Self-tolerance of Human Natural Killer Cells LackingSelf-HLA-specific Inhibitory Receptors

J. Hasenkamp*, A. Borgerding*, M. Uhrberg�, C. Falk�, B. Chapuy*, G. Wulf *, W. Jung*, L. Trumper*& B. Glass*

Introduction

Natural killer (NK) cells recognize and destroy malig-nant, virus infected and haematopoietic allogeneic cellswithout prior immunization [1, 2]. These propertiesmade them a promising tool for anti-cancer immuno-therapy. However, without detailed knowledge about theregulation of NK cell reactivity, clinical approaches tobenefit from NK cell-mediated effects were disappoint-ing [3, 4]. Finding human leucocyte antigen (HLA)-spe-cific inhibitory NK cell receptors, namely lectin-likereceptor CD94 ⁄ NKG2A [5], long-tailed isoforms ofkiller cell immunoglobulin-like receptors (KIR) [6] andleucocyte immunoglobulin receptor (LIR-1) ⁄ immuno-globulin-like transcript (ILT2) [7, 8], resolved the ques-tion how NK cells survey the ‘missing self’ [9]. The‘missing self’ hypothesis is well in line with findingsregarding the graft-versus-leukaemia (GvL) effect afterhaplomismatch haematopoietic stem cell transplantation(HSCT) [10, 11]. This is the strongest evidence of a

clinical relevant NK cell-mediated anti-tumour effect sofar. It may even occur in a non-transplant setting afteradoptive transfer of alloreactive allogeneic NK cells [12]

and may contribute to GvL effects that are seen afterKIR–ligand (HLA-C) mismatched unrelated donorHSCT [13–15]. In addition, stimulatory NK cell recep-tors were found, e.g. natural cytotoxicity receptors(NCR) [16–18], NKG2D [5], DNAM-1 [19] and short-tailed KIR [6], that recognize stress-induced molecules,pathogen-encoded molecules or yet unknown ligands.All together, this lead to the current understanding oftarget cell recognition by NK cells. Whether NK cellsattack or tolerate a target cell depends on integration ofthe stimulatory and inhibitory signals provided by liga-tion of their surface receptors [20]. While stimulatoryNK cell receptors (e.g. NCR) seem to be expressed byall NK cells and some co-receptors are upregulated uponactivation [21], the expression of inhibitory receptors(e.g. KIR) is diverse and clonally distributed [6]. Thisleads to an individual repertoire of NK cells expressing

*Department of Haematology & Oncology,

Georg-August University of Goettingen,

Goettingen; �Institute for Transplantation

Diagnostics and Cell Therapeutics, University

Clinic of Dusseldorf, Dusseldorf; and �NCT –

National Centre for Tumor Diseases and

Institute for Immunology, University of

Heidelberg, Heidelberg, Germany

Received 3 July 2007; Accepted in revisedform 7 November 2007

Correspondence to: Dr J. Hasenkamp MD,

Department of Haematology & Oncology,

Georg-August University of Goettingen,

Robert-Koch-Str. 40, D – 37099 Goettingen,

Germany. E-mail: j.hasenkamp@med.

uni-goettingen.de

Abstract

Natural killer (NK) cells identify cells with altered human leucocyte antigen(HLA) expression as targets through lacking engagement of self-HLA-specificinhibitory receptors (e.g. killer cell immunoglobulin-like receptor, KIR).Thus, they eliminate cells with ‘missing self’ because of viral or malignanttransformation. We performed analysis of HLA, KIR genotypes and KIRreceptor expression patterns at single cell level in NK cells in 17 donors. Thefunction of NK cell subsets is determined by degranulation assays using targetcells expressing self, cognate, control or no HLA class I. Donors could begrouped into three groups: their NK cells possess potential for alloreactivity,autoreactivity based on the presence of NK cells expressing particular KIRonly (mono-KIR) in the absence of its ligand or lack alloreactivity. All donorspossess NK cells lacking all detectable inhibitory receptors. Both potentialautoreactive subpopulations did not respond to HLA class I-positive targetcells. They retain partial reactivity against HLA class I-negative tumour targetcells. Mono-KIR NK cells without the corresponding ligands in the indi-viduals and NK cells lacking all inhibitory receptors behave self-tolerant. Ourresults suggest alternative mechanisms than HLA-specific inhibitory receptorsto control NK cell activity. But HLA seems to be involved in shaping effectorfunction of the NK cell repertoire.

doi: 10.1111/j.1365-3083.2007.02058.x..................................................................................................................................................................

� 2008 The Authors

218 Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 218–229

various numbers of inhibitory receptors specific for HLAmolecules. The broadly expressed non-classical HLA-E isrecognized by heterodimer CD94 ⁄ NKG2A receptor [22].Inhibitory KIRs recognize subgroups of classical HLAclass I: KIR2DL2 and 2DL3 ligand is HLA-C group 1(HLA-CAsn80, C1). HLA-C group 2 (HLA-CLys80, C2) isligand to KIR2DL1. KIR3DL1 recognizes HLA-Bw4and KIR3DL2 ligands are HLA-A 3 and HLA-A 11.Until recently it was assumed that in humans every nor-mal NK cell expresses ‘at least one’ inhibitory receptorspecific for self-HLA to guarantee self-tolerance [23].HLA class I- and KIR-genes are encoded on distinctchromosomes 6p21.3 and 19q13.4 and inherited inde-pendently [24]. This raises questions how self-toleranceof individuals’ whole NK cell repertoire is assuredaccording to the ‘at least one’ hypothesis. Although,over- and under-representation of certain combination ofHLA class I and KIR haplotypes in distinct populationindicate an interrelation of HLA and KIR influencingsurvival [25]. As there is no disorder known which iscaused by autoaggressive NK cells, the mechanism thatcontrols expression of inhibitory receptors for preventingautoreactivity of NK cells must be exceptionally reliable.Moreover, reports of individuals with transporters associ-ated with antigen processing deficiency (resulting infunctional HLA deficiency) do not show an autoaggres-sive NK cell phenotype [26–28]. Recently, two groupsreported independently from NK cells lacking inhibitoryself-MHC-specific receptors in mice [29, 30]. Since then,both groups consider different mechanisms that assureself-tolerance of these NK cells (reviewed by Raulet [31]

