JClin Pathol Ber-ACT8: New monoclonal antibody the mucosa ... · Ber-ACT8 labelled most...

J Clin Pathol 1991;44:636-645

Ber-ACT8: New monoclonal antibody to themucosa lymphocyte antigen

M Kruschwitz, G Fritzsche, R Schwarting, K Micklem, D Y Mason, B Falini, H Stein

AbstractlJsing a newly established HTLV-1positive T cell line as an immunogen, anew monoclonal antibody, Ber-ACT8,was produced. It reacts with in vitroactivated T cells and a small subset ofnormal resting T cells, but not with rest-ing B cells or any of the 29 establishedhuman permanent cell lines tested.Immunohistological analysis of a widespectrum of human tissues showed thatBer-ACT8 reactivity is restricted to afew T cells in the peripheral blood, theextrafollicular areas of lymph nodes andtonsils, and splenic red pulp. In the gutBer-ACT8 labelled most intraepithelialT cells and up to 50% of lamina propriaT cells. The antibody also immuno-stained T cells present in the oral andbronchial mucosa. Double labelling onsplenic cells, fresh blood lymphocytes,and in vitro activated T cells showed thatmost Ber-ACT8 positive cells coexpres-sed CD8. Ber-ACT8 did not react withany of the 14 Hodgkin's lymphomas norany of the 172 non-Hodgkin's lym-phomas tested, with the exception of 10cases of T cell lymphomas, five of whichwere located in the jejunum andassociated with coeliac disease, and oneB cell lymphoma, and most cases ofhairy cell leukaemia tested. Parallelimmunostainings with Ber-ACT8, anti-TCR-f (#F1), and anti-TCR-3 showedthat most Ber-ACT8 positive T cellscarry the TCR of a4 type. Comparison ofBer-ACT8 with HML-1, B-ly7, and LF61showed essentially the same reactivityand an identical molecular target.The molecular structure recognised

seems to be a trimeric molecule withcomponents of 150, 125 and 105 kilodal-tons, with the Ber-ACT8 epitope local-ised on the 150 kilodalton chain. The 150kilodalton molecule contains an 0-linkedcarbohydrate moiety of about 10 kilodal-tons. Because of its very selective dis-tribution, the trimeric antigen is apowerful reagent for the diagnosis of gutT cell-derived T cell lymphomas andother extranodal T cell lymphomas, aswell as hairy cell leukaemia.

Only a small number of true activation mark-ers have been detected-that is, antigens thatare absent from all non-lymphoid cells andfrom peripheral resting T and B cells, as wellas precursor T and B cells, but expressed onin vitro activated (by lectins or viruses) and in

vivo activated T and B cells. At the recentInternational Conference on Leucocyte Dif-ferentiation Antigens, held in Vienna inFebruary 1989, only four antigens-CD25,'2CD30,34 CD69,5 and CDw7O6 fulfilled theaforementioned criteria. Common features ofall four of these activation markers are thatthey can be induced on B and T cells and onCD4 and CD8 subsets of T cells. At present,there is no known activation molecule whoseexpression is restricted to a defined subset ofactivated B or T cells.7 This prompted us tocontinue our search for further activationmarkers using a newly established HTLV-1positive cell line (MAPS16) and monoclonalantibodies. Among the many monoclonalantibodies obtained, there was one designatedBer-ACT88 which recognises an antigen thatis expressed on all in vitro activated CD8 Tcells, but which is absent from most in vitroactivated CD4 T and in vitro B cells.

MethodsIMMUNISATION AND SOMATIC CELLHYBRIDISATIONEight week old female Balb/c mice wereimmunised intraperitoneally with 2 x 107MAPS 16 cells suspended in phosphate bufferedsaline (Gibco) and Freund's complete orincomplete adjuvants at seven day intervals for6 weeks. A final boost without adjuvants wasgiven three days before the spleens wereremoved. The human T cell line MAPS16 wasestablished by co-culturing lectin treated peri-pheral blood lymphocytes with irradiatedHTLV- 1 positive MT2 cells (Fritzsche G,unpublished observations). Cell hybridisationwas performed according to standardprocedures.

ANTIBODY SCREENINGHybridoma supernatants were screened forreactivity by immunostaining frozen sectionsof normal spleen and cytospins of MAPS16,MT-2, and peripheral blood cells before andafter stimulation with phytohaemagglutinin(PHA) using the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. Thetwo hybridomas showing the most selectivereactivity with the immunised cells were clonedby limiting dilution and distributed into 96-well plates (Nunc, Denmark). Ascitic fluid wasobtained by intraperitoneal injection of20 x 106 hybrid cells secreting monoclonalantibodies into nude mice primed one weekpreviously with 0-5 ml pristane.The Ig-subclass was determined by double

immunodiffusion using subclass specific rabbit

Free UniversityBerlin, KlinikumSteglitz, Department.of Pathology,Hindenburgdamm 30,1000 Berlin 45,GermanyM KruschwitzG FritzscheR SchwartingH SteinDepartment ofHaematology, JohnRadcliffe Hospital,OxfordK MicklemD Y MasonIstituto di ClinicaMedica e TerapiaMedica, Policinico,Monteluce, Perugia,ItalyB FaliniCorrespondence to:Dr H SteinAccepted for publication20 March 1991

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637Ber-ACT8: New monoclonal antibody to the mucosa lymphocyte antigen

anti-mouse reagents (Serotec, Wiesbaden,Germany).Normal and neoplastic human biopsy

material was obtained from the Department ofPathology, Steglitz Medical Center, Berlin. Alltissues were snap-frozen in liquid nitrogen andstored at -80'C. Malignant non-Hodgkin'slymphomas (NHL) were classified according tothe updated Kiel classification9 according tomorphological and immunological features.'0

Peripheral blood lymphocytes were isolatedfrom the anticoagulated blood of healthy don-ors by Ficoll-Hypaque density centrifugation.For B cell enrichment T cells were removed bycentrifugation after they had been bound tosheep erythrocytes pre-treated with 2-Amino-ethyl-isothiouironicum bromide. Monocyteswere separated from lymphocytes by plasticadherence.

ISOLATION OF SPLEEN CELLSSpleens were obtained from patients withtraumatic rupture of the spleen. Tissue wasminced with scissors and forced through a250 ,um pore nylon mesh. Erythrocytes wereremoved from cell suspension by Ficoll-Hypaque density centrifugation. Theinterlayers were collected, diluted with RPMImedium, and resuspended in 90% Percoll(Pharmacia, Sweden) and separated fromdebris by centrifugation at 600 x g at 4'C for 20minutes. The recovered pellets were pooledand washed twice in RPMI medium.

