococcal toxins. Invest JDermatol 1992;98:33–37.

13 VanSeventer GA, Newman W, Shimizu Yet al. Analysis of T cellstimulation by superantigen plus major histocompatibility complexclass II molecules or by CD3 monoclonal antibody: costimulation bypurified adhesion ligand VCAM-1, ICAM-1, but not ELAM-1. J ExpMed 1991;174:901–13.

14 Hall WW. Human T cell lymphotropic virus type 1 and cutaneous T cellleukemia/lymphoma. J Exp Med 1991;180:1581–5.

15 Kyoizurni S, Akiyama M, Hirai Yet al.Spontaneous loss and alterationof antigen receptor expression in mature CD4þ T cells. J Exp Med1990;171:1981–99.

16 Uppenkamp M, Pittaluga S, Coupland Ret al. Stable T3¹ and T3þ

subclones derived from the mosaic human T cell leukemia cell lineCEM. J Immunol 1988;140:2802–7.

17 Maecker HT, Levy R. Spontaneous T cell antigen receptor variants of ahuman T leukemia cell line. J Immunol 1988;141:2994–3002.

18 Weiss A, Stobo J. Requirement for the coexpression of T3 and the T cellantigen receptor on a malignant human T cell line. J Exp Med 1984;160:1284–99.

19 Herndier BG, Shiramizu BT, Jewett NEet al.Acquired immunodeficiency

196 S. Sanoet al.


syndrome-associated T-cell lymphoma; evidence for human immuno-deficiency virus type 1-associated T-cell transformation. Blood 1992;79:1768–74.

20 Tsuda H, Takatsuki K. Specific decrease in T3 antigen density in adultT-cell leukemia cells: 1 Flow microfluorometric analysis. Br J Cancer1984;50:843–5.

21 Boutin B, Wagner DK, Nelson DL. Analysis of CD3 antigen expressionin patients with ataxia-teleangiectasia. Clin Exp Immunol 1987;68:320–30.

22 Spertini F, Spits H, Geha R. Staphylococcal exotoxins deliveractivation signals to human T-cell clones via major histocompatibilitycomplex class II molecules. Proc Natl Acad Sci USA 1991;88:7533–7.

23 Kappler J, Kotzin B, Herron Let al.Vb-specific stimulation of human Tcells by staphylococcal toxins. Science 1989;244:811–3.

24 Choi Y, Kotzin B, Herron Let al. Interaction ofStaphylococcus aureustoxin ‘superantigens’ with human T cells. Proc Natl Acad Sci USA1989;86:8941–5.

25 Kotzin B, Leung DY, Kappler Jet al.Superantigens and their potentialrole in human disease. Adv Immunol 1993;54:99–166.

26 McFadden JP, Noble WC, Camp RDR. Superantigenic exotoxin-secreting potential of staphylococci isolated from atopic eczematousskin. Br J Dermatol 1993;128:631–2.

27 Leung DYM, Travers JB, Norris DA. The role of superantigens in skindisease. J Invest Dermatol 1995;105:37–42.

28 Leung DYM, Walsh P, Giorno Ret al. A potential role for super-antigens in the pathogenesis of psoriasis. J Invest Dermatol 1993;100:225–8.

29 Valdimarsson H, Baker BS, Jonsdottir Iet al. Psoriasis: a T-cell-mediated autoimmune disease induced by streptococcal superantigens.Immunol Today 1995;16:145–9.

30 Tokura Y, Yagi H, Ohshima Aet al. Cutaneous colonization withstaphylococci influences the disease activity of Se´zary syndrome:a potential role for bacterial superantigens. Br J Dermatol 1995;133:6–12.

31 Marrack P, Kappler J. The staphylococcal enterotoxins and theirrelatives. Science 1990;248:705–11.

32 Scherer MT, Ignatowicz L, Winslow GWet al.Superantigens: bacterialand viral proteins that manipulate the immune system. Ann Rev CellBiol 1993;9:101–28.



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Immunological study on CD3 defective cutaneous T cell lymphoma cells from a patient with Sézary syndrome