natural killer-type receptors for hla class i antigens are clonally expressed in lymphoproliferative...

British Journal of Haematology, 2000, 110, 525±536

Natural killer-type receptors for HLA class I antigens are

clonally expressed in lymphoproliferative disorders of natural

killer and T-cell type

T. Hoffmann,1 G. de Libero,2 M. Colonna,3 A. Wodnar-Filipowicz,2 J. Passweg,4 G. Favre,4

A. Gratwohl4 and A. Tichelli1 1Haematological Laboratory, 2Department of Research, Kantonsspital,3Basel Institute for Immunology, Basel, and 4Division of Haematology, Kantonsspital, Switzerland

Received 14 November 1999; accepted for publication 16 March 2000

Summary. In recent years, natural killer (NK) cells, as wellas subpopulations of T cells, have been found to expressdiverse NK receptors (NKRs) for HLA class I molecules. Wehave characterized NKR phenotypes in lymphoproliferativedisorders of NK or T-cell type. Peripheral blood of patientswith lymphoproliferative disorders (n � 9) was analysed bymultiparametric immunofluorescence flow cytometry witheight different antibodies against NKRs. Abnormal neoplas-tic cell populations from different types of NK or T-celllymphoproliferative disorders lacked diversity in their NKRrepertoires, i.e. all or none of the abnormal cells expressedindividual NKRs and this expression occurred at single

levels of intensity. This pattern of expression was specific forlymphoproliferative disorders as these resticted NKR reper-toires were not found either in healthy donors (n � 9) or inpatients with viral or autoimmune disease (n � 5). Weconclude that NKRs are clonally expressed in lymphopro-liferative disorders of NK or T-cell origin. NKR repertoiresmay represent a novel tool in diagnosing clonal disorders ofNK and T-cell type.

Keywords: natural killer, HLA receptor, lymphoproliferativedisorder, clonality, diagnosis.

Clonality may be considered necessary, although it is notsufficient, as a prerequisite for defining malignancy. Thus,phenotypic assessment of clonally expressed gene productsis an important tool for diagnosing malignant disease, inparticular lymphoproliferative disorders. Current diagnosisand classification of lymphoproliferative disorders of B-cellorigin use clonality of immunoglobulin (Ig) gene expressionas a tumour marker; similarly, loss of polymorphism in T-cellreceptor (TcR) usage is diagnostic for malignancies of T-cellorigin (Flug et al, 1985; Waldmann et al, 1985; Rockman,1997; Macintyre & Delabesse, 1999). Unlike B and T cells,natural killer (NK) cells lack genetically rearrangedmolecules (Mombaerts et al, 1992; Shinkai et al, 1992),but express diverse receptors for HLA class I molecules,termed natural killer receptors (NKRs), that are alsoexpressed by subpopulations of T cells (Lanier & Phillips,1996). Two major groups of NKRs are known in humans:(i) C-type lectin-like receptors that consist of heterodimers of

NKG2 and CD94 polypeptides and (ii) Ig superfamilyreceptors that include killer Ig-like receptors (KIRs), Ig-liketranscripts (ILTs), NKp44 and NKp46 (Aramburu et al,1990; Karlhofer et al, 1992; Colonna & Samaridis, 1995;D'Andrea et al, 1995; Wagtmann et al, 1995; Samaridis &Colonna, 1997; Sivori et al, 1997; Vitale et al, 1998).According to in vitro data, CD94/NKG2A heterodimers,NKp44 and NKp46 are expressed by most NK cells, whereasCD94/NKG2C heterodimers, KIRs and ILTs are clonallydistributed (Sivori et al, 1997; Lanier, 1998; Vitale et al,1998). Individual NK cells express multiple NKRs (Valianteet al, 1997). The phenotypic diversity of the NKR repertoireof total NK cell populations results from the expression of (i)different types and (ii) different numbers of NKRs byindividual NK cells (Moretta et al, 1990a, 1995; Lanieret al, 1995; Gumperz et al, 1996; Uhrberg et al, 1997;Valiante et al, 1997; Lanier, 1998). Allelic polymorphismand alternative splicing contribute to this phenotypicdiversity (Wong et al, 1991; Dohring et al, 1996a;Selvakumar et al, 1997; Vyas et al, 1998). Furthermore,cytokines have been shown to influence CD94 expression byT cells (Ponte et al, 1998; Bertone et al, 1999).

