Immunophenotypic Characterization of Normal Blood CD56 +lo Versus CD56 +hi NK-Cell Subsets and Its Impact on the Understanding of Their Tissue Distribution and Functional Properties

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Lima et al. Blood Cells, Molecules, and Diseases (2001)27(4) July/Aug: 731–743

doi:10.1006/bcmd.2001.0443, available online at http://www.idealibrary.com on

Immunophenotypic Characterization of Normal Blood CD561lo

Versus CD561hi NK-Cell Subsets and Its Impacton the Understanding of Their Tissue Distributionand Functional PropertiesSubmitted 07/12/01(Communicated by M. Lichtman, M.D., 07/12/01)

Margarida Lima,1,2 Maria dos Anjos Teixeira,1 Maria Luıs Queiros,1 Marina Leite,1

Ana Helena Santos,1 Benvindo Justiça,1 and Alberto Orfaõ3

ABSTRACT: In the present study we have compared the immunophenotypic characteristics of the CD561lo andCD561hi NK-cell subsets in a group of normal healthy adults. Our results show that CD561hi NK-cells display

reater light-scatter properties than CD561lo NK-cells at the same time they have higher levels of CD25CD122 IL-2 chains, together with a higher reactivity for HLA-DR and CD45RO and lower levels of CD4supporting that, as opposed to the majority of the CD561lo population, CD561hi NK-cells might correspond toubset of activated circulating NK-lymphocytes. Higher expression of the CD2 and CD7 costimulatory mound for the CD561hi NK-cells would support their greater ability to respond to various stimuli. In addD561hi NK-cells expressed higher levels of several adhesion molecules such as CD2, CD11c, CD44, CD62L compared to CD561lo NK-cells, supporting a particular ability of these cells to migrate from bloo

issues and/or a potential advantage to form conjugates with target cells. Interestingly, CD561lo and CD561hi

NK-cells showed a different pattern of expression of killer receptors that might determine different acrequirements for each of these NK-cell subsets. For instance, absence or low levels of CD16 expressexplain the lower antibody-dependent cytotoxicity activity of CD561hi NK-cells. On the other hand, the virtuabsence of expression of the CD158a and NKB1 immunoglobulin-like and the greater reactivity for thlectin-like killer receptors on CD561hi in comparison to CD561lo NK-cells might determine different MHC-claspecificities for both NK-cell subsets, a possibility that deserves further studies to be confirmed.© 2001 Academiress

Key Words:NK-cells; immunophenotype; flow cytometry; CD56; blood.

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INTRODUCTION

Natural killer (NK) cells are commonly dfined as CD32/TCR2 large granular lymphocytethat express CD56 and/or CD16 and mediateMHC restricted cytotoxic functions (1). Two mjor NK-cell subsets differing on the expressionCD56 on the cell surface have been describefar: CD561lo and CD561hi NK cells (2). CD561lo

NK-cells predominate in blood whereas CD561hi

NK-cells are more frequently detected in vari

1 Service of Clinical Hematology, Unit of Cytometry, Hospital Geral2 Correspondence and reprint requests to: Margarida Lima, Serviço de HemI, s/n, 4050 Porto, Portugal. Fax: 351-22-6004808. E-mail: mmc.lim
a@clix
3 Servicio de Citometrıá, Universidad de Salamanca and Centro de Investigń

731

organs and tissues including the uterine placdecidua (3–5), as well as in the lymphatic fl(5). Although it was initially suggested thCD561hi could represent precursors of the mnumerous CD561lo NK-cells (6), it was recentlshown that CD561hi NK-cells represent a funionally distinct subset of mature NK-cells whire primarily responsible for cytokine product

n response to monokines (7). In spite of thistill remains unclear whether CD561lo and

to Antońio, Porto, Portugal.ia, Unidade de Citometria, Hospital Geral de Santo Antońio, Rua D Manue.pt.
aciodel Cancer, Universidad de Salamanca, Salamanca, Spain.
1079-9796/01 $35.00Copyright© 2001 by Academic Press

All rights of reproduction in any form reserved

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Blood Cells, Molecules, and Diseases (2001)27(4) July/Aug: 731–743 Limaet al.doi:10.1006/bcmd.2001.0443, available online at http://www.idealibrary.com on