and Yokoyama [32]). While in murine systems both lab-oratories refute the ‘at least one’ hypothesis, but favoureither a (non-)licensing model or hyporesponsiveness dueto an anergy status comparable to T-cell anergy, westarted in vitro experiments in humans describing NKcell repertoires in detail. We identified three distinctgroups in human, healthy adults with regard to theircell surface expression repertoire of inhibitory NK cellreceptors and HLA class I background. The identifiedpattern of cell surface expression of HLA-specific inhibi-tory receptors provide implications for NK cell-basedimmunotherapeutic approaches, as we found functionalconsequences. NK cell repertoires contained NK cellslacking self-HLA-specific inhibitory receptors. This find-ing is well in line with recent first report of human NKcells in peripheral blood lacking inhibitory HLA-specificreceptors. The authors describe occurrence and hypore-sponsive functionality of these cells [33]. We addition-ally investigated reactivity of these NK cells againsttarget cells with physiological HLA class I expression.Our study additionally analyses phenotype and function-ality including self-tolerance of NK cells with or with-out inhibitory KIR surface expression that do not findany appropriate ligands.

Material and methods

Cells and their characterization. Human K562 [34] (noexpression of HLA class I; derived from CML blastcrisis) were obtained from German Collection of Micro-organisms and Cell Cultures (DSMZ, Braunschweig,Germany). B-lymphoblastoid cell lines (B-LCL) werederived by in vitro transformation of normal human Bcells from HLA-typed healthy donors according to stan-dard protocol described elsewhere [35]. Before use theB-LCL were routinely tested for HLA class I surfaceexpression by flow cytometry. K562 and B-LCL weremaintained in RPMI-1640 medium with 25 mM

HEPES and GlutaMAX I (Gibco-BRL, Karlsruhe,Germany) containing 10% heat-inactivated foetal calfserum (Gibco-BRL) and supplemented with penicillin(Sigma, Steinheim, Germany) and streptomycin (Bio-chrom, Berlin, Germany; complete RPMI). Human NKcells were obtained by CD3, CD4, CD14, CD15,CD19, CD36, CD123 and CD235a immunomagneticdepletion (NK cell isolation kit II, Miltenyi, BergischGladbach, Germany) of freshly prepared mononuclearcells (MNC). Bulk NK cells were incubated 18 h with500 U ⁄ ml interleukin (IL)-2 (R&D, Minneapolis, MN,USA). To obtain subpopulations with single orno HLA-specific inhibitory receptors, bulk NK cellswere further depleted by anti-PE beads (Miltenyi) usingthe following monoclonal antibodies (mAb): CD158a-PE(clone HP-3E4), NKAT2-PE (clone DX27), NKB1-PE(clone DX9) (all Becton Dickinson, Heidelberg, Ger-many) and NKG2A-PE (clone Z199, Beckmann-Coulter,Krefeld, Germany). Procedure was followed by CD56-positive selection of NK cells (CD56 MicroBeads, Milt-enyi). For immuno-phenotyping the MNC, untouchedNK cells and depleted cell fractions the following con-jugated mAb were used: CD3-FITC (clone SK7), CD4-PE (clone SK3), CD8-FITC (clone SK1), CD16-FITC(clone NKP15), CD20-PE (clone L27), CD56-PE (cloneNCAM16.2), NKp46-PE (clone 9E2 ⁄ NKP46), CD314-PE (NKG2D, clone 1D11), CD226-PE (DNAM-1,clone DX11), CD337-PE (NKp30, clone p30-15),CD158a-FITC (KIR2DL ⁄ S1, clone HP-3E4), NKAT2-PE(KIR2DL ⁄ S2 ⁄ 3, clone DX27), NKB1-biotin (KIR3DL ⁄S1, clone DX9 with streptavidin-PerCP) (all fromBecton Dickinson) and NKG2A-PE (clone Z199, Beck-man-Coulter). PCR amplification of KIR genes andKIR-specific PCR-SSP typing of NK cell donors wereperformed as described previously [36] with primer pairmodifications [37]. HLA typing of donors includinghigh-resolution HLA-C typing with definite classifica-tion to C group 1 (Asn80, C1) and ⁄ or C group 2(Lys80, C2) was by PCR-Sequence-specific Oligonucleo-tide Probing, using LABType SSO Typing Tests (OneLambda). Student’s t-test was used to determine signifi-cant differences of groups.

J. Hasenkamp et al. Self-tolerance of Human NK Cells 219..................................................................................................................................................................