PURIFICATION OF T CELL SUBPOPULATIONSThe CD4 and CD8 positive subpopulations ofT cells were isolated by using either flowcytometry" or by panning methods, as de-scribed elsewhere.'2 In brief, T cells weretreated with OKT8 (CD8) or OKT4 (CD4)monoclonal antibodies. Cells were washed andincubated at 4'C in Petri dishes coated withpurified rabbit anti-mouse Ig antibodies andthe unbound cells were harvested. For cellsorting experiments, the cells were staineddirectly with fluorescein isothiocyanate (FITC)

Table I Monoclonal antibodies usedfor immunofluorescence studies

CD, target cellsor target molecules Antibody Source

CD la NA1/34 DakoCD 2 T 11 (PE) CoulterCD 3 T 3(PE) CoulterCD 3 OKT3 OrthoCD 4 T 4 (FITC/PE) CoulterCD 4 OKT4 OrthoCD 8 T 8 (FITC/PE) CoulterCD 8 OKT8 OrthoCD 14 Anti-human Leu-M3 (PE) Becton DickinsonCD 15 C3D-1 Author's laboratory/DakoCD 20 B 1 (FITC/PE) CoulterCD 22 Tol5 Dr D Y Mason/DakoCD 25 Anti-human IL-2R (PE) Becton DickinsonCD 25 ACT-1 DakoMacrophages Ber-MAC3 Authors' laboratory/DakoFollicular dendritic cells R4/23 Authors' laboratory/DakoMouse IgG Goat anti-mouse IgG Southern BiotechniquesNegative control Mouse IgG (FITC/PE) CoulterMucosa T cells* HML-1 ImmunotechHairy cells* B-ly7 Dr S PoppemaHairy cells* LF61 Dr B Falini

Activated CD8+ T cells )Gut mucosa T cells > Ber-ACT8 Authors' laboratoryHairy cells J

*This target reactivity was reported by the authors.

or phycoerythrin-labelled CD4 or CD8 mono-clonal antibodies and isolated with an EPICSflow cytometer. Purity and viability of allseparated populations were more than 95%.

LYMPHOCYTE ACTIVATIONFor B cell stimulation experiments, Staphy-lococcus aureus (10-4 v/v) (Calbiochem) wasadded to peripheral blood B cells or spleen cellsuspensions. Activated T cells were obtainedby culturing peripheral blood lymphocytes orsplenic T cells in the presence ofPHA (1% v/v,Gibco) or ConA (1% v/v, Seromed). All cul-tures were established in RPMI 1640 contain-ing 10% heat inactivated fetal calf serum,2 mM glutamine, 500 units/ml penicillin, and100 ,g/ml streptomycin at a concentration of1 x 106/ml in a humidified incubator at 37'Cwith 5% carbon dioxide. Aliquots containing1-2 x 106 cells were removed at various timesand the proliferative response was checked byimmunostaining with Ki-67."' Cytocentrifugeslides were prepared and stored at - 80'C untiluse.The monoclonal antibodies used in this

study are listed in table 1.Frozen sections of normal and neoplastic

tissues as well as cytospin slides, were fixed inacetone or in acetone and chloroform for 10 to30 minutes and immunostained using theAPAAP technique,'4 but slightly modified.'5Monoclonal antibody was purified by affinity

chromatography and subsequently incubatedin 0 1 M Na2CO3/NaHCO3 solution at pH 9with 10% v/v FITC (1 mg/ml in DMSO)(Sigma), followed by dialysis against PBS(Gibco) to remove unbound fluorescent dye.

DIRECT DOUBLE COLOUR IMMUNOFLUORESCENCESTUDIESCultured cells were harvested, washed oncewith medium containing 5% fetal calf serum,and incubated with FITC conjugated Ber-ACT8 and either with CD2, CD3, CD4, CD8,CD20, CD25, or CD 14, for half an hour,washed twice in cold PBS, and analysed on anEPICS flow cytometer. Signals on cells weremeasured by appropriate forward light scatterand 90' light scatter criteria with gate settingsto include small and large lymphocytes and toreduce monocyte contamination. Horizontaland vertical cursor settings were determined bydefining background level with controlantibodies such as mouse IgG, conjugatedeither with FITC or Phycoerythrin.

INDIRECT DOUBLE COLOURIMMUNOFLUORESCENCE STUDIESSuspensions of peripheral blood lymphocyteswere double stained with monoclonalantibodies of different immunoglobulin sub-classes in the following combinations: CD22(To15, IgG2b)/Ber-ACT8 (IgG1); CD22(Tol5, IgG2b)/B-ly7 (IgGI); CD3 (OKT3,IgG2a)/Ber-ACT8 (IgGI); CD3 (OKT3,IgG2a)/B-ly7 (IgGI); CD4 (OKT4, IgG2b)/Ber-ACT8 (IgGI); CD4 (OKT4, IgG2b)/B-ly7 (IgGI); CD8 (OKT8, IgG2a)/Ber-ACT8(IgGl); CD8 (OKT8, IgG2a)/B-ly7 (IgGl).Appropriate isotype specific goat anti-mouse



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Kruschwitz, Fritzsche, Schwarting, Micklem, Mason, Falini, Stein

IgG antisera (Southern BiotechnologyAssociation, Birmingham, Alabama, USA),conjugated with either fluorescein or tetra-methylrhodamine (Southern BiotechnologyAssociation, Birmingham, Alabama), wereused as second reagent for each monoclonalantibody. Negative controls for eachexperiment were performed using mousemonoclonal antibodies unreactive with humandeterminants, followed by the appropriatefluorochrome conjugated goat anti-mouseantibody, or using the latter reagent alone. Thespecificity of the isotype specific goat anti-mouse fluorochrome-labelled antibodies wasproved by their non-reactivity with mono-clonal antibodies of inappropriate isotype.

CROSS-BLOCKING STUDIES OF FLUORESCEINATEDBer-ACT8 WITH HML- 1 AND B-1y71 x 10'MAPS 16 cells were preincubated with100 jpl of unlabelled HML- 1 at concentrationsranging from i0 5 to 0 2 mg/ml and with 100 p1of unlabelled B-ly7 or Ber-ACT8 ascites atvarious concentrations, for 30 minutes. Cellswere washed twice with cold PBS followed byincubation with the FITC labelled antibodyBer-ACT8. As a negative control, a FITClabelled irrelevant antibody was used. Datawere analysed by flow cytometry using anEPICS 752 cell sorter (Coulter, Florida, USA).