CD561hi NK-cells correspond to different typf NK-cells or alternatively they represent diffnt maturational/activation states of the sameell population. Preliminary studies indicated tD561lo and CD561hi NK-cells show differenatterns of expression several surface marker), suggesting that a detailed characterizatio

he molecules expressed on these NK-cell subould potentially contribute to clarify the relatiohip between them; nevertheless, at present iation on the immunophenotypic features of bK-cell subsets is not detailed enough to provcomprehensive view of their tissue distribut

nd functional properties.To gain further insights into the relationsh

etween these subsets of blood NK-cells inresent study we have compared the immunootypic characteristics of CD561hi with those o

the predominating CD561lo NK-cells, as regard tthe expression of killer receptors, activationlated markers, cell adhesion molecules and hing receptors; based on the differences obsewe discuss on the functional relevance of eacthese groups of molecules in determining NK-function and migratory properties.

MATERIALS AND METHODS

Samples

The enumeration of CD561lo and CD561hi

NK-cells and the immunophenotypic analysisthese NK-cell subsets were performed in frEDTA-K3 anti-coagulated blood samples frsix healthy adult individuals (blood donors)three males and three females—aged from 238 years (median age of 32 years) who ginformed consent to enter in this study.

Immunophenotypic Studies

Immunophenotypic studies were performusing a whole blood stain-lyse-and-then-wmethod and a direct immunofluorescence tnique. Briefly, 100ml of whole blood containinbetween 0.5 and 23 106 nucleated cells waincubated for 15 min at room temperature (RT
the darkness, with saturating amounts of the ap-
732

propriate monoclonal antibodies (MoAb). Lyof red blood cells and fixation of the leukocywas then performed by adding 2 ml of FAlysing solution [Becton/Dickinson Bioscienc(BD), San Jose, CA) diluted 1/10 (v/v) in distillwater and incubating the cells for another 10at RT. Cells were then washed once by centriing at 540g and resuspended in 0.5 ml of phphate-buffered saline (PBS) for acquisition inflow cytometer.

The following four-color combinations oMoAb directed against T- and NK-associaantigens conjugated with fluorescein isothcyanate (FITC)/phycoerythrin (PE)/PE-Cyan5 (PE-Cy5) and/allophycocyanin (APC) weused: CD2/CD7/CD56/CD3, CD5/CD7/CD5CD3, CD57/CD11c/CD56/CD3, CD38/CD11CD56/CD3, CD16/NKB1/CD56/CD3, CD158CD161/CD56/CD3, CD94/CD8/CD56/CD3, CD1CD25/CD56/CD3, CD44/CD40L/CD56/CDCD11a/HLA-DR/CD56/CD3, CD45RA/CD45ROCD56/CD3, and CD62L/CD28/CD56/CD3. Tsource and specificity of each of the MoAb reagare shown in Table 1.

Data acquisition was carried out in a FACCalibur flow cytometer (BD) equipped with15-mW air-cooled 488-nm argon laser and625-nm neon diode laser, using the Cell QUEsoftware program (BD). Information on a mimum of 2 3 105 events was acquired for eastaining and stored as list mode data. Foranalysis the Paint-a-Gate PRO software prog(BD) was used. As a first step, NK-cells wselected based on their CD32/CD561 phenotypeand the proportion of CD561lo and CD561hi NK-ells within the total CD561 NK-cell compart-

ment calculated. Then, each NK-cell subsetseparately analyzed for both its light-scatter—scatter (SSC) and forward scatter (FSC)—cacteristics and expression of the surface antirecognized by the MoAb referred above. For eantigenic determinant, the following charactetics were recorded: (i) percentage of positive cevaluated as the percentage of cells stained athe negative control value; (ii) intensity of exprsion, evaluated by the mean fluorescence inte(MFI), expressed in arbitrary relative linear un
of fluorescence scaled from 0 to 10,000; (iii)

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pattern of antigen expression—homogeneoussus heterogeneous—evaluated by the coeffiof variation (CV). In addition, the relative intesity of expression of each antigen on CD561hi

NK-cells compared to CD561lo NK-cells wasvaluated as the ratio between the MFI values

hese NK-cell subsets in each individual sam

tatistical Analysis

For all variables under study, median, metandard deviation, minimum and maximum walculated. To establish the statistical significaf the immunophenotypic differences obseretween CD561lo and CD561hi NK-cells, the

Mann–WhitneyU and thex2 tests were used, fontinuous and dichotomic variables (SPSS