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Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 218–229

Functional assays for NK cell reactivity. Bulk NK cells orsubpopulations were used as effector cells in enzyme-linkedimmunospot (ELISPOT) assay for human granzyme B(GrB) [38]. The assay was performed as described previ-ously [39]: in brief, PVDF membrane 96-well plates(ImmunoSpot M200, C.T.L., Cleveland, OH, USA) werecoated with anti-GrB capture antibody [50 ll per well,10 lg ⁄ ml in phosphate-buffered saline (PBS), BectonDickinson] for 16 h at 4 �C. The plates were blocked with1% bovine serum albumin (BSA; Gibco-BRL) in PBS forat least 1 h at room temperature. After washing with com-plete RPMI, assays were performed by adding cells.1 · 104 target cells (2 · 105 ⁄ ml) were co-incubated withseveral dilutions of effector cells for 4 h at 37 �C and 5%CO2. As negative controls wells on same plate were loadedwith target cells and complete RPMI only. Backgroundspots per well were determined by incubating identicalnumber of effector cells without stimulator cells. As posi-tive control effector cells were stimulated on same platewith K562 or alternatively with phytohaemagglutinin(PHA; Roche, Basel, Switzerland), respectively. Bothresulted always in reasonable spots per well. All sampleswere implemented as triplicates. After washing plates withPBS containing 0.05% Tween (Sigma) 100 ll per well,biotinylated detection antibody (1 lg ⁄ ml in PBS with0.5% BSA; Becton Dickinson) was added and incubatedfor 1 h at room temperature. The plates were washed againand subsequently incubated for another hour with HRP-streptavidin (Becton Dickinson) diluted 1:100 in PBS con-taining 0.5% BSA. Spots were developed using 50 ll perwell freshly prepared 3-amino-9-ethylcarbazole chromogensubstrate in sodium acetate buffer and H2O2 (Sigma).Plates were analysed with automatic ELISPOT-Analyzer(C.T.L.).

In some cases we additionally determined reactivity ofNK cell preparations by CD107a mobilization assay. Theassay was performed as described previously [40] withmodifications: in brief, immuno-magnetically depletedNK cells were incubated with or without target cellsfor 1 h at 37 �C and 5% CO2 in the presence of PE-Cy5-fluorochrome-labelled monoclonal antibody againstCD107a (LAMP-1, Becton Dickinson). Effector to target(E:T) ratio was 1:2. After addition of 50 lM monensin(Sigma) cells were incubated for another hour at 37 �Cand 5% CO2. Cells were then immediately analysed byflow cytometry (FACScan, Becton Dickinson), determin-ing the proportion of CD107a-positive NK cells.

Results

Identification of NK cells lacking inhibitory receptors

specific for self-HLA

To determine whether NK cells generally express at leastone inhibitory receptor specific for self-HLA class I

molecules we studied the NK cell repertoire of 17 volun-teers with known HLA background. After enrichment ofbulk NK cells (purity CD56+CD3): >93%) the expres-sion and co-expression of inhibitory HLA-specific recep-tors were determined using multicolour flow cytometry(Fig. 1). Heterogeneous individual repertoires consistedof all possible inhibitory receptor combinations ofco-expression (Table 1). This included both NK cellsubpopulations lacking expression of any inhibitoryHLA-specific receptors (named hereafter KIR)NKG2A),present in 17 ⁄ 17 donors) and subpopulations withexpression of receptors whose corresponding HLA class Iligands are absent in the particular donors (eight of 17donors). Proportion of KIR)NKG2A) NK cell subpopu-lation ranged from 6.33% to 33.48% (mean 18.1%, SD7.8). In donor 8 and in donors 10–14, KIR genotypeslacked all stimulatory KIR (excepting KIR2DS4). Evenin these individuals the same subpopulations of NK cellswere detected with mAb specific for KIR when comparedto individuals whose KIR genotypes contained stimula-tory KIR (Tables 1 and 3). In some volunteers werepeated determination of phenotypic NK cell repertoireseveral times over periods up to more than 1 year. Therepertoires were stable over time and did not change(data not shown). The frequency of NK cells expressingdifferent KIR was not significantly influenced by thepresence of their particular ligands (HLA type, Table 2).In mean 36.33% (SD 18.06) NK cells showed surfaceexpression of KIR2DL1 in HLA-C2-positive individuals(n = 9) versus 26.82% (SD 12.99) in individuals homo-zygous for HLA-C1 (n = 8). KIR2DL2 ⁄ 3 expressionof NK cells in HLA-C1-positive individuals (n = 15)compared to expression in HLA-C1-negative (n = 2)individuals were 30.35% and 29.88%, respectively.HLA-Bw4-positive (n = 7) and -negative (n = 9) individ-uals NK cells showed mean KIR3DL1 surface expressionof 13.01% and 18.13%, respectively.

Three groups of individuals regarding inhibitory

HLA-specific receptor repertoire

We performed KIR genotyping and HLA typing of thesame 17 healthy volunteers. All individuals except one(donor 2 negative for KIR3DL1) were positive for theinhibitory receptors KIR2DL1, KIR2DL2 and ⁄ or 2DL3,KIR3DL1 and KIR3DL2 specific for HLA-C2, HLA-C1,HLA-Bw4 and HLA-A3,11, respectively. The KIR geno-types and corresponding HLA class I ligands of thesereceptors present in these individuals of donors 1–17 areshown in Tables 2 and 3. According to the constellationof surface expression of inhibitory NK cell receptors(Table 1) and appropriate HLA class I ligands, the donorsare clustered into three groups. In donors of group Ievery KIR phenotype defined subpopulation of NK cells(except KIR)NKG2A) NK cells) finds at least one

220 Self-tolerance of Human NK Cells J. Hasenkamp et al...................................................................................................................................................................