BIOCHEMICAL CHARACTERISATION OF THETARGET ANTIGENCells of cell line MAPS16, HUT-EBV1(established by co-culturing HUT 102 andirradiated BJA-B; Fritzsche G, et al unpub-lished observations) and a case of hairy cellleukaemia were externally radioiodinated using3 mCi ['25J] (Amersham) by the lactoperoxi-dase (Sigma) technique, as described by Yuanet al,16 with modifications. The labelled cellswere lysed with PBS containing 1% Triton X-100 and 2 pmol phenylmethylsulfonyl fluoride(PMSF) (Sigma). For internal labelling,2 x 108 HUT-EBV1 cells were washed twicein methionine-free RPMI 1640 medium(Gibco, Karlsruhe) and then precultured fortwo hours at a concentration of 1 x 107 cells/ml in the same medium supplemented with15% dialysed fetal calf serum. Then the cellswere incubated in 1 mCi/ml of [35S] methionine(specific activity > 600 Ci/mmol, Amersham,Buchler) for 30 minutes. The incorporation ofthe radioactive methionine was terminated byadding 200 ml of prewarmed RPMI 1640 con-taining methionine and supplemented with20% fetal calf serum. For analysis, cells wereharvested at various times, washed three timeswith medium, and the cell pellet was lysed inPBS containing 0-02% NaN3 (Merck, Darm-stadt), 1% NP40 (LKB, Bromma, Sweden),and 1 mmol PMSF (Sigma) for two hours at4°C. Immunoprecipitations from the differentlysates were performed according to themethod of Wano et al."7 The precipitates weresubjected to gradient and non-gradientpolyacrylamide electrophoresis under non-reducing and reducing conditions.

DETERMINATION OF THE CARBOHYDRATE MOIETYImmunoprecipitates were dissolved in 90 M1 ofa 1 M TRIS-HCL buffer (pH 6-8) containing0-3% sodium dodecyl sulphate, 10% glycerol,and 5% mercaptoethanol, and boiled for fiveminutes. After centrifugation the supernatantwas removed and split into 30 p1 aliquots. Onealiquot was incubated in a buffer containing10% n-octylglucoside, 0 1 M McIlvaine buffer(sodium citrate-phosphate buffer) pH 5-5,50 mM EDTA (Merck), and 10 mM1 10-phenanthroline hydrate (Sigma) and30 units/ml endoglycosidase F (Boehringer,Mannheim) for 24 hours at 37°C. The secondaliquot was incubated in a buffer containing0.01 M calcium acetate, 0-02 M sodiumcacodylate buffer (pH 6-5), 0 1 U neura-minidase (Boehringer, Mannheim) and 25 mU0-glycanase (Genzyme) for 24 hours at 37°C.The third aliquot was used as a control and wasincubated for 24 hours at 37°C in 30 pl of a 1 MTRIS-HCL buffer (pH 6 8) without enzymes.The incubated aliquots were analysed by 8 5%sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE).

PURIFICATION OF THE Ber-ACT8 ANTIGEN BYAFFINITY CHROMATOGRAPHYThe Ber-ACT8 antigen was purified by affinitychromatography on protein A sepharose andcoupled to CNBr-activated sepharose at 5 mgIg/ml gel. An NP40 extract of membranesprepared from frozen specific tissue from casesof hairy cell leukaemia was passed through acolumn ofBer-ACT8 sepharose and the boundproteins eluted with glycine buffer pH 2 5containing 0 5% NP40. Eluted material wasanalysed by SDS-PAGE and the gel stainedwith Coomassie Blue. Substitution of pH 2-5elution buffer with 10 mM EDTA was used todissociate the antigen subunits on the antibodycolumn. Material eluted from a Ber-ACT8column was radioiodinated and purified on anSDS-PAGE gel. Material eluted from excisedgel bands was used for protease V8 and trypsindigestion, and the resulting fragments wereanalysed by SDS-PAGE.

SEQUENTIAL IMMUNOPRECIPITATIONLysates of ['25J]-labelled HUT-EBV1 weresequentially immunoprecipitated with HML-1, mAb B-ly7, and Ber-ACT8. Immuno-precipitates were subjected to a discontinuous8-5% polyacrylamide gel for molecular weightdetermination under reducing conditions.Control immunoprecipitations were carriedout with the CD25 ACT-1 before and afterimmunoprecipitation with HML- 1, B-ly7, andBer-ACT8.

ResultsPRODUCTION OF Ber-ACT8Screening of supernatants from nearly 1500hybridomas on cytospins from peripheralblood lymphocytes, PHA blasts, MAPS 16cells, MT2 cells, and tissue sections fromspleen showed two clones unreactive withperipheral blood lymphocytes and MT2 cells,but reactive with 50% of PHA blasts, most

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Figure1 (A) Ber-ACT8 staining offresh, isolated mononuclear blood cells. Only one lymphocyte is positive. Monocytes andgranulocytes areunstained (APAAP); (B) Ber-ACT8 staining ofperipheral blood cells after stimulation with PHA for seven days. About 50% of the blasts are

positive. Note presence ofBer-ACT8 positive and Ber-ACT8 negative mitotic cells (arrowed) (APAAP); (C) Ber-ACT8 staining of normalspleen. Note positive cells distributed in the red pulp and the T zones (frozen section, APAAP); (D) Ber-ACT8 staining of activated monocytesstimulated by plastic adherence for three days (APAAP); (E) Ber-ACT8 staining of the mucosa of normal small intestine. Most intraepithelial Tcells are positive (frozen section, APAAP); (F) Ber-ACT8 staining of a case of a hairy cell leukaemia. Note intensive labelling of the hairy cellsin the absence of staining of any other cell type (APAAP, cytopreparation of isolated mononuclear blood cells).

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Table 2 Reactivity ofBer-ACT8/B-ly7 positive peripheral blood cells with B and Tcell antibodies*

Experimentt

Pairs ofprimary antibodies 1 2 3 4 5 6 7 8 9 10

CD22/Ber-ACT8 0 0 0 0 0 0 0 0 0 0CD22/B-ly7 0 0 0 0 0 0 0 0 0 0CD3/Ber-ACT8 3 1 4 2 3 2 1 2 2 3CD3/B-ly7 3 1 3 2 2 2 1 1 5 2 2-5CD4/Ber-ACT8 0 0 0 0 0 0 0 0 0 0CD4/B-ly7 0 0 0 0 0 0 0 0 0 0CD8/Ber-ACT8 3 1 3 1.5 2 5 2 1-5 2 1.5 2 5CD8/B-ly7 2-5 1 3-5 2 2 2 1 1-5 2 3

*As shown by indirect double colour immunofluorescence assay.tResults are expressed in percentage of cells positive for the first antigen that also coexpressedthe second.