TABLE 1

Specificities, Clones, and Sources of the Monoclonal AntiboUsed in the Present Study

Specificity Clone Conjugate Sourcea

CD2 SFCI3Pt2H9 FITC BCCD3 SK7 APC BDCD5 L17F12 PE BDCD7 3A1/1,7F3; 3A1E-12H7 FITC; PE CLB; BCD8 DK25 PE DKCD11a CLB-LFA-1/2 FITC CLBCD11b D12 PE BDCD11c S-HCL-3 PE BDCD16 3G8 FITC BCCD25 2A3 PE BDCD28 L293 PE BDCD38 LD38 FITC CYTCD44 L178 FITC BDCD45RA L48 FITC BDCD45RO UCHL-1 PE BDCD56 NCAM16.2 PE BDCD57 HNK-1 FITC BDCD62L SK11 FITC BDCD94 HP-309 FITC PHCD122 MIK-b FITC CLBCD154 TRAP1 PE PHCD158a HP-3E4 FITC BDCD161 DX12 PE BDHLA-DR L243 PE BDNKB1 DX9 PE BD

a BC, Beckman Coulter (Miami, FL); BD, Becton/DickinsioSciences (San Jose´, CA); CLB (Amsterdam, The NetherlandYT, Cytognos (Salamanca, Spain); DK, Dako A/S (Gostenmark); PH, Pharmingen (San Diego, CA).

PSS, Chicago, IL).P values less than 0.05 were

733

onsidered to be associated with statisticallyificant differences.

ESULTS

CD561 NK-cells accounted for a mean7 6 8% of the blood lymphocyte compartme

n the blood from the 6 normal individuals iluded in this study, the mean absolute NK-ount being of 2646 183 3 106/L. From theK-cells, the majority showed dim CD56 exprion (CD561lo) (mean of 936 7%, ranging from0 to 99%) while a minor fraction displayedigh reactivity for CD56 (CD561hi) (7 6 7%,

ranging from 1 to 20%). These values diddiffer significantly from those found in a group70 healthy adult individuals used for determition of normal reference values in our lab (9366% versus 76 6%).

Apart from their differential expressionD56 these NK-cell subsets displayed dist

ight-scatter (FSC and SSC) characteristics.ordingly, CD561hi NK-cells showed higher FSP 5 0.041) and SSC (P 5 0.026) valueshan those observed for CD561lo NK-cells, withdentical FSC and SSC mean ratios (1.16 0.1).

From the phenotypic point of view, CD561hi

NK-cells expressed CD56 at levels that were6 1.5-fold higher than those observedCD561lo NK-cells (P 5 0.002)(Fig. 1),whichn turn exceeded those of CD561 T-cells by 3.36 1.5-fold. Interestingly, CD56 expression wextremely homogeneous on CD561hi compared tthe CD561lo NK-cells (P 5 0.002).

Besides the differential expression obserfor CD56, the two NK-cell populations undstudy also differed in the percentage of cellsstained positively (Table 2) and/or on the intenand pattern of expression (Table 3 and Fig. 1a large number of other antigens. Such phenotdifferences are illustrated in Fig. 2 and describelow in more detail.

T-Cell-Associated Markers

None of the two blood NK-cell subsets showsurface expression of CD3. By contrast, both N
cell subsets expressed the CD2 and CD7 antigens,

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although in both cases a CD22 NK-cell populationwas identified which represented a mean of 236 11and 6 6 3% of CD561lo and CD561hi NK-cellubsets, respectively (P 5 0.004). Upon comparinhe reactivity for both antigens in the CD561hi andCD561lo NK-cell subsets it was observed thatformer expressed CD2 and CD7 at higher intensthan those observed on CD561lo cells (ratio o2.6 6 0.6 and 2.96 1.2, respectively;P 5 0.002in both cases). In addition, both CD2 and CD7 wmore heterogeneously expressed on CD561low thanon CD561hi NK-cells (P 5 0.004 and 0.041, r-spectively). A variable proportion of CD561lo NK-ells showed dim CD5 expression (56 7%, rangingrom 0 to 18%), a CD561lo/CD51lo NK-cell popu-lation being clearly identified in 3 out of 6 caswhere they represent 2, 9, and 18% of total CD51lo

NK-cells; in these cases, CD5 expression wasmogeneous, and present at levels 9.6-6 1.2-foldlower than those observed for normal blood T-cIn contrast, CD561hi NK-cells were consistentnegative for CD5 (P 5 0.001).