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 218–229

ligand. Nine of 17 donors (53%) tested belong to donorgroup I. In donors of group II we identified NK cell sub-populations with expression of a single inhibitory KIR(mono-KIR phenotype) missing its ligand in the particu-lar individual (Tables 1 and 2). Eight of 17 donors(47%) were missing at least one HLA class I ligand (2·HLA-C1, 6· HLA-C2, 7· HLA-Bw4) for self-expressedinhibitory KIR and thus harbour NK cells with potentialautoreactivity with regard to the KIR ⁄ KIR–ligand sys-tem. Groups I and II differ in the size of the potentialautoreactive repertoire: 14.52% (SD 6.88) of NK cells(namely KIR)NKG2A) NK cells) in group I individualsare missing a self-HLA-specific inhibitory receptor. Incontrast, group II donors NK cell repertoires consist of34.31% (SD 9.34) NK cells lacking self-HLA-specificinhibitory receptors (P = 0.000154). 85.5% (SD 6.89) ofNK cells in group I donors repertoires find an appropri-ate ligand to their HLA-specific inhibitory receptors incontrast to 65.69% (SD 9.34) in group II donors(P = 0.000153).

The donors of group I could be further subdivided. Ingroup Ia donors [1–6] every KIR–ligand corresponds toNK cell subpopulations exclusively inhibited by this par-ticular ligand. Group Ib donors [7–9] are lacking NKcell subpopulations exclusively inhibited by KIR3DL1.Despite the presence of the KIR–ligand HLA-Bw4, the

presence of the KIR3DL1 gene and co-expression of thereceptor together with other inhibitory receptors, themono-KIR3DL1 NK cell phenotype was not seen inthese individuals (Table 1).

NK cells lacking inhibitory receptors specific for self-HLA

behave self-tolerant

To further study the functional properties of NK cellsubpopulations with potential autoreactivity regardingthe KIR ⁄ KIR–ligand system, we analysed their reactivityagainst targets with natural KIR–ligand (HLA class I)expression and different HLA class I background. Toaddress this subject we first isolated different fractionsfrom bulk NK cells. After immuno-magnetic depletionof NK cells expressing all other HLA-specific inhibitoryreceptors the mono-KIR2DL1 or mono-KIR2DL2 ⁄ 3 sub-populations consisted of about 50% mono-KIR and 50%KIR)NKG2A) cells (Fig. 2). The KIR)NKG2A) NKcells could not be removed as they lack a known specificmarker. To evaluate their impact on the results wedepleted in parallel all NK cells bearing inhibitory HLA-specific receptors resulting in populations with purity of98% negative for all inhibitory HLA-specific receptors(Fig. 2). All effector cell preparations consisted of >97%CD56dimCD3) NK cells. Proportion of CD56bright NK

Figure 1 Evaluation of inhibitory receptor

repertoire by flow cytometry. Highly puri-

fied natural killer cells were stained with

fluorochrome-conjugated mAbs specific for

NKG2A, KIR2DL1, KIR2DL2 ⁄ 3 and

KIR3DL1 (right panels). Control staining

was performed with isotype-matched control

immunoglobulin (left panels). Lower right

dot plot is gated on KIR2DL1 and 2DL2 ⁄ 3negative cells. Given are percentages of

events in indicated areas. Data are from

donor 15, representative for 17 donors that

were phenotyped.

J. Hasenkamp et al. Self-tolerance of Human NK Cells 221..................................................................................................................................................................

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Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 218–229

Tab

le1

NK

cell

ssu

rfac

eex

pre

ssio

nof

HL

A-s

pec

ific

inh

ibit

ory

rece

pto

rs.

Don

orn

um

ber

Inh

ibit

ory

NK

cell

rece

pto

r

NK

cell

s

wit

hse

lf-H

LA-

spec

ific

rece

pto

r

NK

cell

sla

ckin

g

self

-HL

A-

spec

ific

rece

pto

r

NK

G2A

neg

ativ

eN

KG

2Ap

osit

ive

2D

L1

2D

L1

+

2D

L2

⁄32

DL

2⁄3

3D

L1

KIR

neg

ativ

e

2D

L1

2D

L1

+

2D

L2

⁄32

DL

2⁄3

3D

L1

KIR

neg

.+

3D

L1

+3

DL

1+

3D

L1

+3

DL

1+

3D

L1

+3

DL

1

IA

llN

Kce

lls

wit

hH

LA

-sp

ecifi

cin

hib

itor

yre

cep

tor

exp

ress

ion

self

-tol

eran

tan

dal

lo-r

eact

ive*

a1

5.3

54

.53

4.6

52

.48

4.0

03

.03

6.4

87

.43

6.1

13

.14

2.5

51

.80

5.6

42

.06

9.1

93

1.6

49

2.6

57

.43

21

4.3

10

.00

18

.65

0.0

09

.48

0.0

00

.00

16

.93

7.4

70

.00

3.0

00

.00

5.3

40

.00

0.0

02

4.8

28

3.0

71

6.9

3

32

7.5

48

.17

13

.47

8.6

51

0.4

35

.00

0.0

46

.33

2.2

00

.58

2.0

90

.80

0.9

50

.60

1.7

21

1.4

19

3.6

16

.37

47

.57

0.5

41

5.6

91

.58

17

.69

1.6

50

.03

10

.50

9.9

70

.76

6.6

80

.35

0.5

70

.42

1.4

52

4.5

98

9.5

01

0.5

3

50

.00

0.7

21

4.6

63

.33

7.4

82

.85

1.7

01

4.5

42

0.3

20

.86

0.0

00

.59

5.2

11

.40

1.8

92

4.5

48

5.5

41

4.5

4

61

.28

0.2

52

.56

1.6

07

.01

2.8

34

.71

23

.95

1.0

50

.17

0.2

70

.17

5.2

21

.54

4.0

74

3.3

37

6.0

62

3.9

5

b7

7.4

11

.14

10

.67

2.4

16

.85

1.0

40.