MAPS 16 cells, and a significant proportion ofsplenic red pulp lymphocytes. One of these twoclones was designated Ber-ACT8, and chosenfor further characterisation: its isotype is IgG1.

REACTIVITY OF Ber-ACT8 WITH NORMAL ANDACTIVATED PERIPHERAL BLOOD CELLSFigure 1A shows that Ber-ACT8 did not reactwith fresh blood monocytes or granulocytes.Among the lymphocytes, only a small propor-tion (varying between 0-1%-2%, dependingon the donor) were positive. In double colourimmunofluorescence studies the Ber-ACT8positive fresh blood lymphocytes also bound toCD3 and CD8, but not to CD4 or B cellantibodies (table 2).

Reactivity of up to 50% of T cells withBer-ACT8 could be induced by stimulating theT lymphocytes with PHA or ConA. The firstBer-ACT8 positive T blasts appeared on thethird day (fig 1B) and disappeared between the15th and 17th days after stimulation (fig 1B).Flow cytometric double immunofluorescencelabelling experiments with various pairs ofmonoclonal antibodies showed that 80-90% of

Figure 2 Kinetic ofexpression of the Ber-ACT8 antigen onperipheral bloodlymphocytes afterstimulation with PHA.APAAP staining andsingle and two colourimmunofluo escenceanalysis or an EPICSflowcytometer

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Table 3 Reactivity ofBer-ACT8 with permanenthuman cell lines

Cell line Ber-ACT8

T cell derived cell lines:MOLT-4 -

MT-2 -

MOT _HUT 102 -

MAPS16 ++HUT-EBV1 + +Pre-B cell lines:NALM 6 -

NALM 12 -

B cell lines:WEWAKE I -

B-ALL 1 -

B 95-8 -

RAJI -

CESS -

BJA-B -

DAUDIU 266 _JOK _Histiocytic cell line:DHL-1 -

Hodgkin's disease cell lines:L 428 -

L 540 -

L 591 -

COLE _HOLDEN -

KM-H2 -

Myeloid cell lines:KG-1 -

THP-1 -

HL-60 -

K 562 -

U 937 _Carcinoma cell lines:MCF-7 (Mamma Ca)ALA (Mamma Ca)

Key: - no staining; + + strong staining.

Ber-ACT8 blasts were CD8 positive, and lessthan 20% CD4 positive (fig 2). Experimentswith purified activated CD4 T cells andactivated CD8 T cells produced similar results.Ber-ACT8 labelled only a small proportion ofactivated peripheral blood or splenic B cells,generated by incubation with S aureus. Mostperipheral blood monocytes became weaklyreactive with Ber-ACT8 when cultured on aplastic or glass surface for longer than two days(fig ID).Among the many permanent cell lines tested,

the only two reactive with Ber-ACT8 were twonewly established cell lines MAPS16 andHUT-EBVl (table 3).

STAINING OF Ber-ACT8 IN NORMAL HUMANTISSUEIn lymph nodes and tonsils Ber-ACT8 stainedsome scattered small lymphoid cells preferen-tially present in the T zones and in folliclemantles, but not in germinal centres (fig 3). Intonsils Ber-ACT8 additionally strongly stainedmany cells in the oral mucosa and, more

weakly, some lymphoid cells in the underlyingconnective tissue layer (fig 3). In the spleen Ber-ACT8 stained 8-10% of cells mainly dis-tributed in the red pulp; a few Ber-ACT8positive cells were seen in the T zone of thewhite pulp (fig 1C). Double staining withdifferent pairs of monoclonal antibodies on

lymphocytes isolated from spleens identifiedthe Ber-ACT8 positive cells as T cells of CD8type. Most intraepithelial lymphocytes in theoral mucosa, and many lymphoid cells in theepithelium and the submucosa of the oesoph-

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Figure 3 Ber-ACT8staining of a humanslightly hyperplastic tonsil.Scattered Ber-ACT8positive lymphocytespresent in the T zones andfollicle mantle. Insert:tonsillar mucosacontaining many Ber-ACT8 positivelymphocytes (frozensection APAAP).

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Table 4 Reactivity ofBer-ACT8 with T cell lymphomas

Ber-ACT8(positive cases petotal studied caseType of lymphoma

Precusor T cell lymphomas:Lymphoblastic lymphomasPeripheral T cell lymphomas:Nodal pleomorphic T cell lymphomasExtranodal pleomorphic T cell lymphomasCutaneousIntestinalVocal cordMesenterial lymph node and liver

Angioimmunoblastic T cell lymphomasLymphoepithelioid T cell lymphomasImmunoblastic T cell lymphomasNodal anaplastic large T cell lymphomasExtranodal anaplastic large T cell lymphomasTotal

Precursor B cell lymphomas:Lymphoblastic pre-B cell lymphomas (SIg negative)Lymphoblastic B cell lymphomas (SIg positive)Peripheral B cell lymphomasChronic lymphocytic leukaemiaCentrocytic lymphomasCentroblastic-centrocytic lymphomasCentroblastic lymphomasImmunoblastic lymphomasLymphoplasmocytoid lymphomasPlasmocytomasBurkitt's lymphomasLow grade MALT lymphomasHairy cell leukaemiaTotal

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agus and the stomach, expressed the Ber-ACT8 antigen. In the gut Ber-ACT8 labellednearly all, if not all, intraepithelial T cells andabout 20-50% of the lymphoid cells in the

i4:bi lamina propria (fig IE). In double labellingexperiments on isolated lamina propria cells allBer-ACT8 positive cells were positive for pan-T cell markers, such as CD3, CD2, and most ofthem coexpressed CD8. Ber-ACT8 positivecells were also found in the bronchial epi-thelium.

Screening of a wide spectrum of tissues fromAV

various organs (brain, skin, oral mucosa,

oesophagus, stomach, small intestine,pancreas, liver, lung, breast, kidney, urinarybladder, prostate, testes, ovary, thyroid gland,parotid gland, muscle) showed that Ber-ACT8

If4 did not react with any cells other than thosedescribed above.