The mean fraction of CD81 cells within theD561lo (556 16%) and the CD561hi (546 13%)K-cell subsets were similar, CD8 expression be

FIG. 1. Ratio between the intensity of antigen exubsets from six adult healthy individuals. Horizontal linaximum values.

ather heterogeneous in both cases.

734

dhesion Molecules and Homing Receptors

Virtually all NK-cells constitutively expresseomogeneously high levels of CD11a when cared to other circulating lymphocytes, its intity of expression being significantly lowerD561hi NK-cells compared to the predomin-

ng CD561lo NK-cell subset (ratio of 1.46 0.2;P 5 0.002). Incontrast, CD11b was heterogneously expressed on both cell subsets at simlevels (P . 0.05) whereas CD11c expressi

as significantly higher on the latter NK-cell suet (ratio of 1.6-6 0.3-fold; P 5 0.041). Ad-itionally, most CD561hi NK-cells were CD11c1

(92 6 6%) whereas its expression on CD561lo

NK cells was more variable, with a mean fractof 72 6 24% CD11c1 cells (P 5 0.065).Expression of CD11c was relatively heteroneous on both NK-cell subsets, although locoefficients of variation were found on CD561hi

than on CD561lo NK-cells (P 5 0.009). Inontrast, CD57 was expressed in large fractioD561lo NK whereas CD561hi were virtually

negative for CD57 (mean fraction of CD571 cellsof 66 6 14 and 16 1%, respectively;P 5

ion (MFI) obtained on CD561hi and CD561lo blood NK-cellresent median values and vertical lines extend the min

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All NK-cells were CD441, CD44 expressiobeing significantly higher (ratio of 4.16 1.1;P 5 0.002) andmore homogeneous (P 50.032) onCD561hi compared to CD561lo NK-cells. In addition, nearly all CD561lo NK-cellsexpressed CD62L whereas this molecule wasative or dimly positive in only a minor fractionCD561lo NK-cells (1006 0% versus 146 7%;P 5 0.002).

iller Receptors

In contrast to the CD561lo NK-cell compart-ent, in which the majority of cells (986 16%)omogeneously expressed CD16, CD561hi NK-

TABLE 2

Immunophenotypic Characteristics of CD561lo and CD561hi

Peripheral Blood NK-Cell Subsets from Adult HealthyIndividuals (n 5 6)

CD561 NK-cells

P valueCD561lo CD561hi

CD2 776 11 (64–91) 946 3 (91–98) 0.004CD5a 5 6 7 (0–18) 06 1 (0–1) 0.180

D7 996 1 (98–100) 946 5 (85–99) 0.015D8 556 16 (40–72) 546 13 (41–66) 1.000D11a 1006 0 (99–100) 1006 0 (100–100) 1.000D11b 926 13 (68–100) 846 8 (71–90) 0.151D11c 726 24 (28–93) 926 6 (84–100) 0.065D16 986 2 (95–100) 286 16 (10–47) 0.009D25 16 1 (0–2) 226 9 (9–32) 0.002D38 996 2 (95–100) 996 2 (97–100) 0.584D44 1006 1 (97–100) 1006 2 (94–100) 1.000D45RA 1006 0 (100–100) 986 2 (96–100) 1.000D45RO 46 4 (1–12) 126 5 (9–17) 0.026D57 666 14 (52–91) 16 1 (0–2) 0.002D62L 146 7 (4–22) 1006 0 (99–100) 0.002D94 596 11 (40–75) 986 3 (90–100) 0.000D122 1006 0 (100–100) 1006 0 (100–100) 0.937D158a 186 8 (10–25) 16 0 (0–1) 0.037D161 836 11 (63–95) 596 25 (31–96) 0.132LA-DR 23 6 13 (10–46) 836 8 (74–92) 0.002KB1 166 16 (0–47) 06 1 (0–1) 0.026

Note.Results are expressed as means6 standard deviation oercentage of positive cells; range is shown in parentheses.D28, and CD40L were constantly absent in NK-cells fromamples analyzed.

a A CD561lo/CD51lo NK-cell population was clearly identifien three of six cases, where they represent 2, 9, and 18% oD561lo NK-cells, while CD561hi were constantly CD52 (x2 test,

P 5 0.001).