It is thought that HLA class I recognition by inhibitory

q 2000 Blackwell Science Ltd 525

Correspondence: T. Hoffmann, Heinrich Heine University, Insti-

tute for Haemostaseology and Transfusion Medicine, Mooren-strasse 5, D-40225 Duesseldorf, Germany. E-mail: till.hoffmann@

uni-duesseldorf.de

Table IA. Patient characteristics, genetic analyses and phenotypes of lymphocyte subpopulations in patients with lymphoproliferative disorders.

Patient Diagnosis Diagnostic findings Course Genetic analysis

Phenotype of abnormal

cell population*

Phenotype of normal

subpopulations*

LPD1 LGL leukaemia

NK cell type

Multisystemic disease,

increase of atypical LGLs

Rapidly fatal 46, XX, 217, 1mar1, i(7q)

by cytogenetics

CD32 CD56bright CD162

CD72CD32 CD56dull CD161

CD71/±

LPD2 Unclassified LPD

of NK type

Mild neutropenia, increased LGLs,

cutaneous abscesses

Chronic with need

for therapy

Germline TcR rearrangement CD32 CD562 CD8dull

CD161 CD71CD32 CD561 CD81/±

CD161 CD71

LPD3 LGL leukaemia

ab T-cell type

Neutropenia, increased LGLs Chonic without

need for therapy

Clonal TcR rearrangement CD31 CD8dull CD571

CD161 CD561CD31 CD8bright CD572

CD162 CD562

LPD4 LGL leukaemia

gd T-cell type

Splenomegaly, polyarthritis,

increased LGLs

Chronic with need

for therapy

Clonal TcR rearrangement CD31CD42 CD82

CD161 CD571 TcRgd1CD31 CD41/CD81

CD162 CD572 TcRab1

LPD5 LGL leukaemiaab T-cell type

Increased LGLs Chonic withoutneed for therapy

Clonal TcR rearrangement CD31 Vb13´11

CD81 CD161 CD571CD31 Vb13´12

CD81 CD162 CD572

LPD6 LGL leukaemia

ab T-cell type

Neutropenia, increased LGLs Chronic with need

for therapy

Clonal TcR usage CD31 Vb121

CD8dull CD561 CD571CD31 Vb122

CD81 CD562 CD572

LPD7 LGL leukaemia

ab T-cell type


need for therapy

Clonal TcR rearrangement CD31 Vb171

CD81 CD571CD31 Vb172

CD81 CD572

LPD8 LGL leukaemia

ab T-cell type


need for therapy

Clonal TcR rearrangement CD31 Vb91

CD81 CD571CD31 Vb92

CD81 CD572

LPD9 Unclassified LPD

of gd T cells

Lymphocytosis, splenomegaly,

coexisting hereditary elliptocytosis

Chronic without

need for therapy

Clonal TcR rearrangement CD31 TcRgd1

CD161 CD52 CD72CD31 TcRgd2

CD162 CD51 CD71

*Antigens in bold type are those used for gating in the NKR analysis. For definition of `abnormal' and `normal', see PATIENTS and METHODS. LGL, large granular lymphocyte; TcR, T-cell

receptor; nd, no data.

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Table IB. Patient characteristics, genetic analyses and phenotypes of lymphocyte subpopulations in patients with `reactive' disorders.