012

4.0

64

.20

0.0

33

.05

1.0

45

.70

0.4

11

.50

30

.49

75

.95

24

.06

81

.19

0.0

02

.25

0.0

95

.80

0.0

00.

041

7.6

13

.88

0.0

02

.08

0.9

31

5.7

50

.00

0.0

45

0.3

28

1.1

81

8.8

0

94

.12

0.9

74

.06

0.7

22

.77

0.6

00.

018

.05

25

.62

1.8

53

.73

0.7

48

.16

1.4

43

.17

34

.00

91

.96

8.0

5

Mea

n8

5.5

01

4.5

2

SD6

.89

6.8

8

IIP

rese

nce

ofN

Kce

lls

wit

hH

LA-s

pec

ific

inhi

bit

ory

rece

pto

rex

pre

ssio

nin

the

abse

nce

ofit

sli

gan

d(p

oten

tial

auto

-rea

ctiv

ity)

*

10

7.5

31

.70

5.3

02

.19

5.4

02

.76

1.2

21

6.6

11

2.3

41

.42

2.9

40

.43

4.6

10

.96

3.4

73

1.1

17

2.9

32

7.0

6

11

1.6

72

.94

2.8

43

.52

6.7

65

.13

5.4

22

0.5

32

.39

1.7

10

.60

0.1

44

.44

2.6

48

.52

30

.75

69

.44

30

.56

12

6.2

11

.19

11

.41

3.3

22

.98

1.8

71

.51

31

.57

7.1

30

.82

4.2

50

.45

0.1

40

.36

1.8

02

5.0

05

9.5

34

0.4

8

13

2.7

61

.06

4.2

01

.74

11

.72

8.9

49

.61

33

.48

0.7

00

.00

0.1

20

.30

4.2

41

.43

3.2

61

6.4

45

3.0

94

6.9

1

14

7.5

34

.06

8.8

53

.02

17

.17

3.6

89

.24

24

.90

1.8

10

.40

0.4

40

.00

1.8

80

.35

1.0

21

5.6

55

4.2

74

5.7

3

15

12

.84

2.5

91

3.4

61

.89

4.5

30

.98

3.1

01

7.0

86

.72

0.6

14

.00

0.1

10

.00

0.4

11

.81

29

.87

70

.08

29

.92

16

2.7

22

.39

4.6

31

.51

7.0

93

.74

3.9

61

8.7

51

.61

0.2

20

.00

0.3

34

.04

0.3

52

.60

46

.06

66

.46

33

.54

17

9.5

21

.54

15

.59

4.2

32

.98

1.0

10

.96

15

.31

12

.56

1.0

28

.16

1.1

74

.23

0.7

01

.50

19

.52

79

.74

20

.26

Mea

n6

5.6

93

4.3

1

SD9

.34

9.3

4

NK

,n

atu

ral

kil

ler;

HL

A,

hu

man

leuc

ocyt

ean

tige

n;

KIR

,k

ille

rce

llim

mu

nog

lob

uli

n-l

ike

rece

pto

r.

*A

ccor

din

gto

the

rece

pto

r–li

gan

dm

odel

.

NK

cell

sfr

om1

7d

onor

sw

ere

test

edfo

rsu

rfac

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pre

ssio

nof

inh

ibit

ory

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pto

rssp

ecifi

cfo

rH

LAas

illu

stra

ted

inF

ig.

1.

Th

en

um

ber

sre

pre

sen

tp

erce

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lN

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exp

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the

ind

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edre

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tors

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um

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sin

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tesu

bp

opu

lati

ons

wit

hou

tan

app

rop

riat

eli

gan

din

the

don

or(s

eeT

able

2).

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icn

um

ber

sin

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222 Self-tolerance of Human NK Cells J. Hasenkamp et al...................................................................................................................................................................

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Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 218–229

cells ranged from 0.54% to 2.29% (mean 1.28, SD 0.58)with no significant differences between the distinct sub-populations.

Reactivity of the three resulting NK cell subpop-ulations – mono-KIR2DL1, mono-KIR2DL2 ⁄ 3 andKIR)NKG2A) – against target cells expressing different

HLA class I molecules was assessed and compared to thereactivity of bulk NK cells (Fig. 3). Bulk NK cells fromdonor 1 – representative for group I individuals –strongly responded to the HLA class I-negative targetcell line K562. The NK cells showed also significantactivity against B-LCL target cells homozygous for eitherHLA-C1 or HLA-C2 whilst no reactivity was observedagainst targets with co-expression of HLA-C1 andHLA-C2 (Fig. 3A). Mono-KIR2DL1 or mono-KIR2DL2 ⁄ 3 NK cells from donor 1 did not release GrBwhen confronted with target cells expressing their appro-priate inhibitory ligands HLA-C2, HLA-C1 or both,whilst showing reasonable reactivity when stimulated bytargets expressing non-inhibitory HLA, no HLA (HLA Inegative) or by pharmacological stimulants (here PHA;Fig. 3C). The frequency of reactive mono-KIR NK cellsagainst B-LCL expressing non-inhibitory HLA-C groupis approximately only half of the frequency of cells react-ing against the HLA class I-negative control target cellline K562. Having in mind that half of the preparationof mono-KIR NK cells consists of NK cells lacking allinhibitory HLA-specific receptors (Fig. 2) we assumedthat these NK cells do not contribute to the reactivityagainst B-LCL targets. In fact, KIR)NKG2A) NK cellsdid not respond to stimulation by any B-LCL target cellused. Thus, NK cells lacking HLA-specific inhibitoryreceptors behave self-tolerant against HLA class I-express-ing targets, whilst showing conserved reactivity uponPHA stimulation and limited but reasonable reactivityagainst HLA class I-negative cell line K562 (Fig. 3C).