ells either lacked CD16 or showed CD16 expres-

735

ion in a relatively small fraction of cells (mean8 6 16%; P 5 0.009) at anintensity 5.5-6.3-fold lower than that observed for the CD561lo

NK-cell population (P 5 0.009).The expression of the immunoglobulin-li

KB1 and CD158a killer receptors on CD561lo

NK-cells was highly variable (mean percentagpositive cells of 166 16 and 186 8%, rangingrom 0 to 47% and from 10 to 25%, respectivehereas both were constantly negativeD561hi NK-cells (P 5 0.026 and0.037, re-

spectively).In that concerning lectin-like killer recepto

virtually all CD561hi NK-cells expressed CD9whereas only around half (596 11%) of CD561lo

NK-cells showed dim CD94 expression, thetensity of expression for this molecule being 26 0.3-fold higher (P 5 0.007) andmore homogeneous (P , 0.001) onCD561hi comparedo CD561lo NK-cells. In contrast, reactivity foCD161 was relatively heterogeneous in both Ncell subsets, although the intensity of CD161pression was higher on CD561hi NK-cells (P 5

.004).

ostimulatory Molecules, Cytokine Receptorsnd Activation-Related Markers

All NK-cells constitutively expressed higevels of CD45RA. The intensity of CD45Rxpression was 1.7-6 0.7-fold higher (P 5.016) andmore homogeneous (P 5 0.026) onD561lo than on CD561hi NK cells. Although theast majority of NK-cells were CD45RO2, a fewD45RA1/CD45RO1 NK-cells were detecteithin both cell subsets, its percentage beigher among CD561hi NK-cells (126 5% ver-

sus 4 6 4%; P 5 0.026). In both caseD45RO1/CD45RA1 NK-cells expressed low

levels of CD45RA than CD45RA1CD45RO2

NK-cells, expression of CD45RO being costantly dim and heterogeneous.

CD122 was constitutively expressed on vially all NK-cells, its intensity of expression bei2.3- 6 0.6-fold higher on CD561hi NK-cellsP 5 0.002). FewCD561lo NK-cells coex-

pressed CD25 (16 1%) whereas a relatively hig
proportion of CD561hi NK-cells was CD251lo

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(22 6 9%) (P 5 0.002). Themean proportioof HLA-DR1 cells was also higher amo

D561hi NK-cells (836 8% versus 236 13%;5 0.002). Both NK-cell populations dis

layed similar patterns of CD38 expression (P ..05).

The CD28 costimulatory molecule was ctantly absent in the two NK-cell subsets hnalyzed in all blood samples studied, as didD40 ligand (CD154).

ISCUSSION

In the present study we confirm and extrevious observations as regards the presen

he blood from normal individuals of two distinopulations of NK-cells defined by different

evels of CD56 expression. While the CD561lo

subset constantly represent the major fractio

TA

Immunophenotypic Characteristics of CD561lo and CD561hi

(n 5 6): Intensity and Pattern

MFI

CD561lo CD561hi

CD2 1926 70 4766 77CD5 2056 33 NegativeCD7 15856 314 44096 1300CD8 4236 176 2306 66CD11a 5486 109 3436 67CD11b 916 37 616 18CD11c 1626 34 2586 84CD16 2366 161 476 39CD25 246 20 246 14CD38 1106 42 1466 46CD44 386 20 1456 31CD45RA 3086 35 1946 64CD45RO 376 7 636 18CD56 4886 148 30716 461CD57 6986 303 NegativeCD62L 276 7 496 16CD94 606 9 1616 43CD122 306 10 636 12CD158a 276 17 NegativeCD161 486 18 236 7HLA-DR 79 6 23 936 34NKB1 2256 72 Negative

Note.Results are expressed as means6 standard deviation ofof antigen expression on positive cells. MFI are expressed aswere constantly absent in NK-cells from the samples analyz

NK-cells, the CD561hi only accounts for a minor

736

roportion of CD561 NK-cells present in bloodInterestingly, these NK-cell subsets showedtinct light scatter properties, higher FSC and Svalues being found for the CD561hi NK-cells,supporting the existence of differences betwthese NK-cell subsets as regards both size ancell internal complexity/granularity. Additionallboth cell subsets displayed distinct immunopnotypic patterns for a great part of the surfproteins analyzed, which may be related totinct functional and migratory properties (9–1As an example, receptors that regulate adhesivascular endothelium and extracellular mamight contribute to a differential representationthese NK-cell subsets in blood as well as insues; in a similar way, differences on the expsion of killer receptors, NK-cell costimulatomolecules and cytokine receptors may havdeterminant role on the differential capacity