S' TYPE OF T CELL RECEPTOR EXPRESSED BYBer-ACT8 POSITIVE T CELLS

Staining of adjacent sections of the spleen andgut as well as cytospins of PHA blasts withBer-ACT8, ,BF1, and anti-TCR-y6 showed thatthe number ofTCR-yb positive T cells accoun-ted for less than 10% and the TCR-3 positivecells for more than 90% of the Ber-ACT8positive cells. The widely congruent distribu-tion of JIFl positive T cells and Ber-ACT8positive T cells suggests that most Ber-ACT8positive T cells express the ,B chain of the TCR.The Ber-ACT8 positive MAPS16 cells andthree of the four Ber-ACT8 positive T celllymphomas also coexpress the TCR-f chain.

STAINING OF Ber-ACT8 IN REACTIVE DISEASED

LYMPH NODES

Staining with Ber-ACT8 of dermatopathiclymphadenopathy tissue containing sheets ofCDla positive interdigitating cells, confirmedthat Ber-ACT8 is unreactive with these cells.

!S) Some epithelioid type macrophages in smallgranulomas (present in Piringer's lympha-denitis) and large granulomas (present insarcoidosis or tuberculosis) were, however,faintly positive. This staining could not beblocked by preincubation with human IgG.The large granulomas usually contained manyBer-ACT8, CD8 positive T cells.

PROLIFERATIVE ACTIVITY OF Ber-ACT8 POSITIVE

TISSUE CELLS

In tonsil, spleen, and gut the proliferativeactivity of Ber-ACT8 positive cells was

analysed by double labelling with Ber-ACT8and Ki-67. Ki-67 reacts with a nuclear antigenthat is expressed in late G1, S,Mand G2 phases,but not in the Go phase of the cell cycle. Nearlyall Ber-ACT8 positive cells proved Ki-67negative, indicating that they were in theresting phase.

REACTIVITY OF Ber-ACT8 WITH MALIGNANTLYMPHOMASOne hundred and seventy two malignant

s werelymphomas were stained with Ber-ACT8. All68 non-Hodgkin's lymphomas of T cell type

edthe were negative except for five cases of pleomor-phic T cell lymphoma of the jejunum

*The two positive cases showed a preferentially base layer localised epidermotropism.tTwo of the cases coexpressed the CD8 antigen and all were CD4 negative; all caseassociated with coeliac disease.tThe Ber-ACT8 positive case was localised in the left quadriceps and in the minor pelvis.§In two cases only 80-90% of the hairy cells were positive, whereas all hairy cells expresBer-ACT8 antigen in 10 cases.

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Figure 4 SDS-PAGE (5-20% gradient) of surface iodinated Ber-ACT8 antigenprecipitatedfrom the cell line MAPS16 with Ber-ACT8 (lane B), HML-1 (lane C),and B-ly7 (lane D). Positive control using ACT-I in lane A and negative control usingan unreactive monoclonal antibody in lane E. The three precipitated bands show a

molecular mass of 150, 125, and 105 kilodaltons under reducing conditions. Under non-

reducing conditions the moleclar mass of the bands is slightly higher (170, 140 and 120kilodaltons).

associated with coeliac disease and five otherspresenting extranodally or in the abdomen(table 4). Only one B cell lymphoma and 12 outof 12 cases of hairy cell leukaemia tested were

reactive with Ber-ACT8 out of 104 cases (table4); the negative lymphoid neoplasms includedthree cases ofprimary gastric B cell lymphomasof centrocyte-like type-that is, low gradeMALT lymphomas. In three out of 14 cases ofHodgkin's disease most of the admixed reactiveT cells were Ber-ACT8 positive; in theremaining 11 cases only a minority of T cellsbound to the Ber-ACT8 antibody. Hodgkinand Sternberg-Reed cells were consistentlynegative in all 14 cases of Hodgkin's diseasetested.

COMPARISON OF Ber-ACT8 WITH HML-1, B-1y7,AND LF61Immunocytochemical and immunohistologicalstaining with HML-1,'8 B-1y7,'9 and LF6120produced the same results as those obtainedwith Ber-ACT8 in all tissue sections andcytospin preparations, with the only exceptionbeing that HML- 1 tended to stain some

epithelioid-type macrophages and cultivatedmonocytes slightly more strongly than theother two monoclonal antibodies.

Fluoresceinated Ber-ACT8 was titrated to apoint at which preincubation with unlabelled

Figure 5 SDS-PAGE(85%o) of surface-iodinated Ber-ACT8antigen precipitatedfroma hairy cell leukaemia withBer-ACT8 (lane A),negative control using anunreactive monoclonalantibody in lane B. Theelectrophorogram shows athree-band patternidentical in size andsimilar in intensity withthat seen from theMAPS16 cell line.

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Figure 6 SDS-PAGE of sequentialzmmunoprecipitation with Ber-ACT8, HML-1, B-1y7,and a CD25from '25I-labelled HUT-EBVl lysates.Lane 1: precipitate ofACT-1 (CD25) beforeabsorption. Lane 2: precipitate ofBer-ACT8 afterabsorption of the lysate with ACT-1 coated agarosebeads. Lane 3: precipitate ofHML-1 after absorption ofthe lysate with ACT-1 coated agarose beads. Lane 4:precipitate of B-1y7 after absorption of the lysate withACT1 coated agarose beads. Lane 5: precipitate ofHML-1 after absorption of the lysate to Ber-ACT8coated agarose beads. Lane 6: precipitate of B-1y7 afterabsorption of the lysate to Ber-ACT8 coated agarosebeads. Lane 7: negative control using an irrelevantmonoclonal antibody. Lane 8: precipitate ofACT-i afterabsorption of the lysate to Ber-ACT8 coated agarosebeads. The 150, 125, and the 105 kilodalton bands arearrowed.

Ber-ACT8 totally inhibited labelling of thecells, while a control antibody did not. Pre-incubation with B-ly7 and LF61 abolishedlabelling, but HML-1 did not. This indicatesthat Ber-ACT8, B-ly7, and LF61 cross-blockeach other, but that HML- 1 and Ber-ACT8 donot.

IMMUNOBIOCHEMICAL STUDIESThe lysate of surface iodinated cells of the Tcell lines MAPS 16 (fig 4) and HUT-EBV1 andof hairy cell leukaemia (fig 5) were precipitatedwith Ber-ACT8, HML- 1, B-ly7 and LF61 (thelast not shown). SDS-PAGE of all immuno-precipitates gave an identical pattern, showing2 major bands of 150 and 125 kilodaltons, and aminor component of 105 kilodaltons underreducing conditions. Under non-reducing con-ditions the components showed a slightlyhigher molecular mass (170, 140, and 120kilodaltons). In some experiments a weak bandof 210 kilodaltons was precipitated.