We then analysed the reactivity of NK cells fromdonors that harbour NK cells of potential autoreactivitywith regard to the KIR ⁄ KIR–ligand system (group II).Bulk NK cells from representative donor 10 showed sub-stantial reactivity upon stimulation with HLA class I-negative target cells. This individual who is homozygousfor HLA-C1 showed significant NK cell alloreactivityagainst B-LCL target cells homozygous for HLA-C2.Despite the presence of NK cells lacking any inhibitoryreceptor recognizing HLA-C1 we could not detect theirreactivity in bulk NK cells (Fig. 3B). To further analysethe reactivity of NK cells lacking inhibitory receptors forself-HLA class I, we again generated mono-KIR popula-tions. Consistent with results obtained in donor 1, mono-KIR2DL2 ⁄ 3 NK cells that find their ligand in donor 10were stimulated by target cells expressing non-inhibitoryHLA-C2 or no HLA class I (Fig. 3D). Same NK cellsdid not respond if target cells express appropriate ligandHLA-C1. In contrast, mono-KIR2DL1 NK cells fromdonor 10 were inactive not only when stimulated bytarget cells with appropriate ligand HLA-C2, but alsowhen stimulated by B-LCL target cells expressing inap-propriate HLA-C1 only. Mono-KIR2DL1 as well asKIR)NKG2A) NK cells exerted limited reactivity againstHLA class I-negative target cell K562. Remarkably,

Table 2 HLA class I (inhibitory KIR–ligands).

Donor number

KIR Iigands (HLA class I)

A Bw4 Bw6 C1 C2

1 A3, 32 B44 B7 Cw * 07 Cw * 05

2 A1, – B8, 62 Cw * 07 Cw * 04

3 A3, 24 B7, 61 Cw * 07 Cw * 02

4 A2, 3 B7, 35 Cw * 07 Cw * 04

5 A2, 3 B27 B7 Cw * 01,07

6 A24, 26 B38 B35 Cw * 12 Cw * 04

7 A1, – B37 B62 Cw * 03 Cw * 06

8 A2, 24 B44 B62 Cw * 03, 07

9 A1, 2 B44 B8 Cw * 07 Cw * 05

10 A2, 26 B7, 55 Cw * 03, 07

11 A2, 24 B7, 39 Cw * 07

12 A1, 31 B7, 8 Cw * 07

13 A1, 2 B7, 8 Cw * 07

14 A3, 24 B7, 18 Cw * 07

15 A1, 3 B49 B8 Cw * 07

16 A24, 32 B35, – Cw * 04

17 A2, 24 B35, 55 Cw * 04

HLA, human leucocyte antigen; KIR, killer cell immunoglobulin-like

receptor.

All 17 individuals were HLA typed. Bold letters represent appropriate

HLA class I (KIR ligands) to inhibitory KIR. Standard and no letters

indicate absence of ligands to inhibitory KIR.

Table 3 KIR genotypes.

Donornumber

1234567891011121314151617

Inhibitory KIR Stimulatory KIR

2DL1

2DL2

2DL3

3DL1

3DL2

3DL3

2DL4

2DL5

2DS1

2DS2

2DS3

3DS1

2DS4

2DS5

KIR, killer cell immunoglobulin-like receptor.

All 17 donors were KIR genotyped. Dark boxes indicate the presence

of KIR genes. Blank boxes indicate absence of the KIR gene.

J. Hasenkamp et al. Self-tolerance of Human NK Cells 223..................................................................................................................................................................

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Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 218–229

using identical cell numbers of mono-KIR2DL2 ⁄ 3 NKcells response to B-LCL expressing HLA-C2 was half ofthat against HLA class I-negative targets. This againdemonstrates that only the KIR-positive NK cells in themono-KIR2DL2 ⁄ 3 preparation (�50%) were stimulatedby HLA-positive targets, whilst KIR)NKG2A) NK cellsdid not react to HLA class I-positive targets but pre-served reactivity to HLA class I-negative K562 cells.

To further confirm the different reactivity of mono-KIR and KIR)NKG2A) NK cells against HLA class I-negative target cells, we performed CD107a mobilizationassays. In this FACS-based assay KIR)NKG2A) NKcells in the mono-KIR2DL1 preparations can be discrim-inated from mono-KIR2DL1 NK cells by stainingKIR2DL1. Donor 1 does not harbour mono-KIR NKcells with potential autoreactivity with regard to theKIR system. KIR2DL1 expressing cells in mono-KIR2DL1 preparation of donor 1 respond strongly (56%degranulating cells) to K562. Reactivity of theKIR)NKG2A) NK cells is significant (26% degranula-

ting cells) but lower as in KIR+ NK cells (Fig. 4A).K562 stimulation of same NK cell subpopulations butfrom donor 10 (Fig. 4B), whose mono-KIR2DL1 NKcells find no appropriate ligand, resulted in equally lowreactivity of both subpopulations (13% versus 9%degranulating cells). This reveals that both NK cell sub-populations of donors from group II – KIR)NKG2A)

and mono-KIR NK cells missing their ligand in theindividual – show the same functional behaviour. Theyact self-tolerant, but have conserved limited cytotoxicresponsiveness against HLA class I-negative target cells.

Absence of alloreactivity, if appropriate mono-KIR

subpopulation is missing in the NK cell repertoire

In individuals of group Ib, NK cell subpopulationsexclusively expressing KIR3DL1 are absent, despite thepresence of its ligand HLA-Bw4 (Tables 1 and 2). Thisraises the question whether NK cells from these individ-uals lack alloreactivity to HLA-Bw4-negative targets.