Patient Diagnosis Diagnostic findings Course Genetic analysisPhenotype of abnormalcell population*

Phenotype of normal T-/NKcell subpopulations*

RD1 CMV infection Fever, lymphadenopathy, Self-limiting nd CD31 CD81 CD571 CD31 CD81 CD572

hepatomegaly, increased LGLs,

CMV IgM1without specific

therapy

RD2 EBV infection Fever, lymphadenopathy, Self-limiting Germline TcR CD31 CD81 CD51 CD31 CD81 ²/hepatosplenomegaly, atypical without specific rearrangement CD71 CD162 CD572 CD32 CD561

lymphocytosis, EBV IgM1 therapy

RD3 EBV infection Fever, lymphadenopathy, Self-limiting Polyclonal CD31 CD81 CD51 CD31 CD81 ²/hepatosplenomegaly, atypical without specific TcR Vb usage CD71 CD162 CD572 CD32 CD561

lymphocytosis, EBV IgM1 therapy

RD4 Pure red cell

aplasia

Erythropoietic aplasia,

increased LGLs

Chronic

refractory

Germline TcR

rearrangement

CD31 CD81 CD571 CD31 CD81 CD572/

CD32 CD56 1

RD5 Cryo- Pulmonary arterial Chronic Germline TcR CD31 CD81 CD571 CD31 CD81 CD572/globulinaemia hypertension, glomerulonephritis,

lymphocytosis

rearrangement CD32 CD561

*Antigens in bold type are those used for gating in the NKR analysis. For definition of `abnormal' and `normal', see PATIENTS and METHODS. LGL, large granular lymphocyte; CMV,

cytomegalovirus; EBV, Epstein±Barr virus; nd, no data.

²Not distinguishable from abnormal expanded cell population.

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NKRs of cytotoxic cells is a self-defence mechanism thatspares normal self cells but allows destruction of virallytransformed or tumour cells with altered HLA expression(Karre, 1997). In addition, activating NKRs have beendescribed that can potentiate the cytotoxic response of NKcells (Moretta et al, 1995; Campbell et al, 1998).

We have investigated NKR repertoires in patients withNK- or T-cell-type lymphoproliferative disorders using apanel of eight different antibodies against NKRs. Clonal NKRpatterns were found in all neoplastic cell populations but innone of the controls, suggesting that the NKR repertoire is anovel tool for diagnosing clonal disorders of NK or T-cellorigin.

PATIENTS AND METHODS

Patients and controls. The characteristics of patients aregiven in Table IA and B. Nine patients with lymphopro-liferative disorders of NK cell type (LPD1 and LPD2) or T-celltype (LPD3±LPD9), five patients with `reactive' T lympho-cytosis (RD1±RD5) and nine healthy controls were studied.All patients had absolute lymphocytosis in peripheral bloodwith the exception of patient LPD1. This patient presentedwith skin eruptions, arthritis, pulmonary infiltrates, pleur-itis, pancytopenia, renal and cerebral involvement, hepato-pathy and ascites, as well as disseminated intravascularcoagulopathy. Among peripheral blood lymphocytes, thepercentage of CD561 NK cells was increased and cytohisto-logical examination revealed infiltration by granulatedlymphocytes of multiple organs. Patients LPD2±LPD9 hada clinically indolent disease course. Patient LPD9 had beensplenectomized for hereditary elliptocytosis. The spleen wasinfiltrated by T cells and the patient subsequently developedprogressive T lymphocytosis. In eight out of nine patientswith lymphoproliferative disorders, clonality of the diseasewas proven either cytogenetically (patient LPD1), by TcRgene rearrangement analysis [Southern blotting with TcRvariable region (V)b-specific probes in patients LPD3, LPD5,LPD7 and LPD8; polymerase chain reaction (PCR) with Vg-specific probes in patients LPD4 and LPD9; data not shown]or by phenotypic analysis of TcR Vb usage with a panel ofanti-TcRVbantibodies (LPD5±8; data not shown). In patientLPD2, no marker was available to investigate `abnormal' NKcell clonality; the TcR rearrangement was in germlineconfiguration.