In sequential immunoprecipitation experi-ments extensive removal of the radiolabelledBer-ACT8 antigen with Ber-ACT8 led to asubsequent lack of reactivity for HML-1 andB-ly7, and vice versa (data not shown).Unspecific loss of reactivity was ruled out byusing an anti-IL-2 receptor antibody, whichstill precipitated the IL-2 receptor antigenafter absorption (fig 6).

Unlabelled material from hairy cellleukaemia cells prepared by affinitychromatography on a Ber-ACT8-sepharosecolumn, when eluted under conditions of lowpH, comprised material of 150 and 105 kilo-daltons (reduced). The 105 kilodalton material

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could be eluted with EDTA, while the columnretained the 150 kilodalton material (data notshown). This suggests that Ber-ACT8 recog-nises the 150 kilodalton protein chain. The 150and 105 kilodalton bands produced distinctfragments when separately subjected to V8protease or trypsin treatment.To determine the degree and type of glyco-

sylation of the Ber-ACT8 molecule, immuno-precipitates were treated with endoglycosidaseF, or 0-glycanase and neuraminidase. Incuba-tion with 0-glycanase and neuraminidasereduced the molecular mass of the 150 kilo-dalton protein by 10, whereas endoglycosidaseF had no effect on the electrophoretic mobility.To elucidate the - biosynthesis of the Ber-

ACT8 antigen HUT-EBV1 cells were pulselabelled with [35S] methionine for 30 minutes.Samples of cells were taken 0, 15, 30, 60, 120and 300 minutes after the start of the labelling.One 150 kilodalton band could be detected atthe beginning of the chase time. After a chasetime of 15 minutes, one additional band of 125kilodaltons became visible. Identical resultswere obtained after prolongation of the chasetime to 300 minutes-that is, the 150 and the125 kilodalton bands remained unchanged.

DiscussionWe have reported on the production of a newmonoclonal antibody designated Ber-ACT8,with specificity for an antigen associated withlymphoid activation and gut mucosa T cells.The most relevant features of Ber-ACT8 are asfollows:1 It recognises a molecule or molecules consis-ting of three constant components of 150, 125and 105 kilodaltons as shown by investigationof externally labelled cells. In a preparation ofhairy cells the two components of 150 and 105kilodaltons seem to be non-covalently linked ina way dependent on divalent cations, and the150 kilodalton band carries the Ber-ACT8reactive epitope, because the smaller compon-ent can be eluted from the antibody affinitycolumn in the presence of EDTA. Peptidemapping of these two bands showed differentproteolytic fragments, indicating that theyrepresent different polypeptide chains.Enzymatic digestion studies showed that the150 kilodalton molecule contains a smallproportion (less than 10%) of 0-linked carbo-hydrates and sialic acid residues.

Pulse chase experiments showed that themajor 150 kilodalton component and the 125kilodalton band of the Ber-ACT8 molecule arerapidly synthesised and do not have a precusormolecule. The synthesis of the 105 kilodaltonband could not be clarified by the investigationsperformed.2 Ber-ACT8 reacts with 1-3% of fresh,peripheral blood CD8 T cells, but not withperipheral blood granulocytes, monocytes,CD4 positive T cells and B cells. This findingwas confirmed by a subsequent and inde-pendent study.2' Our studies further showedthat Ber-ACT8 strongly binds to nearly allactivated CD8 T cells, but remained unreactivewith more than 80% of activated CD4 T cells

and most B cells. Ber-ACT8 also reacts weaklywith most monocytes activated by adherence.None of the presently known activationantigens show similar restriction in theirexpression to activated CD8 positive T cells.The Ber-ACT8 molecule appears on CD8positive T cells relatively late following theactivation stimulus-that is, on the third day,and reaches its peak of expression on theseventh and eighth days, disappearing betweenthe 15th and 18th days. In this respect theBer-ACT8 antigen is reminiscent of the verylate activation (VLA) antigens.223 With the exception of the two newly estab-lished cell lines MAPS16 and HUT-EBV1,Ber-ACT8 does not stain any of the manyknown cell lines studied, in contrast to all otherantibodies to activation antigens (such asCD25, CD30, CD69, CDw7O), which arepositive on many lymphoid cell lines.4 In lymph nodes and tonsils Ber-ACT8 labelsfew small lymphoid cells in the paracorticalregion and follicle mantle, as well as within theoral mucosa. In the spleen it stains only lym-phoid cells in the red pulp and the T region ofthe white pulp. This distribution pattern of theBer-ACT8 positive cells in tonsils, lymphnodes, and spleen coincides with that seen withCD8. The difference is that CD8 stains more Tcells and, in the spleen, sinus lining cells aswell. By double labelling of suspended spleniccells, it could be shown that most Ber-ACT8positive cells were also CD8 positive, indicat-ing that a proportion of the red pulp CD8 Tcells also express the antigen detected by Ber-ACT8. Studies of extranodal lymphoid tissueshowed that Ber-ACT8 stains more than 90%of intraepithelial lymphocytes and 20-50% oflamina propria T cells in the small intestine. Inother intraepithelial layers such as oral mucosaand bronchial epithelium, Ber-ACT8 alsostains most T cells. Double labelling adjacentsections of lymph nodes, tonsils, small intes-tine, bronchial epithelium and other tissueswith Ber-ACT8 and Ki-67, directed at aproliferation-associated nuclear antigen,showed that nearly all Ber-ACT8 positive cellsin the organs mentioned were in a restingphase. This indicates that the expression of theBer-ACT8 antigen is not only induced byactivation events, but seems to be regulated byspecial tissue environmental signals.Apart from the above mentioned lymphoid

cells, Ber-ACT8 also weakly stains in vitroactivated monocytes and some in vivo activatedmacrophages-that is, some epithelioid typemacrophages and macrophages in some malig-nant lymphomas. No other cells of any othertissues are reactive with Ber-ACT8.5 Extension of the studies to malignant lym-phomas showed that Ber-ACT8 reactivityamong T cell lymphomas is restricted toextranodal types, occurring in the vocal cords,mesenterium, and jejunum-the latter beingassociated with enteropathy. Ber-ACT8proved negative with all B cell lymphomasexcept one extranodal B cell lymphoma ofcentroblastic morphology and 12 cases of hairycell leukaemia. Of interest is the non-reactivityof Ber-ACT8 with anaplastic large cell lym-