Figure 2 Separation of natural killer (NK) cell population expressing no or single inhibitory human leucocyte antigen (HLA)-specific receptor. Repre-

sentative the bulk NK cell repertoire of donor 1 is given in table on top. Subpopulations that were enriched are surrounded. NK cells expressing

unwanted inhibitory HLA-specific receptors (NKG2A, KIR3DL1 and KIR2DL1 or KIR2DL2 ⁄ 3) were depleted. Dot plots show purity of the result-

ing NK cell subpopulations. Numbers indicate percentage of events in each quadrant.

224 Self-tolerance of Human NK Cells J. Hasenkamp et al...................................................................................................................................................................

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Bulk NK cells from the representative donor seven werestimulated in GrB ELISPOT with target cells expressingdifferent pattern of HLA class I ligands to inhibitoryreceptors including HLA-Bw4-negative stimulator cells.As expected, no NK cell alloreactivity is detected, ifHLA-Bw4 is the only inhibitory receptor ligand missingon the target cells. Substantial reactivity occurs againsttargets lacking other HLA class I ligands to inhibitoryreceptors (here C1 or C2) present in the donor (Fig. 3E).

Surface expression of stimulatory receptors on NK cell

subpopulations

A potential mechanism leading to self-tolerance of NKcells lacking inhibitory self-HLA-specific receptor expres-sion is defective presence of stimulatory receptors. Surfaceexpression on NK cell subpopulations described above ofNKp30, NKp46, NKG2D and CD226 (DNAM-1) asimportant stimulatory receptors of NK cells was deter-mined. Virtually all NK cells showed surface expressionof the stimulatory receptors examined. No significantdifferences in phenotype of KIR)NKG2A), mono-KIRfrom group I and II donors NK cells were detectable(data not shown).

Discussion

We here characterize human NK cell repertoires accordingto their inventory of inhibitory HLA-specific receptors andthe presence or absence of their ligands in these individu-

als. We clustered the healthy adults into three groups. Ingroup I individuals (nine of 17) all NK cells with inhibi-tory KIR surface expression match appropriate and presentligands. This group can be further subdivided: in group Ia(six of nine) additionally every ligand is detected by a NKcell subpopulation exclusively and specifically inhibitedthrough the particular ligand. In group Ib (three of nine)mono-KIR3DL1 subpopulations remain absent in the pres-ence of the corresponding inhibitory KIR gene and itsligand. Individuals were assigned to group II (eight of 17)on account of presence of NK cells, which express a singleinhibitory KIR (mono-KIR) not matching any HLA mole-cule of the respective individual. Our functional analysis ofNK cells from group I individuals confirmed the predict-able reactivity – including presence and absence of allo-reactivity – according to the receptor–ligand model [41].In line with previous reports [42, 43], genetic analysis hasto be combined with phenotypic analysis when predictingthe reactivity of NK cell repertoires. Following the recep-tor–ligand model, NK cells from group II individualswould act self-reactive. The three groups allow implica-tions for allogeneic HSCT. Assuming group Ia individualsas donors with KIR–ligand mismatch, occurrence of NKcell alloreactivity can be expected. Whereas group Ibdonors are expected to fail NK cell alloreactivity despiteappropriate KIR ⁄ KIR–ligand mismatch of KIR3DL1 andHLA-Bw4.

Could group II donors’ mono-KIR NK cells possespotential to exert reactivity additionally in KIR–ligandmatch settings? In a small series of patients, assumed

A B

C

E

DFigure 3 Functional reactivity of bulk natu-

ral killer (NK) cells and subpopulations from

group Ia, Ib and II donors. Reactivity of

NK cells were studied in GrB ELISPOT. NK

cells were stimulated with B-lymphoblastoid

cell line expressing the indicated human

leucocyte antigen (HLA) class I, HLA class

I-negative cell line K562, phytohaemaggluti-

nin and obligatory controls (not shown). Dia-

grams show GrB secreting NK cells per 104

above background (NK cells without stimu-

lation) with error bars indicating standard

deviation of triplicates. Data are from donor

1 (A and C), representative for three donors

tested of group Ia; donor 7 (E), representative

for group Ib and donor 10 (B and D), repre-

sentative for three donors tested of group II.

J. Hasenkamp et al. Self-tolerance of Human NK Cells 225..................................................................................................................................................................

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Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 218–229

A

B

Figure 4 Degranulation of natural killer (NK) cell subpopulations stimulated by K562. MonoKIR2DL1 NK cells (see Fig. 2) were stimulated with

(middle) or without (left) human leucocyte antigen class I-negative K562. Dot plots show expression of KIR2DL1 and CD107a. Numbers indicate

percentage of events in each quadrant. Histograms (right) give percentage of degranulating KIR) (upper) or KIR+ (lower) NK cells. Data are from

donor 1, representative for group I (A) and donor 10, representative for group II (B). Experiments were independently replicated twice and data are

representative for three different donors of each group tested.

226 Self-tolerance of Human NK Cells J. Hasenkamp et al...................................................................................................................................................................

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mono-KIR NK cells with potential to self-reactivityaccording to the receptor–ligand model, may protectfrom relapse [44]. Our results from functional analysis ofsuch NK cells argue against a potential of mono-KIRNK cells from group II individuals for immunotherapeu-tic interventions. We here show that they act self-toler-ant and in addition anergic against every used HLA classI-expressing B-LCL target cell. But we cannot excludethat here performed detection and functional analysis ofNK cell subsets in patients undergoing HLA-matchedHSCT would show that developing NK cells after HSCTmay behave different from NK cells extracted fromhealthy individuals.