Five patients with lymphocytosis in the context of viralinfection (RD1±RD3), pure red cell aplasia (RD4) orcryoglobulinaemia (RD5) were collectively considered ashaving `reactive' T-cell disorders. None had clinical evidenceof malignant disease and TcR analyses performed in four outof five patients (Southern blotting analysis with TcR Vb-specific probes in patients RD2, RD4 and RD5; TcR Vb usageby flow cytometry in patient RD3) showed polyclonalpatterns (data not shown).

The nine healthy controls were volunteer donors fulfillingthe Swiss Red Cross criteria for blood donation. Informedconsent of patients and normal donors was obtained. Thestudy was approved by the Ethical Commitee of theUniversity Hospital, Basel.T

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528 T. Hoffmann et al

q 2000 Blackwell Science Ltd, British Journal of Haematology 110: 525±536

Study design. Using three-colour flow cytometry, expres-sion of T- and NK cellular antigens by subpopulations of NK(CD561 CD32) or T cells (CD31 CD81/CD31 CD41) wasestablished. According to the predominant findings inhealthy donors, `normal' NK and T-cell phenotypes weredefined as (CD32 CD56dull CD161 CD71/± CD81/±) and(CD31 CD4bright/CD8bright CD51 CD71 CD162 CD562

CD572 CD252 TcRab1) respectively. Abnormal' pheno-types of expanded cell populations in patients were definedas absence of antigens expressed normally or, conversely, asexpression of antigens normally not present or as expressionof antigens at abnormal intensities. For each patient, two orthree antigens were defined for gating purposes, enablingdistinction between `abnormal' and `normal' subpopula-tions. In addition, in patients LPD5±LPD8, TcR Vb usage bythe `abnormal' subpopulation was demonstrated to berestricted to one single Vb chain, thus allowing for directgating of clonal cells. Subsequently, the pattern of NKRexpression was analysed with eight different antibodies(Table II) in normal, as well as abnormal, cell populationsusing four-colour flow cytometry.

Flow cytometric analysis. In the first step, analysis of T-and NK cell phenotypes was performed using fresh blood

samples. Aliquots of 50±100 ml of heparinized blood wereincubated with combinations of three antibodies specific forCD3, CD5, CD7, CD4, CD8, CD56, WT31 (anti-TcRab),11F2 (anti-TcRgd) (Becton Dickinson, Erembodegem, Bel-gium), CD16 (Dako, Glostrup, Denmark) and CD57 (Serotec,Oxford, UK) labelled with fluorescein isothiocyanate (FITC),phycoerythrin (PE) or peridinin chlorophyll (PerCP). Afterincubation at room temperature for 20 min, erythrocyteswere lysed with 1 ml of FACS lysing solution (BectonDickinson) and cells were washed once in phosphate-buffered saline (PBS). Additionally, in patients LPD5±LPD8, TcR Vbusage was assessed using a panel of antibodiesagainst different TcR Vb chains (anti-Vb1, 2, 3, 5.1, 7, 11,12, 13.1, 13.6, 14, 16, 17, 20, 21.3, 22 from CoulterImmunotech, Marseille, France; anti-Vb5, 8, 9, 23 fromPharmingen, San Diego, CA, USA). Flow cytometry wasperformed with a FACSCalibur cytometer (Becton Dick-inson). In a second step, NKR expression was analysed usingaliquots of the same blood that had been cryopreserved inliquid nitrogen with 10% dimethyl sulphoxide (DMSO).After rapid thawing, cells were preincubated with humanimmunoglobulin (10 g/l; Sandoglobin from Novartis Pharma,Bern, Switzerland) for 10 min at room temperature and

Fig 1. FACS analysis of NKR expression by NK and T cells of a healthy donor. The NKR repertoire of CD561 CD32 NK cells (R1) and CD31

CD81 T cells (R2) was compared by simultaneous gating. Shaded histrograms represent control stainings with isotype-matched control

antibodies and histograms with a bold line show specific stainings with eight different anti-NKR antibodies (see Table II), as indicated. x-axesare 4 log scaled. For details of staining and FACS analysis, see PATIENTS and METHODS.