3

heral Blood NK-Cell Subsets from Adult Healthy Individualspression of Each Antigen Analyzed

alue

CV

P valueCD561lo CD561hi

02 556 8 386 5 0.004— 426 6 Negative —02 816 21 546 15 0.041

200 1156 13 1276 6 0.40002 326 2 316 3 0.240

222 1036 11 1076 18 0.54141 976 12 726 10 0.00909 456 12 656 22 0.132

589 1296 42 1076 20 1.000421 886 23 796 8 0.73002 836 22 486 13 0.03216 346 7 576 16 0.02616 1546 80 1146 12 0.69902 536 7 356 6 0.002— 1106 27 Negative —15 626 11 516 11 0.13207 656 5 386 3 0.00002 626 12 446 12 0.093

— 676 8 Negative —04 656 6 736 6 0.056

589 1536 63 1276 14 0.485— 466 9 Negative —

an fluorescence intensity (MFI) and the coefficient of variatiory relative linear units scaled from 0 to 10,000. CD3, CD28, a

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FIG. 2. Representative dot plots illustrating the distinct immunophenotypic patterns observed for the CD561lo (red dotsand CD561hi (black dots) blood NK-cells subsets from an adult healthy individual. Events painted in gray correspond
T-cells present in the sample.
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either activated or inhibited in response to stimthat trigger and regulate cytotoxic functions.

In that concerning to adhesion moleculesmay favor homing of NK-cells to different tisues, it should be noted that CD62L—the L-lectin adhesion molecule that mediates interacof leukocytes with peripheral lymph node hendothelial venules (HEV) (16, 17)—aCD44—a molecule that mediates adhesionacting as the hyaluronate receptor and promNK-cell homing by binding to mucosal HEV (119)—were preferentially expressed in CD561hi

compared CD561lo NK-cells in accordanceprevious observations (8, 20). These findimight at least in part explain why CD561hi more

fficiently bind to HEV than CD561lo NK-cells20) and could contribute to a preferential homf CD561hi NK-cells into different human tissu

where they are found at higher frequencies (3Other adhesion molecules, such as theb1-

ntegrins (21) have also been shown to beolved in the adhesion to vascular endothelnd transmigration across endothelial cellseans of different interactions, including thoetween CD11a and CD11b and their ligaCD54, CD102, and fibrinogen) (22–30). Inresent study we show, in accordance to prevtudies (8), that CD11a is expressed on NK-ct higher levels than those observed in other bells, a characteristic that probably contributehe greater migratory properties of NK-cells coared to other circulating lymphocyte subsevertheless, differences on the expressioD11a cannot explain the preferential local

ion of CD561hi compared to CD561lo NK-cellsn tissues, as the intensity expression of CD11lightly lower in the former NK-cell subset. Fhe same reasons, differences on the expressD11b can neither account for the higher mig

ory properties of CD561hi NK-cells. The mean-ng of the finding that CD11c is preferentiaxpressed on CD561hi NK-cells is not knownowever, in a similar way to that occurring fD11b (31, 32), both C3bi (33, 34), fibrinog

35) and CD54 (36) have been proposed aands for CD11c and this molecule has blaimed to be involved in adhesion to the en
helium and to the extracellular matrix (36), as
738

f

ell as to play a role in conjugate formatietween NK-cells and target cells (37). Theificance of the exclusive expressionD57—an oligosaccharide with sulfated glucoic acid residues that is expressed on T-curing advanced phases of immune response

hat presumably also mediates cell adhe38)—on CD561lo NK-cells is neither clear.

Besides determining adhesion propertiesigratory capabilities, adhesion molecules mlso be crucial for conjugate formation betwK-cells and target cells, a process that depen the homophilic binding mediated by CD39, 40) and on other pairs of receptors sucD11a/CD54 (41, 42), CD2/CD58 (43, 44), aD8/MHC-class I antigens (45, 46). According

he higher expression of CD56 and CD2 foundD561hi NK-cells may favor a closer interacti

between these NK-cells and their targets.Cytotoxicity depends on a delicate bala

between different groups of receptors that eiactivate or inhibit the NK-cell killer function(47–49) connecting to adapter moleculesturning on/off different signal-transducing paways (50–53).