In all individuals we consistently detected NK cellsubpopulations lacking self-HLA-specific inhibitoryreceptors at all (KIR)NKG2A) NK cells), as a secondpotentially self-reactive NK cell population. Such NKcells readily degranulated after pharmacological stimulusand showed hyporesponsiveness against HLA-negativecells. These findings coincide with those of Anfossi et al.[33], who recently reported KIR)NKG2A) NK cells toshow low activity against HLA-negative target cells inthe human system, and correspond to findings in themouse system in which NK cells lacking self-MHC-spe-cific inhibitory receptors were first discovered [29, 30]. Inaddition to Anfossi et al. we demonstrate for the firsttime that these cells were completely anergic againsttarget cells with natural HLA class I expression.

How can we explain the detection of such NK cells innormal subjects despite the fact that their inhibitoryreceptor equipment would allow autoreactivity?

First, technical issues are unlikely to contribute to ourfindings. We excluded in our experiments – alike others[33] – the known inhibitory receptors KIR3DL2,LIR-1 ⁄ ILT2 ⁄ CD85j and KIR2DL4 specific for HLA-A 3,11 and HLA-G, respectively. FACS analysis ofKIR)NKG2A) NK cells showed that approximately40% were negative for CD85j (data not shown). Unde-tected expression of KIR3DL2 alone, however, cannotexplain our observation, as we found NK cells negativefor all other known HLA-specific inhibitory receptors inHLA-A3- and HLA-A11-negative individuals at similarfrequencies as in HLA-A 3-positive subjects. As forligands to KIR2DL4, HLA-G expression in non-patho-logical conditions is tissue restricted to certain specialsites in humans, i.e. extravillous cytotrophoblast, thymicepithelial cells and cornea [45–47]. So, a possibleco-expression of KIR2DL4 or CD85j in general couldnot ascertain self-tolerance. Even taking into account thatCD85j is able to bind some HLA class I molecules [48]

(including HLA-A2 [49], HLA-B44 [8] and HLA-A0301, HLA-B0702, HLA-B2702, HLA-B2705, HLA-B5101[7]) – but surely to a much lesser extend than toHLA-G or the viral coded equivalent UL18 – this wouldbe irrelevant in individuals negative for such HLA class I

alleles, as were included in our study. Furthermore, themAbs used for KIR cell surface expression analysis donot exclusively detect the inhibitory KIRs, but also bindto their stimulatory, short-tailed isoforms. Nevertheless,detection of stimulatory KIR instead of the inhibitoryisoform does not interfere with the conclusion of identifi-cation of NK cells lacking inhibitory receptors specificfor self-HLA. In addition, individuals were included inthis study lacking stimulatory KIR (except KIR2DS4) intheir genome. Finally, at this point of time we cannotexclude the existence of yet unknown HLA-specificinhibitory receptors as well as non-HLA-specific inhibi-tory receptors that might contribute to self-tolerance.

Secondly, yet other mechanisms of NK cell regulationmay account for the emergence of KIR)NKG2A) andmono-KIR cells. We demonstrated for the first time thatthe identified human NK cell subpopulations behaveself-tolerant, despite lacking inhibitory receptors specificfor self-HLA. The KIR)NKG2A) and mono-KIR NKcells retained, however, functional reactivity against HLAclass I defective target cells. These results are in line withthe observation of others, describing hyporesponsivenessof human KIR)NKG2A) NK cells [33]. Both studiesfound no evidence for defective stimulatory receptorexpression to explain the hyporesponsiveness of such NKcells. Hyporesponsiveness of KIR)NKG2A) NK cellspossibly represent an immature state, as reported recently[50]. Autoreactive NK cells might be specifically sup-pressed by regulatory cells similar to the inhibition ofautoreactive T cells by CD4+CD25+ regulatory T cells(Tregs). The existence of regulatory cells is at leastrestricted to cells with identical phenotype of the poten-tially autoreactive NK cells with regard to the hereexamined surface markers. Other cell types would havebeen depleted by our cell processing. Alternatively theregulatory cells must have delivered previously in vivo anirreversible or long-lasting inhibitory effect.

Our results support two major models to explainself-tolerance of NK cells lacking self-MHC-specificinhibitory receptors: (1) (non-)licensing (or arming) ofNK cells [30] (reviewed by Yokoyama [32]). Only NKcells that acquire self-MHC-specific inhibitory receptorexpression during their development get a license forcytotoxicity. Thus, KIR)NKG2A) NK cells never getlicensed and therefore remain non-reactive against anytarget cell. (2) Anergy ⁄ hyporesponsiveness (or disarm-ing) of NK cells [29] (reviewed by Raulet [31]). NKcells that fail expressing inhibitory receptor specific forself-MHC during their development receive continu-ously stimulating signals because of normal activatingreceptor expression. This leads to a status of hypo-responsiveness or anergy.

The underlying molecular and cellular mechanismsleading to self-tolerance of NK cells lacking self-HLA-specific inhibitory receptors remain incompletely

J. Hasenkamp et al. Self-tolerance of Human NK Cells 227..................................................................................................................................................................

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understood and further elucidation is needed. Under-standing these mechanisms may eventually allowdeveloping strategies for improved recruitment ofKIR)NKG2A) and mono-KIR NK cells to the defenceagainst viral and malignant diseases.

Acknowledgment

We thank Tobias Wommelsdorf, Susan Dingeldein andBarbara Simm for excellent technical assistance. Weappreciate the cooperation of the volunteers involved inthis study.

Supported by grants to J.H., B.G., M.U. and C.F.from Deutsche Krebshilfe, Dr Mildred Scheel Stiftungfur Krebsforschung within the research network ‘Immunetherapy of malignant disease by transplantation of alloge-neic hematopoietic stem cells’.

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