NK-type HLA Receptors in Lymphoproliferative Disorders 529

washed. Eight aliquots of 2±5 � 105 cells each wereincubated with eight different anti-NKR antibodies for30 min at room temperature. The antibodies used (seeTable II) were EB6, GL183 and HP3B1 (all from Coulter/Immunotech, Marseille, France), DX9 (Becton Dickinson),HP3E4 kindly provided by M. Lopez-Botet (Madrid, Spain)and 5.133, 116.2 and GHI/75 (M. Colonna). Cells werewashed again, incubated with a secondary PE-labelled goatanti-mouse antibody (Dako) for 30 min on ice, followed bywashing and blocking of excess secondary antibody byincubation with 10 ml pure mouse serum for 10 min on ice.Subsequently, aliquots were incubated for 20 min on iceusing FITC/PerCP/allophyocyanin (APC)-labelled antibodiesthat were chosen according to the phenotypic featuresdiscriminating abnormal from normal NK or T-cell popula-tions (see first step). In parallel, control stainings withisotype-matched control antibodies were performed. Deadcell exclusion was performed by propidium iodide staining.Analysis was performed with a FACSCalibur cytometerequipped with two lasers. Data were stored as list mode files.

Using the cellquest software (Becton Dickinson),`normal' and `abnormal' populations were gated simulta-neously and NKR expression by these subpopulations wasvisualized as histogram plots. The percentage of NKR1 cellswas calculated using thresholds provided by isotype-matched control stainings.

RESULTS

NKR repertoire in healthy donorsThe percentages of NK cells (CD561 CD32) and cytotoxic Tcells (CD31 CD81) expressing NKRs were determined in theperipheral blood of nine healthy donors. The histogram plotof a typical analysis is shown in Fig 1 and the full results aresummarized in Fig 2. Individual NKRs were expressed byvariable proportions of NK or T cells, but the percentage ofNKR1 cells was higher in NK than in CD81 T-cellpopulations. As a single exception, more CD81 T cells indonor 8 carried NKRs detectable by antibody 5.133. TheCD94 receptor as analysed with antibody HP3B1 wasexpressed by most NK cells, whereas other NKRs wereexpressed by subpopulations of variable size or were absent.CD94 was also the predominating NKR among CD81 Tcells, although in none of the donors were all CD81 Tcells CD941. Subpopulations of CD941 cells weredistinguishable according to dull or bright staining inten-sities in all donors. In some cases, this was also observed forother NKRs, most often with antibodies 5.133 and EB6. NKand CD81 T cells expressed a range of 2±5 and 0±2 NKRsper cell, respectively, as analysed by antibodies with non-overlapping specificities (HP3B1, EB6, GL183, DX9, 116.2and GHI/75).

In summary, our results demonstrate the diversity of NKRexpression in individual healthy donors, resulting fromvariable proportions of NKR1 cells and variable levels ofNKR expression. The diversity of individual NKR repertoirestranslates into clearly different patterns among donors(Fig 2).

NKR repertoire in patients with lymphoproliferative disorders ofNK or T-cell typeAnalysis of cell-surface antigens of NK cells (CD56, CD16,CD7 and CD8) and T cells (CD3, CD4, CD8, CD5 and CD7),as well as TcR and lymphocyte `activation' markers (CD25and CD57), revealed that phenotypically abnormal popula-tions were present in all patients with lymphoproliferativedisorders, co-existing with phenotypically normal cells(Table IA). NKR repertoires were established for both cellpopulations simultaneously.