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phomas and Hodgkin and Stemnberg-Reedcells, which are probably related to activatedlymphoid cells because of their expression ofthe activation markers CD25, CD30, orCDw70. This absence of the Ber-ACT8antigen indicates that Ber-ACT8 keeps itsrestricted specificity even after malignant trans-formation, and indicates that the anaplasticlarge cell lymphoma cells (with rare exceptions)and Steinberg-Reed cells of Hodgkin's diseaseare not related to Ber-ACT8/CD25 positiveactivated T cells.Because reports about the TCR type on

murine gut mucosa T cells23 conflicted with ourinitial findings, we extended our studies on theTCR type of Ber-ACT8 positive cells. Ourresults show that most Ber-ACT8 positiveblood T cells seen following activation ofBer-ACT8 positive T cells in the lymphoidtissue are of TCR-a/,B type. This observationfits in with the finding that the Ber-ACT8positive permanent cell line MAPS 16 and threeof the four Ber-ACT8 positive T cell lym-phomas are also of TCR-a/,B type.Some of the findings described above are

reminiscent of the features of the two recentlypublished monoclonal antibodies, HML-1 andB-1y7.'8 19 HML-1 is described to react with allintraepithelial T cells, 20-50% of laminapropria T cells'8 and most or all cases of gut Tcell lymphomas associated with coeliac dis-ease,2426 and to precipitate two bands of 150and 105 kilodaltons.18 The second antibody,designated B-1y7, is reported to precipitate aband of 144 kilodaltons (unreduced), to reactwith a very small population of normal Blymphocytes and hairy cell leukaemia cells, butwith no other B cell neoplasms, and no T celllymphomas.'9 Although the reported featureswere not totally congruent with those found forthe Ber-ACT8 antigen, the similarities weresufficient to encourage us to compare HML-1and B-1y7 with Ber-ACT8. Both HML-1 andB-ly7 produced a very similar immunostainingin normal and pathological tissues as well aswith fresh resting blood lymphocytes andlectin-stimulated blood T cells. We have des-cribed the latter reactivity of HML-1.27 Whilethe staining results with Ber-ACT8 and B-ly7were identical, HML-1 differed slightly fromthe two other monoclonal antibodies in that itstained more strongly and a higher percentageof in vivo and in vitro activated macrophages.The similar reactivity of the antibodies wasconfirmed by the indistinguishable immuno-precipitation patterns of all three monoclonalantibodies. Sequential immunoprecipitationproved that all three antibodies (Ber-ACT8,B-ly7, and HML-1) react with the same 150/125/105 kilodalton molecular structure. Insubsequent experiments we showed that theantibody LF6 120 is also reactive with the sametrimeric antigen. Cross-blocking experimentsshowed that Ber-ACT8, B-ly7, and LF61 bindto the same or neighbouring epitopes, andHML-1 to a second epitope. This finding mightexplain the slightly different labelling reactivityofHML-1.

Recently, the selective reactivity of themonoclonal HML-1 with coeliac disease

associated T cell lymphomas of the gut waschallenged.28 With the new monoclonal Ber-ACT8, which reacts considerably more weaklywith activated macrophages, however, we con-firmed the absence of the molecule recognisedby HML-l/Ber-ACT8 from all lymph nodalperipheral T cell lymphomas. The frequentreactivity of lymph nodal T cell lymphomasdescribed by Pallesen et al28 thus seems to havebeen due to the use of sticky HML- 1 ascites,29or to positively labelled macrophages erro-neously regarded as tumour cells.

In a recent study30 B-ly7 was reported to bereactive with a case of adult T cell leukaemiaand a case of Sezary's syndrome. These findingsare no different to those presented here or in aprevious study because we also observed reac-tivity of Ber-ACT8 and HML-13' with somecases of cutaneous T cell lymphomas, exhibit-ing a preferentially base layer localised epi-dermotropism.To clarify the marker importance and the

functional properties of the Ber-ACT8 reactivemolecule its expression was correlated in aparallel study2" with the presence of adhesionmolecules (homing receptors), markers ofmaturation and previous activation, and theskin tissue association molecule CLA (cuta-neous lymphocyte antigen).32 These studiesshowed that, with Ber-ACT8 and HECA-452to CLA, three non-overlapping blood T cellpopulations (Ber-ACT8+, CLA-: 1-3%; Ber-ACT8-, CLA+: 10-15%; Ber-ACT8 ,CLA-:80-90%) can be defined, whereby the Ber-ACT8 positive T cells differ from the other Tcell subsets in that the Ber-ACT8 positive Tcells bear homing receptors for gut mucosaltissue but not for peripheral lymph nodes, thatthe Ber-ACT negative T cells carry receptorsfor peripheral lymph nodes but not for mucosaltissue, and that the Ber-ACT8 positive T cellsare found predominantly, if not exclusively, inthe memory or previously activated T cellsubset. These findings are consistent with amucosal derivation or mucosal homingpreference of the peripheral blood Ber-ACT8positive T cells, and make the reactivity ofintestinal T cell lymphomas with Ber-ACT8understandable. As a result of these findings, ithas been proposed that the Ber-ACT8/B-ly7/HML-1/LF61 reactive molecule should bedesignated as mucosa lymphocyte antigen(MLA).2' The clinical importance of MLAexpression on the tumour cells of some cases ofcutaneous T cell lymphomas and hairy cellleukaemia remains to be determined. The datapresented in this paper strongly suggest-incontrast to the paper by Visser et al '9-thatnormal MLA positive blood lymphocytes arenot the long sought physiological equivalents ofhairy cell leukaemia cells because MLApositive blood cells have aT cell and not a B cellphenotype.

I Uchiyama T, Broder S, Waldmann TA. A monoclonalantibody (anti-Tac) reactive with activated and function-ally mature human T cells. Production of anti-Tacmonoclonal antibody and distribution of Tac (+) cells.

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J Immunol 1981;126:1393-7.2 Uchiyama T, Nelson DL, Fleisher TA, Waldmann TA. A

monoclonal antibody (anti-Tac) reactive with activatedand functionally mature human T cells. Expression ofTacantigen on activated cytotoxic killer T cells, suppressorcells, and on one of two types of helper T cells. J Immunol1981;126:1398-403.