CD16—thea-chain of the low-affinity receptor for the Fc portion of IgG (FcgRIII)—activatesytotoxicity on NK-cells through its bindingmmune-complexed IgG and plays a determinole in NK-cell proliferation and both antibodependent and antibody-independent cell cyto

city (54–59). Absence or low intensity of expreion of CD16 may explain at least to a certxtent why both blood and decidual CD561hi

NK-cells are less efficient in mediating noMHC-restricted cytotoxicity than the CD561lo

NK-cells (2, 60, 61). Antibody-independent Ncell cytotoxic activity is usually related to signmediated by multiple immunoglobulin-lik(CD158, NKB1; NKp44; NKp46) and lectin-lik(CD94, CD161) receptors which may act throuan MHC class I dependent (CD94, CD158,NKB1) or independent (CD161) pathway (666). Positive stimulation may be initiated throuthe activation forms of some of the MHC-depdent receptors, such as CD158 and CD94,other non-MHC restricted triggering molecul
such as CD161, whereas suppresser signals derive

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from inhibitory forms of the MHC-dependent rceptors mentioned above. As a consequencthese regulatory mechanisms, differences onexpression of killer receptors may determineferent MHC-dependent activator/suppressivequirements for CD561lo and CD561hi NK-cellubsets. For instance, the fact that CD561hi NK-

cells do not express CD158a and NKB1 whethey strongly express CD94, may determine sificity for non-classical HLA-E moleculewhereas the opposite pattern of expressioCD158a and NKB1 with low reactivity for CD9usually observed on CD561lo NK-cells wouldsuggest a preferential recognition of classMHC class I A, B, and C molecules by this csubset (67–69).

Higher levels of other molecules that exhNK-cell costimulatory functions on CD561hi NK-cells such as CD2 (70–72), CD7 (73, 74), CD(75, 76), and CD44 (77, 78), may also be relato a higher ability of NK561hi NK-cells to re-spond to different stimuli. By contrast, andaccordance to previous reports, we were notto detect surface expression of neither the C(79) nor the CD40L (CD154) (80) costimulatomolecules on NK-cells.

Interestingly, CD561hi NK-cells exhibited ahenotype suggestive of cell activation, asected by a higher fraction of HLA-DR1 andD251 cells (81, 82). The lower intensityxpression of CD45RA and the highest proporf CD45RA1/CD45RO1 cells among CD561hi

NK-cells may also be a signal of NK-cell activtion, since previous studies have shown thatvated NK-cells transiently acquire CD45ROphenomena that is accompanied by a decreaCD45RA expression (83).

NK-cells proliferate and display enhancedtotoxic activity in response to various cytokinand growth factors, including IL-2 (84–89). Cosequently, differences on the expression of ckine receptors would probably contribute todifferent ability of these NK-cell subsets toactivated and to proliferate in response to specytokines. The fact that CD122—IL-2b-chain—is constitutively expressed withhigher intensity on CD561hi NK-cells at the sam
ime that a higher fraction of these cells express
739

D25—IL-2R a-chain—could probably explawhy CD561hi NK-cells display a preferential r-ponse to low doses of IL-2 with increased cyytic activity and proliferation compared to tD561lo NK-cell subset (90–94).

In summary, our results show that CD561hi

and CD561lo NK-cells display distinct pheno-ypic characteristics suggesting that the forells may correspond to a previously activaK-cell subset with a different pattern of recoition of MHC molecules and different requirents to undergo proliferation and activation

ts cytotoxic functions at the same time that tould explain its higher ability to migrate inifferent human tissues.

ACKNOWLEDGMENTS

This work was partially supported by the followirants: Comissaõ de Fomento da Investigaçao em Cuidaos de Sau´de, Ministerio da Sau´de, Portugal (PI 51/99ccao Integrada Luso-Espanhola E31/99, Conselheitores das Universidades Portuguesas, Ministe´rio daducaçao, Lisbon (Portugal); Accioń Integrada Hispanousa HP1998-0091, Direccioń General de Ensenãnzauperior e Investigacioń Cientıfica, Ministerio de Eduacion y Cultura, Madrid (Spain); and FIS 99/12inisterio de Sanidad y Consumo, Madrid (Spain).

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Immunophenotypic Characterization of Normal Blood CD56 +lo Versus CD56 +hi NK-Cell Subsets and Its Impact on the Understanding of Their Tissue Distribution and Functional Properties

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