The results obtained with a patient with NK cell largegranular lymphocyte (LGL) leukaemia are shown in Fig 3.Abnormal NK cells (CD56bright CD162) were clearlyexpanded compared with normal NK cells. These abnormalcells were characterized by a uniform pattern of NKRexpression, i.e. all or none of the abnormal cells expressedindividual NKRs. Furthermore, stainings with HP3B1 andDX9 antibodies that detected the NKR expressed in thispatient were at single levels of intensity. Analysis of normalNK cells (CD56dull CD161), present in this patient in small

Fig 2. Percentages of NKR1 NK and T cells in nine healthy donors.

NKR expression was analysed with eight different anti-NKRantibodies, as indicated. Bars from the central column to the right

and left give the percentage of NKR1 NK and T cells respectively. For

legibility, identification numbers of every second donor only are

given within the central coloumn. NKRs expressed at different levelsof intensity are marked by #. nd, no data.



numbers only, showed a diverse NKR repertoire, similar tothe findings in healthy donors.

The results obtained with a patient with T-cell LGLleukaemia are shown in Fig 4. Abnormal T cells (CD8dull

CD571) uniformly expressed most of the NKRs analysed andexpression was at single levels of intensity. This pattern wasin sharp contrast to normal counterpart T cells in thispatient (CD8bright CD572), which were mostly NKR2. Onlysmall subpopulations expressed ILT-2 or CD94. Dull andbright subpopulations of CD941 cells were present.

Uniformity of the NKR pattern was also found in all of theother seven patients including LPD5±LPD8, in whom NKRexpression could be directly related to clonal T cells.

Table III summarizes the results in all nine patients withlymphoproliferative disorders (LPD1±LPD9). Expression ofNKRs by abnormal cell populations had two characteristicfeatures. First, individual NKRs were either present on allcells within the population or were entirely absent. In otherwords, co-existence of subpopulations expressing or lackingone individual NKR, which occurred in any of nine healthydonors, was never observed in the 71 NKR analyses ofabnormal cells from LPD patients. Second, single levels ofintensity were found for any NKR expressed, againcontrasting with findings in healthy donors. Furthermore,

these patterns of NKR expression by abnormal cells inpatients with NK- or T-cell-type lymphoproliferative dis-orders were different not only from NKR patterns in healthydonors but also from those of normal cells in the samepatients.

NKR repertoire in patients with `reactive' disordersTo test further the specificity of the `all or none' pattern ofNKR expression in patients with lymphoproliferative dis-orders, we analysed the NKR repertoire in diseases leadingto `reactive' T-cell expansion. Phenotypic profiles of T cells inthese patients are summarized in Table IB and a typicalexample of NKR analysis is shown in Fig 5. The proportionof NKR1 T cells was significantly higher among abnormalcells than in normal counterpart cells. However, none of theNKRs tested was expressed by all abnormal T cells. CD94expression was distributed among subpopulations withbright and dull levels of intensity. In all patients, onlyfractions of T cells were NKR1. This was also true for NKcells in these patients with `reactive' disorders, with theexception of CD94 being expressed by all NK cells frompatients RD4 and RD5 (Table III). Again, this CD94expression was distributed among subpopulations withbright and dull levels of intensity.

Fig 3. NKR expression in a patient with NK cell LGL leukaemia (LPD1). Non-T cells (CD32, gate R1) were gated according to the CD56bright

CD162 phenotype (abnormal NK cells, gate R2) or to the CD56dull CD161 phenotype (normal NK cells, gate R3). The NKR repertoire of thesetwo NK populations was compared.