3 Schwab U, Stein H, Gerdes J, et al. Production of amonoclonal antibody specific for Hodgkin and Stemberg-Reed cells of Hodgkin's disease and a subset of normallymphoid cells. Nature 1982;299:65-7.

4 Stein H, Gerdes J, Schwab U, et al. Identification ofHodgkinand Steinberg-Reed cells as a unique cell type derivedfrom a newly-detected small cell population. Int J Cancer1982;30:445-59.

5 Corte G, Moretta L, Damiani G, Mingari MC, Bargellesi A.Surface antigens specifically expressed by activated T cellsin humans. Eur J Immunol 1981;11:162-5.

6 Stein H, Gerdes J, Schwarting R, Froese P, Lemke H. In:McMichael AJ, ed. Leukocyte typing III. Oxford: OxfordUniversity Press, 1987:574.

7 Knapp W, Dorken B, Rieber EP, et al. Leucocyte typing IV:White cell differentiation antigens. Oxford: OxfordUniversity Press, 1990.

8 Schwarting R, Dienemann D, Kruschwitz M, Fritzsche G,Stein H. Specificities of monoclonal antibodies B-ly7 andHML-1 are identical. Blood 1990;75:320.

9 Stansfield AG, Diebold J, Kapanci Y, et al. Updated Kielclassification for lymphomas. Lancet 1988;i:292-3.

10 Stein H, Gerdes J, Falini B. Phenotypic and genotypicmarkers in malignant lymphomas: Cellular origin ofHodgkin and Steinberg-Reed cells and implications forthe classification of T-cell and B-cell lymphomas. In:Seifert G, Hubner K, eds. Pathology of cell receptors andtumor markers. Application of immunocytochemistry andhybridization in tumor diagnosis. Stuttgart: Gustav FischerVerlag, 1987:121-44.

11 Blue ML, Daley JF, Levine H, Schlossman SF. Coexpres-sion of T4 and T8 on peripheral blood T cells demon-strated by two-color fluorescence flow cytometry.J Immunol 1985;134:2281-6.

12 Wysocki LJ, Sato VL. "Panning" for lymphocytes: Amethod for cell selection. Proc Natl Acad Sci USA1978;75:2844-8.

13 Gerdes J, Schwab U, Lemke H, Stein H. Production of amonoclonal antibody reactive with a human nuclearantigen associated with cell proliferation. Int J Cancer1983;31:13-20.

14 Cordell J, Falini B, Erber ON, et al. Immunoenzymaticlabeling of monoclonal antibodies using immunecomplexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J HistochemCytochem 1984;32:219-29.

15 Stein H, Gatter K, Asbahr H, Mason DY. Use of freeze-dried paraffin-embedded sections for immunohistologicstaining with monoclonal antibodies. Lab Invest 1985;52:676-83.

16 Yuan D, Vitetta ES. Biosynthetics, surface labeling, andisolation of membrane immunoglobulin. MethodsEnzymol 1984;108:426-33.

17 Wano Y, Uchiyama T, Fukui K, Maeda M, Uchino H,

Yodoi J. Characterization ofhuman interleukin 2 receptor(Tac-antigen) in normal and leukemic T cells: Co-expres-sion of normal and aberrant receptors on HUT-102 cells.J Immunol 1984;132:3005.

18 Cerf-Bensussan N, Jarry A, Brousse N, Lisowska-Grospierre B, Guy-Grand D, Griscelli C. A monoclonalantibody (HML-1) defining a novel membrane moleculepresent on human intestinal lymphocytes. Eur J Immunol1987;17: 1279-85.

19 Visser L, Shaw A, Slupsky J, Vos H, Poppema S. Mono-clonal antibodies reactive with hairy cell leukemia. Blood1989;74:320-5.

20 Flenghi L, Spinozzi F, Stein H, Kruschwitz M, Pileri S,Falini B. LF61: a new monoclonal antibody directedagainst a trimeric molecule (150 kDa, 125 kDa, 105 kDa)associated with hairy cell leukaemia. Br J Haematol1990;76:451-9.

21 Picker LJ, Terstappen LWMM, Rott LS, Streeter PR, SteinH, Butcher EC. Differential expression of homing-associated adhesion molecules by T cell subsets in man.J Immunol 1990;145:3247-55.

22 Hemler M, Jacobson JG, Brenner MB, Mann D, StromingerJL. VLA-1: a T cell surface antigen which defines a novellate stage of human T cell activation. Eur J Immunol1985;15:502-8.

23 Goodman T, Lefrancois L. Expression of the y-3 T-cellreceptor on intestinal CD8 intraepithelial lymphocytes.Nature 1988;333:855-8.

24 Isaacson PG, O'Connor NTJ, Spencer J, et al. Malignanthistiocytosis of the intestine: a T cell lymphoma. Lancet1985;ii:688-91.

25 Spencer J, Cerf-Bensussan N, Jarry A, et al. Enteropathy-associated T cell lymphoma (malignant histiocytosis oftheintestine) is recognized by monoclonal antibody (HML- 1)that defines a membrane molecule on human mucosallymphocytes. Am JPathol 1988;132:1-5.

26 Stein H, Dienemann D, Sperling M, Zeitz M, Riecken EO.Identification ofa T cell lymphoma category derived fromintestinal mucosa-associated T cells. Lancet 1988;ii:1053-4.

27 Schieferdecker HL, Ullrich R, Weiss-Breckwoldt AN, et al.The HML-1 antigen of intestinal lymphocytes is anactivation antigen. J Immunol 1990;144:2541-9.

28 Pallesen G, Hamilton-Dutoit SJ. Monoclonal antibody(HML-1) labelling of T-cell lymphomas. Lancet 1989;i:223.

29 Brousse N, Jarry A, Peuchmaur M, et al. Monoclonalantibody (HML-1) reactivity of T-cell lymphomas.Lancet 1989;ii:1 107-8.

30 Mulligan SP, Travade P, Matutes E, et al. B-ly-7, amonoclonal antibody reactive with hairy cell leukemia,also defines an activation antigen on normal CD8+ T cells.Blood 1990;76:959-64.

31 Sperling M, Kaudewitz P, Braun-Falco 0, Stein H.Reactivity of T cells in mycosis fungoides exhibitingmarked epidermotropism with the monoclonal antibodyHML-1 that defines a membrane molecule on humanmucosal lymphocytes. Am J Pathol 1989;134:955-60.

32 Picker LJ, Michie SA, Rott LS, Butcher EC. A uniquephenotype of skin-associated lymphocytes in humans.Am JPathol 1990;136:1053-68.

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