DISCUSSION

Using a panel of eight different antibodies against naturalkiller-type HLA receptors (NKRs) in multiparametric flowcytometry, we have studied the phenotypic NKR repertoiresof subpopulations of T and NK cells in different clinicalconditions. Our data, together with published findings(Moretta et al, 1995; Gumperz et al, 1996; Uhrberg et al,1997; Valiante et al, 1997), clearly show that the NKRrepertoire in healthy subjects is heterogeneous and diverseamong individuals. This diversity results from (i) variableproportions of NK or T cells expressing individual NKRs and(ii) NKR expression at different levels of intensity. Incontrast, expanded NK or T-cell subpopulations in patientswith lymphoproliferative disorders display uniform NKRrepertoires, with respect both to proportions of NKR1 cellsand to levels of cell-surface expression. This was true forall the patients studied with different types of lymphopro-liferative disorders. As eight out of nine patients hadproven clonal disease and as homogeneous NKR patternswere directly related to clonal T-cell populations in fourpatients, we conclude that NKRs are clonally expressed inlymphoproliferative disorders of NK or T-cellular origin.

Earlier studies have shown interindividual diversity ofNKR repertoires in patients with lymphoproliferativedisorders of granular lymphocytes (Zambello et al, 1993;Cambiaggi et al, 1996). These studies investigating p58receptors used two-colour flow cytometry and thereforeNKR expression by abnormal and normal cells could not bedistinctively separated. A significant proportion of patientshad no detectable p58 expression, suggesting clonalexpansion of p582 cells. As major subpopulations of NKand T cells in healthy donors also lack p58 expression, noconclusion with respect to clonal patterns of NKR expres-sion could be drawn. Our study, using eight different anti-NKR antibodies in four-colour flow cytometry, detectedclonal expression of at least two NKRs by the abnormal cellpopulation in each of nine patients. Furthermore, five of thepatients (LPD2, LPD4, LPD6, LPD7 and LPD9) had beensuffering from a lymphoproliferative disorder for yearsbefore cells were taken for the present investigation. Thisindicates that the malignant clone had undergone extensivecell division in vivo and had been exposed to variousalterations of the milieu, including intercurrent infections orimmunosuppressive therapy (LPD4 and LPD6). Despite suchenvironmental influences, a strictly homogeneous NKR

Fig 4. NKR expression in a patient with T-LGL leukaemia (LPD3). T cells (CD31, gate R1) were gated according to the CD81 CD571 phenotype

(abnormal T cells, gate R2) or to the CD81 CD572 phenotype (normal T cells, gate R3). The NKR repertoire of the two populations wascompared.



repertoire was found. Thus, comprehensive analyses of theNKR repertoire allowed detection of clonality in differenttypes of lymphoproliferative disorders of NK or T-cell origin,irrespective of the stage of the disease.

In the present study, the NKR repertoire of abnormal cellsin patients with clonal disorders was compared with theNKR repertoire of abnormal cell populations in patients with`reactive' T-cell expansion in the context of viral orautoimmune disease. Although NKR expression wasincreased in `reactive' T-cell populations, none displayed auniform NKR repertoire, which was true for NK cells also.Thus, patterns of NKR expression distinguish neoplasticdisease not only from normal but also from `reactive'disorders. Uniformity of the NKR repertoire as a clonalmarker would be of particular importance in lymphoproli-ferative disorders of NK cell type as, at present, diagnosisand therapy are hampered by the lack of such markers(Loughran, 1993; Zambello et al, 1993; Chan et al, 1997). Itis tempting to suggest that assessment of the NKR repertoire,which can be established within hours by multiparametricflow cytometry, may represent a new and specific diagnostictool in lymphoproliferative disorders of NK or T-cell origin.

Although the number of patients in our study was small,characteristic abnormalities of the NKR repertoire weredetected in all patients with lymphoproliferative disordersand in none of the controls, thus arguing for the relevance ofNKR repertoire assessment as a diagnostic tool.

The NKR family includes inhibitory as well as activatingisoforms, which may be co-expressed by the same cell(Lanier, 1998). Activatory and inhibitory heterodimers ofCD94/NKG2 receptors may differ with respect to staininglevels with anti-CD94 antibodies (Moretta et al, 1997). Invivo clonally expanded populations of granular lymphocytesreacting with anti-p58 antibodies have been shown toexpress either inhibitory or activatory isoforms (Cambiaggiet al, 1996). Our screening of NKR repertoires in differenttypes of lymphoproliferative disorders has shown uniformitynot only with respect to expression or absence of NKRs butalso with respect to levels of staining intensity. At present, itis not clear whether such phenotypic uniformity of the NKRrepertoire reflects the presence of only the activatory orinhibitory NKR forms. According to our data, furtherfunctional analyses in NK or T-cell lymphoproliferativedisorders will have to address not only functional pheno-

Table III. NKR repertoire as established with eight different anti-NKR antibodies in nine patients with lymphproliferative disorders (LPD1±

LPD9) and in five patients with reactive lymphocytosis (RD1±RD5).

NKR1 cells (%)

HP3B1 EB6 GL183 DX9 5.133 HP3E4 116.2 GHI/75

Patient a n a n a n a n a n a n a n a n

LPD1 100 100 0 26 0 29 100 23 0 27 0 12 0 0 0 62

LPD2 100 71 100 25 0 11 100 29 100 29 100 26 0 9 100 47LPD3 100 17 100 0 100 0 0 0 100 0 100 0 0 0 100 9

LPD4 100 56 0 0 0 0 0 0 0 0 0 0 0 0 100 35

LPD5 100 11 0 0 0 0 0 0 100 14 0 0 0 0 100 26

LPD6 100 53 0 0 0 7 0 0 0 8 0 0 0 0 100 44LPD7 100 15 0 0 0 0 0 0 0 0 0 0 0 0 100 7

LPD8 100 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0

LPD9 100 90 0 0 0 20 0 14 100 18 0 66 0 63 nd nd

NKR1 cells (%)

HP3B1 EB6 GL183 DX9 5.133 HP3E4 116.2 GHI/75

Patient a n NK a n NK a n NK a n NK a n NK a n NK a n NK a n NK

RD1 86 57 nd 14 0 nd 20 6 nd 9 0 nd 20 6 nd 11 0 nd 0 0 nd 33 14 nd

RD2 66 x 92 0 x 25 0 x 30 nd nd nd 0 x 23 0 x 34 0 x 29 nd nd nd

RD3 54 x 90 0 x 42 0 x 37 nd nd nd 27 x 24 32 x 20 28 x 0 nd nd ndRD4 82 56 100 10 0 8 10 0 10 0 0 0 0 0 0 0 0 8 0 0 0 82 34 21

RD5 81 72 100 8 0 33 0 0 28 0 0 10 10 12 53 0 0 28 0 0 0 30 10 47

a, abnormal population; n, normal counterpart; NK, natural killer cells (for definition see Table I and Patients and methods). nd, no data; x,

no data because normal cells were not distinguishable from abnormal expanded cells. Values $ 95% are given as 100% and values # 5% are

given as 0%.



types of NKRs but also co-signalling by several receptorsexpressed by the same cell.

It remains to be demonstrated whether functionallydefined NKR patterns in lymphoproliferative disorderscontribute to the biology of a given tumour and thus tothe clinical course. T-LGL leukaemia and NK-LGL leukaemiarepresent opposite extremes of malignant disease. Whereasthe natural course of NK-cell LGL leukaemia is extremelyprogressive and invariably leads to death within weeks, T-cell LGL leukaemia presents with limited tumour load and astable course for long periods of time (Loughran, 1993;Richards et al, 1995; Chan et al, 1997); indeed, there isongoing debate as to whether clonal expansions of largegranular T lymphocytes are truly leukaemic (Lamy et al,1998; Melenhorst et al, 1999). As it is now possible todetermine clonal NKR expression, lymphoproliferative dis-orders of NK or T-cell type thus seem to represent anattractive model for the study of NKR function in humandiesase.

ACKNOWLEDGMENTS

This work was supported by grant no. 5/98 of the Krebsliga

beider Basel, and by the Stiftung fuÈ r medizinische For-schung, Basel. We thank V. Meier, D. Zimmermann and H. J.Mueller for performing the genetic analyses.

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